In Silico Identification of Potential Therapeutic Agents for COVID-19 Based on Molecular Docking Study of Main Protease and Receptor Binding Domain of Spike Protein.


 Severe acute respiratory syndrome coronavirus (SARS-CoV-2) enter the cell by interacting with human angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) of S-protein. In the cell the viral 3-chymotrypsin-like cysteine protease (3CLp) enzyme is essential for its life cycle and controls coronavirus replication. Therefore the S-RBD and 3CLp are hot targets for drugs discovery against SARS-CoV-2. This study was to identify repurposing drugs using in-silico screening, docking and molecular dynamics simulation. The study identified Dibenzoyl Thiamine, Folic Acid and Vitamin B12 against the RBD of S-protein and Dibenzoyl Thiamine, Folic Acid, Fursultiamine and Riboflavin to 3CLp. The strong and stable binding of these safe and cheap vitamins at the important residues (R403, K417, Y449, Y453, N501 and Y505) in S-protein –ACE2 interface and 3CLp active site residues (His 41 and Cys 145), indicating that they could be valuable repurpose drugs for inhibiting SARS-CoV-2 entry into the host and replication.


Introduction
The ongoing covid-19 pandemic has resulted in over 71,051,805 affected individuals and 1,608,648 deaths by the end of 14 December 2020 (World Health Organization) [1]. Therefore, it is vital to control and prevention this spreadable disease.
The 3-D structure of 3CLpro enzyme, also called Main protease (Mpro), contains three domains. Domain I (residues 1-100) and domain II (residues 101-183) have a two-β-barrel fold, which is alike to chymotrypsin, whereas the third domain (residues 198-303) consists of ve α-helical structures. The substrate binding cleft, located between domain I and II, with a catalytic dyad of His41 and Cys145, in which the cysteine thiol allow the nucleophilic attacks and His acts as a proton acceptor (Fig. 1B).
The 3′ terminal of a coronavirus genome encodes structural viral proteins: nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein and spike (S) protein (Fig. 1C). The spike protein localizes on the virion surface and consists of ectodomain region (ED), intracellular domain and TM region [8]. The ED region (S1 + S2) (aa 13-1273) includes S1 subunit (aa 13-685) mediating SARS-CoV attachment to host angiotensin-converting enzyme 2 receptor via receptor-binding domain (aa 319-541) and S2 chain (aa 686-1273), which serving host and viral membrane fusion as a result, potentiate the CoV to release its RNA genome in host cell. [8][9]. Several studies have recently, clarifying the residues are involved in interaction between RBD and human ACE2. The key residues of Spike RBD interact with ACE2 receptor are as follow: Arg403, Glu406, Gly446, Lys417, Tyr449, Tyr453, Ala475, Asn487, Gln493, Tyr495, Gly496, Phe497, Gln498, Thr500, Asn501 and Tyr505 [10][11] Among the above residues Lys 417, Tyr 449, Tyr 489, Gln 493, Asn 501 and Tyr 505 play crucial role in anchoring RBD to ACE2 receptor (Fig. 1B) [11]. It therefore appears designing drugs for inhibiting the interaction of the S-protein with its receptor, to be also another attractive strategies for controlling of SARS-cov-2 In this study to nd the potent drug molecules to inhibit main protease and also prevent the spike protein interaction with the host receptor, virtual screening was performed against vitamins library in Selleckchem Inc (WA, USA) and subsequently molecular dynamics simulation carried out on selected ligands. Dibenzoyl Thiamine, Folic Acid, Ribo avin, Fursultiamine, Benfotiamine, Calcipotriene, Cocarboxylase and Ergosterol are discovered as potent inhibitor against 3CLp and Vitamin B12 and Dibenzoyl Thiamine, Folic acid and Benfotiamine were found to inhibit RBD of spike protein.

Receptors and Ligands preparation:
The crystal structures of receptor-binding domain/RBD of the spike protein (PDB ID.6M0J_E) and 3CLprotease (PDB ID. 6LU7_A) were obtained from the Protein Data Bank [12] and after a cleaning with Discovery Studio 4.1 [13], minimized and changed to pdbqt format using AutoDockTools 1.5.6 [14].
For docking against RBD of the spike protein and 3CL-protease, 47 FDA approved vitamins were collected from Selleckchem Inc and the 3D structure of the ligands were retrieved from PubChem database [15] in SDF le format and converted to pdb and pdbqt format using Avogadro [16] and AutoDockTools-1.5.6, resprctively,

Molecular Docking And Post Docking Analysis
To compute binding a nity between protein and ligands, AutoDock vina in PyRx 0.8 [17] was used to perform the docking based virtual screening over 47 candidate compounds against the 3-D structure of 6lu7 amd 6M0J proteins. For 3CLp inhibition calculation, the grid box was set at 26 A° x 26 A° x 26 A° (x, y and z) and center − 15.518 A° x 21.151 A° x 66.865 A° (x, y and z) with grid point spacing of one Angstrom. The grid box was set into the His41, Cys145 (catalytic dyad) at ligand binding site.
For the RBD domain of S-protein inhibition calculation, the default parameters for grid box were set to 36 A° x 55 A° x 32 A° (x, y and z) and center − 33.688 A° x 30.917 A° x 7.143 A° (x, y and z) to cover the possible binding sites of the SARS-CoV-2 S-protein with the ACE-2 human receptor, in agreement with previous results [11].
The ligands with the highest a nity binding scores -i.e., the most negative binding energies-were selected for more analysis. The protein-ligand complexes were visualized by Discovery Studio 4.1. The hydrophobic interactions and hydrogen bonds of the selected docked complexes were analyzed by Discovery Studio and LigPlot+ (v 1.4.5) [18].

