3.1 De novo design of inhibitory peptides against RBD
RBD domain of spike glycoprotein mediates the entry of SARS-CoV-2 into the human respiratory cells by interacting with cell surface receptor ACE2 . Therefore, blocking the interaction residues of RBD might block its interaction with ACE2, hence making it unable to infect human cells. The RBD of SARS-CoV-2 and SARS-CoV-1 is highly conserved  and mainly uses residues 417, 453, 458, 490, 493-495, 498, 501 and 502 for binding to ACE2 [29,7] . Another study also reported same residues of SARS-CoV-2 RBD involved in binding with another cell-surface receptor (Glucose Regulated Protein 78 (GRP78) for entry into the cells , which indicates that SARS-CoV-2 interacts with different receptors located on human cell surface, using almost same binding residues of RBD. Therefore, blocking the binding residues of RBD through inhibitory peptides can potentially block entry of SARS-CoV-2 into the human cells and can also be useful against future pandemic if caused by newly emerged coronaviruses due to the conserved nature of RBD .Thereby, targeting the RBD to block its interaction with ACE2 is ideal choice for SARS-CoV-2 drug discovery.
Previously, we targeted these nine residues of RBD to be blocked through tACE2 . However, the current study involved re-designing the binding interface of tACE2 to produce shorter peptide with more binding affinity and covering all the binding residues of RBD . Short therapeutic peptides have gain interest due to its specificity and low immunogenicity response. The RBD binding residues 490, 493-495, 498, 501, 502 are clustered at one region (region1) while 417 and 458 are clustered slightly distal from the later (region2).Therefore either two peptides can block these two regions or single peptide with extended binding network can hinder interaction between RBD and cell surface receptors.
The residues of ACE2 at amino acid position 21-40 (scafold1) and 65-76 (scafold2) were re-designed and produced 10 de novo sequences for each scaffold. Two best sequences, Pep1 and Pep2 were selected from top-10 de novo sequences produced by EvoDesign from scaffold1 and 2, respectively. The TM score 0.61 of Pep1 indicate its similar fold to that of scaffold1, while Pep2 Tm-score was 0.16 indicating its different fold than the scaffold2 structure. The Lower RMSD of Pep1 (0.58 A˚) are in agreement with its Tm score, while Pep2 showed RMSD 2.12, which indicate slight deviation of secondary structure from their scaffold (Fig 1). Similarly, the amino acid sequence of Pep1 showed 30% similarity while Pep2 showed 20% similarity with their corresponding native sequences (Table 1). The designed peptides with high similarity to their native sequence usually exhibit higher binding affinity towards its partner protein . We further investigated the binding pattern and affinity of the designed peptides for RBD through protein-protein docking.
3.2 Protein-protein docking
To test the binding properties, protein-protein docking of the designed peptides with RBD was performed through HADDOCK. The HADDOCK-Score of Pep1 and Pep2 was -119 and -111, respectively, when docked with RBD (The more negative the better binding affinity). The haddock score of Pep1 is greater than that of the intact ACE2 (-111) docked with RBD . The docking RMSD of Pep1 and Pep2 in complex with RBD were 0.6 and 0.8, respectively, showing the high likelihood of the docked complexes with native one .
Our docking results showed that nine residues Ala2, Lys7, Ans10-Asp14, Ser16 and Phe20 of Pep1 interact with Arg403, Lys417, Tyr453, Lys458, Gln493-Gly496, Gln498, Thr500, Asn501 and Tyr505 residues of RBD (Fig.2 A-C), while Leu67-Asp69, Thr72 and Glu75 of Pep2 interact with Arg404, Lys417, Tyr495 and Tyr 505 of RBD (Fig 2D-E). These results confirm that Pep1 cover almost all the binding residues of RBD involved in interaction with human ACE2 and GRP78, making this peptide ideal for further investigation for its therapeutic potential.
Previous studies has shown that binding residues of RBD are located at two distinct position, region 1 (490, 493-495, 498, 501, 502) and region 2 (417 and 458) [8,10], which demands two different peptides to be block. Interestingly, our de novo designed peptide Pep1 showed binding with region 1 as well as region 2 residues (Fig. 2C). The superimposition of docked Pep1 with its scaffold showed that redesigning changed the Phe20 into Arg with favorable side chain positioning for interaction with Lys458 of RBD, while mutation Ala16Ser results in interaction with Tyr417 of RBD (Fig. 1). Both of these residues are located at region 2 and reported to be critical for interaction with RBD . The de novo design approach created optimum mutation which increased binding network of the designed peptide Pep1, resulting in successful blocking of the RBD binding residues required for interaction with human cell surface receptors.
