Computational design for the development of natural molecules as compelling inhibitors against the target SARS-CoV-2: An in-silico attempt

A threat to the global human population has been established by the COVID-19 pandemic in 2020 and it is quite challenging to identify innovative medications in this epidemic. These are zoonotic and can potentially create massive outbreaks of illnesses that can result in morbidity and death. As a consequence, natural therapies for the anticipation and dealing of COVID-19 are widely acknowledged as a quick means to find successful therapeutic choices that can be found through in-silico drug screening tests. RNA-dependent RNA polymerase (RdRp), a vital precursor involved in the virus’s life cycle, is present in SARS-CoV-2. Blocking the formation of the RdRp–RNA complex inhibits viral replica and boosts the immune response of the host. In our present research, with the use of a SuperNatural Database, we started the high-throughput virtual screening method to recognize inhibitors aiming for SARS-CoV-2 RdRp. According to extra-precision docking data, two compounds, SN00293542 and SN00391842 had −14.79 and −14.65 kcal/mol docking scores, respectively. In addition, Prime molecular mechanics generalized bond surface area research has identified hydrophobic energy and Van der Waal energy footings as significant contributions towards total binding free energy. Additionally, a hundred nanosecond Molecular dynamics simulation of the SN00391842/7D4F complex was run to determine its dynamic behavior.


INTRODUCTION
The large genus of enclosed coronaviruses is a singlestranded positive-sense RNA virus (CoVs) (V'kovski et al., 2021) that can infect people and are zoonotic and have the probability to create massive outbursts of illnesses that can cause morbidity and mortality.The viral pneumonia outbreaks in Wuhan in 2019 and 2020 are instigated (Lakshmi and Suresh, 2020) by an anew discovered Coronavirus illness.It is mainly spread with droplets produced by infested individuals while coughing, sneezing, or exhaling.Simple colds to serious respiratory conditions including severe acute respiratory syndrome are among the symptoms of the recently identified coronavirus (Adithya et al., 2021) COVID-19.There are approximately 4 million fatalities and 175 million sick cases documented globally.With 44 million active belongings and 0.53 million fatalities (WHO, 2023), India is experiencing a daily increase in coronavirus infections.In the face of a viral pandemic that is scattering as wildfire and an inadequate treatment reserve, we needed to quickly find novel beneficial agents with clinical applications (Umakanthan et al., 2020).Natural products have been generally accepted as chemical entities for therapeutic application from their inception as a foundation and source of modern medications.Natural chemicals' pharmacological and therapeutic properties with low toxicity play a significant role in the creation of additional effective medications (Rajagopal et al., 2019).Herein current study, we want to high spot the unrealized potential of plant-based natural compounds as antiviral drug medications.The nucleocapsid (N), spike (S), membrane protein (M), and envelop proteins (E), as well as proteases, hemagglutinin esterases, helicases, and other proteins involved in the viral entrance, the existence, and reproduction, were used to build the viral structure (Kumar et al., 2021;Mielech et al., 2014).The nonexistence of proteins in human equivalents makes them interesting targets (Raj, 2021;Yadav et al., 2021).The SARS-CoV-2 core Rd-RNA polymerase (RdRp) is made up of the non-structural protein nsp12, and two accessary subunits (nsp7 and nsp8).The SARS-CoV-2 virus contains RdRp, an essential enzyme involved in the viral life cycle (Lei et al., 2018;Lim et al., 2000;Osipiuk et al., 2021).Blocking the formation of the RdRp complex inhibits viral replica and enhances the immune response of the host, so limiting its spread (Yin et al., 2021).We have attempted to design and assessed several molecules for their biological activities such as anticancer, anti-SARS CoV-2, and others as a component of our ongoing research and utilized in silico and wet lab approaches for the discovery of dynamic molecules (Kalirajan et al., 2017) for various biological activities (Kalirajan, 2020;Kalirajan et al., 2012aKalirajan et al., , 2012bKalirajan et al., , 2018Kalirajan et al., , 2019aKalirajan et al., , 2019bKalirajan et al., , 2020)).The current work attempts to find inhibitors against RdRp utilizing the high-throughput virtual screening (HTVS) procedure by utilizing the SuperNatural Database, which contains 4,00,000 natural chemicals (Rajagopal et al., 2021).The binding modes were identified using consecutive docking of HTVS, standard precision (SP), and Extra-Precision (XP) modes.To recognize natural drug-bound patterns in the RdRp active site, further molecular mechanics generalized bond surface area (MM-GBSA) and Molecular dynamics (MD) simulations (Ram et al., 2022).