Molecular Dynamic Simulation
In order to assess the stability of the protein-ligands complexes, molecular dynamics (MD) simulation was performed on docked complexes using GROMACS package 2019.6 [19]. Amber99SB-ILDN [20] force eld was applied for simulations. The aqueous environment were created using simple point charge TIP3P water model in a cubic box with a distance of 0.8 nm from the box to the surface of the protein.
Steepest descent algorithm was utilized for energy minimization. For each simulation, 50000 steps of energy minimization were performed. The systems were equilibrated using NVT and NPT ensembles for 200 ps. The pressure and temperature were set to 1 bar and 300 K. Finally the 100 ns MD simulation production runs were carried out on ligand-receptor complexes. All simulations were repeated three times.
GROMACS in-built tools were applied to analyzing the MD trajectories to calculate the root-mean-square deviation (RMSD), root-mean-square uctuation (RMSF) and, Xmgrace was used for plotting graphs. Molecular dynamic was done on dimer form of 6lu7, which was retrieved using PyMOL [21].
In another attempt for comparing the variation of RMSD, the RMSD standard deviation (RMSD-SD) was calculated for each residue through MD simulation among the three repeated MD trajectories.

Result And Discussion
Virtual screening of vitamins against the key viral proteins and analysis.
Many of the current researches focused on repurposing FDA approved drugs for anti-SARS drug development. In this work 47 approved FDA vitamins were virtual docked against 3CL-protease and S-RBD of coronavirus2.
For the S-RBD, the Vitamin B12, Folic Acid, Dibenzoyl Thiamine and Benfotiamine displayed the highest a nity (Table 1) and good characterization results.
The selected compounds were visually inspected using Discovery Studio to determine their residues which involved in interactions with 3CLpro and S-RBD. The results in Table 1 show that all selected ligands interact with the catalytic dyad residues (Cys-145 and His-41) as well as signi cant interactions with the most ligand -binding residues, id; Thr24, Thr25, Thr26, Met49, Asn142, Gly143, His164, Glu166 and Gln189 in the binding pocket of 3Clp.
For spike protein, the ligands covers the major part of the RBD-ACE2 interface and interact with important residues in RBD-ACE-2 interface, such as: R403, K417, Y449, Y453, S494, N501 and Y505. Although the 487 and 475 don't fall into traps, but the cover is created over S-RDB seems a barrier for interaction between Spike RBD and human ACE2 2 interface (Table 1).

