Problem Solving Approaches It is reasonable to assume that HR1 may also be a good target for the development of fusion inhibitors of highly pathogenic HCoV viruses. The second domain to be inhibited is the RBD domain, which binds to the enzyme ACE2 (Angiotensin-converting enzyme 2). In this case, ACE2 binds to the receptor binding motif (RBM) in the receptor binding domain (RBD) of SARS-CoV and functions as a receptor for SARS-CoV. Recently, it has been discovered that human ACE2 facilitates the penetration of SARS-CoV-2 into cells [Crystal Structure of the Receptor-Binding Domain from Newly Emerged Middle East Respiratory Syndrome Coronavirus]. The RBD domain of SARS-CoV-2 interacts with human ACE2. Thus, ACE2 is defined as a receptor for SARS-CoV-2 and a suitable target for inhibiting the virus in the cell. Thus, to solve this problem, it is necessary to develop inhibitors of the HR1 and HR2 regions, blocking the merger of two subunits S1 and S2. The fusion region of HR1 and HR2 is a convenient target for inhibiting viral fusion. The second domain is the RBD site, which is responsible for binding to the ACE2 enzyme, which is a cellular receptor.
Project Stages Analysis and selection of glycoprotein protein structures taking into account mutations in various conformations of subunits S1 and S2. One of the directions of the proposed project is to identify common features and differences between different types of SARS-Cov viral protein, which is prone to acquire mutations. Moreover, differences can be observed both within different types of protein, as well as between representatives of the same type of proteins due to various experimental methods used to calculate the three-dimensional structure of the protein.
The study of previous developed inhibitors, as well as therapeutic approaches based on small molecules. In this case, successful and unsuccessful trends and approaches in protein inhibition, mobile and conservative interaction sites, flexible sites and sites prone to mutations and conformational
restructuring. Available data will be divided into groups:
Small chemical molecule inhibitors (1 week)
Inhibitors in the form of D-peptides, L-peptides, alpha-helices, amino acid
sequences with famous structure (3 weeks)
unknown structure. In this case, we will reconstruct the three-dimensional structure of previously found inhibitors, the antiviral activity of which was detected during the experiments, but did not reach clinically significant levels. (6 weeks)
Transition to a detailed study of HR1, HR2, and RBD plots in proteins of various types with and without mutations (wild type).
Development of mathematical methods and algorithms for modeling alanine scanning of selected areas and calculation of potential energies between pairwise taken amino acid residues of each protein. And also based on the developed mathematical methods, we will analyze the change in affinity, calculate the range of variation of the dissociation constant for point substitutions amino acid residues with alanine in proteins, which will significantly save on biological material, payment and use of research equipment. In molecular biology, alanine scanning is a method used to determine the contribution of a particular residue to the stability or function of a given protein. Alanine scanning mutagenesis of protein-protein interfacial residues can be applied to a wide range of protein complexes to understand structural and energy characteristics of hot spots.
The results of the work performed at this stage will produce the following results:
- Determination of the stability of protein complexes with point substitutions of amino acid residues, taking into account the three-dimensional structure of the biocomplex, based on the developed project criteria for the stability of the biological complex.
- The range of changes in the affinity of the dimeric complex will be obtained with each replacement. The biophysical approach developed in the project allows qualitatively determining key amino acid residues in the interaction of two polypeptide chains, as well as assess the range of Kd changes when replacing key amino acid residues
in peptides when they are bound to the target protein. Thus, it is possible to predict the stability of protein complexes, as well as qualitatively determine the range of variation of the dissociation constant, synthesize peptides with a given dissociation constant for various proteins, while at the same time allowing to increase
the selectivity of peptides, increasing the affinity for one protein and decreasing for others, which is an important condition for improving peptide therapy.
- Determining the measure of change in differential entropy for each replacement of the amino acid residue as a result of a model alanine scan.
- Development of three-dimensional maps of the potential energy of the electrostatic interaction of two wild-type proteins. Moreover, the obtained maps of the potential energy of the electrostatic interaction of the amino acid residues of the involved proteins will allow us to visualize the nature of the formation of the protein complex, identify the maxima and minima of the potential energy between the amino acid residues of the two proteins, and identify the key amino acid residues that take into account the maximum values of the potential
energy. Particular attention will be paid to nature interacting amino acid residues (hydrophobic, hydrophilic, charged). The analysis of interacting proteins was carried out taking into account the three-dimensional structure, where the areas with the greatest potential energy of electrostatic interaction will be revealed.
Mutagenesis based on the mathematical methods developed in the project (replacing amino acid residues in wild-type proteins in the HR1, HR2 region with 4 other amino acid residues) alpha-helical and flexible regions HR1, HR2 for the selected two types of S-glycoprotein structures, as well as their mutant analogues. The results of work at this stage will supplement the data on the previously performed model alanine scanning:
Determination of the stability of protein complexes with mutations of amino acid residues, taking into account the three-dimensional structure of the biocomplex
The range of changes in the affinity of the dimeric complex before and after the mutations will be obtained with each replacement.
The values of the potential energy of the potential energy of interaction between all amino acid residues of one protein and amino acid residues of another protein for each mutation.
A measure of the differential entropy change for each mutation.
Theoretical calculation of the range of variation of Kd values.
Three-dimensional maps of potential energy of electrostatic interaction
two wild-type proteins and a mutant protein. Moreover, maps of the potential energy of the electrostatic interaction of amino acid residues of the involved proteins, which will visualize the nature of the formation of the protein complex
Selection of inhibitors for selected areas, taking into account the three-dimensional structure of proteins, using the programs developed in the project. Analysis of the resulting structures, the selection of structures characterized by a minimum of potential energy, a minimum measure changes in differential entropy, the minimum value of lg (cond (w)). Using off-the-shelf molecular docking programs, computing clusters and servers to select the best inhibitors.
EXPERIMENTAL PART Experimental verification of the molecules found. Synthesis of inhibitors, acquisition of an S-glycoprotein molecule, verification of in vitro affinity of the found inhibitors to S-glycoprotein. Determination of Kd (dissociation constant) and IC50 (concentration of half-maximal inhibition, — an indicator of the effectiveness of the ligand with inhibitory biochemical or biological interaction. Note that IC₅₀ is a quantitative indicator that shows how much is needed. ligand inhibitor to inhibit the biological process by 50). An in vivo experiment on cell lines. Preparatory phase (synthesis of inhibitors, supply of molecules, ordering and supply of protein S-glycoprotein, checking the purity and suitability of the drug) 1.5 months The main stage of the experiment In vitro and in vivo (on cell lines).
Processing received data Expected results.
The results of the completed project will allow identifying area inhibitors HR1 and HR2 of the viral S-glycoprotein protein of the SARS-CoV-2 virus, which by their biological functions will lead to blocking the fusion of the two viral subunits S1 and S2. In this case, a second group of inhibitors will be developed, the effect of which will be directed to the inhibition of the second domain, which is the site of the RBD (receptor-binding domain) of the S-glycoprotein protein, which binds to ACE2 cell enzyme (Angiotensin-converting enzyme 2), which is the receptor of the host cell. The results of this project will allow the development of a therapeutic vaccine against Covid-19 based on the inhibitors found.