Design of peptide inhibitors
In the structure of ACE2 and SARS-CoV-2 (PDB: 6LZG) we analysed the interacting amino acids of ACE2 with SARS-CoV-2. In total 23 amino acids of ACE 2 interact with SARS-CoV-2 viz. 19 (S), 24 (Q), 27 (T), 28 (F), 30 (D), 31 (K), 34 (H), 35 (E), 37 (E), 38 (D), 41 (Y), 42 (Q), 45 (L) are in α1, 79 (L), 82 (M), 83 (Y) are in α2, 325 (Q), 329 (E), 330 (N), 353 (K), 354 (G), 355 (D), 357 (R) and 393 (R) are in β1 + β2. Nine inhibitors with three residues have been designed. Combinations of peptides were designed, which are depicted in table 1
Molecular docking study
The designed inhibitors' binding affinity with the spike potein was investigated.It has been reported that SARS-CoV-2-CTD displays a stronger affinity for hACE2. The docking results were given in terms of moldock score, rerank score, H-bond energy and interacting amino acid residues present at the predicted active site of the protein. It has been reported that ligands that possess the lowest or minimal energy create better interactions with proteins [15]. All the designed small peptide inhibitors have a binding energy of less than -120 kcal/mol and bind at the active site of protein. But out of all the proteins, HisGluAsp showed the highest binding affinity to the predicted active site of the protein, with a moldock score of −146.98 kcal/mol and a rerank score of -116.48 kcal/mol. From all the directed non-covalent interactions between biomolecules, hydrogen bonding is considered the most important one because it facilitates protein-ligand binding. With each additional hydrogen bond, the binding affinity of the ligand increases by one order of magnitude [24]. Moreover, HisGluAsp formed three H-bonds with active amino acids; two with Lys 417 and one with Gln 498 residues present at the predicted active site of the protein (Fig. 1a). Compounds with the highest moldock score are inserted into the cavity of the protein's activity site (the best 03 compounds are shown in fig. 1b). Steric interactions play an important role and can influence the binding of protein and ligand. It was observed that HisGluAsp forms steric interactions with various amino acids, including the most active amino acids. In contrast, SerGlyThr showed the lowest binding affinity to the predicted active site of protein with a moldock score of -124.58 kcal/mol, a rerank score of -80.39 kcal/mol and an H bond interaction of -7.20. It was observed that SerGlyThr forms no H-bond with active amino acids. PheAsnLys showed considerable binding affinity in comparison with HisGluAsp with a moldock score of 144.95 kcal/mol, a rerank score of -108.08 kcal/mol and an H bond interaction value of -8.13. It forms two hydrogen bonds with active amino acids Gln 498 and Lys 417 (Table 2). Table 3 elicits the distance, energy, and interaction of atoms from ligand and protein in hydrogen bond interactions of HisGluAsp.
Molecular dynamics
Root mean square deviation is widely used as a similarity measure in the analysis of macromolecular structures and dynamics. Figure 4 depicts the mean RMSD values obtained for the MD simulations trajectory and time. RMSD after 100 ns MDs of HisGluAsp was 0.17. This value indicates the marked stability of the compound with the protein [25, 26].
Physicochemical properties of peptides
Peptide-based and small-molecule drugs fulfil different chemical spaces due to variations of native amino acids in their side chains, putting peptides outside the established descriptor cutoffs. The molecular weight of the most potent compound in relation to binding affinity follows Lipinski’s molecular weight rule with a value of 396.33. The number of hydrogen bond donors and acceptors is a fundamental molecular descriptor to predict the oral bioavailability of small drug candidates. It is generally assumed that hydrogen bond donors and acceptors impact passive diffusion across cell membranes, a fundamental event during drug absorption and distribution. In the case of Lipinski hydrogen bond acceptors (LHBAs) the HisGluAsp does not follow the Lipinski rule, having a value of 13. The Lipinski hydrogen bond donors (LHBDs) are determined by counting the numbers of OH and NH bonds in each molecule. HisGluAsp follows the trend of LHBD with a value of 1. Previous reports have revealed that while physical models of permeability for conventional drug-like molecules are well established; the descriptors of membrane diffusion for molecules are not completely understood, hindering the rational design of cell-permeable, "beyond-Rule- of-Five" (bRo5) molecules as therapeutic agents. Other properties validated for HisGluAsp include molar refractivity (40–130), rotatable bonds (1–10), total polar surface area (140), and total atoms (20–70) (Table 4) [27-29].
Energy inspector of peptide
Ligand Energy Inspector allows to get detailed information about the energy interactions of a given ligand with a given protein. In table 5 the energy contribution of each atom of HisGluAsp with maximum binding affinity is depicted. The short-range interactions promote short-range conformational order, i.e., short segments of the chain have higher probabilities of assuming the same local conformations as in the native conformation. In table 5 ligand energy inspector of the most potent molecule, HisGluAsp, is depicted and both short- and long-range interactions are in the negative range, which suggests promising interactions between ligand and protein and also the interaction of an atom with protein (Table 4).
Boiled egg model
The boiled egg model is proposed as an accurate predictive model that works by computing the lipophilicity and polarity of small molecules. The BOILED-Egg model delivers a rapid, intuitive, easily reproducible yet statistically unprecedented method to predict the passive gastrointestinal absorption and brain access of small molecules useful for drug discovery and development. The white region is the physicochemical space of molecules with the highest probability of being absorbed by the gastrointestinal tract, and the yellow region (yolk) is the physicochemical space of molecules with the highest probability of permeating the brain. Yolk and white areas are not mutually exclusive. In fig. 2a the radar graph represents the value of various physicochemical properties, and in fig. 2b the most potent molecule has been shown to be highly absorbable in the gastrointestinal tract [30].