In silico exploration of binding potentials of anti SARS-CoV-1 phytochemicals against main protease of SARS-CoV-2

The phytochemicals can play complementary medicine compared to synthetic drugs considering their natural origin, safety, and low cost. Phytochemicals hold a key position for the expansion of drug development against corona viruses and need better consideration to the agents that have already been shown to display effective activity against various strains of corona viruses. In this study, we performed molecular docking studies on potential forty seven phytochemicals which are SARS-CoV-1 Mpro inhibitors to identify potential candidate against the main proteins of SARS-CoV-2. In Silico Molecular docking studies revealed that phytochemicals 16 (Broussoflavan A), 22 (Dieckol), 31 (Hygromycin B), 45 (Sinigrin) and 46 (Theaflavin-3,3′-digallate) exhibited excellent SARS-CoV-2 Mpro inhibitors. Furthermore, supported by Molecular dynamics (MD) simulation analysis such as Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of gyration (Rg) and H-bond interaction analysis. We expect that our findings will provide designing principles for new corona virus strains and establish important frameworks for the future development of antiviral drugs.


Introduction
Current high outline, global outbreaks of viral diseases from the coronavirus family have been caused by enveloped viruses. Acute respiratory tract inflammation caused by SARS-CoV-2 is an infectious disease, often fatal, that is characterized by the rapid and unexpected spread. Worldwide, the COVID-19 pandemic has recorded, as of 1st March 2022, 437 million cases, 369 million recovered cases with 5.97 million deaths, and the numbers continue to increase progressively [1] (https://covid19.who.int/). Furthermore, patients with preexisting kidney dysfunction, immune-compromised persons, pulmonary disease and diabetes are the most susceptible community with higher mortality rates from SARS-CoV-2 infection. Coronavirus families are able to cause a number of diseases, such as hepatitis, gastroenteritis, bronchitis, systemic diseases, and even death in birds, humans, and other animals [2]. The contagion effect of such epidemic could possibly bring key challenges to worldwide health systems and have farreaching significances on the global economy if it is not controlled effectively.
The SARS coronavirus is the viable microorganism accountable for the worldwide outburst of a severe disease that caused several deaths [3]. To design the anti-SARS drug, the coronavirus main protease (M pro ), recognized as the utmost attractive target due to its crucial role in facilitating viral transcription and replication [4]. Near about 30,000 nucleotides comprised in the SARS-CoV-2 genome: the gene of SARS-CoV-2 namely, replicase encodes pp1a and pp1ab two overlapping polyproteins which are essential for viral replication and transcription [5]. Polyproteins excreted the functional polypeptides by extensive proteolytic processing, mostly by the 33.8-kDa M pro (also known as 3C-like protease). Polyprotein digested by M pro at least 11 conserved sites, initially the autolytic cleavage of this enzyme itself from pp1a and pp1ab [6]. In the viral life cycle M pro plays functional importance, shared with the absence of closely associated homologues in humans, recognize M pro as an attractive target for the design of antiviral drugs [7].
Herein we describe the in silico molecular docking results that intended quickly discovery of lead compounds for clinical use, by assimilation structure-assisted drug design, virtual drug screening. This in silico study focused on identifying drug leads that target main protease (M pro ) of SARS-CoV-2. Jin and coworker identified a mechanism-based inhibitor by computeraided drug design, and then determined the crystal structure of M pro of SARS-CoV-2 [8]. We have screened 47 SARS-CoV-1 M pro active phytochemicals for high binding affinity and interaction to the conserved residues of the substratebinding pocket of SARS-CoV-2 M pro using molecular docking-based virtual screening. Our results demonstrated the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Molecular docking analysis
Molecular docking study was performed to explore the binding potential of the selected phytochemicals against the M pro of SARS-CoV-2. Structure of the M pro of SARS-CoV-2 (PDB ID: 6W63) [9] was downloaded from the free protein database www.rcsb.org.
The phytochemical analyzed in this work are showed profound activity against SARS-CoV-1 and their possible mode of action is via inhibition of the main protease. SARS-CoV-2 is mutated form of the main protease of SARS-CoV-1 and has up to 96% similarity [10,11]. Table 1 shows structures of phytochemicals utilized in current study. In virtue of all these reports, we thought virtual analysis of the molecules with profound activity on the SARS-CoV-1 on main protease of SARS-CoV-2 will be an attractive strategy for identification and development of potent inhibitors against viruses. Thus, phytochemicals with reported activity against SARS-CoV-1 were selected for the docking analysis [12][13][14].Grip based docking simulation was performed and best molecules were analyzed on the basis of docking score and binding interactions with M pro of SARS-CoV-2.
Theaflavin-3,30-digallate (46) was found to be another active molecule in docking simulation with showing docking score of À76.85 and hydrogen bond interaction with THR26 (1.

