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AHB2 was designed using an ACE2 helix scaffold, while LCB1 and LCB3 were designed completely from scratch, attempting to make the best possible helix with the greatest affinity for the spike protein receptors . Although LCB1 was designed before LCB3, LCB3 was less effective at neutralizing the viral response with a high IC<sub>50</sub> Value <ref name="Longxing">PMID:32907861</ref> .
AHB2 was designed using an ACE2 helix scaffold, while LCB1 and LCB3 were designed completely from scratch, attempting to make the best possible helix with the greatest affinity for the spike protein receptors . Although LCB1 was designed before LCB3, LCB3 was less effective at neutralizing the viral response with a high IC<sub>50</sub> Value <ref name="Longxing">PMID:32907861</ref> .
These minibinders are small proteins, modeled similarly to the ACE2 and SARS-CoV-2 spike protein. There were two strategies utilized. One strategy included directly incorporating the ACE2 helix of the RBD and creating more interactions, increasing the binding affinity of the minibinders <ref name="Longxing">PMID:32907861</ref>. The other strategy was designing the minibinders completely from scratch, completely dependent on the RBD <ref name="Longxing">PMID:32907861</ref>. AHB2 utilized the first method, incorporating the ACE2 helix, while LCB1 and LCB3 utilized the second method <ref name="Longxing">PMID:32907861</ref> .
These minibinders are small proteins, modeled similarly to the ACE2 and SARS-CoV-2 spike protein. There were two strategies utilized. One strategy included directly incorporating the ACE2 helix of the RBD and creating more interactions, increasing the binding affinity of the minibinders <ref name="Longxing">PMID:32907861</ref>. The other strategy was designing the minibinders completely from scratch, completely dependent on the RBD <ref name="Longxing">PMID:32907861</ref>. AHB2 utilized the first method, incorporating the ACE2 helix, while LCB1 and LCB3 utilized the second method <ref name="Longxing">PMID:32907861</ref> .
===Potency of the minibinders===
Examining the IC<sub>50</sub> values of the various mini binders gives quantitative data to the effectiveness of the proteins in preventing an immune response. The highest IC<sub>50</sub> was AHB2 (15.5 nM), followed by LCB3 (40.1 pM) LCB1 (23.5 pM) <ref name="Longxing">PMID:32907861</ref>. The higher IC<sub>50</sub> indicates a larger concentration of mini binder required to inhibit the biological process. Both LCB1 and LCB3 proved to be significantly more effective than AHB2,  LCB1 and LCB3 were within 3-fold of the most potent anti-Spike monoclonal antibodies described to date <ref name="Longxing">PMID:32907861</ref>.


===Structure===
===Structure===
The goal of designing these minibinders was to create a molecule with a higher binding affinity with the RBD than ACE2, meaning they had to be designed with specific residues that form stronger connections with the same binding pockets that ACE2 would bind to <ref name="Longxing">PMID:32907861</ref>. This section highlights some important residue differences between the minibinders and ACE2 that give the minibinders a higher affinity.
These minibinders were designed to form stronger connections to the spike protein RBD than ACE2<ref name="Longxing">PMID:32907861</ref>. This section highlights some important residue connections with the RBD that give the minibinders higher affinities for the spike protein than ACE2.  
 
The glutamine-493 (Q493) residue is an important residue in showing the differences in strength between ACE2 and the minibinders <ref name="Longxing">PMID:32907861</ref>. ACE2 doesn’t make use of this residue when binding to the RBD, the nearest residues, Glu-35 and Lys-31 <scene name='10/1075250/Q493_ace2/5'>don’t form any interaction with Q493</scene>. Comparing this to the AHB2 minibinder, which <scene name='10/1075250/Q493-ahb2/3'>forms a Hydrogen bond with the Q493 residue</scene>, the AHB2 minibinder makes better use of the RBD’s residue than ACE2, helping it have a higher affinity to the spike protein. LCB1 makes even better use of the Q493 residue, <scene name='10/1075250/Q493_lcb1/4'>forming two hydrogen bonds with two different residues</scene>, giving it the highest affinity based on the Q493 residue.  


