This article describe a detailed method for selecting inhibitors based on modified natural peptides for the SARS-CoV protein spike glycoprotein. The selection of inhibitors is carried out by increasing the affinity of the peptide to the active center of the protein. The article also provides a step-by-step guide to the analysis of affinity of interaction by comparing 3 criteria, presents an analysis of energy interactions between the active center of a protein and the wild-type peptide interacting with it and taking into account modifications of the latter.

1. Previously reported SARS-CoV infectivity was reduced over 10,000-fold through pre-incubation with two peptides, while it was completely inhibited in the presence of three peptides.Were reported that four 20-mer synthetic peptides (S protein fragments), designed to span these sequence variation hotspots, exhibited significant antiviral activities in a cell line. [Synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of SARS-associated coronavirus]

2. Another study reported that Peptide P8 (P2 + P6 + P10) exhibited the strongest antiviral activity, Synergistic antiviral effects mediated by peptide combinations. [Synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of SARS-associated coronavirus]

3. In the study [Synthesized peptide inhibitors of HIV-1 gp41-dependent membrane fusion. Curr Pharm Des 390 19: 1800-9] peptides derived from the HR1 and HR2 sequences of the class I viral fusion proteins have been demonstrated to possess antiviral activity through binding to the prehairpin intermediate thus blocking the formation of viral 6-HB core.

4. Cholesterylated peptide exhibits greatly increased α-helical stability and target-binding affinity [Design of 1 potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity]

5. Successful peptide inhibitors of virus penetration into the host cell have been successfully developed in the case of HIV-1, Ebola virus, paramyxoviruses (SV5), respiratory syncytial virus (RSV). So, the Ebola virus was 99% inhibited when using the HIV peptide C-peptide conjugate as an inhibitor. The sequence found is characterized by the highest activity against the Ebola virus [Inhibition of Ebola Virus Entry by a C-peptide Targeted to Endosomes]

6. Inhibitor molecules included D peptides, synthetic peptides from the N and C ends of a protein virus molecule that could bind the HR1 viral domain and effectively inhibit viral infection.

In this paper, we propose a stepwise increase in the affinity of a natural peptide for a target viral protein. we have developed a phased, five-point method for determining the most suitable modified peptide. The method also includes a phased selection of the alpha-helical region for such modifications that lead to an increase in the stability of the dimer complex compared to the wild-type protein dimer, selection criteria are presented and described.

Three-dimensional structure of the dimeric complex of the peptide "blue" Spike-glycoprotein and the "green" protein Spike-glycoprotein. We will replace amino acid residues in the "blue" peptide. Interacting sites are represented by alpha-helical sites.

_______This article described a method for phasing the affinity of a natural peptide to a target viral protein by modifying the peptide. The selection criteria for the most suitable modifications were given, among which the following can be listed: the stability of the dimer complex by calculating the value of log (cond (W)) and comparing the obtained value with the value of log (Cond (W)) obtained for the wild-type dimer and other modified dimers .
_______The next indicator was the measure of the change in differential entropy ∆Н, which characterizes the ordering of the system. Moreover, the values of lg (cond (W)) and ∆Н are interrelated: the minimum number lg (cond (W)) must correspond to a negative value of ∆Н, otherwise, the dimeric complex does not satisfy the necessary criteria.
________As an example of the selection of suitable modifications of natural peptides, the Spike-glycoprotein viral protein was selected, which is responsible for the attachment of the virus to the cell and the incorporation of the viral genome into the cell. In this work, we analyzed the Central helix region of this protein, performed mutagenesis, and calculated the stability of the formed dimeric complexes upon modification of the natural peptide in the region of the Central helix region. The calculation results showed that the modification of the site from 995 a.a. till 1014 a.a. most likely to affect the affinity of the modified peptide for the Spike-glycoprotein viral protein. In the course of the numerical simulation, wild-type peptide mutagenesis was performed, the calculation of the values of log (cond (W)) and ΔH, the minimum values of log (cond (W)) were compared with negative values of ΔH, since these values are analyzed in pairs. If the log (cond (W)) values take the minimum values, and the ΔH value is in the range of positive values, then this modified peptide does not pass the test according to the criteria.
________To reduce false results, we remove from further analysis all dimeric complexes that are characterized by a ∆H value in the positive range of values. We also believe that an important aspect affecting the results of the modification of a natural peptide is the environment of the modified portion of the polypeptide chain with other amino acid residues, the location of which in turn depends on the solvent (water, impurities, salts, temperature, pH). in three-dimensional space is characterized by the distribution of potential energy of interaction with other amino acid residues in three-dimensional space. Replacing even one amino acid residue in a polypeptide chain can serve as a substantial redistribution of the potential energy of interaction with all amino acid residues of the adjacent polypeptide chain, which in turn will affect the stability of the dimer complex and the entropy of the dimer complex, and more precisely, entail measures to change the differential entropy of the dimer complex ∆H , which we calculate for each replacement.
______By analyzing the Spike-glycoprotein protein peptide in this way, we determined amino acid residues, the replacement of which can affect the affinity, namely, the increase in affinity due to increased stability of the dimeric complex and ordering of the system of two proteins. The aim of this work was to develop and describe in detail a methodology for increasing the affinity of a natural peptide for a target protein by modifying the peptide, namely, replacing amino acid residues. In the course of the work done, several key amino acid residues found were replaced with 997, 998, 1002, 1006, 1008, see fig. on MET (M), GLN (), ASN ().
________The results presented in fig. and in the table it was possible to determine the most suitable modification of the peptide to increase the affinity of interaction with the Spike-glycoprotein viral protein. These modifications are two substitutions ALA997MET and ALA1002 MET, since two simultaneous substitutions significantly reduced the value of log (cond (W)), and the value of ∆Н was characterized by the lowest negative value. A good result was also obtained with the simultaneous replacement of two amino acid residues in the peptide ALA997MET and ALA1002GLN, as well as ALA997MET and ALA1008MET. The worst result was obtained with the simultaneous replacement of ALA998MET and LEU1006GLN, since the value of log (cond (W)) was obtained in a high range of values, and the value of ΔН was in the range of positive values.
To identify a high affinity modified peptide for the wild-type virus virus Spike-glycoprotein, it is necessary to perform large-scale numerical calculations and, based on the results, obtain the most suitable modifications of the peptide for further refinement by increasing the resistance of peptides against protease cleavage and increasing the circulation time in the body to achieve a significant therapeutic effect .