Molecular dynamics (MD) simulation analysis
The molecular docking study revealed the most promising phytochemical inhibitor molecules against SARS-CoV-2 M pro protein and so these docked complexes were selected for simulations. MD simulations were performed with control Table 1 The chemical structures of known phytochemical compounds acting against SARS-CoV-1.
(continued on next page) (M pro -X77) and different M pro -phytochemical docked complexes to assess their stability after the phytochemical inhibitor molecule binds to it. The stability was checked using Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of gyration (Rg) and H-bond interaction analysis [15][16][17].
The RMSD value is the representation of equilibration of MD trajectories [18][19]. The RMSD values of the protein backbone atoms are usually plotted as a function of time over entire simulation, to analyze the stability of each system. As illustrated from Fig. 8A the RMSD values of control and different M pro -phytochemical inhibitor complexes are determined RMSF value is a marker of flexibility observed of the residues throughout the simulation [18,19]. In present study the flexibility in the residues of SARS-CoV-2 M pro protein was analyzed after binding of different phytochemical inhibitor molecules and compared it with control. In consideration of the average position of the residues, high RMSF value signifies high flexibility, whereas, the low RMSF value indicates lower flexibility throughout the simulation. RMSF values observed for all M pro -phytochemical inhibitor complexes are more or less similar and with significantly lesser fluctuations as like control (Fig. 8B). The active site residues HIS41, SER144, CYS145, HIS163 and GLU166 in all M pro -inhibitor complexes displayed RMSF values of 0.05, 0.07, 0.06, 0.04 and 0.07 nm respectively similar to control (Fig. 8B). Thus, comparison of RMSF value of control and M pro -phytochemical inhibitor docked complexes indicates that the residues bound to phytochemical molecules, 4, 5, 16, 22, 31, 45 and 46 are rigid and has limited flexibility in same manner to control (Fig. 8B).
Radius of gyration helps to investigate the changes observed in the conformation of SARS-CoV-2 M pro in terms of compactness after binding of different inhibitor (phytochemical) molecules [18,19]. By meaning, Rg corresponds to  mass weighted root mean square distance of a collection of atoms from their common center of mass. Therefore the overall conformation of the protein could be analyzed by calculating the Rg values. Fig. 8C indicates the Rg values of control and different M pro -inhibitor docked complexes over entire simulation period. The Rg values for control and different M proinhibitor complexes were found to be in the range of 2.2 nm to 2.26 nm (Fig. 8C). From this it is apparent that there is no change in the conformation of the M pro protein after binding of different phytochemical inhibitors and also the compactness of M pro structure was found to be similar in presence of experimental inhibitor X77 and computationally identified phytochemical inhibitors (Fig. 8C).
In order to have a stable protein-inhibitor interaction the formation of hydrogen bonds is of supreme importance. For this we have illustrated the number of hydrogen bonds formed between SARS-CoV-2 M pro protein and different phytochemical inhibitor molecules viz; 4, 5, 16, 22, 31, 45, 46 and compared it with hydrogen bonding pattern observed in control (Fig. 9A-H). Thus, it can be clearly seen from Fig. 9B-H that significant hydrogen bonding pattern is observed in all M proinhibitor docked complexes in comparison to control (Fig. 9A). This is an indication of the fact that there is a strong interaction between SARS-CoV-2 M pro and phytochemical inhibitor molecules viz; 4, 5, 16, 22, 31, 45, 46. As seen from   In silico exploration of binding potentials of anti SARS-CoV-1 phytochemicals against main protease of SARS-CoV-2 comparatively effective interactions with SARS-CoV-2 M pro as also illustrated from molecular docking study (Table 2).

