Proteopedia - User contributions [en]

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-28T18:11:32Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|300 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1078124/Ace2andspikecomplex/1'>binds to ACE2</scene>, giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of [https://docs.rosettacommons.org/docs/latest/Home antibodies]. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/4'>preventing association of the viral cell with ACE2.</scene>
[[Image:AHB2_Method.png|300 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]
[[Image:LCB_Method.png|300 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]
===The Process of Discovery===
The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the [https://docs.rosettacommons.org/docs/latest/Home Rosetta Blueprint] protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.
[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]
De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

All in all, minibinders designed to inhibit binding of ACE2 to the spike protein have revealed promising potential. The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD, and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine, as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability, and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2 was that it was specifically made from ACE2, making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors is identifying the residues that will increase affinity to the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-28T18:11:01Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|300 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1078124/Ace2andspikecomplex/1'>binds to ACE2</scene>, giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of [https://docs.rosettacommons.org/docs/latest/Home antibodies]. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/4'>preventing association of the viral cell with ACE2.</scene>
[[Image:AHB2_Method.png|300 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]
[[Image:LCB_Method.png|300 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]
===The Process of Discovery===
The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the [https://docs.rosettacommons.org/docs/latest/Home Rosetta Blueprint] protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.
[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]
De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.<ref name="Cao">DOI:10.1126/science.abd9909</ref>

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

All in all, minibinders designed to inhibit binding of ACE2 to the spike protein have revealed promising potential. The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD, and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine, as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability, and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2 was that it was specifically made from ACE2, making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors is identifying the residues that will increase affinity to the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-28T18:01:40Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|300 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1078124/Ace2andspikecomplex/1'>binds to ACE2</scene>, giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of [https://docs.rosettacommons.org/docs/latest/Home antibodies]. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/4'>preventing association of the viral cell with ACE2.</scene>
[[Image:AHB2_Method.png|300 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]
[[Image:LCB_Method.png|300 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]
===The Process of Discovery===
The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the [https://docs.rosettacommons.org/docs/latest/Home Rosetta Blueprint] protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.
[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]
De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

All in all, minibinders designed to inhibit binding of ACE2 to the spike protein have revealed promising potential. The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD, and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine, as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability, and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2 was that it was specifically made from ACE2, making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors is identifying the residues that will increase affinity to the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-28T18:00:44Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|300 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1078124/Ace2andspikecomplex/1'>binds to ACE2</scene>, giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of [https://docs.rosettacommons.org/docs/latest/Home antibodies]. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/4'>preventing association of the viral cell with ACE2.</scene>
[[Image:AHB2_Method.png|300 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]
[[Image:LCB_Method.png|300 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]
===The Process of Discovery===
The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the [https://docs.rosettacommons.org/docs/latest/Home Rosetta Blueprint] protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.
[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]
De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

All in all, minibinders designed to inhibit binding of ACE2 to the spike protein have revealed promising potential. The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD, and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine, as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability, and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2 was that it was specifically made from ACE2, making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors is identifying the residues that will increase affinity to the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-28T17:57:03Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|300 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1078124/Ace2andspikecomplex/1'>binds to ACE2</scene>, giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of [https://docs.rosettacommons.org/docs/latest/Home antibodies]. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/4'>preventing association of the viral cell with ACE2.</scene>
[[Image:AHB2_Method.png|300 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]
[[Image:LCB_Method.png|300 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]
===The Process of Discovery===
The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the [https://docs.rosettacommons.org/docs/latest/Home Rosetta Blueprint] protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.
[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]
De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