3.3 Binding affinity of designed peptides for ACE2
We further determined the binding affinity of the designed peptides for RBD and complex stability. The binding affinity showed by Pep1 for RBD was -13.2 kcal mol-1 at 36 ºC as optimum temperature which is greater than the binding affinity of intact ACE2 (-10.7 kcal mol-)  and GRP78 (-9.8 kcal mol-1) for RBD . The binding affinity of Pep1 for RBD was also higher than the binding affinity (-11kcal mol-1) of therapeutic peptides proposed previously for blocking RBD of SARS-CoV-2 . It seems that the favorable mutations and side chain rearrangement resulted in dramatic increase in binding affinity of Pep1 for RBD. We further determined the dissociation constant Kd values of peptide-receptor complexes. The Pep1-RBD complex showed Kd value 3.9 × 10−10 M, which is lower than the previously reported Kd values of inhibitory peptides proposed for S protein of SARS-CoV-2  and intact ACE2-RBD complex . The smaller Kd value indicates high stability and strong binding affinity between protein-protein complex .The lower Kd value of Pep1-RBD complex suggest that the designed peptide Pep1 are tightly bound to the corresponding region of RBD. Binding affinity and Kd values of Pep2-RBD complex was found lower than the Pep1-RBD complex. This indicates that region 21-40 has important role in binding with RBD.
3.4 MD simulation showed stability of designed peptides-RBD complex
To investigate the structural stability and dynamic behavior of the designed peptides in complex with RBD, we performed MD simulation of the RBD in complex with Pep1 and Pep2. The docking conformation with lowest energy was subjected to MD simulation. To investigate structural stability of the complex, RMSD plot of the complex backbone was produced. The RMSD values of Pep1-RBD complex remained 0.2-0.25 nm initially for 40ns and then increased up to 0.4-0.5 nm for 60-100 ns of MD run. Similarly, the RMSD values of Pep2-RBD complex remained 0.3-0.4 nm during initial 90 ns while slightly increased up to 0.95 nm during 90-100 ns (Figure 3A). In general, the RMSD <0.3 nm during a 20 ns MD run indicate strong complex stability [33,34]. Overall, a uniform lower RMSD of Pep1-RBD complex indicates that Pep1 bind more tightly to RBD than the Pep2. The RMSD value of Pep1-RBD complex is also lower than the previously reported therapeutic peptides for SARS-CoV-2 treatment [31,10].
Root mean square fluctuation (RMSF) determined in the docked complexes shows residues flexibility with high RMSF values indicate the mobility of residue side chains in relation to their average position . The RMSF plot of Pep1-RBD complex shows that the residues of RBD at position 358, 417 and 490-500 showing lower fluctuation (nm) than the Pep2-RBD complex. The overall RMSF value of Pep1-RBD complex is less than 0.2 nm in region 1 & II window, which is lower than the RMSF value (0.35nm) of RBD when bound to intact ACE2. The residues involved in binding interaction with lower RMSF values indicates the most stable region of the complex . The lower RMSF values of RBD binding residues indicate that Pep1 form a stable complex with RBD, as RMSF value <0.4 nm reveals complex stability .
The Radius of gyration (Rg) was determined to describe the structural integrity and folding behavior of the designed peptides in complex with RBD. A low Rg value reveals better structural integrity and folding behavior [37,38]. Pep1-RBD complex showed a uniform and stable Rg value between 1.80-1.84 nm throughout a 100 ns MD run, while the Rg value of Pep2-RBD complex increased to 2.23 nm during 90-100 ns. Overall the Rg values for both peptides remained >1.84, which is lower than the Rg value (2.2nm) showed by intact ACE2-RBD complex , which indicates structural integrity of Pep1- and Pep2-RBD complex (Figure 3C). Overall, the MD simulation results suggests that the de novo designed peptides form a stabilized complex with RBD and propose their potential to block the SARS-CoV-2 Spike glycoprotein for interaction with human cell surface receptors.