Инсило-исследования для понимания взаимодействий флавонового ингибитора с nsp12-РНК-зависимой РНК-полимеразой SARS-CoV-2 и его гомологами.

Introduction

COVID19 has been a major global health concern for the last few years. While the pandemic in 2020 caused mortality and morbidity, it continues to be an epidemic that needs to addressed. Though there have been multiple modalities of treatment for COVID19, each approach has been riddled with lacunae [ [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] ]. From a Biophysical perspective, (1) these nucleoside analogue inhibitors are hindered by the proof reading activity which can excise the incorporated inhibitors conferring resistance to SARS-CoV-2 [ 15 , 16 ]; (2) Cyano group substituted at the 1’ position will sterically clash with side chain of Ser861 preventing translocation of RNA, and a possible mutation at this position may allow processing of RNA synthesis and thereby survival of this virus [ 17 ]. Clearly, the clinical compliance of the patients and biophysical pitfalls of nucleoside inhibitors emphasize the need to develop more potent, non-covalently binding non-nucleoside inhibitor molecules.

The replicase gene encoding the non-structural proteins ( nsp s) occupies two-thirds of the genome, about 20 kb, as opposed to the structural and accessory proteins. The replicase polyproteins of the virus comprises of four domains that interact to form RNA Dependent RNA Polymerase (RdRp), that functions in (i) production of negative strand RNA (–RNA); (ii) new genome molecules and, (iii) generation of sub-genomic (sg) messenger RNAs (mRNAs) [ [18] , [19] , [20] ]. Owing to these important attributes, along with the fact that it lacks a human homologue makes the SARS-CoV-2 RdRp an important and attractive target for the rational design of potent inhibitory molecules [ [21] , [22] , [23] , [24] ]. Flavonoid molecules in addition to their diverse structure and drug-relevant features unsurpassable by any synthetic compound, mimic nucleosides making them ideal for a physio-chemical complementarity at the NTP binding site of nsp12 -RdRp [ [25] , [26] , [27] , [28] , [29] ].

A detailed structural characterization of drug targets is known to be useful in the designing of potent inhibitory molecules [ 30 ]. Our group has been actively involved in structure based drug design against targets in microbes causing human infections [ 31 ]. In this study, we have conducted comprehensive structural analysis and inhibition studies of the nsp12 -RdRp of SARS-CoV-2 using computational methods, exploring natural flavonoids as potential anti-viral agents against SARS-CoV-2 and its homologs.

Methodology

Sequence analysis

Primary structure of RdRp from SARS CoV-2 (Accession no.: NC_045512.2 ) was obtained from NCBI [ 32 ]. Homologous sequences were searched using BLAST [ 33 ]. Sequences of RdRp from SARS-CoV-1 ( NC_004718.3 ) , MERS-CoV ( NC_038294.1 ) and Bat-CoV ( NC_048212.1 ) were selected from NCBI and multiple sequence alignment was done using CLUSTAL-W [ 34 ].

Modelling and validation of the RdRp enzymes and protein preparation

Cryo-EM structure of Replication Transcription Complex of SARS-CoV-2 bound to ATP, in pre-catalytic state ( PDB Id: 7UO7 ) was obtained from ‘Research Collaboratory for Structural Bioinformatics-Protein Data Bank’ (RCSB PDB) [ 35 ]. Structure of RdRp-nsp-12 subunit with primer-template RNA, magnesium and zinc ions was energy minimised. This structure with the mentioned ligands was then validated using PROCHECK, VERIFY 3D and ERRAT and considered for further experiments [ [36] , [37] , [38] , [39] ]. Homology modelling was performed on RdRp sequences of SARS-CoV-1, MERS-CoV , and Bat-CoV obtained from the NCBI database to create their respective models based on experimental structures of RdRp from SARS CoV-2 and SARS CoV-1 available on RCSB PDB using software tools such as Discovery Studio, Geno 3D, SWISS-MODEL and PHYRE [ [40] , [41] , [42] , [43] ]. The conformational correctness and reliability of the generated models were analysed using the same validation tools mentioned earlier. Validated structure of RdRp SARS-CoV-2 with ATP was prepared by adding hydrogen atoms, assigning bond borders and energy minimised using Protein Preparation Wizard from Schrodinger suite [ 44 ]. Also, RNA, two zinc ions and one magnesium ion were added to the generated RdRp models of SARS-CoV-1, MERS-CoV , and Bat-CoV from Discovery Studio and structures were prepared using same methodology.