All in all, minibinders designed to inhibit binding of ACE2 to the spike protein have revealed promising potential. The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD, and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine, as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability, and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2 was that it was specifically made from ACE2, making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors is identifying the residues that will increase affinity to the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-28T17:48:35Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|300 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1078124/Ace2andspikecomplex/1'>binds to ACE2</scene>, giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of [https://docs.rosettacommons.org/docs/latest/Home antibodies]. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/3'>preventing association of the viral cell with ACE2.</scene>
[[Image:AHB2_Method.png|300 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]
[[Image:LCB_Method.png|300 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]
===The Process of Discovery===
The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the [https://docs.rosettacommons.org/docs/latest/Home Rosetta Blueprint] protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.
[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]
De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

All in all, minibinders designed to inhibit binding of ACE2 to the spike protein have revealed promising potential. The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD, and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine, as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability, and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2 was that it was specifically made from ACE2, making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors is identifying the residues that will increase affinity to the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-28T17:42:11Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|300 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1078124/Ace2andspikecomplex/1'>binds to ACE2</scene>, giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/3'>preventing association of the viral cell with ACE2.</scene>
[[Image:AHB2_Method.png|300 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]
[[Image:LCB_Method.png|300 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]
===The Process of Discovery===
The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the [https://docs.rosettacommons.org/docs/latest/Home Rosetta Blueprint] protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.
[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]
De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

All in all, minibinders designed to inhibit binding of ACE2 to the spike protein have revealed promising potential. The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD, and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine, as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability, and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2 was that it was specifically made from ACE2, making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors is identifying the residues that will increase affinity to the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-22T20:04:12Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|300 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1078124/Ace2andspikecomplex/1'>binds to ACE2</scene>, giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/3'>preventing association of the viral cell with ACE2.</scene>
[[Image:AHB2_Method.png|300 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]
[[Image:LCB_Method.png|300 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]
===The Process of Discovery===
The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.
[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]
De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

All in all, minibinders designed to inhibit binding of ACE2 to the spike protein have revealed promising potential. The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD, and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine, as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability, and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2 was that it was specifically made from ACE2, making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors is identifying the residues that will increase affinity to the spike protein<ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

File:ACE2andRBD general.pdb

2025-04-22T19:34:28Z

Matthew Pereira: ACE2 in complex with spike protein trimer

ACE2 in complex with spike protein trimer

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-22T19:18:04Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/3'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/2'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/2'>LCB1</scene> and <scene name='10/1078124/Lcb3_general/1'>LCB3</scene> mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

The limitation behind the design of AHB2, it was specifically made from ACE2 making it an inhibitor that can't be universally used to fight other viruses. The biggest limitation regarding the de novo designed inhibitors, is there is a large protein bank that is used as reference when creating the inhibitors. With an abundance of inhibitors created with varying mutations, difficulty arises when determining which mutations will be most effective for the specific virus being neutralized in a steadfast manner <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-22T19:12:03Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/3'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/1'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the <scene name='10/1078124/Lcb1_general/1'>LCB1</scene> and LCB3 mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-22T19:07:11Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/3'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/1'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking method with in silico mini-proteins was used with a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the different LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/4'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine as it still had a higher binding affinity to the RBD compared to ACE2. All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>. The use of mini-inhibitors through the de novo protein design has also shown results in being able to control cell function and detecting protein activity within the cell <ref name= "Weinberg">PMID:38293112</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

[6]https://www.rcsb.org/structure/7JZM - SARS-CoV-2 spike in complex with LCB3 (local refinement of the RBD and LCB3)

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-22T18:54:11Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

[https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], better known as Covid-19, sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/3'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called <scene name='10/1078124/Ahb2_general/1'>AHB2</scene>. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/3'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1078124/Lcb3/1'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==
[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>. While AHB2 wasn't as effective as LCB1 and LCB3 at neutralizing the virus, it still showed results of being an effective vaccine as it still had a higher binding affinity to the RBD compared to ACE2.All three inhibitors show more advantages as a vaccine compared to antibodies due to their small size, stability and their ability to be quickly modified <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-22T18:43:25Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, [https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/3'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1078124/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1078124/Ahb2/1'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1078124/Lcb1/1'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

[[Image:Comparison.jpeg|400 px|right|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders.

