Preparation of ligand and target structure for computational analyses. The selected chemical structure of anti-HIV protease inhibitor drugs and phyto-flavonoids with individual PubChem with physicochemical properties such as molecular weight (g/mol), number of H-bond acceptors, H-bond donors, octanol/ water partition coefficient (XlogP), topological polar surface area (tPSA in Å), molar refractivity (mol/m3), number of rotatable bonds were recorded (Table S1 & Table S2).. Currently, all cited parameters are collectively known as the Lipinski rule five (RO5) in the current drug development module. However, all drugs do not follow the standard RO5 regulations, while a maximum number of phyto-flavonoids are obeying the RO5 rules. Currently, the RO5 is a fundamental physicochemical parameter based standard rule to filter the possible drug-able compound in the early stage of ‘lead drug’ selection. On the other hand, from fold recognition analysis with the selected target SARS-CoV–2-Mpro (ID: 6Y2E) confirmed that a >92 % structural portion with previously recognized SARS-CoV-Mpro (2DUC) was conserved (Fig. 1).. Concomitantly, from sequence-level analysis, twelve substituted amino acids/ mutations also found. From which nine variations/ mutations were conservative in between both CoV-Mpro. Thus, the pandemic SARS-CoV–2-Mpro is structurally-sequentially conserved, but the mutated amino acids may be associated with the development of severity or resistance to applied treatments.
Molecular docking of both drug and phytochemicals. The molecular docking score of selected 20 ligands (ten anti-HIV protease inhibitors drugs or n = 10 and ten phyto-flavonoids or n = 10) against the energy minimized and a stable (in 50 ns MDS) allosteric target, SARS-CoV–2- Mpro was recorded (Table 1 & Table 2; Figs. S1 to S3).. Among all anti-HIV drugs, darunavir with docking score, –10.25 kcal/mol and tipranavir with docking score, –10.14 kcal/mol were two most potent drugs against SARS-CoV–2-Mpro, based on recorded docking score (Table 1).. Similarly, from the phyto-flavonoid side, quercetin–3-rhamnoside with docking score, –10.90 kcal/mol and LPRP-Et–97543 with docking score, –10.11 kcal/mol were recorded (Table 2).. From molecular interactions study, the potent antiviral drug darunavir has formed three hydrogen bond interactions with amino acids with LYS5 and LEU282 (Fig. 2).. In comparison, quercetin–3- rhamnoside has assembled eight potent H-bond interactions at amino acids, LYS5, ALA7, GLN127, LYS137 and GLU290 (Fig. 3),, respectively against SARS-CoV–2-Mpro during docking study. Herein confirmed that the phyto-flavonoid quercetin–3-rhamnoside was more potent and stable than anti-HIV drugs in molecular docking analysis. As per the hypothesis of the combination drug approach, a total of four candidates (n = 4), from which two potent anti-HIV drugs, darunavir and tipranavir (n = 2) and LPRP-Et–97543 and quercetin–3-rhamnoside (n = 2), were selected. Hypothetically, the double-docking score is comparatively higher than the individual docking score of each selected ligands. Fortunately, among four double-docking complexes, the darunavir-quercetin–3-rhamnoside (n = 1) combination was the most effective combination with total docking score, –14.83 (double docking score, –10.95 kcal/mol and ligand- ligand docking score, –3.88 kcal/mol) than other double docking complexes (Table 3; Fig. 4).. Above all docking analysis, three docking complexes, SARS-CoV–2-Mpro-darunavir, SARS- CoV–2-Mpro-quercetin–3-rhamnoside and SARS-CoV–2-Mpro-darunavir-quercetin–3-rhamnoside were selected for stability analyses with MDS at 30 ns.
Molecular dynamic simulation study with most effective protein-ligand complexes. Towards understand the structural stability of native protein with selected protein-ligand docking complexes through the root mean square deviation (RMSD)-protein backbone, root mean square fluctuation (RMSF)-C-alpha and Radius of gyration (Rg) of protein were analyzed by MDS. The explained RMSD plot of single protein determined that continuous unorthodoxy in backbone protein has appeared during 50 ns time. But between 32 to 46 ns, absolute mere stability was found from the analyzed plot (Fig. S1).. Similarly, the RMSF-plot describes the c-alpha and Rg- plot of the whole protein including c-alpha, backbone and side-chain described the inconsistency to maintain the stability during 50 ns in extracted plots (Figs. S2 & S3).
