PMC:7755033 / 30391-104009 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7755033","sourcedb":"PMC","sourceid":"7755033","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7755033","text":"3. Results\n\n3.1. Virtual screening of prospective antiviral candidates from WS phytoconstituents on the basis of physicochemical parameters and Lipinski’s rule of five (PASS analysis)\nIn the drug discovery context, it is generally believed that an orally active drug candidate cannot have more than one violation of Lipinski’s criteria otherwise it might compromise its bioavailability (Balakrishnan et al., 2014).\nBased on Lipinski's rule of five, WS phytoconstituents were previously screened and selected for their drug like properties (Table 2). As is evident from Table 2, none of the selected WS phytoconstituents exhibited Lipinski’s violation. Interestingly, standard reference drugs cinacalcet and poziotinib displayed 1 violation each of Lipinski’s rule of five.\nTable 2. PASS analysis of WS phytoconstituents versus FDA-approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib).\nPhysicochemical properties\nS. No. Ligands Lipinski’s rule of 5 parameters\n% Absorption(\u003e50%)a Topological polar surface area (Å)2(TPSA)b(\u003c160 Å) MW (\u003c500) clog Pc(\u003c5) Hydrogen bond donors(NOHNH)(≤5) Hydrogen bond acceptors(NON)≤10) Number of rotatable bonds(≤10) Lipinski’s violation (LV)\n1. Withaferin A 75.76 96.36 470.61 2.49 2 6 3 0\n2. Withanolide A 75.76 96.36 470.61 2.56 2 6 2 0\n3. Withanolide B 82.74 76.13 454.61 3.42 1 5 2 0\n4. Withanolide D 75.76 96.36 470.61 2.56 2 6 2 0\n5. Withanolide E 68.78 116.59 486.61 1.77 3 7 2 0\n6. Withanone 75.76 96.36 470.61 2.60 2 6 2 0\n7. Viscosalactone B 68.78 116.59 488.62 1.92 3 7 3 0\n8. Anaferine 94.81 41.12 224.35 1.47 2 3 4 0\n9. Withasomnine 102.85 17.83 184.24 2.65 0 2 1 0\n10. Losartan 77.09 92.5 422.9 3.95 2 5 8 0\n11. Procainamide 88.85 58.4 235.33 0.93 2 3 6 0\n12. Cinacalcet 104.86 12 357.4 5.65 1 4 6 1\n13. Arbidol 81.4 80 477.4 4.17 1 5 8 0\n14. Hydroxychloroquine 92.31 48.38 335.88 3.08 2 4 9 0\n15. Oberadilol 67.95 119 484 2.80 4 7 10 0\n16. Poziotinib 82.57 76.6 491.3 5.29 1 7 6 1\nRule: aPercentage absorption was calculated as: % absorption = 109 – [0.345 × topological polar surface area].\nbTopological polar surface area (defined as a sum of surfaces of polar atoms in a molecule).\ncLogarithm of compound partition coefficient between n-octanol and water. WS phytoconstituents were further analyzed using additional filters viz. Ghose, Veber, Egan, Muegge and Leadlikeness filters (Table 3). The selected phytoconstituents showed no violations of Veber, Egan and Muegge filters thereby indicating their drug-like character. The drug cinacalcet showed 1 and 2 violations of Egan and Muegge filters, respectively, whereas drug poziotinib exhibited 1 violation of Muegge filter.\nTable 3. Drug-like character of WS phytoconstituents versus FDA-approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib).\nS. No. Ligands GNo. of vio.a VNo. of vio.b ENo. of vio.c MNo. ofvio.d Leadlikeness\n1. Withaferin A 1 0 0 0 2\n2. Withanolide A 1 0 0 0 1\n3. Withanolide B 1 0 0 0 2\n4. Withanolide D 1 0 0 0 1\n5. Withanolide E 2 0 0 0 1\n6. Withanone 1 0 0 0 1\n7. Viscosalactone B 1 0 0 0 1\n8. Anaferine 0 0 0 0 1\n9. Withasomnine 0 0 0 0 1\n10. Losartan 0 0 0 0 3\n11. Procainamide 0 0 0 0 1\n12. Cinacalcet 1 0 1 2 2\n13. Arbidol 0 0 0 0 3\n14. Hydroxychloroquine 0 0 0 0 2\n15. Oberadilol 2 0 0 0 2\n16. Poziotinib 2 0 0 1 2\nRule: aGhose filter.\nbVeber filter.\ncEgan (Pharmacial) filter.\ndMuegge (Bayer) filter.\n\n3.2. admetSAR analysis of selected WS phytoconstituents\nGood ADME and toxicity properties are as critical as therapeutic properties. Human intestinal absorption (HIA), Caco-2 cell permeability, Blood–brain barrier (BBB) penetration, and Ames test were calculated for the chosen phytoconstituents and reference drugs using admetSAR version 1.0 (Table 4).\nTable 4. admetSAR prediction of selected WS phytoconstituents versus FDA-approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib).\nLigands Human intestinal absorption (HIA) Caco-2 permeability P-glycoprotein inhibitor Blood–brain barrier penetration (BBB) Ames mutagenesis Subcellular localization Biodegradation Acute oral toxicity (kg/mol)\n± p value ± p value ± p value ± p value ± p value ± p value ± p value\nWithaferin A + 0.9729 – 0.6673 + 0.6132 + 0.9537 – 0.6900 Mitochondria 0.7714 – 0.8750 3.276\nWithanolide A + 0.9829 – 0.6006 + 0.6554 + 0.8333 – 0.8700 Mitochondria 0.6830 – 0.8000 5.165\nWithanolide B + 0.9829 – 0.5605 + 0.7494 + 0.9128 – 0.8000 Mitochondria 0.6784 – 0.8750 4.099\nWithanolide D + 0.9750 – 0.6274 – 0.4303 + 0.8345 – 0.7800 Mitochondria 0.7352 – 0.8250 3.66\nWithanolide E + 0.9640 – 0.6455 – 0.4399 – 0.5510 – 0.7100 Mitochondria 0.6273 – 0.9250 5.292\nWithanone + 0.9829 – 0.6472 + 0.6845 + 0.8333 – 0.8300 Mitochondria 0.6830 – 0.8500 4.775\nViscosalactone B + 0.9480 – 0.7386 – 0.4906 + 0.9214 – 0.7500 Mitochondria 0.7598 – 0.8250 3.059\nAnaferine + 0.9064 + 0.5418 – 0.9112 + 0.9929 – 0.7300 Mitochondria 0.7672 – 0.7000 2.517\nWithasomnine + 0.9932 + 0.9586 – 0.9813 + 0.9966 – 0.9100 Mitochondria 0.5372 – 0.8750 2.41\nLosartan + 0.9883 – 0.9373 + 0.8124 – 0.9930 – 0.5200 Mitochondria 0.7540 – 0.9250 3.322\nProcainamide + 0.9795 + 0.9185 – 0.9721 + 0.9707 – 0.5900 Lysosomes 0.8295 – 0.6000 2.59\nCinacalcet + 0.9911 + 0.7035 + 0.5803 + 0.9974 – 0.5000 Lysosomes 0.9070 – 1.0000 3.492\nArbidol + 0.9684 + 0.6814 + 0.6810 + 0.9739 + 0.5300 Lysosomes 0.5338 – 0.9000 2.753\nHydroxychloroquine + 0.9934 + 0.5313 – 0.7900 + 0.9878 + 0.6400 Lysosomes 0.8067 – 0.8500 2.665\nOberadilol + 0.9820 – 0.7895 + 0.7739 + 0.9693 – 0.6300 Mitochondria 0.8157 – 0.7250 3.747\nPoziotinib + 0.9852 – 0.6765 + 0.8852 + 0.9900 – 0.5600 Mitochondria 0.5163 – 0.9000 3.121\n\n3.2.1. Human intestinal absorption (HIA)\nAn orally administered drug is absorbed primarily in the intestine. All WS phytoconstituents and standard reference drugs exhibited positive results, thereby indicating their absorption and assimilation in human intestine.\n\n3.2.2. Caco-2 permeability\nCaco-2 is a human colon epithelial cancer cell line and is used as a model for human intestinal assimilation of drugs and other compounds. In the present study, whereas anaferine and withasomnine exhibited positive results indicating Caco-2 permeability, the remaining seven WS phytoconstituents displayed negative results. In case of standard reference drugs, procainamide, cinacalcet, arbidol and hydroxychloroquine displayed good permeability characteristics for Caco-2 (Table 4).\n\n3.2.3. Blood–brain barrier (BBB) penetration\nAn important consideration for drug candidates is their ability to cross the BBB. All of the chosen WS phytoconstituents displayed positive results for BBB penetration except withanolide E. In case of standard reference drugs, only losartan displayed inability to penetrate the BBB (Table 4).\n\n3.2.4. Ames test\nIn the present study, none of the chosen WS phytoconstituents were predicted to have any mutagenic effect in contrast to standard reference drugs arbidol and hydroxychloroquine which tested positive for their ability to induce mutations (Table 4).\n\n3.3. Docking studies of WS phytoconstituents with respect to selected target proteins\nDocking studies of the selected WS phytoconstituents were carried out with human ACE2 receptor, SARS-CoV and SARS-CoV-2 specific proteins. The catalytically active sites of SARS-CoV-2 specific proteins were targeted in order to obtain the binding energy involved in the complex formation and to discover the molecular mechanisms responsible for specific inhibition of targets. Tables 5–11 summarize the predicted binding energies and dissociation constants (Kd) of WS phytoconstituents with respect to specific human ACE2 receptor, SAR-CoV and SARS-CoV-2 spike glycoproteins as well as the two main SARS-CoV-2 proteases viz. 3CL-pro and PL-pro. The binding sites of the WS phytoconstituents on the selected viral target proteins as well as the interacting amino acids were predicted to be almost the same by the three molecular docking softwares (Tables 5–11). The common interacting amino acids between the three softwares have been written in italicized form in Tables 5–11. As is evident from Tables 5–11, most of the WS phytoconstituents exhibited potent binding kinetics to the above-mentioned proteins. Docking analyses using AutoDock 4.0/ADT version 4.2.6 program revealed that the binding affinities of the WS phytoconstituents for the human ACE2 receptor decreased in the order withanolide B \u003e withanolide A \u003e withanolide E \u003e viscosalactone B \u003e withaferin A \u003e anaferine \u003e withanolide D \u003e withanone \u003e withasomnine. Withanolide B exhibited a 1000× stronger binding to human ACE2 receptor (Table 5; BE: −10.21 kcal/mol, Kd: 32.78 nM) as compared to standard reference drugs, arbidol (Table 5; BE: −6.69 kcal/mol, Kd: 12.47 µM) and losartan (Table 5; BE: –6.72 kcal/mol, Kd: 11.86 µM). Withanolide B also exhibited potent binding to papain like protease of SARS-CoV-2 (Table 8; BE −10.3 kcal/mol, Kd: 28.32 nM) as compared to procainamide (Table 8; BE −5.03 kcal/mol, Kd: 206.96 µM) and cinacalcet (Table 8; BE −6.44 kcal/mol, Kd: 19.17 µM).