==Conclusion==

The inhibitors LCB1 and LCB3 were shown to be the most effective at inhibiting the virus by having the highest affinity for the RBD and as well as the highest neutralization effect as seen in figure 5 <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:KdValues.jpg|400 px|right|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3<ref name="Cao">DOI:10.1126/science.abd9909</ref>.]]

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

User:Matthew Pereira/Sandbox 1

2025-04-22T18:39:44Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>
===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/3'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Conclusion==

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

User:Matthew Pereira/Sandbox 1

2025-04-22T18:34:02Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/3'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Conclusion==

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-22T18:31:37Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme [https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme angiotensin converting enzyme 2] <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, [https://en.wikipedia.org/wiki/SARS-CoV-2 SARS-CoV-2], enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/3'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Conclusion==
[[Image:KdValues.jpg|400 px|left|thumb|Figure 5: The binding affinity values (Kd) and the half maximum inhibitory concentration values (IC50) are shown for ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB - SARS-CoV-2 spike in complex with AHB2-2GS-SB175 (local refinement of the RBD and AHB2)

[2]https://www.rcsb.org/structure/8YZC - Structure of BA.2.86 spike protein in complex with ACE2

[3]https://www.rcsb.org/structure/7JZL - SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]https://www.rcsb.org/structure/6LZG - Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]https://www.rcsb.org/structure/7CDI - Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

User:Matthew Pereira/Sandbox 1

2025-04-22T18:10:58Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. Image created using BioRender.]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid [https://www.who.int/europe/emergencies/situations/covid-19 transmission and infection]. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be interrupted <ref name=”Zhu”>PMID:36682464</ref>.

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/4'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/7'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name=”Zhang”>PMID:36934742</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2 <ref name="Cao">PMID:32907861</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao"/>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/3'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Conclusion==

</StructureSection>

==References==
<references/>

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-15T19:43:51Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/2'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1078124/Spike_protein/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/2'>preventing association with ACE2</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-15T19:38:18Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/2'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">PMID:36562937</ref>. The virus, SARS-CoV-2, enters our bodies containing a <scene name='10/1077473/Rbd_only/2'>spike protein</scene> protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name="Jackson">PMID:34611326</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/2'>preventing association with ACE2</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-15T19:36:26Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/2'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Ace2andrbd2/1'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/2'>preventing association with ACE2</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-15T19:30:08Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/2'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_monomer/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/2'>preventing association with ACE2</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-15T19:20:17Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/2'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/2'>preventing association with ACE2</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-15T19:15:49Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/2'>preventing association with ACE2</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-15T19:12:12Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1078124/Spikeblockedbyminibinder/1'>preventing association with ACE2</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-15T18:57:10Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL):SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells. Receptor binding domains (RBDs) are highlighted at the top of each monomer.' scene='10/1078124/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

User:Matthew Pereira

2025-04-15T18:15:18Z

Matthew Pereira:

* Full Real Name: Matthew Pereira

* Position: Student

* Institution (NO ABBREVIATIONS): Butler University

* City, State/Province, Country: Indianapolis, Indiana, United States

* Field of Expertise or Study: Biochemistry

[[User:Matthew Pereira/Sandbox 1]]

[[Engineered Protein Inhibitors of SARS-CoV-2 Entry]]

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:53:35Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these De Novo proteins is their high stability, allowing for less delicate forms of administration. Additionally, it was found that the Rosetta built minibinder had a lower thermal stability than the completely De Novo proteins. Looking at the <scene name='10/1078124/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>, there are only 4 key internal residues significantly contributing to stability, and they are not oriented directly towards the center of the protein for optimal interaction. Comparatively, the <scene name='10/1078124/Lcb1_internal_stability/1'>nonpolar core of LCB1</scene> and <scene name='10/1078124/Lcb3_internal_stability/1'>LCB3</scene> had 5 key internal residues more centrally directed contributing to stability.