From the single docking complex of SARS-CoV–2-Mpro-darunavir, SARS-CoV–2-Mpro- quercetin–3-rhamnoside, the RMSD plot of quercetin–3-rhamnoside was comparatively more stable than the darunavir complex (Fig. 5).. Protein backbone of SARS-CoV–2-Mpro-quercetin–3- rhamnoside (green color bar in Fig. 5), showing the least deviation in between 0 to 29 ns, while SARS-CoV–2-Mpro-darunavir (blue color bar in Fig. 5) displayed the variation for first 20 ns and the rest 10 ns gradually less fluctuate in plotted RMSD. Similarly, the RMSD plot of double docking was comparative higher fluctuation at an upper length, > 0.25 nm between 10 to 15 ns (orange color bar in Fig. 5). From the above RMSD plot analysis, quercetin–3-rhamnoside having the least fluctuation within 30 ns (Fig. 5).. Correspondingly, garnered individual RMSF- and Rg- plots of and overplayed for judgment the variation with simulation time in between three docking complexes (Figs. 6 & 7).. Based on the RMSF-analyses, both anti-HIV drug and double docking complex (SARS-CoV–2-Mpro-darunavir-quercetin–3-rhamnoside) exhibited the most diverged value in corresponding to C-alpha residues than phyto-flavonoids. Furthermore, Rg-plots showed the compactness or solidity of the quercetin–3-rhamnoside with more squeezed Rg-values rather than other complexes (Fig. 7).. Additionally, H-bond interaction analyses exposed that, eight strong H-bond interactions in both SARS-CoV–2-Mpro-quercetin–3-rhamnoside (green color bar in Fig. 8) and SARS-CoV–2-Mpro-darunavir-quercetin–3-rhamnoside (in the orange color bar), were found (Fig. 8).. In the form of activity based on strong H-bond interactions points of view, the quercetin–3-rhamnoside was a most active component than the anti-HIV drug darunavir; as a result, the flavonoid combined drug formulation exhibited the most therapeutic potency towards inhibition of SARS-CoV–2.
Possible toxicity and drug-ability prediction for both anti-HIV drug and phytochemicals. The toxicity or side effect is a significant concern for implementation of therapeutic agents/ drugs. Currently, a maximum number of lead-drug candidates unable to express the ideal safety pieces of stuff and later on, withdrawn from the clinical trial. Herein, from the predicted toxicity profile records, phyto-flavonoids were comparatively safer than the anti-HIV drugs (Tables 4 & 5).. However, except for hepatotoxicity and immunotoxicity, anti-HIV drugs are safer like phytochemicals. Mainly selected darunavir showed a moderately reliable under class-III category with severe hepatotoxicity (Table 4).. On the other hand, phyto-flavonoid, the quercetin–3- rhamnoside, was reasonably safer with class-V, as a more reliable/ inoffensive drug-able compound (Table 5)..
In the same way, the overall drug-likeness or possibility for a drug molecule score for each ligand was recorded (Tables 1 & 2).. As per the drug-ability plot, positive drug-likeness value is good; a score between 0.60 to 1.20 is an ideal score to be a successive drug molecule. The drug candidates presented perfect drug-likeness scores, while it was discernible that all phyto-flavonoids too exhibited drug-like scores as a recommended anti-HIV drugs, computationally. The drug-ability score of the quercetin–3-rhamnoside had the most potential score, 0.82, while the anti-HIV drug darunavir score, 0.60 (Figs. S4 & S5). Thus, as per the predicted drug-likeness score, it also supported the proposed phyto-drug combination.
Possible pharmaceutical profiles prediction for both anti-HIV drug and phytochemicals. At the close stage of drug validation, the pharmacokinetics profile plays a crucial during recommendation. Moreover, advanced computational tools also able to provide some statistical- based reports for each ligand from its training set documents at an early stage. Herein, both anti- HIV drugs and phyto-flavonoids pharmacokinetics profiles were recorded (Tables S3 & S4).. As per reports, except for lopinavir and indinavir, rest for anti-HIV medicines have lower gastrointestinal absorption (GI-abs.), including darunavir (Table S3).. Similarly, all drugs are unable to cross the blood-brain barrier (BBB) report. On the other hand, pharmacokinetics profiles of phyto-flavonoids displayed in a changeability manner of GI-abs., P-gp substrate, etc., where the quercetin–3-rhamnoside was similar to anti-HIV drug profile along with, all phytochemical showed the same negative BBB cross report. (Table S4).. The overall pharmacokinetics also reports of all ligands were presented, graphical (Figs. S6 & S7).. From the above analysis reports, all ligands are some deviate profiles, mainly phytochemicals. As a result, a combination of a mainstream drug with a phytochemical may have maintained the activity as well as pharmacokinetics in the presence of one with another during treatment.