\nTable 5. Binding energies of WS phytoconstituents with human ACE2 receptor (PDB ID: 1O8A) in comparison to the FDA approved standard reference drugs (Arbidol and Losartan).\n  AutoDock v4.2.6 AutoDock vina iGEMDOCK v2.1\nS. No. Ligands BE (kcal/mol) K d Interacting amino acids BE(kcal/mol) K d Interacting amino acids TE(kcal/mol) VDW HB EI Interacting amino acids  \n1. Withaferin A –8.44 647.61 nM Glu123, Met223, Trp220, Pro519, Arg522, Ser517, Val518, Glu411, His410, Ser355, Ala356, His387, Phe391, Glu403 –10.4 23.67 nM His353, Val518, His513, Tyr523, Glu411, Arg522, Phe512, Ala356, Ser355, Ser516, Glu143, Asn70, Asn66, Tyr69, Leu140, Leu139, Leu81, Asn85, Tyr62, Asn136, Arg124 –111.33 –90.07 –21.26 0 Ser284, Tyr287, Val291, Asp288, Asn285, Glu376, Leu375, Asn374, Thr302, Thr301, Asp300, Met299, Ser298, Pro297, Pro294, Ala296  \n2. Withanolide A –10.13 37.44 nM Asp453, Thr282, Gln281, Phe457, Glu376, Val379, Val380, Asp415, His383, Glu384, Ala354, His353, His387, Glu411, His513, Phe512, Tyr523 –10.6 15.57 nM His410, Ala353, His387, Trp357, Phe391, Asn66, Asn70, Ser516, Glu143, Phe512, Val518, His353, Ser355, His513, Tyr523, Glu411, Arg522 –98.26 –78.36 –19.90 0 Leu375, Lys449, Tyr287, Ser298, Met299, Asp300, Thr301, Thr302  \n3. Withanolide B –10.21 32.78 nM Phe391, His387, Ala356, His383, Ser355, Ala354, His353, Glu403, Gly404, His410, Glu411, Pro407, Tyr523, Arg522, Phe512, His513, Met223 –10.7 10.60 nM Asn85, Tyr62, Arg124, Asn66, Phe512, His353, Val518, His513, Tyr523, Arg522, Glu411, Leu81, Leu140, Leu139, Tyr69, Glu143, Asn70, Ser516, Ser355, Ala356, His387 –97.61 –86.31 –11.29 0 Thr226, Glu225, Pro227, Tyr224, Leu229, Ser222, Arg221, Asp218, Tyr213, Asn211, Asp121, Gln120, Lys117  \n4. Withanolide D –8.23 934.88 nM Phe391, Ala356, Ser355, His387, Glu384, Ala354, His353, Glu411, Tyr523, Val518, His383, Val380 –11.3 600 pM Glu411, Tyr523, Arg522, His410, Phe391, Ala356, His387, Asn66, Asn70, Tyr69, Trp357, Glu143, Val351, Ser516, Phe512, His353,Val518, Ser355 –101.56 –82.00 –19.56 0 Arg124, Leu140, Leu81, Glu143, Asn66, Asn70, Tyr69, His353, Ala354, Ser355, Ala356, Glu384, His387, Glu411, Tyr523, Val518, His513, Phe512  \n5. Withanolide E –9.75 71.2 nM Phe391, His410, His387, Glu411, Arg522, Tyr523, Val518, His513, Phe512, His353, Ala354, Ser355, Glu384, Ala356, Lys368, Asn70 –10.6 15.45 nM Arg124, Leu140, Leu139, Leu81, Asn70, Tyr69, Glu143, Ser516, Val351, Trp357, Phe512, His353, Ser355, Ala356, Glu384, His387, Phe391, Val518, Tyr62, Asn66 –104.39 –90.97 –13.41 0 Glu239, Arg235, Leu236, Asp232, Ser228, Thr226, Pro575, Trp574, Pro585, Asn586, Met587  \n6. Withanone –8.12 1.12 µM Ser422, Phe527, Lys454, Tyr523, Val379, His383, Glu384, Val380, His513, Gln281, Thr282, Ser284, Phe457, Asp453 –10.2 31.56 nM Lys449, Val291, Pro297, Ser298, Asp300, Met299, Ser284, Asn285, Asn374, Glu376, Thr302, Leu375, Thr301, Tyr287, Asp288 –100.15 –79.22 –20.92 0 Asn374, Leu375, Thr302, Thr301, Asp300, Met299, Ser298, Pro297, Ala296, Pro294, Val291, Tyr287, Asn285  \n7. Viscosalactone B –8.83 339.1 nM Ala356, His387, Ser355, Glu384, Ala354, His353, His513, Phe512, Tyr523, Thr282, Asp453, Ser284, Glu376, Val379, Val380, His383, Glu411 –11.1 760 pM Tyr62, Asn85, Asn136, Leu81, Glu143, Leu139, Leu140, Asn66,Asn70, Phe512, Ala356, Ser355, Ala354, His353, Tyr523, His383, His387, Glu411, Arg522, Val518, Arg124 –119.88 –92.25 –27.63 0 Tyr62, Leu81,Asn136, Asn66, Tyr69, Leu139, Leu140, Glu143, Ser516, Asn70, His513, His353, Ala354, Tyr523, Ser355, Ala356, Trp357, His387, Glu411, Glu384, His383  \n8. Anaferine –8.25 890.86 nM Glu411, Tyr523, His383, His387, Glu384, Val380, Ala356, Ser355, Ala354, His353 –6.7 12.50 µM His383, His387, Ala354, Glu384, Ser355, Ala356, Val518, His513, Phe512, His353, Tyr523, Tyr520, Gln281, Phe457, Phe527 –77.85 –74.35 –3.5 0 Phe570, Met223, Asn406, Glu403, Gly404, Pro407, His410, Phe391, Glu411, His387  \n9. Withasomnine –4.99 218.08 µM Tyr523, His513, Val380, His383, Glu384, His387, Ala356, Ser355, Ala354, His353 –6.7 12.59 µM Phe291, Tyr394, His410, Arg522, Gly404, Pro407, Glu403, Met223, Phe570, Asn406 –74.24 –64.10 –10.14 0 Leu122, Thr92, Ala125, Arg124, Ala89, Ile88, Asn85, Asn136, Trp59  \n10. Arbidol –6.69 12.47 µM Ala354, His353, Glu162, Lys511, Trp279, Gln281, Thr282, Phe457, Asp453, Tyr523, Lys454, Phe527, Asp415, His383, Val380, Glu384 –8.3 880.23 nM Phe570, Met223, Asn406, Gly404, Glu403, Arg402, Tyr394, Tyr360, Phe391, Asp358, Trp357, Ala356,His387, His410, Glu411, Arg522, Pro407 –86.22 –73.55 –12.67 0 Phe391, His410, Glu411, His387, Trp357, Ala356, Ser355, Ala354, His353, Asn66, Ala63, Tyr62, Val518, Tyr523  \n11. Losartan –6.72 11.86 µM Asn406, Met223, Arg522, Pro407, Gly404, Glu403, His410, Glu411, Tyr523, Phe391, His387, Ala356, Val518 –9.4 129.59 nM Met223, Pro519, Arg522, His410, His387, Tyr523, Glu411, Ser355, Val518, Ala356, Asp358, Tyr360, Phe391, Gly404, Glu403, Asn406, Pro407 –101.28 –85.24 –16.04 0 Asp121, Leu122, Thr92, Trp220, Glu123, Arg124, Ala89, Ile88, Pro519, Tyr62, Ser517, Tyr135, Ile204  \nTable 6. Binding energies of WS phytoconstituents with SARS-CoV spike glycoprotein (PDB ID: 5WRG) in comparison to the FDA approved standard reference drugs (Arbidol and Hydroxychloroquine).\n  AutoDock v4.2.6 AutoDock vina iGEMDOCK v2.1\nS. No. Ligands BE (kcal/mol) K d Interacting amino acids BE(kcal/mol) K d Interacting amino acids TE(kcal/mol) VDW HB EI Interacting amino acids  \n1. Withaferin A –8.39 702.21 nM Ile979, Leu983, Thr980, Gly981, Gln984, Tyr738, Leu983, Gln984, Thr980 –10.3 28.30 nM Lys715, Ala753, Ala754, Asp757, Arg761, Leu846, Arg758, Pro651, Ile652, Leu843, Leu597, Gln599, Ala633, Val581, Tyr300, Thr302 –104.25 –83.69 –20.56 0 Arg453, Glu452, Asp454, Ser456, Arg449, Arg441, Val458, Pro459, Arg444, His445, Phe460, Pro466, Gly464, Ser461, Lys465  \n2. Withanolide A –9.78 67.23 nM Gln984,Leu983, Thr980, Gly981, Arg977, Phe952, Tyr738 –10.5 19.50 nM Ile299, Ala754, Ser750, Thr302, Ala753, Val581, Leu843, Gln599, Ile652, Arg758, Lys715, Gly653, Asp757, Tyr300, Arg761, Pro651, Leu846 –97.67 –85.32 –12.35 0 Ile652, Pro651, Thr302, Tyr300, Ile299, Gln301, Ser750, Ala754, Arg747, Gly751, Lys715  \n3. Withanolide B –9.4 129.59 nM Gln984, Thr980, Leu983, Gly981, Arg977, Phe952, Tyr738, Asp976 –10.4 23.67 nM Ser950, Asn951, Gly981, Arg977,Thr980, Asp976, Asp976, Phe952,Gln984, Leu983, Gln947, Phe741, Ser985, Tyr738 –100.62 –94.31 –6.32 0 Lys715, Asp757, Ala753, Arg761, Arg758, Ala754, Leu846, Gly653, Ile652, Pro651, Tyr300  \n4. Withanolide D –9.1 212.78 nM Thr980, Gly981, Leu983, Tyr738, Phe952, Gln984, Asp976, Ile979 –10.7 11.45 nM Ile299, Ala754, Pro651, Arg758, Arg761, Asp757, Leu846, Leu597, Lys715, Val581, Ile652, Ala633, Gln599, Leu843, Tyr300, Ser750, Ala753, Thr302 –103.3 –84.53 –18.77 0 Ala926,Leu927, Ser924, Gly928, Thr925, Gln931, Asn935, Lys297, Asp296, Ile295, Glu294, Val290  \n5. Withanolide E –7.13 5.93 µM Thr980, Gly981, Gln984, Arg977, Tyr738, Leu983 –9.5 99.21 nM Lys715, Ala753, Ser750, Pro844, Thr302, Leu843, Ala633,Gln599, Ile652, Leu597, Val581, Ile299, Pro651, Ile650, Tyr300, Ala754 –99.34 –93.84 –5.49 0 Leu597,Ala633, Gln599, Ile652, Val581, Pro651, Thr302, Ile650, Tyr300, Ile299, Pro844, Leu843, Lys715, Ser750, Ala753, Ala754  \n6. Withanone –6.49 17.46 µM Thr980, Gly981, Gln984, Leu983, Phe741,Tyr738 –9.5 99.56 nM Arg747, Gly751, Ala754, Pro651, Ile652, Lys715, Gln599, Val581, Tyr200, Leu843, Ala753, Thr302, Ser750, Ile299, Gln939, Gln301 –104.09 –85.03 –19.07 0 Leu810, Ala938, Asn942, Lys946, Thr943, Gln744, Arg747, Thr743, Ser289, Gln301, Val290, Lys291  \n7. Viscosalactone B –7.6 2.69 µM Gln987, Gln984, Leu983, Thr980, Asp976, Phe952, Gly981, Gln984, Thr988 –10.2 32.60 nM Leu994, Ile995, Glu999, Ala998, Ile752, Arg1001, Ala997, Glu755, Gln936, Asp932, Ile299, Asp296, Tyr300, Gln301, Gln939, Arg747, Ala748, Gly751, Arg996 –121.82 –89.39 –32.43 0 Arg563, Arg315, Phe551, Gln550, Asn530, Gly531, Asn505, Leu504, Leu503, Ser380, Phe379, Cys378, Arg965, Asn530, Gly551  \n8. Anaferine –6.94 8.16 µM Gln947, Phe741, Ser985, Gln984, Gly981, Tyr738, Phe952, Leu983, Thr980, Ile979, Asp976 –7.0 6.40 µM Arg747, Thr943, Gln744, Thr988, Gln987, Phe741, Gln947, Ser985, Tyr989, Leu944, Ala940, Gln992 –84.12 –69.13 –14.99 0 Arg965, Leu374, Lys373, Tyr352, Cys378, Leu503, Phe501, Met417, Val369, Ser370  \n9. Withasomnine –5.87 49.69 µM Gln947, Phe741, Ser985, Gln984, Gly981, Leu983, Phe952, Asn951, Tyr738, Ser950 –7.1 6.25 µM Leu963, Ser728, Cys725, Val958, Leu948, Phe558, Asp557, Phe837, Asn960, Thr535, Thr533, Asp727, Leu959 –80.03 –72.43 –7.60 0 Cys278, Phe262, Met263, Val276, Cys288, Lys287, Ser292  \n10. Arbidol –4.91 251.65 µM Phe952, Gln947, Gly739, Phe741, Tyr738. Gly981, Arg977, Thr980, Asp976, Thr980, Leu983, Gln984 –7.7 2.20 µM Ala754, Ser750, Asn746, Arg747, Thr302, Gln599, Gln301, Val581, Tyr300 –83.02 –78.02 –5 0 Leu859, Pro1035, Phe1034, Ser1033, Cys1014, Val1015, Gln883, Leu1016, Pro879, Gly862  \n11. Hydroxychloroquine –5.25 142.18 µM Gln984, Leu983, Thr980, Ile979,Ser985, Gly981, Phe741, Gln947, Tyr738, Phe952 –7.0 7.0 µM Ala753, Ser750, Pro651, Lys715, Leu843, Gly653, Ile652, Val581, Tyr300, Thr302, Gln599, Leu597, Ile299, Ala754 –76.22 –68.13 –8.09 0 Lys1027, Gly1026, Val1022, Ser1012, Val1015, Gln766, Lys768  \nTable 7. Binding energies of WS phytoconstituents with SARS-CoV-2 spike glycoprotein (PDB ID:6VXX) in comparison to FDA approved standard reference drugs (Arbidol and Hydroxychloroquine).