Another important binding site on the RBD includes the Lysine-417 (K417) and Arginine-403 (R403) residues. While <scene name='10/1075250/417_403-ace2-needmeasure/1'>ACE2 does form a hydrogen bond interaction with the K417 residue</scene> using its own D30 residue, LCB1 forms <scene name='10/1075251/D30_lcb1/1'>H bond interactions with both of them</scene>, using its own D30 residue, forming a very strong interaction that is hard to break.  
ACE2 binds to the spike protein RBD using <scene name='10/1075251/Ace2_allbinding/3'>these residues</scene>. The minibinders are smaller than ACE2, but still form strong connections to the spike protein. This means the minibinders interact with the RBD residues more efficiently than ACE2 giving them a higher affinity for the spike protein <ref name="Longxing">PMID:32907861</ref>. AHB2 was designed to mimic ACE2 while making better use of the RBD residues giving it a higher affinity for the spike protein with an IC<sub>50</sub> value of 15.5 nM<ref name="Longxing">PMID:32907861</ref> (<scene name='10/1075251/Ahb2_all/3'>AHB2 and RBD binding residues</scene>). LCB1 was designed from scratch to bind the most efficiently to the RBD residues giving it the highest affinity with the lowest IC<sub>50</sub> value of 23.5 pM<ref name="Longxing">PMID:32907861</ref> (<scene name='10/1075251/Lcb1_binding_residues/3'>LCB1 and RBD binding residues</scene>). LCB3 was designed after LCB1 with the goal of making the minibinder even more efficient, however it ended up having a lower affinity than LCB1 with a higher IC<sub>50</sub> value of 40.1 pM<ref name="Longxing">PMID:32907861</ref> (<scene name='10/1075251/Lcb3_all/2'>LCB3 and RBD binding residues</scene>).


It is important to note that these highlighted residues aren’t the only residues that differ between the minibinders, and it is a compilation of all the residue interactions that give each minibinder different affinities. For example, <scene name='10/1075250/Q493-lcb3/3'>LCB1 forms no interactions with the Q493 residue</scene> of the RDB previously mentioned, yet it still has a higher affinity to the RBD than AHB2 which forms a hydrogen bond with the Q493 residue <ref name="Longxing">PMID:32907861</ref>.
The Glutamine-493 residue on the spike protein is an important residue in showing the differences in strength between ACE2 and the minibinders <ref name="Longxing">PMID:32907861</ref>. ACE2 does not make use of this residue when <scene name='10/1075250/Q493_ace2/12'>binding to the RBD</scene>. The nearest residues of ACE2, Gln35 and Lys31, do not form any interaction on Gln493 of the spike protein. The AHB2 minibinder, instead forms a <scene name='10/1075251/Q493-ahb2/1'>hydrogen bond</scene> on the Gln493 of the spike protein residue of the spike protein with its Glu41, increasing affinity to the spike protein. LCB1 forms <scene name='10/1075250/Q493_lcb1/9'>two hydrogen bonds</scene> on Gln493 of the spike protein using its Asp17 and Arg14 residues, giving it the highest affinity based on the Gln493 residue. However, these interactions are not required. LCB1 forms <scene name='10/1075251/Q493-lcb3/1'>no interactions</scene> with the Gln493 residue of the RDB previously mentioned, yet it still has a higher affinity to the RBD than AHB2.  


===Function===
Similarly, LCB1 forms <scene name='10/1075250/D30_lcb1/3'>two hydrogen bonds</scene> with both the Lys417 and Arg403 of the spike protein from its Asp30 residue. This is a very strong interaction, further contributing to the high affinity of LCB1 to the spike protein<ref name="Longxing">PMID:32907861</ref>. ACE2, however, forms only <scene name='10/1075250/417_403-ace2-needmeasure/4'>one hydrogen bond</scene> with the Lys417 from its Asp30 residue.  
The effect of the minibinders’ higher affinity to the spike protein RBD is that ACE2’s binding site is now sterically blocked, meaning, ACE2 cannot bind to the spike protein and initiate the infection pathway. This effectively hinders the effects of the virus, which accomplishes the goal of creating these minibinders<ref name="Longxing">PMID:32907861</ref>.  


==Implications==
==Implications==  
===Potency of the minibinders===
Examining the IC<sub>50</sub> values of the various mini binders gives quantitative data to the effectiveness of the proteins in preventing an immune response. The highest IC<sub>50</sub> was AHB2 (15.5 nM), followed by LCB3 (40.1 pM) LCB1 (23.5 pM) <ref name="Longxing">PMID:32907861</ref>. The higher IC<sub>50</sub> indicates a larger concentration of mini binder required to inhibit the biological process. Both LCB1 and LCB3 proved to be significantly more effective than AHB2,  LCB1 and LCB3 were within 3-fold of the most potent anti-Spike monoclonal antibodies described to date <ref name="Longxing">PMID:32907861</ref>.
===Results from mice study===
===Results from mice study===
The effectiveness of the most potent minibinder was examined in mice. LCB1 was administered to the mice via nasal delivery. As expected, compared to control mini protein, the LCB1 was significantly more effective at reducing the viral burden, diminishing the immune cell infiltration, and inflammation <ref name="Case">PMID:34192518</ref>. The virus was not detected in the lungs 4-7 days post-infection, and the spleen, heart, and brain had viral RNA at very low concentrations <ref name="Case">PMID:34192518</ref>.
The effectiveness of the most potent minibinder was examined in mice. LCB1 was administered to the mice via nasal delivery. As expected, compared to control mini protein, the LCB1 was significantly more effective at reducing the viral burden, diminishing the immune cell infiltration, and inflammation <ref name="Case">PMID:34192518</ref>. The virus was not detected in the lungs 4-7 days post-infection, and the spleen, heart, and brain had viral RNA at very low concentrations <ref name="Case">PMID:34192518</ref>.
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