Ligand library preparation

ATP and four natural molecules predicted to inhibit RdRp from SARS-CoV-2 were chosen as scaffold templates [ [45] , [46] , [47] ]. A structural similarity search with a Tanimoto coefficient of 0.5 was performed at the COCONUT Natural Products database to shortlist 100 molecules for each of the submitted scaffolds [ 48 ]. These molecules were taken up for ligand preparation by generating different stereochemistries, tautomers, and ionization states using LigPrep module of Schrödinger suite [ 49 ].

Virtual screening workflow

Prepared structure of RdRp from SARS CoV-2 was considered as a receptor with ATP as the centroid for grid generation. This was used to identify possible inhibitors from the curated library using virtual screening workflow program using Glide software from the Schrodinger suite [ 50 , 51 ]. This comprised of an initial library screening using High Throughput Virtual Screening (HTVS). Top 10 % of ranked compounds from this screening were then re-screened using Standard Precision (SP) mode, and the subsequent top 10 % of compounds were further analysed using the Extra Precision (XP) mode. Based on the docking score, top ranked compound amongst these was considered as a potential inhibitor of the enzyme. Dissociation constant (Kd) was estimated using the equation ΔG = -nRT.lnKd, where ΔG stands for change in free energy of binding, R stands for gas constant in calorie/mol/Kelvin, T stands for temperature in Kelvin, ln stands for natural log, and n stands for number of moles (calculated for 1 mol).

Validation of docking methodology

The co-bound ligand (ATP) was extracted from the prepared Cryo-EM structure (PDB Id: 7UO7 ) while preserving the original co-ordinates. Using the same methodology, ATP was redocked into the binding site of the enzyme. Docked complex was superimposed on the native complex and RMSD was evaluated.

Preparation of RdRp-Inhibitor model complexes

Co-ordinates of the inhibitor from the complex of RdRp of SARS-CoV-2 was taken and added to RdRp model structures of SARS-CoV-1, MERS-CoV , and Bat-CoV using PyMOL [ 52 ]. Modelled RdRp-inhibitor complexes from SARS-CoV-1, MERS-CoV and Bat-CoV RdRp were pre-processed, energy minimised, and taken for further analysis.

Molecular dynamics

The docked RdRp-Inhibitor complex of SARS-CoV-2 and the RdRp-Inhibitor complexes of homologs obtained through co-ordinate replacement were subjected to molecular dynamics simulation to understand the stability and dynamics of the binding interactions. Simulation was performed using the Desmond suite of programs using the OPLS3 force field parameters [ 53 , 54 ]. RdRp-Inhibitor poses were solvated using the TIP3P explicit water model in an orthorhombic box. Net electrostatic charge on solvated models were neutralized by adding Na + and Cl − ions at a concentrations of 0.15 mol/L. Systems were then subjected to molecular dynamics simulation for 500 ns at a temperature of 300K and a pressure of 1 atm, using the Nose-Hoover thermostat and Martyna-Tobias-Klein barostat [ 55 , 56 ]. The simulation was carried out with a time step of 2 fs (fs), incorporating LINCS harmonic constraints, while the motion was integrated using the RESPA integrator [ 57 , 58 ]. The Simulation Interaction Diagrams program from Schrodinger was utilized for analysis of the trajectories post MD [ 53 ].

Principal Component Analysis (PCA) and free energy landscaping (FEL)

Principal Component Analysis (PCA) of the RdRp-Inhibitor complex trajectories was performed using the MDTraj program [ 59 ]. A covariance matrix was constructed from the Cartesian coordinates of the C-α atoms of the protein and the heavy atoms of the ligands. Eigenvalues and eigenvectors of this covariance matrix were computed, enabling the extraction of the ten principal components associated with the eigenvectors that had the highest eigenvalues [ 60 ]. A 2D histogram was generated based on Principal Component 1 (PC1) and Principal Component 2 (PC2). The free energy landscape was created from the histogram using the Boltzmann equation: ΔE = −k b Tln(n i /n max ); where n i represents the number of conformations in the ith bin, n max indicates the number of conformations in the most populated bin, ΔE denotes the energy difference between these conformations, k b is the Boltzmann constant, and T is the temperature in Kelvin.