\n    AutoDock v4.2.6 AutoDock vina iGEMDOCK v2.1\nS. No. Ligands BE (kcal/mol) K d Interacting amino acids BE(kcal/mol) K d Interacting amino acids TE(kcal/mol) VDW HB EI Interacting amino acids\n1. Withaferin A –6.11 33.39 µM Leu303, Tyr313, Thr302, Gln314, Thr315, Ser316, Asn317, Arg319 –8.6 447.56 nM Gln218, Phe59, Gly219, Phe220, Thr33, Asp287, Phe58, Val289, Ser297, Lys300, Asp294, Leu296, Asn606, Leu293 –84.45 –66.52 17.93 0 Thr430, Asp428, Phe515, Ser514, Glu516, Tyr396, Arg355, Phe464\n2. Withanolide A –7.18 5.48 µM Glu309, Tyr313, Thr302, Leu303, Lys304, Gln957 –8.4 650.76 nM Leu977, Cys743, Val976, Asn978, Leu966, Ser975, Ser967, Arg44, Ser45, Val47, Asn856, Arg1000, Tyr741, Ile742 –83.67 –61.91 –21.76 0 Arg273, Pro272, Cys291, Thr274, Cys301, Gln52, Lys304, Ser50, Thr301, Thr315, Ala292, Glu298\n3. Withanolide B –6.81 10.26 µM Gln31, Tyr313, Thr302, Leu303, Lys304, Gln957, Asn960, Lys964 –8.0 771.67 nM Arg319, Thr572, Thr573, Asp571, Arg567, Leu546, Thr547, Gly548, Phe541, Thr549, Pro589, Cys590, Phe592, Arg319 –86.89 –78.89 –7.9 0 Asn556, Ile584, Leu582, Arg577, Lys557, Lys558, Phe559, Leu560, Pro561\n4. Withanolide D –6.9 8.68 µM Gln957, Thr961, Lys964, Gln965, Ser967, Ser968, Leu303, Lys304, Thr302 –8.7 398. 84 nM Val976, Arg1000, Phe855, Ser975, Lys854, Val963, Asn856, Ser967, Leu966, Asn978, Leu977, Asp745, Met740, Gly744, Tyr741 –82.91 –69.68 –13.23 0 Gln954, Ala958, Thr961, Leu962, Arg1014, Glu1017, Ile1013, Gln1010, Tyr1007, Thr1006, Ser1003, Gln965\n5. Withanolide E –6.94 8.17 µM Gln957, Lys964, Lys304, Leu303, Thr302 –8.0 771.57 nM Asn30, Phe59, Thr33, Phe58, Phe306, Val289, Ala288, Lys300, Ser297, Leu296, Leu293, Asp294 –91.24 –82.26 –8.98 0 Glu340, Gly339, Phe338, Cys336, Ala363, Asp364, Val362, Leu335\n6. Withanone –7.15 5.75 µM Gln309, Ser305, Leu303, Lys304, Thr302, Lys964, Gln957 –8.0 771.87 nM Asn978, Val963, Lys964, Ser967, Asn856, Ser975, Val976, Leu966, Tyr741, Leu977, Arg1000, Gly744 –84.14 –69.21 –14.92 0 Pro793, Pro792, Thr791, Lys790, Pro897, Thr883, Ile896, Gln895\n7. Viscosalactone B –6.02 38.89 µM Gln957, Ser305, Lys304, Leu303, Thr302, Thr315, Gln31, Ser316, Asn317 –8.2 935.88 nM Asn856, Gly744, Arg1000, Val976, Ser975, Ser967, Leu966, Asn978, Leu977 –98.52 –80.77 –17.75 0 Phe86, Asn87, Ile235, Asp88, Asn234, Pro272, Ile233, Leu54, Asn196, Asp53, Gln52, Ile197, Gly199, Asp198\n8. Anaferine –3.86 1.47 mM Tyr313, Glu309, Leu303, Lys304, Gln957 –5.0 213.05 µM Ser967, Ser975, Val976, Leu977, Arg1000, Asn978, Gly744, Asp745, Met740, Phe855, Tyr741, Leu966, Asn856, Val963, Lys964 –72.56 –58.62 –13.94 0 Lys933, Ser929, Ile934, Ala930, Gln926, Thr719, Ser721, Ile720, Val722\n9. Withasomnine –4.51 494.91 µM Ser316, Thr315, Glu298, Cys291, Thr302, Cys301, Thr274, Lys304, Ser50 –6.2 29.91 µM Ser1003, Leu962, Tyr1007, Gln957, Arg1014, Gln1010, Ala958, Thr1006, Gln965, Thr961 –73.49 –65.71 –7.78 0 Glu1111, Gln1113, Thr1105, Gln1106, Val1104, Thr912, Asn1119, Glu1092\n10. Arbidol –3.14 4.99 mM Glu309, Tyr313, Gln314, Leu303, Thr302, Gln957, Thr961, Lys964 –5.8 56.89 µM Phe59, Thr33, Phe220, Thr286, Asp287, Ala288, Val289, Asp290, Leu293, Asp294, Leu296, Ser297, Lys300 –78.93 –72.29 –6.64 0 Thr618, Glu619, Gln644, Asn616, Val615, Asp614, Phe592, Cys590, Pro589, Thr589\n11. Hydroxychloroquine –2.48 15.11 mM Ser50, Cys301, Glu298, Thr302, Thr315, Ser316 –6.1 35.2 µM Asp294, Leu296, Leu293, Ser297, Lys300, Val289, Asp287, Phe306, Thr33, Asn606, Thr602 –72.42 –62.92 –9.5 0 Val722, Thr724, Ile726, Ile934, Ser937, Leu938, Thr941, Ala944, Ser943, Ile726\nTable 8. Binding energies of WS phytoconstituents with papain like protease of SARS-CoV-2 (PDB ID: 6W9C) in comparison to the FDA approved standard reference drugs (Procainamide and Cinacalcet).\n    AutoDock v4.2.6 AutoDock vina iGEMDOCK v2.1\nS. No. Ligands BE (kcal/mol) K d Interacting amino acids BE(kcal/mol) K d Interacting amino acids TE(kcal/mol) VDW HB EI Interacting amino acids\n1. Withaferin A –8.74 393.84 nM Asn109, Leu162, Gly160, Gln269, Glu161, Val159, His89, Thr158 –9.8 61.88 nM His89, Val159, Gly160, Glu161,Leu162, Gln269, Asn109, Thr158 –101.80 –86.23 –15.57 0 Trp106, Ala107, Asn267, Asp108, Leu162, Gly163, Lys157, Asp164, Glu167, Tyr264, Pro248, Pro247\n2. Withanolide A –10.19 34.04 nM Gln269, Asn109, Leu162, Glu161, Gly160, Val159, Thr158 –10.2 32.78 nM Asp286, Asn267, Asp164, Tyr264, Tyr273, Pro248, Gly163, Leu162, Lys157, Asp108, Ala107, Trp106, Gly266, Ala288, Lys105, Tyr268, Leu289 –103.61 –84.71 –18.90 0 Asn109, Val159, Thr158, Glu161, Gly160, Gln269, Leu162\n3. Withanolide B –10.3 28.32 nM Gln269, Asn109, Leu162, Glu161, Gly160, Val159, His89, Thr158 –10.4 22.51 nM His89, Thr158, Gly160, Asn109,Gln269, Leu162, Val159 –104.57 –96.14 –8.43 0 Thr158, Leu162, Glu161, Gly160, His89, Asp108, Ser85, Ala86, Gly160, Val159, Asn109\n4. Withanolide D –9.56 98.21 nM Gly160, Glu161, Asn109, Gln269, Cys270, Leu162, Cys160, Val159, Thr158, His89 –10.1 38.9 nM Thr158, His89, Glu161, Gly160, Asn109, Gln269, Leu162, Val159 –99.07 –91.72 –7.35 0 Pro59, Ala68, Arg65, Phe69, Thr74, Thr75, Pro77, Ile44, Lys45, Pro46, Met23, His47, Asn48\n5. Withanolide E –9.05 231.88 nM Asn109, Gln269, Leu162, Gly160, Asp108, Glu161, His89 –10.6 15.57 nM Asp108, Thr158, Glu161, Val159, His89, Gly160, Leu162, Asn109, Gln269 –107.44 –76.48 –30.96 0 Thr158, Asn109, Gly160, Glu161, Leu162, Asp108, Val159, His89\n6. Withanone –9.09 218.4 nM Val159, Gly160, Glu161, Asn109, Leu162, Gln269, Gln269 –10.1 38.83 nM Trp106, Glu167, Ala107, Trp93, Lys92, His89, Asp108, Lys157, Asp164, Tyr264, Gly163, Tyr273, Val165, Thr301, Pro248 –109.36 –87.06 –22.31 0 Asn109, Thr158, Gly160, Gln269,Glu161, Leu162, Val159,His89\n7. Viscosalactone B –9.02 243.41 nM Ser85, Ala86, His89, Va159, Gly160, Asp108, Asn109, Gln269, Leu162,Glu161 –9.7 70.23 nM Lys92, His89, Val159, Trp93, Asp108, Leu162, Gly163, Tyr273, Tyr264, Pro248, Pro247, Thr301, Asp164, Lys105, Glu167, Trp106, Ala107, Glu161, Lys157 –102.08 –86.12 –15.96 0 Leu162, Glu161, Gly160, Val159, Thr158,Asn109, Asp108, Ala107, Trp106\n8. Anaferine –6.43 19.24 µM Gly160, Asp108, Ala107, Trp93, Leu162, Glu161, Lys157 –6.0 39.69 µM Thr301, Tyr273, Tyr264, Trp106, Asp164, Pro248, Pro247, Met208, Arg166 –75.28 –68.35 –6.93 0 Ala68, Thr74, Phe79, Asp76, Pro77, Lys43, Arg65, Pro59, Leu58\n9. Withasomnine –5.56 84.41 µM Asn109, Gln269, Cys270, Leu162, Gly160, Cys270 –7.2 4.50 µM Asp76, Pro59, Phe79, Leu80, Thr74, Ala68, Thr75, Arg65, Pro77, Leu58 –69.24 –69.24 0 0 Asp76, Pro77, Thr75, Leu80, Leu58, Pro59, Arg65\n10. Procainamide –5.03 206.96 µM Glu161, Asn109, Leu162, Asp108, Gly160, Gln269, Cys270 –6.3 24.06 µM Asn109, Gln269, Leu162, Val159, His89, Asp108, Glu161, Gly160 –76.81 –66.66 –10.15 0 Leu162, Glu161, Gly160, Gln269,Asp108, Asn109,Cys270\n11. Cinacalcet –6.44 19.17 µM Val159, Asp108, Gly160, Glu161, Leu162, His89, Val159 –8.8 344.50 nM Gly160, Asn109, Leu162, Gln269, Cys270, Glu161, Asp108 –81.40 –81.40 0 0 Thr158, Leu162, Glu161, Gly160, His89, Ser85, Ala86, Asn109, Val159, Asn108\nTable 9. Binding energies of WS phytoconstituents with SARS–CoV main protease/3CL-pro (PBB ID: 1P9U) in comparison to the FDA approved standard reference drugs (Oberadilol and Poziotinib).