Comparative inhibition analysis of wild type and G671S catalytic site mutant of the SARS-CoV-2 RNA-dependent RNA polymerase


 Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) has wrecked a global havoc, after its emergence in Wuhan, Hubei province of China. It is known to have a distressing effect on the respiratory tract and has a high mortality rate. Till to date (25 Dec 2021), total number of cases (279,362,428) have been reported across the globe. Variable mutations have been reported on various geographical levels. In this study, we have analyzed G671S mutation of RdRp of the SARS-CoV-2, which has been reported in various strains globally, but importantly delta variant cases of Pakistan and has a serious impact on the protein structure. To study the conformational impact, we screened a Tibetan medicinal compound/Sowa Rigpa library against RdRp and compared the best docked compound (Kaempferol 3-O-gentiobioside) to the wild type and mutant RdRp against Remdesivir. A short simulation was used to validate the findings. Both remdesivir and our screened compound showed better inhibition for mutant, compared to the wild type RdRp.


Introduction
In December, 2019, an unknown viral outbreak, with pneumonia-like symptoms was reported in China (Wuhan, Hubei Province). It caused severe respiratory distress and was fatal in many cases. This deadly zoonotic virus led the World Health Organization (WHO) to declare a pandemic and lockdown was implemented in various parts of the world to alleviate spread (Rangayasami et al., 2021). Since the pandemic began, millions of cases have been reported, all over the world. Demographic studies estimated that approximately 100,871 people have been affected in China, 52,986,307 in America, and 1,293,081 in Pakistan till todate (25 December, 2021) [1].
SARS-CoV-2 is an RNA based viral strain, belonging to the subfamily ortho-coronavirinae, family coronaviridae and order Nidovirales (Fan et al., 2019). Its genomic sequence has 79.6% similarity with SARS-CoV and is 96% homologous to the bat corona virus RaTG13 (Zhou et al., 2020). SARS-CoV-2 has mutated over time span and altered its genomic structure. This variation impacts its spread and severity in different regions of the world (Dong et al., 2020). It follows lysogenic cycle based on RNA dependent RNA polymerase (RdRp). RdRp is composed of ORF1a, ORF1b, nsp7, nsp8 and nsp12 (a catalytic subunit), involved in virion replication and transcription . Mutation based irregularity or dysfunction in viral machinery may lead to strengthen the e cacy of infection. Apart from phylogenetic and other sequence analysis, present study aimed to investigate whether this mutation in uenced the binding of drugs to the mutant RdRp, compared to the wild-type control. In this study, bioinformatics methods like molecular docking, dynamics simulation and MM/PBSA energy analysis were used to analyze the impact.

Energy changes
Electrostatic Energy (ELE), Van der Waals contribution (VDW), Total Gas Phase energy (GAS), Non-polar and Polar contributions to solvation (PBSOL/GBSOL), binding free energy ΔPB/ΔGB, PBTOT/GBTOT, and entropy (TS) was also calculated (kcal/mol). The binding free energy (ΔGbind) was calculated based on binding energy (ΔEbind), solvation entropy (-TΔSsol) and conformational entropy (-TΔSconf). The enthalpy and the entropy were calculated by the MM/PB(GB)SA method (Wang et al., 2018) and empirical method, respectively. In order to gain a detailed pro le of protein-ligand interactions and the contribution of each residue, the binding energy was decomposed per residue by using the decomposition module of the AMBER16 program.

Dynamics simulation
Top-scoring docking compounds were subjected to dynamics simulation for nding enhanced complex stability and identify interactions among them. High performance molecular dynamic simulation software Desmond from Schrodinger LLC was used to analyze dynamics, with OPLS3e force-eld to resolve geometries. This was followed by energy minimization. The parameters in Desmond were: water solvation model, TIP3P; boundary conditions: orthorhombic box shape; size of box with a buffer donor/acceptor (H-bond distances distance =2.5Å each). This was neutralized by adding Na + ions and 0.15 M salt with Na + /Cl ions. The runtime for simulation was 15 ns for each complex, keeping the recording interval of 50 ps trajectory and an energy equivalent to 1.2. Ensemble class was NPT with a temperature of 300K and a pressure of 1.01325 bar. Once the simulation was done, then interaction analysis was initiated and manually interpreted.

Molecular docking and energy analysis
In SARS-CoV-2, RNA dependent RNA polymerase (RdRp) enzyme plays a pivotal role in genetic procreation by replicating the strain and then transcribe it into a protein sequence . It is the topographic (fundamental surface) point for antiviral drug to stop or slow down the viral replication via remdesivir binding . We studied its binding with reference to Sowa Rigpa and mutant G671S RdRp. Ligand-receptor complex docking scores for RdRpremdesivir, RdRp-3-O-gentiobioside, G671S-remdesivir and G671S-kaempferol 3-O-gentiobioside were -6.9, -9.1, -7.3, and -7.3, respectively. These values show that RdRp-kaempferol 3-O-gentiobioside interaction was more stable than RdRpremdesivir. It is pertinent to mention that Kaempferol 3-O-gentiobioside occurs in Meconopsis horridula. Its anti-tumor activity has previously been noted (Fan et al., 2015).
Mutant values were even lower for RdRp-ligand binding, hinting at a better drug binding with mutant than the native protein. N-terminus and H-bond interactions of wild type and mutant RdRp were considerably altered ( Fig. 2A, C)

Dynamics simulation
G671S RdRp-remdesivir shows a more stable interaction as RMSD of native RdRp shows increased value (>2.8 Å) after 8 ns, whereas it stays within pan of 2.5 Å for G671S RdRp, even after 14 ns (Fig. 3). Similarly for native RdRp binding with kaempferol 3-O-gentiobioside, RMSD values were >3 Å, after 10 ns, whereas they were less than or round about 2.8 Å for G671S RdRp-kaempferol 3-O-gentiobioside complex. This depicts that mutant protein was better inhibited by studied compounds. Hydrogen bonding plays a crucial role in protein-ligand interaction. A varied pattern of hydrogen bonding, hydrophobic, and ionic interactions was also observed for studied complexes. Mutant protein made no ionic interactions, while hydrogen bonds were also decreased in mutant-ligand complex formation (Fig. 4).
We conclude that Kaempferol 3-O-gentiobioside has equal potential of inhibiting G671S mutant RdRp as remdesivir, while mutant has high a nity towards these inhibitors when compared to the wild type.

Declarations
Competing interests: The authors declare no competing interests.