HTVS and molecular docking
Molecular docking is a bioinformatic modeling approach used to predict the three-dimensional conformer   et al., 2020).Using Prime at pH 7.0, missing side chains and loops were added to produce protonation and tautomeric states for acidic and basic residues.Protein was optimized using the molecular force field OPLS4 (potential optimized for liquid simulations) and the crystallographic heavy atom root mean square deviation (RMSD) was set to 0.30 Å.A grid box (x = 52.5;y = 31.1;z = −0.61)was constructed at the active site around the cocrystal using a van der Waals scaling of 0.80 for the receptor, and 0.16 was used as the partial charge limit.We obtained threedimensional conformers of 4,00,000 natural chemicals (Dunkel et al., 2006) using the SuperNatural database.The computer-generated roadmap started by preparing ligands using Ligprep and using the prefilter option to remove excess.Using default settings for HTVS, SP, and XP modes, ligands were sequentially docked into RdRp catalytic pocket (7D4F.pdb).The ideal docked position was selected using hydrogen bond, glide score, and glide energy evaluations.

Total free energy calculation using prime MM-GBSA
The generalized-born surface area (GBSA) continuum solvent model and prime MM-GBSA methods were used to calculate the ligand-protein complex binding contributions of enthalpy and entropy-associated mechanisms (Naresh et al., 2020).The equation was used to estimate (in kcal/mol) molecular mechanics energy, and polar and nonpolar solvation contributions from components (Shivakumar et al., 2010).
ΔG bind = G complex -G protein -G ligand ΔG bind = binding free energy of complex Calculated.G complex = Binding free energy of complex minimized.G protein = Binding free energy of receptor.G ligand = Binding free energy of ligand unbound. ./ Journal of Applied Pharmaceutical Science 13 (Suppl 1); 2023: 034-040

MD simulation study
We employed the Schrödinger, LLC, New York, Desmond module to perform MD simulation to analyze the binding behavior of top-ranked molecules at the nuclear level and to comprehend the molecular interface investigation (Bowers et al., 2006;Jorgensen et al., 1983).The complex SN00391842/7D4F was solvated using the TIP4P water model (Essmann et al., 1995) with orthorhombic periodic boundaries and a 10 Å of buffer zone between protein atoms and box edges.The generated system was neutralized with the addition of 0.15 Molar NaCl counter ions.The OPLS4 force field settings were then used to minimize the system (Harder et al., 2016).With a 1e-09 tolerance, long-range electrostatic interactions were calculated using the Smooth Particle Mesh Ewald method.At a cut-off radius of 9.0 Å, the short-range Vander Waals and Coulomb interactions were estimated.In an isothermal-isobaric ensemble, 100 ns of MD simulations at 2 fs a time step were run at 300 Kelvin and 1 bar of pressure [Simulation of system based on constant number (N), and constant-temperature (T), but pressure (P) is regulated].The Martyna-Tobias-Klein barostat and Nose-Hoover thermostat chain thermostat techniques are merged at 100 and 200 ps (Martyna et al., 1992(Martyna et al., , 1994)).Reference system propagator algorithm multiple time-step algorithms were employed for bonded, non-bonded short-range, and long-range electrostatic forces, respectively.Data was gathered, and the resulting trajectories were analyzed for every 100 ps (Kalirajan, 2020;Kalirajan et al., 2017Kalirajan et al., , 2020)).

Molecular docking and total binding free energy calculation
Using a structural-based virtual screening technique from the Schrödinger suite, the RdRp (7D4F.pdb)enzyme was utilised to screen a library of 400,000 molecules from the SuperNatural Database.Initial prefilters were used in the virtual screening method to exclude ligands containing reactive functional groups according to Lipinski's rule.Three accuracy stages of the sequential docking methodology (HTVS, SP, and XP docking) were carried out while maintaining default constraints.A final 21 compounds were identified after visually analyzing the bound postures and hydrogen bond establishment for the highranked hits during XP mode.With the help of the virtual screening protocol, several different scaffold topologies were discovered, such as 2-phenyl chromene rings allied with sugar moieties, pyrazinyl hexanoic acid, cyclohexyl dihydrogen phosphates allied with sugars, tetrahydroxy hexanal allied with pyran, benzyloxy benzoates allied with pyranoacetates and dicarbamimidamido pyran trihydroxy benzoates, etc.In the present research work, the docking protocol was validated by performing the re-dock with the co-crystal structure in the catalytic pocket of 7D4F and from the re-docked results, the RMSD for the superimposition of native and docked conformer revealed as 3.7 Å (Fig. 1).Thus, the docking protocol was considered good enough for replicating the docking results similar to the co-crystal structure and can consequently be applied for further molecular docking analysis.Tables 1  and 2 provided the glide scores and MM-GBSA energy scores.Selected first top 5 XP docked pose hits showed 5-10 hydrogen bonding, the most observed bonds by all the phytoconstituents (Table 3).Table 1 shows that the glide score ranges from −14.79  2. The hits SN00293542 and SN00391842 had the highest glide scores of −14.79 and −14.65 kcal/mol.Conferring to the binding free energy calculation MMGB-SA approach, compounds SN00299979 and SN00391842 had the highest binding affinity with −164.84 and −155.86 kcal/mol respectively.Figure 2 shows the 2D interactions of the top 10 hits.Compound SN00391842 had taken for further study by considering the both docking score and binding affinity.