\n    AutoDock v4.2.6 AutoDock vina iGEMDOCK v2.1\nS. No. Ligands BE (kcal/mol) K d Interacting amino acids BE(kcal/mol) K d Interacting amino acids TE(kcal/mol) VDW HB EI Interacting amino acids\n1. Withaferin A –7.56 2.87 µM Thr47, Leu164, Pro188, Gln187, Glu165, Ser189, Met190, Gln191, Leu192, Asn168, Gly167, Leu166 –9.4 129.60 nM Gly126, Leu3, Cys284, Glu286, Arg4, Phe287, Lys5, Tyr280, Ser282, Ser279, Lys136 –116.60 –96.85 –19.75 0 Thr288, Thr290, Glu291, Arg294, Val299, Thr143, Tyr117, Cys116, Gly122, Ala115, Ser123, Gln8, Val150, Leu151, Glu152, Met6, Ser110\n2. Withanolide A –8.93 285.01 nM Gly182, Gly183, Tyr184, Glu185, Leu192, Gln191, Met190, Ser189, Gln187, Val50 –10.9 345 nM Leu3, Arg4, Glu286, Tyr280, Phe287, Lys5, Cys284, Lys136, Ser279 –100.1 –84.54 –15.56 0 Phe272, Gly271, Leu268, Lys270, Asn269, Ser282, Leu283, Cys284, Asp285, Glu286, Lys136, Met198, Asn196\n3. Withanolide B –8.12 1.12 µM Glu185, Gly183, Gly182, Tyr184, Ser189, Met190, Gln191, Leu192 –9.7 72.23 nM Lys270, Arg267, Leu268, Trp217, Phe218, Arg275, Gln256, Arg216, Thr254, Gly214 –104.12 –93.39 –10.74 0 Gly154, Asn153, Leu151, Glu152, Gly122, Tyr117, Ser123, Ala115, Thr143, Met6, Ser138, Ile140, Gln295, Arg294, Gly298\n4. Withanolide D –7.68 2.34 µM Gly182, Tyr184, Glu185, Ser189, Met190, Gln191, Leu192, Glu193 –9.3 145.58 nM Glu54, Asn52, Tyr53, Arg40, Tyr81, Met57, Gln132, Ser131, Arg80, Val197, Thr239, Gly83 –111.44 –98.83 –12.61 0 Gly154, Asn153, Leu151, Glu152, Gln8, Gly122, Ser123,Cys116, Tyr117, Ala115, Thr143, Ile140, Ser138, Gln295, Met6, Arg294\n5. Withanolide E –7.72 2.19 µM Glu185, Tyr184, Gly182, Gln132, Glu193, Gly194, Leu192, Gln191, Met190, Ser189 –9.0 240.98 nM Lys82, Gly83, Lys234, Ser131, Gln132, Ala107, Glu54, Tyr81, Arg80, Met57, Val197, Arg40, Thr239, Glu240 –104.61 –85.73 –18.88 0 Met6, Asn112, Ser110, Gln8, Val150, Glu152, Glu109, Thr288, Glu291, Gln295, Thr290, Arg294\n6. Withanone –7.41 3.7 µM Glu193, Leu192, Gln191, Met190, Ser189, Glu185, Tyr184, Gly183, Gly182, Gln132 –9.1 212.78 nM Thr254, Arg216, Trp217, Arg275, Phe218, Lys260, Gln256, Leu268, Arg267, Lys270 –109.22 –100.63 –8.59 0 Arg294, Gln295, Ile140, Glu291, Glu152, Gly122, Ala115, Ser123, Met6, Gln8, Ala7, Ser110, Phe111, Val150, Asn112, Tyr149\n7. Viscosalactone B –7.34 4.14 µM Leu192, Gly167, Leu166, Glu165, Leu164, Gln191, Met190, Ser189, Pro188, Gln187 –9.5 107.69 nM Lys5, Phe287, Glu286, Lys136, Met198, Asp285, Cys284, Leu283, Ser282, Arg4, Leu278, Ser279, Tyr280, Leu3 –100.67 –85.43 –15.24 0 Tyr125, Met6, Gly126, Lys5, Arg4, Leu3, Lys136, Leu278, Ser279, Tyr280, Cys284, Glu286, Phe287\n8. Anaferine –6.19 29.14 µM Gly183, Glu185, Tyr184, Gly182, Gln191, Leu192, Glu193, Gln132 –6.8 10.34 µM Arg294, Gln295, Glu291, Gln8, Ser110, Lys5, Gly126, Ala7, Met6, Ser123, Gly122 –74.07 –60.38 –13.68 0 Tyr280, Leu3, Arg4, Lys5,Gly126, Tyr125, Val124\n9. Withasomnine –6.06 36.09 µM Tyr53, Ile51, Ser189, Gln191, Gln187, Pro188, Asp186, Thr47, His41, Leu164, Glu165 –7.1 6.20 µM Arg294, Met6, Gln295, Glu291, Lys5, Asn112, Phe111, Gln8, Val150, Ser110, Leu151 –71.58 –63.14 –8.44 0 Ala79, Val60, Gly194, Met57, Ser58, Glu193, Gln132, Val197, Arg80, Thr195\n10. Oberadilol –4.54 469.45 µM Glu185, Tyr184, Gly183, Gly182, Gln132, Glu193, Leu192, Gln191, Met190, Ser189 –8.7 401.24 nM Glu54, Tyr53, Arg40, Gly83, Val197, Arg80, Ser131, Thr239, Ala107, Glu240, Lys234, Lys82, Tyr81 –95.18 –81.58 –13.60 0 Trp217, Arg267, Leu268, Lys270, Phe272, Gly271, Gly273, Gly274, Arg275, Val219\n11. Poziotinib –6.41 20.18 µM Thr195, Gly194, Glu193, Leu192, Gln191, Met159, Tyr184, Gly182, Gln132 –8.4 710.54 nM Met190, Gly167, Leu166, Ser189, Glu165, Ala141, Ile140, Phe139, Cys144, Thr47, His41, Pro188, Leu164, Gln187, Gln191 –103.87 –94.39 –9.49 0 Arg275, Gly274, Gly273, Trp217, Thr220, Asn221, Gly271, Lys270, Thr222, Arg267, Leu263\nTable 10. Binding energies of WS phytoconstituents with SARS-CoV-2 main protease/3CL-pro (PDB ID: 6LU7) in comparison to the FDA approved standard reference drugs (Oberadilol and Poziotinib).\n    AutoDock v4.2.6 AutoDock vina iGEMDOCK v2.1\nS. No. Ligands BE (kcal/mol) K d Interacting amino acids BE(kcal/mol) Kd Interacting amino acids TE(kcal/mol) VDW HB EI Interacting amino acids  \n1. Withaferin A –2.85 8.21 mM Tyr126, Gln127, Gly138, Va171, Gly170, Lys137, Lys5 –8.4 670.89 nM Arg105, Ile106, Val104, Ser158, Phe294,Phe112, Phe8, Thr111, Asp295, Asn151,Gln110, Thr292, Gln107 –98.51 –78.64 –19.87 0 Gly143, Ser144, Cys145, His163, Met165, Glu166, Gln189, Thr190, Asn142, Leu141, Phe140  \n2. Withanolide A –5.26 139.0 µM Lys5, Gln127, Tyr126, Val125, Ser139, Lys137, Glu288, Glu290 –8.5 592.38 nM Asp289, Thr199, Glu290, Arg131, Lys137, Tyr239, Leu272, Leu287, Leu286, Met276, Gly275 –91.61 –70.18 –21.43 0 Gly179, Asn180, Phe181, Asn84, Cys85, Arg40, Phe185, Tyr54, Asn53, Pro52, Arg188  \n3. Withanolide B –5.67 69.79 µM Ser139, Gly138, Lys137, Cys128, Glu290, Gln127, Tyr126, Val125 –8.3 845.65 nM Leu287, Tyr239, Tyr237, Leu272, Leu286, Asn238, Thr199, Asp289, Glu288, Arg131, Glu290, Lys137 –89.17 –72.53 –16.64 0 His163, His164, Met165, Cys145, Ser144, Phe140, Leu141, Asp142, Gly143, Thr25, Met149, Thr45, Ser46, Thr24  \n4. Withanolide D –5.55 85.19 µM Tyr126, Gln127, Gly138, Ser139, His172, Gly170, Lys137, Lys5 –8.7 424.04 nM Tyr239, Leu287, Val204, Leu272, Tyr237, Asn238, Arg131, Lys137, Thr199, Leu286, Asp289 –104.35 –85.29 –19.06 0 Thr26, Leu27, Cys145, Gly143, Ser144, His164, Asn142, Met165, Glu166, Pro168, Thr190, Gln189, Arg188, Met49,His41  \n5. Withanolide E –5.2 154.68 µM Tyr126, Gln127, Lys5, Glu290, Lys137, Gly138, Ser139 –8.1 1.16 µM Asn238, Asp289, Arg131, Lys137, Thr199, Leu286, Leu287, Tyr239, Leu272, Tyr237 –97.42 –70.98 –26.44 0 Arg131,Asn133, Ala194, Gly195, Asp289, Asp197, Thr196, Thr199, Thr198, Asn238  \n6. Withanone –6.14 31.77 µM Val125, Tyr126, Gln127, Cys128, Ser139, Gly138, Lys137, Ala129, Glu290 –8.4 707.34 nM Leu286, Tyr239, Lys137, Asp197, Arg131, Thr198, Thr199, Asn238, Tyr237, Met276, Gly275, Leu272, Leu271, Leu287 –100.18 –88.69 –11.49 0 Thr24, Thr25, Thr26, His41, Gly143, Cys145, Ser144, His164, His163, His172, Phe140, Glu166, Leu141, Asn142, Met49, Ser46  \n7. Viscosalactone B –4.86 274.4 µM Val104, Asn151, Phe8, Arg298, Gln127, Asp295, Phe294, Thr111, Thr292, Gln110, Gln107, Ile106, Arg105 –8.2 969.04 nM Gly278, Lys137, Asp289, Arg131, Tyr239, Thr199, Leu287, Leu286, Leu272, Gly275,Met276, Asn274, Asn277 –97.17 –67.45 –29.72 0 Tyr239, Asn238, Tyr237, Thr199, Leu287, Leu286, Ala285, Gly278, Asn277, Met276,Gly275  \n8. Anaferine –5.13 174.99 µM Glu290, Cys128, Lys5, Gln127, Tyr126, Lys137 –5.7 66.02 µM Cys145, Asn142, Leu141, Phe140,Glu166, Ser144,His172,Met165, His164, Gln189, Asp187, Arg188, Met49, His41 –76.27 –67.75 –8.52 0 Thr25, Thr26, Leu27, Cys145, His163, Met165, Glu166,His172, Phe140, Leu141,Ser144, Gly143  \n9. Withasomnine –5.22 149.2 µM Ser158, Ile152, Asp153, Asn151, Thr111, Asp295, Phe294, Thr292, Gln110 –6.2 28.68 µM Ile152, Phe8, Val104, Ile106, Gln110, Thr111, Asn151, Asp153, Phe294 –71.96 –60.03 –11.94 0 His41, His163, His164, Met165, Cys145, Ser144, His172, Glu166, Phe140, Leu141  \n10. Oberadilol –2.23 23.18 mM Lys137, Glu290, Gln127, Tyr126, Lys5 –6.9 9.00 µM Leu286, Tyr239, Lys137, Asp197, Arg131, Thr198, Thr199, Asn238, Tyr237, Met276, Gly275, Leu272, Leu271, Leu287 –100.96 –94.41 –6.55 0 His163, Cys145, His164, Met165, Asn142, Glu166, Leu141, Leu167, Pro168, Gln189, Met49, Asp187, His41  \n11. Poziotinib –4.49 513.87 µM Tyr126, Gln127, Cys128, Lys5, Ala129, Glu290, Lys137, Gly138, Ser139 7.7 2.20 µM Gly278, Ala285, Gly275, Tyr237, Leu272, Asn238, Tyr239, Thr199, Asp289, Arg131, Leu286, Lys137, Leu287, Met276 –111.32 –96.66 –14.66 0 His41, Cys145, His164, Asp187, Arg188, Met165, Gln189, Glu166, Thr190, Ala191, Gln192, Pro168, Phe140, Leu141, Asn142  \nTable 11. Binding energies of WS phytoconstituents with Nsp-10/Nsp-16 complex from SARS-CoV-2 (PDB ID: 6W75) in comparison to the FDA approved standard reference drugs (Losartan and Hydroxychloroquine).\n    AutoDock v4.2.6 AutoDock vina iGEMDOCK v2.1\nS. No. Ligands BE (kcal/mol) K d Interacting amino acids BE(kcal/mol) K d Interacting amino acids TE(kcal/mol) VDW HB EI Interacting amino acids  \n1. Withaferin A –9.33 144.58 nM Asp6906, Asn7095, Ser6907, Thr6908, Leu7093, Leu6909, Val7092, Ser7089, Ser7090, Val7092 –10.0 40.98 nM Lys6968, Glu7001, Ser6999, Thr6970, Ser7000, Asn6996, Lys6844, Asn6841, Tyr6930, Gly6871, Asp6928, Pro6932, Ser6872, Met6929, Gly6869, Leu6898, Asp6897, Asn6899 –99.04 –82.04 –17 0 Tyr6828, Ser6999, Ser7000, Thr6970, Glu7001, Asn6996, Lys6844, Lys6968, Asn6841, Asp6928, Tyr6930, Gly6871, Ser6872  \n2. Withanolide A –10.38 24.67 nM Ala6905, Asp6906, Lys4346, Ser6907, Thr6908, Leu6909, Ile6910, Ser7090, Leu7093, Val7092, Ser7090 –10.4 23.67 nM Pro6932, Tyr6930, Asn6899, Asp6931, Leu6898, Asp6897, Met6929, Gly6869, Ser6872, Gly6871, Asn6841, Lys6844, Lys6968, Glu7001, Thr6970, Ser6999 –101.39 –78.76 –22.63 0 Gly6871, Gly6869, Asn6899, Asp6897, Leu6898, Asp6912, Cys6913, Phe6947, Asp6931, Lys6933, Pro6932, Met6929, Asp6928  \n3. Withanolide B –10.09 39.9 nM Lys4346, Cys4330, His4333, Val6902, Ser6903, Phe6901, Asp6900, Thr6908, Ile6910, Leu6909, Val7092, Ser7090 –11.0 1.98 nM Lys6844, Asn6841, Asp6928, Lys6968, Tyr6930, Gly6871, Gly6869, Asp6931, Leu6898, Cys6913, Met6929, Phe6947, Asp6897, Asn6899, Ser6872, Pro6932 –103.08 –95.57 –7.51 0 Val4274, Ile4334, Asp4335, Ile4334, Cys4332, Arg4331, Thr4292, Ile4291, Pro4290, Gln4289, Ala4271, Phe4272, Ala4273  \n4. Withanolide D –9.58 94.36 nM