Docking of top ranked compound to ATP-binding human proteins

Crystal structures of DNA ligase (PDB ID: 6P0D ), PI3K (PDB ID: 5DXU ), and hexokinase (PDB ID: 1QHA ) were retrieved from the RCSB. The ligands were removed from the crystal structures, the proteins were prepared and the inhibitor of RdRp from SARS-CoV-2 was docked at the ATP binding sites using the same methodology mentioned earlier. The docked complexes were then analysed to calculate the Dissociation constant (Kd).

Results and discussion

Sequence analysis of RdRp from SARS-CoV-2 and its homologs

Primary sequences of the RdRp enzymes from SARS-CoV-2, SARS-CoV-1, MERS-CoV and Bat-CoV show at least 80 % similarity. Understandably SARS-CoV-1 shares a closer amount of identity as compared to the MERS-CoV and Bat-CoV sequences ( Fig. 1 ). While key catalytic residues Asp 618 and Asp 623 are conserved on the motif DXXXXD, Ser 759, Asp 760 and Asp 761 are conserved on the motif SDD, on all the four sequences. Though the length of the primary sequences is approximately the same the number of cysteines vary, with MERS-CoV having a maximum of 32 and Bat-CoV having a minimum of 28. Interestingly only 19 of these are in homologous positions. The enzymes could therefore be stabilized by the di-sulphide bond across these cysteines. There are residue variations on the RdRp of SARS-CoV-2 as compared to the residues conserved on the other three sequences. Notable variations at specific positions, such as Phe77, Val233, Thr252, Lys281, Thr739, Asn743, Phe766, Thr769, and Ser784, may impact the overall stability of the 3D structures, which will be further examined in the structural analysis.

Modelling of the homologs and validation of RdRp enzymes

Structure of SARS-CoV-1 RdRp (PDB id: 6NUR ), had 161 residues less and so we modelled the complete structure and generated two models. Since MERS-CoV and Bat-CoV did not have any experimental RdRp structure as of date, they were modelled as well to obtain complete structure and a total of 17 models were generated for each. These structures were evaluated for stereochemical quality and statistical potentials ( Table S1 ). The top models for RdRp from SARS-CoV-1, MERS-CoV, and Bat-CoV had 99.5 % (926 residues), 99 % (930 residues) and 99.4 % (926 residues) in allowed regions of the Ramachandran plot, respectively. The ERRAT and Verify 3D also shows the accuracy of these models. The energy minimised Cryo-EM structure of SARS-CoV-2 RdRp (PDB Id: 7UO7 ) had 99.6 % (926 residues) in the allowed regions of Ramachandran plot, thereby enabling to benchmark the other three generated models.

Structural analysis of RdRp enzymes

The superimposition of the four RdRp structures showed that RMSD of Cα chains of SARS-CoV-1 with SARS-CoV-2 is 1.4 Å, with MERS-CoV is 0.9 Å, and with Bat-CoV is 1.4 Å ( Fig. 2 A). This reveals that there are no major variations in the secondary structural features and the minor differences noted are in the flexible loop regions connecting the helices and sheets. Comparative analysis at certain residue positions in RdRp of SARS-CoV-2 revealed that residues at certain conformations were better suited and made favourable interactions due their inherent biophysical properties and individually contributed to the overall compactness of the protein. Three representative examples of Thr252, Phe77, and Phe766 are provided here for the sake of comparison ( Fig. 3 ).