Md Simulations And Ligplot Analysis
RMSD analysis of the protein evaluate its conformational and examines whether simulation has equilibrated.
The RMSD plots shown in Figs. 2A and 2B, demonstrated 6lu7-ligand complex structures, except 6lu7ligand 2, reached a steady state during rst few nanoseconds and remained stable throughout the simulation. For 6lu7-ligand 2 very mild variation observed during simulation. This slight instability may be due to the elongated structure of ligand 2, which couldn't not ts well into the 6lu7' binding pocket.
For 6m0j-ligand complexes, due to the special structure of it which doesn't have speci c binding pocket, shows a variety of behaviors with different ligands. In relation to 6m0j-ligand 1, a relatively sharp deviation was seen about 20 nanoseconds for two repeats, and then the system reached equilibrium. (Fig. 2B). The simulation of 6m0j-ligand 2 also showed deviation for three repeats, which could be explained by the special properties of the substrate and the ligand. 6m0j-ligand 5 exhibits a steady increase during the rst 20 ns, and reached a stable state throughout equilibration. The 6m0j-vitamin B-12 (ligand6) complex presented an acceptable equilibration during simulation.
Comparison of the standard deviation of backbone RMSD among the three repeated simulation of all protein-ligand complexes revealed small SD values (< 1Aº), which indicated all simulated protein-ligand complexes are stable ( Fig. 2A-B).
RMSF measures the uctuations of each residue during simulation. The RMSF plot for each residue of the 6lu7-ligand complexes indicates that the protein was not uctuated in the 100 ns simulation periods (Fig. 2C). Comparatively, the RMSF plot of the 6m0j complexes (Fig. 2D) demonstrating overall stability of these constructs, with the exception of a few residues in C-terminal domains that show greater uctuations. Investigation in 6m0j 3D-structure indicated that residues in loop region are more uctuated during the simulation. This indicates that the complexes were not uctuated in the simulation periods.
The top 10 compounds including the compounds selected for the MD study, were subjected to ligplot analysis.
Investigation in the LigPlot of the 6lu7-Dibenzoyl Thiamine, 6lu7-Fursultiamine, 6lu7-Folic Acid and 6lu7-Ribo avin shows that the catalytic dyad residues of both chains of dimer molecule bind to Folic Acid and Dibenzoyl Thiamine ligands. These results are comparable and consistent with the results of LigPlot before MD and Docking results (Fig. 3, Table 1and Supplementary Fig. 1).
Examination and comparison of the two-dimensional representation of 6moj interactions with selective ligands after and before MD show the compatibility of the amino acids involved in the interaction with the residues present in the 6moj-S-RBD interface (Fig. 3, Table 1).
These results indicate the stability of the interaction between the selected ligands with the respective receptors.
The above results suggest that vitamins compounds might be the potential 3CLpro and S-RBD inhibitors and could probably be used for treating SARS-CoV-2.
The description of selected compounds are as below: Benfotiamine is a thioester that is a synthetic S-acyl derivative of thiamine (vitamin B1). Benfotiamine may also be bene cial for the treatment and preventing diabetic nephropathy and type 2 diabetes mellitus .
Cocarboxylase is a thiamine (vitamin B1) derivative which is produced by the enzyme thiamine diphosphokinase (Selleckchem). Thiamine pyrophosphate were found less than toxic in comparison to thiamine and thiamine monophosphate. Thiamine pyrophosphate chloride used in food supplements, as sources of vitamin B1 [25].
Calcipotriene (Calcipotriol, MC903) is a synthetic derivative of calcitriol, a form of vitamin D (Selleckchem). Calcipotriene is used as cream or solution drug in controlling of in psoriasis, and lead to normalization of epidermal growth [27]. The side effects of this drug is very limited [28].
Dibenzoyl Thiamine, a lipophilic derivative of vitamin B (thiamine), is a kind of food additive that can be rapidly absorbed into the body and converted to thiamine (Selleckchem). It appears to break down incompletely to thiamine [22], therefore there will always be some of it in our body intact and may be able to inhibit 3Clp. Bentiamine has been approved as a food additive in Japan [22]. The toxicity, teratogenicity and mutagenicity studies have been showed no side effects for Bentiamine [23].
Folic Acid, a B vitamin, plays an important role in cell division and in the synthesis of amino acids and nucleic acids like DNA (Selleckchem). Currently, this vitamin is prescribed as an oral supplement for all women planning, or capable of, pregnancy. Also, due to the function of folate for detoxi cation of arsenic through arsenic methylation, folic acid can be used as an effective supplement in promoting health in all age and gender groups [24].
Fursultiamine is a nutritional supplement and vitamin B1 derivative, with potential antineoplastic activity.
Fursultiamine can be used for vitamin B 1 de ciency .
Ergosterol is a sterol and a biological precursor (a provitamin) to vitamin D2 (Selleckchem). In recent years, the ergosterol rich extract presented notable antioxidant and antimicrobial properties, besides showing no hepatotoxicity. In fact, ergosterol is a type of plant sterol found in mushrooms. Mushrooms have been widely consumed as food, especially by the Chinese and Japanese [26].
Ribo avin (Vitamin B2) is a heat-stable and water-soluble vitamin found in food and used as a dietary supplement to prevent and treat ribo avin de ciency. This vitamin is essential for healthy skin, nails, and hair .
Vitamin B12 is a water soluble vitamin with a key role in the normal functioning of the brain and nervous system (Selleckchem). It is better to use natural resources to meet the body's need for vitamin B12, but doctors sometimes prescribe forti ed foods and supplements for people over the age of 50 [29].

Conclusion
In this study, virtual screening was successfully used to identify FDA 47 approved vitamins, which having therapeutic activity against the viral Mpro enzyme and S-RBD spike protein of COVID-19. It was observed that Dibenzoyl Thiamine, Folic Acid, Ribo avin, Fursultiamine, Benfotiamine, Ergosterol, Cocarboxylase and Calcipotriene were found to be potential inhibitors against the viral Mpro enzyme and Vitamin B12, Folic Acid, Dibenzoyl Thiamine and Benfotiamine for S-RBD spike protein of COVID-19.

Declarations
Funding: No funding was received for this work.
Con icts of interest/Competing interests: The authors declare that there is no con ict of interest Availability of data and material: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability: The software used during this study are freely available and are also available from the corresponding author on reasonable request.
Authors' contributions: This manuscript has been done by myself and there was no co-author  Figure 1 Schematic representation of the SARS-CoV-2 structure and its genomic organization. A) Schematic representation of genome sequence of SARS-CoV-2 and proteome showing different polyproteins (pp1a and pp1b) along with the structural and accessory proteins. B) Structure of 3CLp with catalytic residues and S-RBD which represented the ACE2 binding residues. C) Diagrom of coronavirus structure showing M (membrane) protein, S (Spike) protein, E (envelope) protein, N (nucleocapsid) protein & RNA along with the ACE2 receptor [30].

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.