Val7092, Ser7089, Ser7090, Asp7091, Leu6909, Thr6908, Ser6907, Asp6906, Lys4346 –10.5 27.89 nM Lys6844, Asn6841, Asp6928, Ser6872, Tyr6930, Gly6871, Met6929, Gly6869, Leu6898, Asp6897, Asp6899, Pro6932, Asp6931, Lys6968, Thr6970, Glu7001, Ser6999 –110.77 –85.34 –25.43 0 Gly4341, Phe4342, Cys4343, Asp6906, Lys4346, Gly4347, Lys4348, Val4310, Arg6884, Thr6889, Gly6890, Asn7096  \n5. Withanolide E –8.96 272.55 nM His4333, Phe6901, Val6902, Leu6909, Ser6903, Thr6908, Lys4346, Ser6907, Asp6906, Val7092 –9.8 62.56 nM His4333, Cys4332, Phe4272, Thr4292, Ile4291, Ala4271, Gln4289, Asn4293, Arg4331, Val6876, Lys6874, Gly6875, Val6902 –104.92 –92.21 –12.72 0 Tyr7020, Leu6820, Glu6821, Lys6822, Asp6942, Trp6974, Asn6941, Glu6940, Glu6971, His6972, Lys6939  \n6. Withanone –10.17 34.9 nM Cys4330, His4333, Val6901, Asp6900, Phe6901, Thr6908, Ile6910, Leu6909, Ser7090, Val7092 –9.8 63.56 nM Val7092, Asp7091, Thr6915, Leu7093, Asp6912, Gly6911, Leu6898, Ser7089, Ile6910, Leu6909,Ser7090 –105.42 –91.03 –14.39 0 Ala4273, Cys4332, Val4274, Ile4334, Ala4276, Asp4335, Pro4337, Tyr4329, His4336, Asn4338, Pro4339  \n7. Viscosalactone B –9.03 238.8 nM Asp6900, Leu7093, Val7092, Gly6911, Asp7091, Ser7090, Ile6910, Leu6909, Thr6908, Ser6907, Asp6906 –9.9 57.78 nM His4336, Ile4334, Asp4335, Tyr4329, Cys4330, His4333, Val6902, Ile6910, Asp6900, Val7092, Leu6909, Ser7089, Ser7090, Thr6908 –119.11 –103.53 –15.58 0 Thr4292, Ile4291, Pro4290, Gln4289, Tyr4283, Ala4271, Phe4272, Arg4331, Cys4332, Ile4334, Asp4335, Tyr4329, Val4274, Ala4273  \n8. Anaferine –7.1 6.25 µM Asp4335, Asp6900, Phe6901, Ile6910, Thr6908 –6.3 22.67 nM Leu6819, Tyr7020, Val7021, Asp7018, Leu6820, Met6818, Arg6817 –86.35 –74.35 –12 0 Gly6911, Asp6912, Cys6913, Phe6947, Leu6898, Asp6897, Gly6871, Gly6869, Met6929, Tyr6930, Asp6928, Phe6947  \n9. Withasomnine –6.06 36.03 µM Val7092, Asp7091, Ser7090, Ile6910, Leu6909, Ser7089 –8.0 1.23 µM Tyr7020, Leu6820, Met6818, Leu6819, Ala7024, Arg6817, Val7021, Asp7018 –74.52 –65.96 –8.56 0 Thr4292,Arg4331, Ile4291, Pro4290, Gln4289, Tyr4283, Phe4272, Ala4271,  \n10. Losartan –6.49 17.54 µM Thr6908, Leu6909, Ile6910, Asp6900, Ser7090, Ser7089, Val7092 –8.3 780.68 nM Ala4273, Val4274, Asp4335, His4333, Ala4271, Ile4334, Pro4290, Gln4289, Thr4292, Ile4291, Cys4332, Phe4272, Arg4331, Gly6875, Lys6874, Val6876 –104.17 –75.33 –28.84 0 Gln6957, Gln6956, Glu7062, Lys6958, Lys6921, Ile7080, Leu6959, Leu6961, Tyr7009, Ile6955, His6984  \n11. Hydroxychloroquine –4.93 244.14 µM Thr6908, Ile6910, Ser6907, Leu6909, Ser7090, Ser7089, Val7092 –6.7 11.70 µM Gly6869, Gly6871, Asn6841, Tyr6930, Pro6932, Ser6872, Asp6873, Asn6899, Asp6897,Leu6898, Met6929 –80.22 –69.44 −10.78 0 Lys6968, Asp6928, Gly6871, Gly6869, Met6929, Asp6897,A sn6899, Leu6898, Tyr6930, Pro6932, Ser6872   Withasomnine was found to bind near or at the active site of SARS-Co-V main protease 3CL-pro (PDB ID: 1P9U; Table 9), whereas anaferine was found to interact with the active site residues Cys145, Glu166, Ser144, Met165, His163, His164, Gln189, Asp187, Arg188, Met49 and His41 present at the active site of SARS-CoV-2 main protease 3CL-pro (PDB ID: 6LU7; Table 10). The 3CL-pro active site has been found to be evolutionarily conserved between SARS-CoV and SARS-CoV-2 (Báez-Santos et al., 2015; Chen et al., 2020; Guy et al., 2005; Zhang et al., 2020). In the same manner, the other seven phytoconstituents also displayed potent binding to the active site of SARS-CoV-2 3CL-pro except viscosalactone B as predicted by AutoDock vina and iGEMDOCK. The active site residues have been written in bold in Tables 9 and 10. As far as viral PL-pro and human ACE2 are concerned, WS phytoconstituents displayed allosteric binding to these enzymes.\nOn the other hand, withanolide A displayed strong binding to SARS-CoV spike glycoprotein (Table 6; BE: −9.78 kcal/mol, Kd: 67.23 nM), SARS-CoV-2 spike glycoprotein (Table 7; BE: −7.18 kcal/mol, Kd: 5.48 µM), SARS-CoV 3CL-pro main protease (Table 9; BE: –8.93 kcal/mol, Kd: 285.01 nM) and SARS-CoV-2 Nsp10/Nsp-16 complex (Table 11; BE: −10.38 kcal/mol, Kd: 24.67 nM). Interestingly, withanolide A exhibited almost 1000× times stronger binding to SARS-CoV main protease as compared to standard reference drugs arbidol (Table 6; BE: −4.91 kcal/mol, Kd: 251.65 µM) and hydroxychloroquine (Table 6; BE: −5.25 kcal/mol, Kd: 142.18 µM). The same binding profile was observed for withanolide A with respect to SARS-CoV-2 spike glycoprotein as compared to standard reference drugs arbidol (Table 7; BE: −3.14 kcal/mol, Kd: 4.99 mM) and hydroxychloroquine (Table 7; BE: −2.48 kcal/mol, Kd: 15.11 mM). Withanolide A also displayed a 1000× stronger binding to Nsp-10/Nsp-16 complex from SARS-CoV-2 in comparison to losartan (Table 11; BE: −6.49 kcal/mol, Kd: 17.54 µM) and hydroxychloroquine (Table 11; BE: −4.93 kcal/mol, Kd: 244.14 µM)\nWithanone also displayed significant binding to SARS-Cov-2 main protease (Table 10; BE: −6.14 kcal/mol, Kd: 31.77 µM) in comparison to standard reference drug oberadilol (Table 10; BE: −2.23 kcal/mol, Kd: 23.18 mM). The best docking poses of the WS phytoconstituents with respect to the human ACE2 receptor and viral target proteins have been depicted in Table 12 (Tables 12.1–12.7). Binding studies on WS constituents to unbound spike receptor-binding domain (RBD) of SARS-CoV-2 (PDB ID: 6M0J) and binding of WS phytoconstituents with SARS-CoV-2 spike receptor-binding domain (RBD) bound with ACE2 have been provided as supplementary data files ST1, SFI, ST2 and SF2, respectively.\nTable 12. Best docking poses of human and viral target proteins with selected WS phytoconstituents.\nLigands AutoDock v4.2.6 AutoDock vina iGEMDOCK v2.1\n12.1. Best docking poses of WS phytoconstituents with human ACE2 receptor (PDB ID: 1O8A) in comparison to the FDA approved standard reference drugs (Arbidol and Losartan)\nWithaferin A\nWithanolide A\nWithanolide B\nWithanolide D\nWithanolide E\nWithanone\nViscosalactone B\nAnaferine\nWithasomnine\nArbidol\nLosartan\n12.2. Best docking poses of WS phytoconstituents with SARS-CoV spike glycoprotein (PDB ID: 5WRG) in comparison to the FDA approved standard reference drugs (Arbidol and Hydroxychloroquine)\nWithaferin A\nWithanolide A\nWithanolide B\nWithanolide D\nWithanolide E\nWithanone\nViscosalactone B\nAnaferine\nWithasomnine\nArbidol\nHydroxychloroquine\n12.3. Best docking poses of WS phytoconstituents with SARS-CoV-2 spike glycoprotein (PDB ID: 6VXX) in comparison to FDA approved standard reference drugs (Arbidol and Hydroxychloroquine)\nWithaferin A\nWithanolide A\nWithanolide B\nWithanolide D\nWithanolide E\nWithanone\nViscosalactone B\nAnaferine\nWithasomnine\nArbidol\nHydroxychloroquine\n12.4. Best docking poses of WS phytoconstituents with papain like protease of SARS-CoV-2 (PDB ID: 6W9C) in comparison to the FDA approved standard reference drugs (Procainamide and Cinacalcet)\nWithaferin A\nWithanolide A\nWithanolide B\nWithanolide D\nWithanolide E\nWithanone\nViscosalactone B\nAnaferine\nWithasomnine\nProcainamide\nCinacalcet\n12.5. Best docking poses of WS phytoconstituents with SARS-CoV main protease/3CL-pro (PBB ID: 1P9U) in comparison to the FDA approved standard reference drugs (Oberadilol and Poziotinib)\nWithaferin A\nWithanolide A\nWithanolide B\nWithanolide D\nWithanolide E\nWithanone\nViscosalactone B\nAnaferine\nWithasomnine\nOberadilol\nPoziotinib\n12.6. Best docking poses of WS phytoconstituents with SARS-CoV-2 main protease/3CL-pro (PDB ID: 6LU7) in comparison to the FDA approved standard reference drugs (Oberadilol and Poziotinib)\nWithaferin A\nWithanolide A\nWithanolide B\nWithanolide D\nWithanolide E\nWithanone\nViscosalactone B\nAnaferine\nWithasomnine\nOberadilol\nPoziotinib\n12.7. Best docking poses of WS phytoconstituents with Nsp-10/Nsp-16 complex from SARS-CoV-2 (PDB ID: 6W75) in comparison to the FDA approved standard reference drugs (Losartan and Hydroxychloroquine)\nWithaferin A\nWithanolide A\nWithanolide B\nWithanolide D\nWithanolide E\nWithanone\nViscosalactone B\nAnaferine\nWithasomnine\nLosartan\nHydroxychloroquine\n\n3.4. Bioavailability radar and score as parameters for analysis of pharmacokinetic properties of WS phytoconstituents\nPharmacokinetics and pharmacodynamics are two interlinked terms in drug development having a mutual influence on each other. Bioavailability radar offers a first glimpse into the pharmaceutical properties of a prospective drug candidate. By convention, the pink area represents the optimal biological range for each physiochemical property including lipophilicity (XLOGP3 range 0.7–5.0), size (MW range 150–500), polarity (TPSA range 20–130 Å2), solubility (log S ≤ 6), saturation (fraction of carbons in sp3 hybridization ≤0.25), and flexibility (≤9). The Abbot Bioavailability Score62 is identical, but attempts to determine whether a compound is likely to have oral bioavailability score of at least 10% in rats and/or Caco-2 permeability (Martin, 2005). As is evident from Figure 2A and B, all withanolides from WS exhibited a significant bioavailability radar and score as comparable to the standard reference FDA-approved drugs.