Folic Acid and its derivatives can prevent the interaction of S protein with ACE-2 receptor
Prepared structure of the S protein (Receptor Binding Domain)- ACE2 complex (PDB ID: 6LZG) was first analyzed in the native conformation to investigate the major interactions between spike protein and ACE-2 receptor. In the analysis, it was found that the TYR41, ASP38, GLU35, ASP30 of ACE-2 receptor was making the hydrogen bond with THR500, GLY496, GLN493 and LYS417 of S protein respectively (Fig.1) .Further, the complex was taken for the screening of nutraceuticals against it. For the docking of the ligands, the grid was generated, taking interacting residues in the center of the box. The coordinates of the grid X = -35.07, Y = 25.72, Z = 0.66 and the dimension of the grid was 25 Angstrom3. The ligands having the best binding affinity towards the complex were taken further for the analysis of their interacting residues. It was found that, out of 106 nutraceuticals screened; Folic Acid had the strongest binding energy of -9.0 Kcal/mol, followed by tetrahydrofolic acid (-8.9 Kcal/mol) and 5-methyltetraydrofolic acid (-8.6 Kcal/mol). The interaction analysis further showed that these nutraceuticals have the potential to break or prevent the interactions between the protein complex. Folic acid could break the hydrogen bond interaction Of ASP30 of ACE-2 with S protein; similarly, GLY496 of S protein, which previously interacting with ACE-2, was interacting with folic acid after docking. Likewise, GLY493, LYS417 of spike protein, was interacting with Folic acid (Fig 2 A). These significant reductions of the major interacting residues between the spike protein and ACE2 showing the potential of folic in the prevention of viral entry. Similarly, when we analyzed the Tertrahydrofolic acid interactions, we found that it was also able to break the crucial interactions and was making interactions with GLN493, GLN496 of spike protein and ASP38 of ACE-2 receptor (Fig. 2B). Furthermore, 5-methyl-tetrahydrofolic acid was making the hydrogen bond with ASP30 of ACE-2 and LYS417 of S protein and was making various non-polar interactions with the crucial amino acids, which were previously helping the interaction of spike protein with ACE-2 receptor (Fig 2 C) . All these analyses of the binding energy and interactions with crucial residues of the complex indicating the possibility of folic acid and its derivatives to be lead molecules against SARS-CoV-2 entry inside the human cells ( Supplementary Table 1).
Folic acid and it's derivative and Ginkgolide A against PLpro
PLpro, a crucial papain-like protease of the SARS-CoV2, helps in the cleavage of viral polyproteins and making them functional. The inhibition of the functional activity of this protease could inhibit the viral replication inside the host cell. So, the virtual screening was done with all the 106 nutraceuticals compounds, and the grid was generated at the substrate-binding site. Grid was generated around main catalytic residues ASP164, VAL165, CYS270, LYS274, VAL303 at catalytic site  for docking of compounds. The center of the Grid was X= -30.19, Y = 31.61, Z= 31.04 with dimension of 25 Angstrom3. After screening, it was found that 5-methyl-hydrofolic acid had the best binding energy (-7.1Kcal/mol) followed by Ginkgolide -A(-7.0 Kcal/mol), a terpene lactone compound mainly found in Ginkgo biloba and Folic acid (-6.9 Kcal/mol). The 5methylhyrofolic acid had the highest number of hydrogen bond interactions with residues (ASP164, ARG166, ALA246, THR301) at the catalytic site of PLpro (Fig 3 A). While, Gingkoglide was making the hydrogen bond with GLY163, in the best binding pose. And, Folic acid was making the hydrogen bond with GLY163 and ASP164 (Fig 3B).The ASP164 has been reported to be a main catalytic residue of PLpro, which helps in proteolysis of the substrate (Fig 3C).Here we could see that all tree best compounds had multiple polar as well as non-polar interactions at the catalytic site , but 5-methyl-hydrofolic acid and Folic acid were making contact with ASP164. All these analyses were again indicating the value of the folic acid and its derivative and Ginkgolide -A potential against the inhibition of COVID-19 ( Supplementary Table 2).
Water-soluble Vitamins against Mproof SARS-CoV-2
Another crucial protease enzyme for SARS-CoV-2 is Main protease or Cysteine like protease, which helps in the cleavage of polyproteins and make them functional. The structure of the main protease (6LU7) docked with an N3 inhibitor at the substrate-binding site. The main interacting residues of N3 was taken for creating a grid for the molecular docking of 106 Nutraceutical compounds. So, Grid was generated around PHE140, ASN142, GLY143, HIS164 and Centre of grid was, X= -19.50, Y = 19.24, Z= 64.35; the dimension was 25 *25*25 Angstrom. Magnesium Ascorbate, a buffered (non-acidic) form of Vitamin C (Ascorbic acid), was found to be the top lead compound among 106 nutraceuticals. It had binding energy of -8.1 Kcal/mol and was making the hydrogen bond with LEU141, SER144 and HSI164 (Fig 4A). Followed by Folic acid and Riboflavin had a similar binding affinity towards Mpro with the binding energy of -7.7 Kcal/mol. Folic was making polar contact with TYR54, ASP187, ARG188 and THR190 (Fig 4B), while Riboflavin with LEU141, GLY143, CYS145 in their best binding pose at the catalytic site of Mpro (Fig 4C).Virtual screening of the nutraceutical against Mpro shown that water-soluble vitamins could be helpful in preventing the functional activity of the enzyme and hence COVID19 infection ( Supplementary Table 3)
Folic Acid and its derivative and NADH can prevent the endoribonuclease activity
Once the virus enters inside the host cell, it uses the host translational machinery to produce various Nsps, which helps virus replication. One such Nsp is Nsp15, which is RNA uridylate specific endoribonuclease associated with the viral RNA processing. Inhibition of the functional activity of Nsp15 could help in the inhibition of viral replication inside the body. So the grid was generated at the substrate-binding site, Grid was generated around LEU246, GLY248, LEU249, TYR343 . The center of grid was X= -62.80, Y = 67.89, Z= 34.93 and the dimension was 25 *25*25 Angstrom. The result of the screening shown that NADH, Folic acid, and 5-methyl-hydrofolic acid with the binding energy of -7.2, 7.1 and 7.1 Kcal/mol, respectively, were the top lead compounds against Nsp15. At the best binding pose, NADH was making one hydrogen bond with TYR343 (Fig 5A). Folic acid had 3 three hydrogen bond interactions with ASP240, HIS243, and TYR343(Fig 5B). 5-methyl-hydrofolic acid was also making 3 hydrogen bonds with ASP240, GLU340 and TYR343 (Fig 5C). These binding energy and number of interacting residues together were indicating that folic acid and its derivatives had a good binding affinity towards Nsp15, which could be lead compounds in the prevention of COVID-19. (Supplementary Table 4).