Preparation of protein structure
The structure of the M pro protein of SARS-CoV-2 (PDB ID: 6W63) [9] was downloaded from the free protein structure database RCSB (www.rcsb.org) with resolution of 2.1 Å . The downloaded protein structure was prepared for the docking analysis using V life MDS 4.6 via addition of missing hydrogen atoms. This prepared protein structure was utilized for the docking analysis.

Preparation of phytochemical structures
The structures of the phytochemical compounds were drawn by using molecule builder module of the V life MDS 4.6 and converted into the 3D structures. These developed structures were then optimized via energy minimization using merck molecular force field (MMFF). These optimized structures of phytochemical compounds were utilized for docking analysis.

Molecular docking study
The molecular docking study of the phytochemical compounds with receptor SARS-CoV-2 M pro protein was performed using biopredicta module. Redocking was performed using native ligand X77 of SARS-CoV-2 M pro , to ascertain the docking protocol applied [15][16][17]. Grip based docking analysis was performed keeping ligand structures in the flexible conformation. For docking analysis the rotational angle was kept at 10°and   total number of rotation to 30. The best docking pose of used phytochemical compounds bound to SARS-CoV-2 M pro was selected on the basis of the docking score and type of interactions.

Molecular dynamics (MD) simulation
Molecular dynamics (MD) simulation was performed to study the dynamic behavior and assess the stability of SARS-CoV-2 M pro protein bound to different phytochemical compounds. The crystal structure of M pro protein of SARS-CoV-2 bound with non-covalent inhibitor X77 (PDB ID: 6 W63) [9] served as a control system in the MD simulation study. The reliability of the binding mode and conformation of predicted best docked phytochemical molecules were confirmed using molecular dynamic simulation of docked complexes by GROMACS 2018.3 (www.gromacs.org) software package [20].  [21], whereas topology files of all inhibitor molecules was generated using PRODRG server [22]. After generating topology of each complex, further each complex was centered in the system of cubic box by keeping periodic distance of 1 nm between complex and edge of the box. All complexes were then solvated with SPC216 water molecules to fill the defined box for each complex. The solvated systems were neutralized by addition of suitable number of Na + ions to maintain electro-neutrality of the system. The Particle-Mesh-Ewald (PME) method [23] was used for calculation of long-range electrostatic interactions of all the systems. A 50,000-step energy minimization was performed with the steepest descent (SD) method at 300 K by applying periodic boundary conditions (PBC) in all directions. Berendsen thermostat temperature coupling and Parrinello-Rahman pressure coupling for each 500-ps run were used to keep all the systems in equilibrated environment 300 K and 1 bar, respectively. The leap-frog algorithm was used for integrating Newton's equation in molecular dynamics (MD) simulation of all the systems. All the bond lengths were constrained using the LINCS algorithm [24], and the time step was set to 0.002 ps. Finally, a 20-ns MD simulation was carried out for all eight systems. The simulation trajectories obtained after 20 ns MD simulations were analyzed using gmx_rms, gmx_rmsf, gmx_gyrate and gmx_hbond tools from the GROMACS 2018.3 package [20] and visualized using UCSF Chimera molecular visualizing software [25].

Conclusion
In the present study, we extensively analyzed the binding potential and mechanism of inhibition of phytochemical towards the SARS-CoV-2 M pro having profound inhibition towards the SARS-CoV-1 M pro . Molecular docking analysis was used to find the number of hydrogen bonds formed between SARS-CoV-2 M pro protein and different inhibitor molecules. Molecular docking analysis along with molecular dynamics simulation analysis showed 16 (Broussoflavan A), 22 (Dieckol), 31 (Hygromycin B), 45 (Sinigrin) and 46 (Theaflavin-3,3 0 -digallate) have high binding affinity and interaction to the conserved residues of the substrate-binding pocket of SARS-CoV-2 M pro . Thus, these phytochemicals offer preventive and complementary medicine in the fight against viruses due to their natural origin, safety and low cost compared to synthetic drugs. This study provides foundation for the identification of SARS-CoV-2 M pro inhibitors by structural manipulation of active phytochemicals to develop antiviral drugs for future.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.