\nFigure 2. (A) Bioavailability radar and score prediction of WS phytoconstituents using SwissADME. (B) Bioavailability radar and score prediction of FDA–approved reference standard drugs using SwissADME.\n\n3.5. Druglikeness and Bioactivity score (BAS) analysis\nBiological targets of prospective drug candidates can be classified into ion channels, proteases, kinases, G-protein coupled receptors (GPCRs), nuclear receptors and enzymes. The BAS of WS phytoconstituents was determined using web-based software Molinspiration (www.molinspiration.com). As a general rule, it is known that if the BAS \u003e 0.0, then the drug candidate is physiologically active; if it is in the range −5.0 to 0.0; then the drug candidate is moderately active, and if the BAS\u003c −5.0, then the drug candidate is inactive.\nIt is evident from Table 13, that most of the WS phytoconstituents had positive values with respect to the following receptors.\n\n3.5.1. As GPCR ligands\nAll WS phytoconstituents were active except withanolide E, anaferine and withasomnine which were predicted to be moderatively active. Most of the reference drugs also had positive values for GPCR except procainamide and arbidol which were predicted to be moderately active.\n\n3.5.2. As ICMs\nAll WS phytoconstituents had positive values except withasomnine which was found to be moderately active. Standard reference drugs losartan, cinacalcet, hydroxychloroquine, oberadilol and poziotinib were all found to be active whereas procainamide and arbidol were found to be moderately active.\n\n3.5.3. As KIs\nAll WS phytoconstituents displayed moderate activity except withasomnine that displayed significant activity. Standard reference drugs losartan, hydroxychloroquine and poziotinib were found to be active whereas procainamide, cinacalcet, arbidol and oberadilol were found to be moderately active.\n\n3.5.4. As NRLs\nWithaferin A, withanolides A, B, D and E, withanone and viscosalactone B possessed significant BAS scores whereas anaferine and withasomnine were found to be moderately active. All standard reference drugs were predicted to have moderate BAS scores as NRLs.\n\n3.5.5. As PIs\nWithaferin A, withanolides A, B, D and E, withanone and viscosalactone B had positive BAS scores indicating their potential as protease inhibitors. On the other hand, anaferine and withasomnine were found to have moderate activity as protease inhibitors. Interestingly, most withanolides especially withanolide B and withanolide A showed potent binding to papain like protease of SARS-CoV-2 (PDB ID: 6W9C), SARS-CoV 3CL-pro main protease (PDB ID: IP9U) and SARS-CoV-2 Nsp10/Nsp-16 complex (PDB ID: 6W75) thus supporting their role as potential viral protease inhibitors. On the other hand, losartan, cinacalcet and hydroxychloroquine also displayed positive values as protease inhibitors whereas procainamide, arbidol, oberadilol and poziotinib displayed moderate potential as protease inhibitors.\n\n3.5.6. As EIs\nMost of the WS phytoconstituents including Withaferin A, withanolides A, B, D and E, withanone, viscosalactone B and anaferine had positive BAS scores indicating their potential as enzyme inhibitors whereas withasomnine displayed moderate potential. This observation was further validated by the fact that most of the phytoconstituents including Withaferin A, withanolides A, B, D and E, viscosalactone B and anaferine showed potent binding to human ACE2 receptor in the nanomolar range which was about 1000× times greater than the binding of known standard reference drugs arbidol and losartan (Table 4). This finding lends support for targeted use of withanolides from WS as SARS-CoV-2 entry blocking agents by virtue of their preferential binding to human ACE2, thereby blocking or inhibiting it. Losartan, cinacalcet, hydroxychloroquine, oberadilol and poziotinib also displayed significant potential as enzyme inhibitors whereas procainamide and arbidol displayed moderate potential.\nDruglikeness of a compound can be predicted by comparing its structural features with those of marketed drugs. All WS phytoconstituents showed molar lipophilicity (cLog P) \u003c5 thereby indicating good permeability across cell membranes (Figure 2A). Withaferin A, withanolide D, viscosalactone B and withasomnine had positive values of druglikeness which indicated that these compounds contain fragments that are present in marketed drugs. Out of the standard reference drugs, procainamide, hydroxychloroquine and oberadilol exhibited positive scores for druglikeness (Table 14).\nTable 13. Bioactivity scores and Druglikeness of WS phytoconstituents versus FDA-approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib).\nS. No. Ligands Parameters of bioactivity score\nGPCR ligand Ion channel modulator (ICM) Kinase inhibitor (KI) Nuclear receptor ligand (NRL) Protease inhibitor (PI) Enzymeinhibitor (EI)  \n                 \n1. Withaferin A 0.07 0.14 –0.49 0.76 0.15 0.94  \n2. Withanolide A 0.04 0.32 –0.43 0.71 0.15 0.86  \n3. Withanolide B 0.07 0.24 –0.47 0.79 0.15 0.76  \n4. Withanolide D 0.05 0.30 –0.50 0.73 0.16 1.07  \n5. Withanolide E –0.70 0.16 –0.50 0.61 0.06 0.89  \n6. Withanone 0.00 0.27 –0.38 0.71 0.12 0.78  \n7. Viscosalactone B 0.03 0.04 –0.51 0.78 0.19 0.84  \n8. Anaferine –0.08 0.17 –0.60 –0.58 –0.14 0.08  \n9. Withasomnine –0.49 –0.43 0.58 –0.10 –0.58 –0.17  \n10. Losartan 1.06 0.16 0.03 0.01 0.33 0.44  \n11. Procainamide –0.09 0.01 –0.10 –0.70 –0.20 –0.04  \n12. Cinacalcet 0.22 0.15 –0.0.8 0.00 0.17 0.02  \n13. Arbidol –0.19 –0.44 –0.39 –0.34 –0.46 –0.07  \n14. Hydroxychloroquine 0.35 0.30 0.44 –0.12 0.12 0.15  \n15. Oberadilol 0.04 –0.47 –0.43 –0.37 –0.02 0.02  \n16. Poziotinib 0.04 –0.17 0.53 –0.35 –0.27 0.01  \nRule: BAS \u003e0: Active;\nBAS –5.0–0.0: Moderately active, moderately active and inactive.\nBAS ≤5.0: Inactive;\n\n3.6. Toxicity risk assessment\nIn silico prediction of drug-like properties has now become a norm for pharmaceutical industries for investing in and classifying drug compounds and their product potential. The toxicity risk evaluation is an important consideration to prevent undesirable substances with adverse effects to undergo further drug screening (Balakrishnan et al., 2015). Potential drug candidates are analyzed for their toxicity parameters like tumorigenic, mutagenic, irritant and for their effects on the reproductive system. In the present study, toxicity risk assessment of WS phytoconstituents was calculated using OSIRIS data warrior. The software estimates the toxicity potential of the compounds based on similarities between the phytoconstituents being examined and the compounds present in its in vitro and in vivo database (Sander, 2001).\nThe obtained results have been presented in Table 15. As is evident from Table 15, none of the analyzed WS phytoconstituents had any mutagenic effects in contrast to standard reference drugs hydroxychloroquine and poziotinib which displayed high mutagenicity. Most of the WS phytoconstituents displayed little to no tumorigenicity in comparison to standard reference drugs cinacalcet and oberadilol which exhibited a high tendency for tumorigenicity and poziotinib which exhibited a mild tumorigenicity. The irritant and reproductive effects of the WS phytoconstituents were also predicted to be from negligible to none, in contrast to standard reference drugs procainamide which was predicted to possess high adverse effects and poziotinib that was predicted to have mild irritant and reproductive effects.\nTable 14. Drug like properties of WS phytoconstituents versus FDA–approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib).\nS. No. Ligands Druglikeness (DL)   clogS\n1. Withaferin A 1.69   –4.47\n2. Withanolide A –0.63   –4.53\n3. Withanolide B –1.04   –4.98\n4. Withanolide D 0.14   –4.53\n5. Withanolide E –0.41   –4.03\n6. Withanone –0.63   –4.53\n7. Viscosalactone B 1.83   –4.29\n8. Anaferine –0.69   –2.48\n9. Withasomnine 4.16   –2.81\n10. Losartan –6.63   –4.99\n11. Procainamide 7.96   –1.72\n12. Cinacalcet –4.58   –5.65\n13. Arbidol –1.16   –4.75\n14. Hydroxychloroquine 5.73   –3.55\n15. Oberadilol 3.49   –6.12\n16. Poziotinib –4.70   –6.72\n\n3.7. Ligand-based target prediction analysis\nSimilarity in structures of ligands or distribution of electrostatic potential may result in an identical effect leading to the probability of interaction with similar targets (Wirth \u0026 Sauer, 2011). These predictions also indicate how a drug candidate can be chemically altered in order to maximize its effect on a given target by comparing it to known ligands having similar structure. Thus, this prediction analysis can help harness natural ligands for use as therapeutic adducts. From the pie-chart representation, it is evident that most of the withanolides possessed broad-spectrum of bioactivity against several targets present in humans (Figure 3).