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:43:35Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor (7JZL & 7UHB).]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors (7JZL).]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078124/Ahb2_internal_stability/1'>Nonpolar core of AHB2</scene>

<scene name='10/1078124/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:38:59Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078124/Ahb2_internal_stability/1'>Nonpolar core of AHB2</scene>

<scene name='10/1078124/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:37:25Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078124/Ahb2_internal_stability/1'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:34:33Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/3'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/3'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/5'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:23:17Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
De novo design of picomolar SARS-CoV-2 miniprotein inhibitors - PubMed https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Advances in developing ACE2 derivatives against SARS-CoV-2 - PubMed https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:11:17Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.

[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)

[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2

[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:10:14Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB
[2]RCSB PDB - 8YZC: Structure of BA.2.86 spike protein in complex with ACE2.
[3]RCSB PDB - 7JZL: SARS-CoV-2 spike in complex with LCB1 (2RBDs open)
[4]RCSB PDB - 6LZG: Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2
[5]RCSB PDB - 7CDI: Crystal structure of SARS-CoV-2 antibody P2C-1F11 with RBD

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T03:01:43Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

</StructureSection>

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

Engineered Protein Inhibitors of SARS-CoV-2 Entry

2025-04-14T02:54:10Z

Matthew Pereira: New page: <StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the vi...

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

</StructureSection>

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

User:Matthew Pereira

2025-04-14T02:53:56Z

Matthew Pereira:

User:Matthew Pereira

2025-04-14T02:49:12Z

Matthew Pereira:

New Test

2025-04-14T02:41:30Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein <scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

</StructureSection>

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

New Test

2025-04-14T02:34:46Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein<scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078118/Lcb3_internal_stability/1'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

</StructureSection>

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

New Test

2025-04-14T02:29:10Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein<scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078099/Lcb3_internal_stability/2'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

</StructureSection>

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

New Test

2025-04-14T02:26:05Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein<scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078099/Lcb3_internal_stability/2'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

</StructureSection>

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

New Test

2025-04-14T02:24:12Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein<scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078118/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>

<scene name='10/1078099/Lcb3_internal_stability/2'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

New Test

2025-04-14T02:21:23Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein<scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/2'>Nonpolar core of AHB2</scene>

<scene name='10/1078099/Lcb1_internal_stability/2'>Nonpolar core of LCB1</scene>

<scene name='10/1078099/Lcb3_internal_stability/2'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

New Test

2025-04-14T02:09:54Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein<scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scene name='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/1'>Nonpolar core of AHB2</scene>

<scene name='10/1078099/Lcb1_internal_stability/2'>Nonpolar core of LCB1</scene>

<scene name='10/1078099/Lcb3_internal_stability/2'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

New Test

2025-04-14T02:00:46Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein<scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scenename='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.

<scene name='10/1078099/Ahb2_internal_stability/1'>Nonpolar core of AHB2</scene>

<scene name='10/1078099/Lcb1_internal_stability/2'>Nonpolar core of LCB1</scene>

<scene name='10/1078099/Lcb3_internal_stability/2'>Nonpolar core of LCB3</scene>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

New Test

2025-04-14T01:09:17Z

Matthew Pereira:

<StructureSection load='7jzl' size='350' frame='true' side='right' caption='SARS-CoV-2 Spike Protein (7JZL): A trimer responsible for interacting with host ACE2 receptors to deliver the virus into host cells.' scene='10/1078099/Spike_protein/1'>

==Introduction==
[[Image:Illustration.png|400 px|right|thumb|Figure 1: The process of the SARS-CoV-2 virus entering human cells. The image was created using the program biorender. ]]

SARS-CoV-2 better known as Covid-19 sent the world into a global pandemic due to its rapid transmission and infection <ref name=”WHO”>https://www.who.int/europe/emergencies/situations/covid-19</ref>. In order to create a vaccine, it was essential to understand how SARS-CoV-2 infected us.