MD simulation
Both H3U(cocrystal)/7D4F and the docked pose SN00391842/7D4F complexes underwent 100 nanoseconds of MD simulation.During simulation, from Figure 3A and B, it was observed that the protein Cα atoms were for both H3U/7D4F and the docked pose SN00391842/7D4F stabilized throughout the simulation with a minimum RMSD of 2.3 to 3.2 Å 2.1 to 2.7 Å respectively and in Figure 3A for the ligand during the initial 42 ns, minimal fluctuations were observed and after that stabilized up to 80 ns and again fluctuations were observed with RMSD 5.6 to 7.2 Å whereas in Figure 3B for the ligand during the initial 15 ns, minimal fluctuations were observed and after that stabilized up to 75 ns and again fluctuations were observed with RMSD 4.7 to 7.2 Å.From Figure 4A and B, we can observe that fluctuations with root mean square fluctuations (RMSF) of 0.6 to 4.2 Å and 0.5 to 4.7 Å respectively maintained throughout the simulation but whenever the ligand atoms bound to the particular amino acids there, we can notice the minimum fluctuations i.e. from 500 to 580, 700 to 750, 800 to 900 amino acids with 0.8 to 1.4, 0.6 to 1.3 and 0.7 to 1.3 Å respectively.In Figures 5A and B, most amino acids in the catalytic pocket have maintained their interaction as hydrogen bonds and water bridges, and most of them have demonstrated multiple interactions.Figure 6A and B, Asn496, Asn497, Arg569, Gln573, Lys577, Tyr689, Gly 590, Thr591, Gly683, Tyr689, and Cys813 are these particular   ./ Journal of Applied Pharmaceutical Science 13 (Suppl 1); 2023: 034-040 amino acids that maintained continuous and multiple contacts with the ligand atoms throughout the simulation.From the deposited data, we can notice that the SN00391842/7D4F complex attained more stability while performing the triplicate with a different random seed number.With this, we may suggest that these natural chemical entities would act as scaffolds for the development of new drugs in the future for the management of COVID-19.

CONCLUSION
There have been approximately reported 174 million cases of infection and 3.75 million fatalities worldwide.There are currently 4 million coronavirus cases active in India, and there have been 0.5 million fatalities.The current investigation aims to find inhibitors that target the RdRp enzyme, which is essential for viral replication and growth.Using the SuperNatural database of 400,000 natural chemicals as part of an HTVS process, we used HTVS, SP, and XP docking modes.Then sequential docking technology was introduced.The top 28 hit compounds were found after docking optimization.According to the XP docking results 2 molecules, SN00293542 and SN00391842 had glide scores of −14.79 and −14.65 kcal/mol, respectively.According to the MM-GBSA investigations, VanderWaal energy (ΔVdW) ranges from −36.01 to −64.25 kcal/mol while the hydrophobic energy terms (ΔLipo) range from −6.46 to −27.43 kcal/mol.According to 100 ns MD simulations, the SN00391842/7D4F complex was maintained in the catalytic pocket by hydrogen-bonding, hydrophobic, and water-bonding interactions.MD simulations for the trajectory SN00391842/7D4F were validated by performing the dynamic simulations in triplicate with random seed and the data was deposited.To further the development of possible SARS-CoV-2 inhibitors, in silico studies that are now underway might be beneficial.

.no Compound code Glide score Glide model Glide evdw Glide_ecoul Glide_energy
(Yin et al., 2021)13)nt possible conformers that are ranked and grouped using a scoring function in the software.Docking simulations predict optimized docked conformers based on the system's total energy(Dar and Mir,  2017).Using the protein preparation wizard of Schrödinger Suite LLC,(Sastry et al., 2013)the three-dimensional X-ray crystal structure of RdRp with co-crystal suramin (7D4F.pdb)(Yinetal., 2021), which was retrieved from the research collaboratory for structural bioinformatics, was further created.Removed crystalized water and modified bond ordering with hydrogen additions, the protein was produced (Sukumaran / Journal of Applied Pharmaceutical Science 13 (Suppl 1); 2023: 034-040 of any complex

Table 3 .
The number of hydrogen bonds and intermingling amino acid residues for the top 5 hits in the catalytic pocket of RdRp enzyme (7D4F.pdb).