\nFigure 3. Ligand–based target prediction analysis of WS phytoconstituents versus FDA–approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib) using SwissTargetPrediction.\n\n3.8. Identification of SOMs in WS phytoconstituents\nBiotransformation refers to a biochemical modification process of xenobiotics inside the living system involving the utilization of special enzymes. In pharmaceutical industry, this term is equivalent to ‘drug metabolism’. Drug metabolism influences drug-like properties of prospective drug molecules which may contribute to the production of metabolites with drastically altered pharmacological and toxicological parameters. The recognition of SOMs containing specific atom(s) in the molecule which are oxidized by CYP isozymes, provides knowledge for the design and optimization of potent candidates in early stage. Cytochrome P450s are accountable for more than 90% of the pharmaceutical drugs to undergo phase I metabolism. Therefore, having prior knowledge about the metabolic liabilities of prospective drug candidates could have important ramifications in drug discovery process. The primary, secondary and tertiary predicted SOMs for selected WS phytoconstituents versus FDA-approved standard reference drugs have been shown in Figure 4. The figure is a graphical output for a combination of all nine isozymes of cytochrome P450. The results indicated that WS phytoconstituents and standard reference drugs were predicted to possess SOMs likely to undergo phase I metabolism.\nFigure 4. Prediction of cytochrome P450–mediated SOMs on WS phytoconstituents versus FDA–approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib) using RS–WebPredictor.\n\n3.9. Structure activity relationship (SAR)\nWS is known to harbor a wide variety of secondary metabolites having low MWs viz. terpenoids, flavonoids, tannins, alkaloids and resins. Withanolides, alkaloids, flavonoids and tannins are the major chemical constituents that include compounds of diverse chemical structures (Dhar et al., 2015; Kumar et al., 2015). Of these, withanolides are attributed with diverse and widely known biological activities. In the present study, most of the predicted pharmacological activity against the chosen biological target(s) was found to be associated with two main withanolides, viz. withanolide A and B, as well as withanone, a WS phytoconstituent with structural similarity to withanolide D. Nearly 40 naturally occurring withanolides have been reported till date comprising of C-28 steroidal lactone triterpenoids assembled on an integral or reorganized ergostane structure, in which C-22 and C-26 are oxidized to form a six-membered lactone ring (Jain et al., 2012). The withanolide backbone is chemically classified as 22-hydroxy ergostane-26-oic acid 26, 22-lactone (Mirjalili et al., 2009). The withanolides consist of several oxygen atoms and are thought to be synthesized via oxidation of all carbon atoms in a steroid nucleus.\nThe parent configuration of withanolides and ergostane-type steroids is one C-8 or C-9 side chain with an either six or five membered lactone or lactol ring. A carbon-carbon bond or oxygen bridge is responsible in attaching the lactone ring with the carbocyclic part of the molecule (Mirjalili et al., 2009). Withanolides have a varying distribution in the fruits and vegetative parts of the plant such as leaves, roots and stem (Sangwan et al., 2008). However, withanolides are mainly localized in the leaves, in low concentrations (0.001–0.5% of dry weight) which is the main drawback for their use as drugs. Geographical, environmental and seasonal factors as well as growth conditions are also known to contribute to modulation of the content of withanolides (Dhar et al., 2013).\nIn the present study, the differential binding kinetics obtained for withanolide A (C28H38O6), withanolide B (C28H38O5), withanolide E (C28H38O7) and withanone (C28H38O7) might be attributed to the varying number of oxygen atoms in their structures which might affect hydrogen bonding within the binding site of the target protein(s). Another explanation for differential SAR obtained for the above withanolides might be due to various kinds of structural rearrangements (A or B) involving oxygen substituents like bond scission, new bond formation, ring aromatization, etc. which help in formation of novel structural variants and compounds with novel structures (Figure 5) often described as modified withanolides or ergostane type steroids (Misico et al., 2011). The structural rearrangement as seen in withanolide A and B might be responsible for a better complementary fit of the phytoconstituent in the binding pocket of the target protein(s).\nFigure 5. Structural differences in Withanolide A (R1 = OH, R2 = H); Withanolide B (R1 = H, R2 = H); Withanolide E (5β, 6β–epoxy) and Withanone (17α–OH, R1 = H, R2 = H).\n\n3.10. Principle component analysis\nPCA is one of the most familiar methods of multivariate analysis which attempts to model the total variance of originally formed data set with the unrelated principal components. Absorption rate, TPSA, MW, clog P, NOHNH, NON, number of rotatable bonds and Lipinski’s violations were the various variable properties on which PCA was performed using linear correlation as shown in Figure 6A and 6B. PCA analysis was also performed on leadlikeness (Table 14; Figure 7) as well as for bioactivity score parameters using linear correlation between the variables (Table 13; Figure 8)\nFigure 6. PCA of physiological properties of WS phytoconstituents versus FDA–approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib). (A) Scatter Plot (B) 3D Point Plot.\nFigure 7. PCA of leadlikeness of WS phytoconstituents versus FDA–approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib). (A) Scatter Plot (B) 3D Point Plot.\nFigure 8. PCA of bioactivity score prediction of WS phytoconstituents versus FDA– approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib). (A) Scatter Plot (B) 3D Point Plot.\nTable 15. Toxicity risk assessment of WS phytoconstituents versus FDA-approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib).\nS. No. Ligands Mutagenic Tumorigenic Reproductive effective Irritant\n1. Withaferin A None None Mild None\n2. Withanolide A None Mild Mild Mild\n3. Withanolide B None Mild Mild Mild\n4. Withanolide D None None Mild None\n5. Withanolide E None None Mild None\n6. Withanone None Mild Mild Mild\n7. Viscosalactone B None None None None\n8. Anaferine None None None None\n9. Withasomnine None None None None\n10. Losartan None None None None\n11. Procainamide None None None High\n12. Cinacalcet None High None None\n13. Arbidol None None None None\n14. Hydroxychloroquine High None None None\n15. Oberadilol None High None None\n16. Poziotinib High Mild Mild Mild As is evident from Figures 6–8, all WS phytoconstituents fall close in 3D to the standard reference drugs used in the present study, thereby denoting their ‘drug-like’ character. Tables 16, 17 and 18 represent the Bravais–Pearson (linear correlation) coefficients of WS phytoconstituents versus FDA-approved standard reference drugs.\nTable 16. Bravais–Pearson (linear correlation) coefficient of WS phytoconstituents versus FDA-approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib) for physicochemical properties.\nProperties   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16\n% AB 1   –0.719 –0.867 0.387 –0.807 0.0443 –1 0.274 0.99 –0.0259 0.037 –0.0781 0.0966 –0.0537 0.0186 5.38E–05\n(NOHNH) 2 –0.719   0.582 –0.357 0.399 0.243 0.719 –0.466 –0.779 0.214 0.45 –0.346 0.156 0.032 –0.0146 –1.39E–08\n(NON) 3 –0.867 0.582   0.0762 0.938 –0.0124 0.867 0.11 –0.893 –0.397 –0.147 –0.119 –0.00919 –0.0307 0.0873 2.76E–09\nLV 4 0.387 –0.357 0.0762   0.0607 0.182 –0.387 0.76 0.35 –0.822 –0.0907 –0.427 –0.103 –0.00279 –0.0306 1.09E–08\nMW 5 –0.807 0.399 0.938 0.0607   0.012 0.807 0.278 –0.821 –0.491 –0.203 0.157 0.0848 –0.0985 –0.0469 5.89E–10\nRB 6 0.0443 0.243 –0.0124 0.182 0.012   –0.0443 0.363 0.0185 –0.386 0.905 0.157 –0.0754 –0.0364 0.00653 4.45E–09\nTPSA 7 –1 0.719 0.867 –0.387 0.807 –0.0443   –0.274 –0.99 0.0259 –0.0371 0.0782 –0.0964 0.0537 –0.0186 5.38E–05\nclog Pc 8 0.274 –0.466 0.11 0.76 0.278 0.363 –0.274   0.264 –0.931 –0.0242 0.205 0.112 0.0929 0.00734 –1.71E–08\npc1 9 0.99 –0.779 –0.893 0.35 –0.821 0.0185 –0.99 0.264   9.62E–09 –8.89E–09 5.33E–09 –1.73E–08 6.60E–09 –5.23E–09 1.16E–08\npc2 10 –0.0259 0.214 –0.397 –0.822 –0.491 –0.386 0.0259 –0.931 9.62E–09   –5.16E–09 –9.75E–09 –1.46E–08 –1.70E–09 3.02E–10 –8.98E–10\npc3 11 0.037 0.45 –0.147 –0.0907 –0.203 0.905 –0.0371 –0.0242 –8.89E–09 –5.16E–09   1.02E–08 –8.09E–09 –2.00E–09 –2.34E–08 –1.94E–08\npc4 12 –0.0781 –0.346 –0.119 –0.427 0.157 0.157 0.0782 0.205 5.33E–09 –9.75E–09 1.02E–08   –7.30E–09 9.18E–09 –6.69E–09 5.02E–09\npc5 13 0.0966 0.156 –0.00919 –0.103 0.0848 –0.0754 –0.0964 0.112 –1.73E–08 –1.46E–08 –8.09E–09 –7.30E–09   4.55E–09 9.35E–09 2.03E–09\npc6 14 –0.0537 0.032 –0.0307 –0.00279 –0.0985 –0.0364 0.0537 0.0929 6.60E–09 –1.70E–09 –2.00E–09 9.18E–09 4.55E–09   6.33E–09 –1.14E–08\npc7 15 0.0186 –0.0146 0.0873 –0.0306 –0.0469 0.00653 –0.0186 0.00734 –5.23E–09 3.02E–10 –2.34E–08 –6.69E–09 9.35E–09 6.33E–09   –1.39E–08\npc8 16 5.38E–05 –1.39E–08 2.76E–09 1.09E–08 5.89E–10 4.45E–09 5.38E–05 –1.71E–08 1.16E–08 –8.98E–10 –1.94E–08 5.02E–09 2.03E–09 –1.14E–08 –1.39E–08  \nTable 17. Bravais–Pearson (linear correlation) coefficient of WS phytoconstituents versus FDA–approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib) for drug like properties and leadlikeness.