RdRp from the four viruses SARS-CoV-2, SARS-CoV-1, MERS-CoV , and Bat-CoV have a multi-domain globular structure comprising of 34–36 alpha helices and 21–23 β-sheets. Conserved catalytic motifs in the RdRp is responsible for the orchestrated movement of the template-primer RNA and NTP into the active site for catalysis ( Fig. 2 A). From a functional perspective, the catalytic reaction involves transfer of the alpha phosphate of the incoming nucleotide to the deprotonated 3′ OH group of the terminal nucleotide in the product strand incorporating it and pyrophosphate leaving the active site. This is enabled by the RdRp enzymes that have well defined channels for RNA entry, NTP entry and RNA exit [ [61] , [62] , [63] ]. Structural elements that are crucial for the incorporation of NTP to the elongating RNA are: (1) Asp 618, Tyr 619, Asp 760, Asp 761 that make coordination bonds with magnesium ions along with alpha phosphate oxygen of incoming nucleotide and 3′OH of the terminal nucleotide; (2) Recognition of incoming nucleotide by Lys 621, Cys 622, Asp 623 and Ser 682; (3) Basic residues Lys 545, Lys 551, Arg 553 and Arg 555 for positioning of negatively charged phosphates of NTP and RNA; (4) Arg 555 forms a pi-stacking with the nucleobase aligning it for catalysis as well as base pairing with the corresponding nucleotide in the template strand; (5) Basic residue Lys 545 forms a hydrogen bond with nucleobase, while Lys 551 interacts with the gamma phosphate of the NTP; (6) Residues Cys 813 and Ser 814 keeps the primer stand at the active site [ 21 ]. The above-mentioned structural features are presented in Fig. 2 B.

Screening of inhibitors at NTP binding site of RdRp from SARS-CoV-2

Cryo-EM structure of SARS-CoV-2 RdRp in a pre-catalytic conformation with ATP at the NTP binding site (PDB Id: 7UO7 ) was considered for modelling studies. Structure of RdRp of SARS-CoV-2 enables the incorporation of NTP to the product RNA chain. NTP binding site, where the incoming nucleotide is recognized and positioned for catalysis, makes it an ideal site on the RdRp target for inhibitor screening. This NTP binding site of SARS-CoV-2 RdRp was screened against natural molecule library. In the Virtual Screening Workflow, High-Throughput Virtual Screening identified 663 molecules. These were further refined using Standard Precision docking that yielded 66 top hits. Extra Precision docking was performed narrowing the selection to top 6 molecules. From among these Coconut ID: CNP0127177.0 which had the best docking score was chosen for further experiments. It occupies the NTP binding site in the vicinity of the elongating RNA and Magnesium ion ( Fig. 4 A). From a chemical perspective, this molecule is 3,3′,4′,5,5’,8-Hexahydroxyflavone, a flavonoid with molecular weight of 318.2Da, present in coffee. For the sake of convenience, this molecule will henceforth be referred to as HHF318 in the rest of the manuscript. Structurally, it comprises of two aromatic rings and one heterocyclic pyrone ring with six hydroxyl groups and one carbonyl group ( Fig. 4 B). As per the flavonoid nomenclature, the benzene rings are named Ring A and Ring B, and the heterocyclic ring is named Ring C [ 64 ]. Based on binding energies (ΔG), Dissociation constant (Kd) was estimated to be 1.8 nM. Interestingly, it is seen to be much more potent than the currently used nucleoside analogue inhibitors such as Remdesivir (5236 nM) and Favipiravir (159 nM). Also, the potency is better than the ATP, an NTP which has a Dissociation constant of 170 nM. Complexes of RdRp from SARS-CoV-1, MERS-CoV , and Bat-CoV with HHF318 were generated. These four RdRp complexes with HHF318 were subjected to 500ns of Molecular Dynamics simulation and stable conformation of the complexes were obtained. Trajectory analysis of MD simulation indicates that system quickly reached at target temperature of 300K and pressure of 1 bar and remained uniform throughout the equilibration stage ( Fig. 5 A–C). This indicated that the solvent and the ions had attained the right orientation around RdRp enzymes during period of equilibration. Under an NPT ensemble with constant number of atoms, pressure, and temperature, RdRp with the inhibitor molecule HHF318 closely replicates an experimental condition. Total energies were constant for all the four protein complexes indicating stability of the systems ( Fig. 5 , D). The RMSD plot was stable with average deviation of less than 2 Å from the mean indicating that the system of protein and solvent had reached a stable state with protein target in an optimum conformation ( Fig. 5 E and F). Protein RMSD plots for the complexes show that the backbone of the proteins from SARS CoV-2 and MERS-CoV converged around 200ns, while that of SARS-CoV-1 and Bat-CoV converged around 300ns. RMSD of HHF318 in the complexes of SARS-CoV-2 and SARS-CoV-1 remained stable throughout the simulations after convergence while that of MERS-CoV and Bat-CoV varied persistently indicating that HHF318 was not stably bound to the complexes. Values of potential energy indicates that the systems are stabilized and equipped for the molecular dynamics phase ( Fig. 5 , G). The free energy landscapes (FEL) of the first two principal components of the MD trajectory frames for the four complexes are shown in Fig. 5 H–K, respectively. The FEL of SARS-CoV-2 has a single well-defined deep blue basin indicating a stable conformational state with low energy compared to the other three enzyme complexes. Structures from the energy minima of each FEL, indicated by dark blue region were used for analysis of the interaction of HHF318 with each of the four RdRp enzymes. The Cryo EM structure of SARS-CoV-2 with the ATP ( PDB Id: 7UO7 ) was used for validating the modelling methodology. RMSD of docked complex of RdRp: ATP with the Cryo-EM structure is 0.4 Å ( Fig. 6 ). This validates the docking methodology used in this study.

Interactions of HHF318 with RdRp

HHF318 binds to RdRp at the NTP binding site which has the 3’ strand of elongating RNA and co-factor magnesium ion. HHF318 forms an array of interactions with residues of RdRp from SARS-CoV-2 . The details of the interactions are: Two hydroxyl groups from Ring B are involved in magnesium coordination along with residues Asp 618, Tyr 619, Asp 760. Hydroxyl group on Ring B forms a hydrogen bond with oxygen of phosphate group from the primer strand. Hydroxyl group in Ring A forms hydrogen bond with Lys 545. Carbonyl group on Ring C forms hydrogen bond with Asp 623, and hydroxyl group on Ring A makes hydrogen bond with the backbone of Gly 683. In addition, hydrophobic interactions exist between Carbon atoms of Ring A with main chain Carbon of Ser 682, and side chain of Val 557. Arg 555 makes cationic-π interaction with Ring A. Ring A additionally makes stacking interaction with nucleobase of G35 ( Fig. 7 A). The collated effect of these interactions is responsible for the strong binding affinity of HHF318 against RdRp of SARS-CoV-2 . In the complex of SARS-CoV-1 RdRp with HHF318, the major interactions include coordination of magnesium along with Asp 618 and Asp 760. Hydrogen bonds with Ser 759, Pro 620, and U-20 on the template strand. Hydrophobic interactions are made with Cys 622, Asp 623, Arg 555, Thr 687, and ribose sugar of G35 ( Fig. 7 B). Complex of MERS-CoV RdRp with HHF318 shows magnesium coordination along with Asp618, Asp760, and Asp761. Hydrogen bonded interactions exist between ligand and Asp 761 and Arg 555. In addition, hydrophobic interactions are with Ser549 and nucleobase of G35 ( Fig. 7 C). Lastly, Complex of Bat-CoV RdRp with HHF318 shows magnesium coordination along with Asp618 and Asp761. Hydrogen bonded interactions are seen with Asp760, Arg 553, Ser 682, and Asp 618. Hydrophobic interactions are with Lys798, Arg553, and G35 ( Fig. 7 D).

Comparison of superimposed structures of RdRp of SARS-CoV-2 with ATP and SARS-CoV-2 with HHF318

For a better understanding of the inhibition of SARS-CoV-2 RdRp by HHF318, the structure complex was superimposed on the structure complex of SARS-CoV-2 RdRp enzyme with ATP at the NTP binding site. It is seen that the main chain RMSD of the two complexes was 2 Å. Also, the two molecules in the two complexes too were superimposed on one another, adjacent to the magnesium ion for coordination ( Fig. 8 ). The other interesting findings are: (1) Benzene (Ring A) with two hydroxyl group of HHF318 is within 3 Å distance from the adenine base of ATP; (2) Both molecules, HHF318 and ATP are positioned adjacent to the guanine base of the RNA in the primer strand; (3) heterocyclic pyrone (Ring C) of the HHF318 is superimposed on the ribose sugar of ATP, and (4) Benzene (Ring B) with three hydroxyl group comes to lie adjacent to the hydrophilic phosphate group ( Fig. 8 ). This clearly establishes that the physio-chemical features of HHF318 makes it a potential competitive inhibitor of nucleotide binding at NTP binding site of RdRp.

Physico-chemical and pharmacokinetic properties of HHF318

Physico-chemical and pharmacokinetic properties of a ligand is the main stay for its development into a drug molecule. Molecule HHF318 was evaluated for the same and the estimated Physico-chemical and pharmacokinetic properties is presented in Table 1 . Some of the notable aspects are: (1) its highly polar nature makes it water soluble; (2) compound is moderately lipid soluble explaining the low GI absorption and blood brain barrier non-permeability; (3) High aromatic heavy atom count could possibly help in forming strong interactions with biological target, but and also increase inhibition of CYP1A2, CYP2D6, and CYP3A4 enzymes in the liver that could cause drug-drug interactions; (4) compound being a non-P-glycoprotein substrate could improve its bioavailability; and (5) The Lipinski rule compliance, moderate bioavailability score, no violations in lead-likeness and relative easiness in synthesizing makes HHF318 an ideal candidate for further optimisation in drug discovery against COVID19.

Interactions of HHF318 with secondary drug targets in the human body

In order to analyse the specificity of HHF318 binding to RdRp enzyme, the molecule was docked with certain ATP binding human proteins like DNA Ligase, PI3K and Hexokinase. The molecule binds at the ATP binding site of the proteins ( Fig. 9 ). The theoretical Kd of binding of HHF318molecule with these enzymes are 6.7 μM, 15 nM and 4 μM, respectively. These values are higher than 1.8 nM which we have estimated for the same molecule against RdRp from SARS-CoV-2 . This is indicative of the fact that the molecule is not only potent but also very specific against the drug target of RdRp from SARS-CoV-2 .

Limitation of the study

This is a computation based structural bioinformatic study. Crystallization experiments, enzyme inhibition assays and ex-vivo studies would help to validate the credentials of the identified molecule for it to be translated into therapeutics for COVID 19 disease.

Conclusion

RNA Dependent RNA polymerase enzymes across the Coronaviridae family have similar sequence and three-dimensional fold. However, variations in analogous residue positions play a role in the compactness thereby imparting greater stability of RdRp enzyme from SARS-CoV-2 compared to its homologs. Structure Based Drug Screening helped to identify the flavonoid molecule with Coconut ID: CNP0127177.0 (HHF318) that has binding affinity in low nano-molar range against RdRp from SARS-CoV-2. The strong affinity stems from the cumulative effect of an array of interactions including cationic-π, and π-stacking with protein and RNA, respectively. This molecule engages with residues that are crucial for functions such as magnesium coordination, nucleotide recognition and positioning, and catalysis. Additionally, it has a lower potency for human ATP binding proteins, making it an ideal drug candidate for COVID19.

CRediT authorship contribution statement

Shamiya Anwar Kizhakkiniyakath: Methodology. Tejaswini Choudhury: Methodology. Madhan Vishal Rajan: Methodology. Sagar Rathee: Methodology. Basant Meena: Methodology. Gururao Hariprasad: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Data availability statement

The models are available in ModelArchive (modelarchive.org) with the accession codes: RdRp from SARS-CoV-2 with molecule HHF318 (ma-6pwl6); RdRp from SARS-CoV-1 with molecule HHF318 (ma-tknqc), RdRp from MERS-CoV with molecule HHF318 (ma-z5jqd), RdRp from Bat-CoV with molecule HHF318 (ma-gcwge).

Declaration of competing interest

Authors report no conflict of interest in this work.

Footnotes

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrep.2025.101975 .

Supplementary data

The following is the Supplementary data to this article:

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Associated Data

Supplementary Materials

Multimedia component 1

Data Availability Statement

The models are available in ModelArchive (modelarchive.org) with the accession codes: RdRp from SARS-CoV-2 with molecule HHF318 (ma-6pwl6); RdRp from SARS-CoV-1 with molecule HHF318 (ma-tknqc), RdRp from MERS-CoV with molecule HHF318 (ma-z5jqd), RdRp from Bat-CoV with molecule HHF318 (ma-gcwge).