2.1.  Molecular docking studies

2.1.1.  Protein preparations
The crystal structure of SARS-CoV-2 RdRp (PDB ID: 6M71) (Yan et al., 2020) was retrieved from the protein databank (www.rcsb.org) (Berman et al., 2000). The crystal structure was prepared individually by adding hydrogen atoms and computing the Gasteiger charge using the AutoDock v4.2 program (Morris et al., 2009). Subsequently, the file was saved as .pdbqt format in preparation for molecular docking. Schematic representation of the work-flow for selecting potential natural polyphenolic inhibitors for the SARS-CoV-2 RdRp is shown in Figure 2.
Figure 2.  Flow chart of the methodology for shortlisting the best natural polyphenolic inhibitor of the SARS-CoV-2 RdRp.

2.1.2.  Ligand preparations
The SDF structures of GTP, remdesivir, and selected hundred polyphenols (see Table S1 in Supplementary Information) were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) (Kim et al., 2019). The compounds were converted into PDB format, and conformational energies of all the compounds were minimized by using UCSF Chimera (Pettersen et al., 2004).
Table 1.  Binding energy (kcal/mol) of the natural polyphenols along with the control compounds (GTP and remdesivir) against RdRp of the SARS-CoV-2 (PDB ID: 6M71) by molecular docking study.
S. No.  Compound Name  Binding energy (kcal/mol)  S. No.  Compound Name  Binding energy (kcal/mol)
1   TF3   −9.9  52  Cyanidin  −6.3
2   TF2b   −9.6  53  Daidzein  −6.3
3   TF1   −9.6  54  Glycitein  −6.3
4   TF2a   −9.3  55  Wogonin  −6.3
5   Hesperidin   −8.8  56  Phloretin  −6.3
6   EGCG   −7.3  57  Catechin  −6.2
7   Myricetin   −7.2  58  Urolithin B  −6.2
8   Quercetagetin   −7.0  59  Angolensin  −6.2
9  Quercetin  −6.9  60  Pinosylvin  −6.2
10  Curcumin  −6.9  61  Formononetin  −6.2
11  Dihydrorobinetin  −6.8  62  Liquiritigenin  −6.2
12  Peonidin  −6.8  63  Prunetin  −6.2
13  Fisetin  −6.8  64  Alpinetin  −6.2
14  Robinetin  −6.7  65  Biochanin A  −6.2
15  5-Deoxygalangin  −6.7  66  Rhapontigenin  −6.1
16  Kaempferol  −6.7  67  Genistein  −6.1
17  Scutellarein  −6.7  68  Chrysin  −6.1
18  (-)-Epicatechin  −6.7  69  6-Hydroxyflavone  −6.1
19  Purpurin  −6.7  70  Equol  −6.1
20  Isorhamnetin  −6.7  71  Piceatannol  −6.1
21  Tricetin  −6.6  72  Isorhapontigenin  −6.0
22  Gossypetin  −6.6  73  Resveratrol  −5.8
23  Norathyriol  −6.6  74  Danshensu  −5.7
24  Coumestrol  −6.6  75  Eugenin  −5.6
25  Isosakuranetin  −6.6  76  Sinapic acid  −5.5
26  Pectolinarigenin  −6.6  77  Pterostilbene  −5.5
27  Tangeritin  −6.6  78  Ferulic acid  −5.4
28  Nobiletin  −6.6  79  Caffeic acid  −5.4
29  Pratensein  −6.6  80  Isoferulic acid  −5.4
30  Hispidulin  −6.6  81  Dihydrocaffeic acid  −5.4
31  Baicalein  −6.5  82  Gentisic acid  −5.3
32  Apigenin  −6.5  83  Pyrogallol  −5.3
33  Morin  −6.5  84  4-Hydroxycinnamic acid  −5.2
34  Urolithin A  −6.5  85  Resacetophenone  −5.2
35  Acacetin  −6.5  86  Salicyclic acid  −5.1
36  Pelargonidin  −6.5  87  Syringic acid  −5.1
37  Irilone  −6.5  88  2-Hydroxybenzoic acid  −5.1
38  Naringenin  −6.5  89  Gallic acid  −5.0
39  Pinocembrin  −6.5  90  3-Hydroxybenzoic acid  −5.0
40  Kaempferide  −6.5  91  4-Hydroxybenzoic acid  −5.0
41  Malvidin  −6.5  92  Vanillin  −5.0
42  Luteolin  −6.4  93  p-Coumeric acid  −4.9
43  Dalbergin  −6.4  94  Vanillic acid  −4.8
44  Butein  −6.4  95  Paeonol  −4.8
45  Biochanin A (1-)  −6.4  96  Cinnamic acid  −4.7
46  Fustin  −6.4  97  Protocatechuic acid  −4.6
47  5-Hydroxyflavone  −6.4  98  4-Ethylphenol  −4.5
48  Pinostrobin  −6.4  99  Catechol  −4.5
49  Pinobanksin  −6.4  100  Tyrosol  −4.5
50  Datiscetin  −6.3  101   GTP   −7.9
51  Galangin  −6.3  102   Remdesivir   −7.7

2.1.3.  Docking studies using AutoDock Vina
The energy-minimized structure of all the natural polyphenols, remdesivir, and GTP were docked with the receptor (RdRp of SARS-CoV-2) using AutoDock Vina 1.1.2 (Trott & Olson, 2010). The ligand files were further saved in PDBQT file format, a modified PDB format containing atomic charges, atom type definitions for ligands, and topological information (rotatable bonds). A grid box (30 Å × 30 Å × 30 Å) centered at (121, 120, 125) Å for the SARS-CoV-2 RdRp, was used in the docking experiments. After the receptor-ligand preparation, docking runs were started from the command prompt. The lowest binding energy and best-docked conformation were considered as the ligand molecule with maximum binding affinity.

2.1.4.  Protein-ligand interactions
LigPlot+ was used to investigate protein-ligand interactions for a given .pdb file containing the docked conformation and also the final simulated conformation (Wallace et al., 1995). The LigPlot+ program self-generated schematic 2D representations of protein-ligand interaction. The output file represents the intermolecular interactions and their strengths, including hydrogen bonds, hydrophobic contacts, and atom accessibilities. H-bonds are shown in green dotted lines, whereas residues involved in hydrophobic interaction are represented in the red semicircle.