\nProperties   1 2 3 4 5 6\nLL 1   –0.669 0.631 0.858 –0.51 –0.066\nclog Pc 2 –0.669   –0.754 –0.912 –0.173 –0.372\nclogS 3 0.631 –0.754   0.896 0.311 –0.316\npc1 4 0.858 –0.912 0.896   –1.95E–09 1.70E–09\npc2 5 –0.51 –0.173 0.311 –1.95E–09   8.57E–10\npc3 6 –0.066 –0.372 –0.316 1.70E–09 8.57E–10  \nTable 18. Bravais–Pearson (linear correlation) coefficient of WS phytoconstituents versus FDA–approved standard reference drugs (Losartan, Procainamide, Cinacalcet, Arbidol, Hydroxychloroquine, Oberadilol, Poziotinib) for bioactivity score prediction.\nProperties   1 2 3 4 5 6 7 8 9 10\nEI 1   0.0219 0.658 0.923 0.689 0.901 0.37 0.114 –0.0254 0.193\nGPCR 2 0.0219   0.317 –0.0436 0.573 0.354 –0.894 0.229 –0.148 0.0199\nICM 3 0.658 0.317   0.543 0.762 0.849 –0.13 –0.501 –0.101 –0.0238\nNRL 4 0.923 –0.0436 0.543   0.599 0.836 0.456 0.244 –0.106 –0.151\nPI 5 0.689 0.573 0.762 0.599   0.905 –0.318 0.0426 0.276 –0.0383\npc1 6 0.901 0.354 0.849 0.836 0.905   1.08E–09 8.12E–09 –3.79E–09 1.67E–08\npc2 7 0.37 –0.894 –0.13 0.456 –0.318 1.08E–09   7.21E–09 1.03E–09 –4.17E–09\npc3 8 0.114 0.229 –0.501 0.244 0.0426 8.12E–09 7.21E–09   4.11E–09 3.79E–10\npc4 9 –0.0254 –0.148 –0.101 –0.106 0.276 –3.79E–09 1.03E–09 4.11E–09   9.73E–09\npc5 10 0.193 0.0199 –0.0238 –0.151 –0.0383 1.67E–08 –4.17E–09 3.79E–10 9.73E–09  \n\n3.11. Molecular dynamics simulation\nFigures 9 and 10, respectively, depict molecular simulation analysis of SARS-CoV-2 spike receptor-binding domain (PDB ID: 6M0J) bound with withanolide A and SARS-CoV-2 papain-like protease (PDB ID: 6W9C) bound with withanolide B. Both MD simulations showed an acceptable stability profile at a temperature of 300 K. Root mean square deviation (RMSD) is one of the most important fundamental properties to establish protein stability and its conformation to experimental structure (Kuzmanic \u0026 Zagrovic, 2010; Laskowski et al., 1997). RMSD is a measure of the deviation of the 3D or tertiary structure of a protein and is applied in order to get an insight into the stability of the protein in a biological system during a MD simulation. SARS-CoV-2 spike receptor-binding domain-withanolide A complex displayed constant RMSDs (0.5–2.0 angstrom) of both protein side chains and Cα atoms from the initial structure (before equilibrium) throughout the 3 ns time scale (Figure 9.1). Similarly, SARS-CoV-2 papain-like protease-withanolide B complex also exhibited constant RMSDs (0.8–2.9 angstrom) of both protein side chains and Cα atoms from the initial structure throughout the 3 ns time scale (Figure 10.1). Figures 11.1–11.3 and 12.1–12.3, respectively depict MS dynamics analyses of SARS-CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B and SARS-CoV-2 main protease (PDB ID: 6LU7) with withanolide A.\nFigure 9. Molecular simulation of SARS–CoV–2 spike receptor–binding domain bound (6M0J) with withanolide A using Playmolecule open server (Table 1). Figures 9.1–9.2, Tables 2–4 here corresponds to the tables of MD simulation statistics. RMSD values were obtained as a function of time obtained at 300 K. Values were calculated with the use of Cα atoms. Figures 9.3–9.4. Average RMSF values obtained as a function of amino acid sequence numbers at 300 K. Values were calculated with the use of Cα atoms.\nFigure 10. Molecular simulation of papain–like protease (6W9C–A chain) with withanolide B using Playmolecule open server. Figures 10.1–10.2, Tables 2–4 here corresponds to the tables of MD simulation statistics. RMSD values were obtained as a function of time obtained at 300 K. Values were calculated with the use of Cα atoms. Figures 10.3–10.4. Average RMSF values obtained as a function of amino acid sequence numbers at 300 K. Values were calculated with the use of Cα atoms.\nFigure 11.1. MD simulation of SARS–CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B using LARMD online server. (A) Ligand–protein conformation, (B) RMSD of receptor and ligand (C) RMSD histogram of receptor (D) RMSD histogram of ligand (E) Radius of gyration—Rg value (F) Fraction of native contacts analysis of SARS-CoV–2 PL-pro (PDB ID: 6W9C) with withanolide B over a time frame of 4000 ps (4 ns) (G) RMSF value of each residue (H) B–factor value (changing from blue to red with increase in value) and (I) B–factor analysis of defined complex.\nFigure 11.2. PCA of SARS–CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B (A) PCA results for Trajectory (B) Simple clustering in PC subspace(C) Table data showing residue–wise loadings for PC1, PC2 and PC3 and residue number at each position (D) Clustering dendogram based on PC1, PC2 and PC3 (E) Dynamical residue cross–correlation map; the correlated residues are in blue, anti–correlated residues are in red; the pairwise residues with higher correlated coefficient (\u003e0.8) and with higher anti–correlated coefficient (≤0.4) are linked with light pink and light blue (Int_mod) (F) Residue–wise loadings for PC1, PC2 and PC3 (G) Table showing pairwise cross–correlation coefficients; higher correlated coefficient value is \u003e0.8 and higher anti–correlated coefficient value is ≤0.4.\nFigure 11.3. Energy, hydrogen bond analysis and decomposition analysis of SARS–CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B (A) MM/PB(GB)SA result consists of electrostatic energy (ELE), van der Waals contribution (VDW), total gas phase energy (GAS), non–polar and polar contributions to solvation (PBSOL/GBSOL) (B,C) Statistics of hydrogen bonds (D) energy decompose of protein–ligand complex (Kcal/mol) (E) Graphical representation of decompose result (F) Showing the heatmap of decompose.\nFigure 12.1. MD Simulation of SARS-CoV-2 main protease (PDB ID: 6LU7) with withanolide A using LARMD online server. (A) Ligand-protein conformation (B) RMSD of receptor and ligand (C) RMSD histogram of receptor (D) RMSD histogram of ligand (E) Radius of gyration- Rg value (F) Fraction of native contacts analysis of SARS-CoV-2 PL-pro (PDB ID: 6W9C) with withanolide A, over a time frame of 4000ps (4 ns) (G) RMSF value of each residue (H) B-factor value (changing from blue to red with increase in value) and (I) B-factor analysis of defined complex.\nFigure 12.2. PCA of SARS-CoV-2 main protease (PDB ID: 6LU7) with withanolide A (A) PCA results for trajectory (B) Simple clustering in PC subspace(C) Table data showing residue-wise loadings for PC1, PC2 and PC3 and residue number at each position (D) Clustering dendogram based on PC1, PC2 and PC3 (E) Dynamical residue cross-correlation map; the correlated residues are in blue, anti-correlated residues are in red; the pairwise residues with higher correlated coefficient (\u003e0.8) and with higher anti-correlated coefficient (≤0.4) are linked with light pink and light blue (Int_mod) (F) Residue-wise loadings for PC1, PC2 and PC3 (G) Table showing pairwise cross-correlation coefficients; higher correlated coefficient value is \u003e0.8 and higher anti-correlated coefficient value is ≤0.4.\nFigure 12.3. Energy, hydrogen bond analysis and decomposition analysis of SARS-CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B (A) MM/PB(GB)SA result consists of electrostatic energy (ELE), van der Waals contribution (VDW), total gas phase energy (GAS), non-polar and polar contributions to solvation (PBSOL/GBSOL) (B,C) Statistics of hydrogen bonds (D) Energy decompose of protein–ligand complex (Kcal/mol) (E) Graphical representation of decompose result (F) Showing the heatmap of decompose.\nVibrations around the equilibrium are not random, but depend on the local structure flexibility. In order to calculate the average fluctuation of all residues during simulations, the root mean square fluctuation (RMSF) of the Cα atoms of both target proteins were plotted from the primary structure of both proteins as a function of residue number (Kuzmanic \u0026 Zagrovic, 2010). 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