The enzyme angiotensin converting enzyme 2 <scene name='10/1077473/Ace2_only/1'>(ACE2)</scene> is attached to our cell membranes and then can be bound to a receptor binding domain <scene name='10/1077473/Rbd_only/2'>(RBD)</scene> <ref name="Borkotoky">DOI:10.1007/s11033-022-08193-4</ref>. The virus, SARS-CoV-2, enters our bodies containing a spike protein protruding from the viral membrane. The RBD on the end of the spike protein<scene name='10/1076049/Ace2andrbd/1'>binds to ACE2</scene> giving SARS-CoV-2 the ability to enter our host cells <ref name=”Jackson”>DOI:10.1038/s41580-021-00418-x</ref>. Once the spike protein has access to our host cells, it is able to further infect our cells and spread the virus throughout our bodies, causing us to get sick. Figure 1 demonstrates the path SARS-CoV-2 takes to get into our cells. In order to create an effective vaccine, the pathway between the spike protein and the RBD needed to be [https://www.sciencedirect.com/science/article/pii/S0166354223000190 interrupted].

==Vaccines==
A [https://en.wikipedia.org/wiki/Vaccine vaccine] activates the body’s immune system by containing weakened parts of viruses. This activation stimulates the production of antibodies. <scene name='10/1077473/Antibody_overview/2'>Antibodies</scene> then compete with the ACE2 protein for the binding of viral spike proteins. In the case of SARS-CoV-2 spike proteins, once they enter the body they <scenename='10/1077473/Antibody/4'>bind</scene>
to the Fab fragment of the antibody, a 90% neutralizing response for targeting the RBD is created. In this scene only the Fab fragment is being shown due to the fact that the Fab fragment is the part of the antibody that interacts with the RBD. With this being said, this method of treatment is difficult for long term use due to the evolution of the viral cells <ref name="Zhang">DOI:10.1016/S2666-5247(23)00011-3</ref>.

==Protein Inhibitor Development==
Protein inhibitors were thought of as a new idea for creating vaccines due to their smaller size and better stability compared to antibody vaccines<ref name="Cao">DOI:10.1126/science.abd9909</ref>. These protein inhibitors, also referred to as mini-binders, interact with the receptor binding domain of the spike protein, <scene name='10/1075220/Spikeblockedbyminibinder/2'>preventing association of the viral cell with ACE2.</scene>

===The Process of Discovery===
[[Image:AHB2_Method.png|400 px|right|thumb|Figure 2: The use of the Rosetta Blueprint protein design to create the AHB2 inhibitor.]]

The first mini-binder to be created to combat COVID-19 is called AHB2. In order to ensure that the mini-binder would bind to the same RBD that the ACE2 was bound to, AHB2 was designed by looking at the specific sequence of ACE2 to find the alpha-helix that makes interactions with the spike receptor binding domain. This design process is referred to as the Rosetta Blueprint protein design. Figure 2 shows the RBD trimer with one part of ACE2 being used for the reference alpha helix to create AHB2. <ref name="Cao">DOI:10.1126/science.abd9909</ref>.

[[Image:LCB_Method.png|400 px|right|thumb|Figure 3:The use of the De Novo protein design to create the LCB1 and LCB3 inhibitors.]]

As the AHB2 inhibitors were tested and found to be effective, it was then time to manipulate the mini-binders to create a more effective vaccine. A rotamer interaction field docking with in silico mini-proteins were used by using a scaffold library to generate binders to more distinct regions of the RBD surface <ref name="Cao">DOI:10.1126/science.abd9909</ref>. This method is known as the de novo protein design and it is how the LCB1 and LCB3 mini-binders were created. Figure 3 shows the difference LCBs pulled from the scaffold library to create the different LCB inhibitors.

