Engineered Protein Inhibitors of SARS-CoV-2 Entry: Difference between revisions

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<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'>
<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==
==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.]]
[[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.  
[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>.
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==
==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>
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>.  
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 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>
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 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 [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>.


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 [https://en.wikipedia.org/wiki/In_silico 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.  


[[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).]]
===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 [https://en.wikipedia.org/wiki/Thermal_shift_assay 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>.


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==
==Binding Site and Interactions==
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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.  
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.]]
[[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.  
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.  
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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>.
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 that 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>.  
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>
</StructureSection>
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