===Stability===
One of the most important findings with these DeNovo proteins is their high stability, allowing for less delicate forms of administration.
<scene name='10/1077473/Lcb1_internal_stability/1'>Nonpolar core of LCB1</scene>,
<scene name='10/1077473/Lcb3_internal_stability/1'>nonpolar core of LCB3</scene>, and
<scene name='10/1077473/Ahb2_internal_stability/1'>nonpolar core of AHB2</scene>
</StructureSection>

==Binding Site and Interactions==

In order to best target the RBD of the spike protein, the minibinders reveal a wide range of interactions to compete with <scene name='10/1075219/Ace2/1'>ACE2 binding</scene>.

The first design method, Rosetta, created AHB2 based on the single interacting helix of ACE2. With <scene name='10/1075219/Ahb2/2'>AHB2 binding</scene>, we see two alpha helices mimicking ACE2. Hydrogen bonding interactions between N36, D11, K43, E41, and E30 of the minibinder interact with residues K417, R403, Y449, Q493, and N487, respectively, in the spike protein.

De novo designed proteins, as discussed previously, focused on computational design to determine residues best able to interact with the spike protein. We will focus on LCB1 and LCB3. <scene name='10/1075219/Lcb1/4'>LCB1 binding</scene> reveals hydrogen bonding between D30 of the minibinder and both K417 and R403 of the spike protein, in addition to D17 and R14 of the minibinder interacting with Q493 of the spike protein. Similarly, <scene name='10/1075219/Lcb3/5'>LCB3 binding</scene> reveals hydrogen bonding between D11 of the minibinder to K417 and R403 of the spike protein.

Within all four binding sites, we see two conserved residues throughout: K417 and Q493. This finding reveals the importance of these residues in both binding and stability.

Furthermore, an interesting difference between the two design methods is the location of hydrogen bonding interactions. In AHB2, similar to ACE2, we see interactions spanning the entirety of the helices in contact with the spike protein. De novo design, on the other hand, reveals the main hydrogen bonding interactions occurring within the center residues of the helices.

Figure 4 shows the sequence comparison between the interacting helix of ACE2 and the helices of the three minibinders. [[Image:Comparison.jpeg|400 px|left|thumb|Figure 4: The sequence differences between ACE2, AHB2, LCB1 and LCB3.]]

==Protein Inhibitor Effectiveness==

==Limitations==

==References==
Cao, L., Goreshnik, I., Coventry, B., Case, J.B., Miller, L., Kozodoy, L., Chen, R.E., Carter, L., Walls, A.C., Park, Y., Strauch, E., Stewart, L., Diamond, M.S., Veesler, D., & Baker, D. De novo design of picomolar SARS-CoV-2 mini protein inhibitors. Science 370, 426-431 (2020). https://doi.org/10.1126/science.abd9909

https://www.who.int/europe/emergencies/situations/covid-19

https://pmc.ncbi.nlm.nih.gov/articles/PMC9786537/#:~:text=The%20receptor%2Dbinding%20domain%20(RBD,that%20initiates%20the%20viral%20transmission.

https://www.nature.com/articles/s41580-021-00418-x#citeas

https://www.science.org/doi/10.1126/science.abd9909

Zhang, Haoran et al. Advances in developing ACE2 derivatives against SARS-CoV-2. The Lancet Microbe, Volume 4, Issue 5, e369 - e378 (2023). https://doi.org/10.1016/S2666-5247(23)00011-3

https://www.cdc.gov/vaccines/basics/explaining-how-vaccines-work.html\

https://en.wikipedia.org/wiki/Vaccine

===PDB Files===
[1]https://www.rcsb.org/structure/7UHB

==Student Contributors==
*Giavanna Yowell
*Shea Bailey
*Matthew Pereira

User:Matthew Pereira

2025-04-14T00:38:27Z

Matthew Pereira:

User:Matthew Pereira

2025-04-14T00:34:04Z

Matthew Pereira: