Exploiting the antiviral properties of selected phytochemicals from leaf extracts of Spondias mombin (Linn): A computational repurposing approach toward the discovery of potential SARS-CoV-2 inhibitors.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a pneumonia-like disease with a pattern of acute respiratory symptoms, remains a major public health concern that is causing tremendous human suffering. There is no approved drug for the direct treatment of the disease, although several vaccines have been approved for use. Exploring medicinal plants and their phytochemicals has emerged as possible therapeutic alternatives since they are affordable and present minimal toxicity effects. This study sought to investigate the potential of the phytochemical compounds isolated from ethanolic leaf extract of Spondias mombin as potential therapeutic agents against SARS-CoV-2. From a select list of bioactive compounds extracted from the leaf of Spondias mombin with known antiviral properties, we identi�ed Geraniin and 2-O- Caffeoyl-(+)-allohydroxycitric acid as potential SARS-CoV-2 inhibitors targeting SARS-CoV-2 RNA-dependent polymerase, the receptor-binding domain (RBD) of SARS-CoV-2 viral S-protein and the 3C-like main protease (3CLpro). Analysis of the binding mechanism of these compounds is characterized by the formation of high-anity intermolecular interactions with respective binding site residues of SARS-CoV-2 RNA-dependent polymerase, 3CLpro, and RBD of viral S-protein, which subsequently contributed to favourable binding a�nity. Using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MMPB-SA) approach, Geraniin exhibited a binding free energy (ΔGbind) of -25.87kcal/mol and − 21.74kcal/mol, respectively, whereas 2-O- Caffeoyl-(+)-allohydroxycitric acid exhibited a ΔGbind of -32kcal/mol towards 3CLpro. Molecular Dynamics (MD) simulations revealed crucial structural changes induced by the identi�ed inhibitors, which possibly interfered with enzyme functions. The molecular insights provided regarding the inhibitory potency of the two phytochemicals warrants further experimental evaluation towards discovering novel SARS-CoV-2 therapeutics.


Introduction
Reported in Wuhan, China, in early December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has spread worldwide and was eventually declared a pandemic by the World Health Organisation(WHO) causing the COVID-19 disease with a 2.2% mortality rate and over 72 million infected persons so far.
To augment the existing therapeutic options, the focus of several recent reports has included the exploration of the anti-SARS-CoV-2 potential of medicinal plants and their phytochemical extracts.Many of these reports have particularly sought to repurpose natural products with proven antiviral properties towards coronavirus treatment [40][41][42][43].The prominence of medicinal plants for therapeutic purposes is attributed to their affordability, effectiveness, safety, cultural preferences, and ample accessibility and when it is needed [44][45][46].One of such medicinal plants that have been extensively investigated for antiviral properties is Spondias mombin (S. mombin), [41][42][43]; however, its therapeutic potential against SARS-CoV-2 remains unexplored.

System Preparation
From the Protein Data Bank [97], the X-ray crystal structures of SARS-CoV-2 RNA-dependent polymerase (PDB:7BTF) [98], SARS-CoV-2 3C-like main protease (3CL pro ) (PDB:6LU7) [99], and the receptor-binding domain of viral S-protein.(PDB:6M17) [100] were retrieved and prepared for molecular docking using UCSF Chimera [101].To reduce computational cost and resources, DNA, Zn 2+ , and Mg 2+ were all deleted.Using UCSF Chimera, hydrogen ions were added before proceeding to perform molecular docking.This was to cater for any missing hydrogen that may not have been present in the retrieved PDB structures.In addition, the de nition of at least the polar hydrogens allows the establishment of hydrogen bonds that may be present between the therapeutic targets and the compounds.

Retrieval and preparation of investigated phytochemicals
The two-dimensional (2D) structures of the phytochemicals isolated from ethanolic extracts of the leaves of S. mombin selected for this study were generated using Marvin Sketch [102].Subsequently, the Avogadro 1.2.0 software [103] was employed to perform energy minimization and optimization of the phytochemicals using the UFF force eld and the steepest descent algorithm [100].Afterward, 3D conformations of the structures were generated and saved as mol2 les for further investigations.of the molecular docking were viewed using the ViewDock function incorporated in UCSF Chimera.The docking results were validated by superimposing generated docked complexes with the retrieved cocrystallized structures of the target SARS-CoV-2 therapeutic targets.

In silico exploration of Drug-likeness of hits
We predicted the physicochemical and pharmacokinetic properties of the studied phytochemicals using SwissADME [105].These properties provided insights into each of the phytochemicals' absorption, distribution, metabolism, excretion, and toxicity (ADMET).Although these properties could be evaluated using experimental methods, these are usually time-consuming and expensive, hence applying SwissADME.The physicochemical and pharmacokinetic properties of the phytochemicals predicted were also used to ascertain their possible adherence to Lipinski's rules of ve, a set of rules widely employed in assessing the drug-likeness chemical compounds [106][107][108] .

Molecular dynamics (MD) simulations
To reveal the conformational and structural changes that accompany the binding of the identi ed bioactive compounds to their respective SARS-CoV-2 therapeutic targets, we performed an atomic-scale MD simulation using the AMBER 18 GPU with an integrated PMEMD module [109,110].This is because these structural changes could inform their possible inhibitory mechanism of the identi ed compounds.Any additional cocrystallized molecules such as crystal water were removed from the enzyme structures to minimise computational resources before the beginning of the MD simulation.The ANTECHAMBER module was then used to parameterize the inhibitors, in which atomic partial charges (AM1BCC) were added [111].The FF14SB AMBER force eld was also used to parameterize the enzymes [112].Protonation of histidine residues was then performed using the pdb4amber script at a constant pH (cpH) to ensure compatibility of the prepared SARS-CoV-2 therapeutic target models with the LEAP module.Subsequently, the Leap module was then employed to solvate and neutralize the entire prepared systems.The counter ions, Na+ or Cl -were used to neutralize as systems whereas TIP3P orthorhombic box size of 12Å of water molecules was added to solvate each system [113].Topology and coordinate les of the bioactive compounds, SARS-CoV-2 therapeutic targets, and the resultant complexes were then generated and saved.The prepared bound complexes and the unbound enzymes were then subjected to an initial 2000 minimization steps at a restraint potential of 500kcal/mol just to minimize the positions of the water and ions.Afterward, a 1000 steps steepest descent minimization with no restraint was performed.The systems were gradually heated from 0K to 300K for 50ps.After heating, a 500ps equilibration was performed at a constant pressure of 1bar.The pressure was maintained constant using Berendsen barostat [114].The SHAKE algorithm was employed to constrict all atomic hydrogen bonds, after which a 200ns MD simulation was performed on all simulated models using a 1fs time step [115].Coordinates for generated MD trajectories were saved at 1ps interval.These generated trajectories were further analysed using the PTRJ and CPPTRAJ modules of AMBER [116].
Graphical plots for analysis of the generated trajectories created with the Microcal Origin analytical software [117].

Binding Free Energy Calculations
Computer-based binding free energies of the identi ed phytochemicals were calculated using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MMPB-SA) techniques [118,119].MMPB-SA as a reliable technique is used to evaluate docking poses, determine structural stability, and predict binding a nities and hotspots.Also, MMPB-SA can be employed to analyse the energy contributions from individual residues through energy decomposition analysis.In this report, the binding free energies of the identi ed inhibitors were calculated using some known inhibitors of the respective targets as a control.This allowed for an assessment of the inhibitory potential of the identi ed compounds.MMPB-SA has been applied widely in protein-ligand interactions with proven reliability over the years.The binding free energy (DGbind) was determined by the equations: DG bind = DH -TDS = DE MM + DG sol -TDS (2) in which G non_polar = gSASA + β (5) where ΔE MM , ΔG sol , and −TΔS are the changes in the gas phase molecular mechanics (MM) energy, solvation free energy, and conformational entropy upon ligand binding respectively.DE MM is also the sum of the internal energy terms DE int (bond, angle, and torsion), van der Waals (DE vdw ) and the non-bonded electrostatic energy component (DE elec ).The solvation free energy, G sol , on the other hand, is a summation of the electrostatic solvation energy ΔG PB (polar contribution) and the nonpolar contribution ΔG SA between the solute and the continuum solvent.G SA is calculated from the solvent assessable surface area (SASA), obtained utilizing a 1.4 A° water probe radius whereas the polar contribution is calculated using PB.γ and β represented empirical constants for 0.00542 kcal/(mol•Å 2 ) and 0.92kcal/(mol•Å 2 ), respectively.To explore the inhibitory potential of the phytochemical compounds from ethanolic leaf extract of S. mombin against SARS-CoV-2 therapeutic targets, molecular docking was performed.The docking scores which gave insights into the possible binding a nity of the compounds against the studied targets were calculated as presented in Table 2. Docking scores allow for the determination of the most favourable binding orientation of a compound within a given binding pocket.A favourable binding orientation, of a ligand within a given pocket consequentially informs the nature of binding interaction and hence in uences overall binding a nity [120].The lower the docking score, the more favourable the corresponding binding orientation [120].As shown in Table 2, molecular docking of all the studied compounds at the active site of SARS-CoV-2, revealed that Geraniin exhibited the most favourable binding orientation at the inhibitor binding sites of both SARS-CoV-2 RdRp and the RBD of viral S-protein with the highest docking score of -10.4kcal/mol and -7.3kcal/mol respectively.Also, at the inhibitor binding site of 3CL pro , 2-O-Caffeoyl-(+)-allohydroxycitric acid exhibited the highest docking score of -5.6kcal/mol against binding to 3CL pro in comparison to the other studied compounds.

Results And Discussion
Therefore from the molecular docking, the results indicate that Geraniin and 2-O-Caffeoyl-(+)allohydroxycitric acid bind favourably to their respective targets when compared to other phytochemicals and could thus eventually modulate the activities of their target enzymes for therapeutic purposes upon further investigations.[122] reported that His163 is essential to the inhibition of 3CL pro since the mutation of its homologous residue His162 in SARS protease inactivates 3CL pro [122].As such, the conventional hydrogen bond interaction exhibited between 2-O-Caffeoyl-(+)allohydroxycitric acid and His163 con rms the cruciality of this residue and also predicts a possible - inhibitory potential of 2-O-Caffeoyl-(+)-allohydroxycitric acid against 3CL pro .Also, 2-O-Caffeoyl-(+)allohydroxycitric acid is shown to elicit a conventional hydrogen bond interaction with Cys145, one of the catalytic dyad (Cys145 and His41) [123] of 3CL pro further suggesting its possible inhibitory activity against 3CL pro .These strong interactions observed could have contributed to the favourable binding orientation of 2-O-Caffeoyl-(+)-allohydroxycitric acid and its consequential high docking score as calculated in the molecular docking process.Considering previous in vitro reports on the antiviral activity of 2-O-Caffeoyl-(+)allohydroxycitric acid against Coxsackie and Herpes simplex viruses [49], the molecular insights provided in this report about its possible activity against SARS-CoV-2 3CL pro warrant its further experimental validations.

SARS-CoV-2 RNA Dependent RNA Polymerase-Geraniin complexes
As shown in Figure 2, Geraniin, which exhibited the highest docking score amongst the studied phytochemicals against SARS-CoV-2 RdRp is bonded within the binding pocket by a pi-alkyl interaction with Arg550, a conventional hydrogen bond with both Arg555 and Ala553 while a pi-cation interaction is engaged with Arg836.In addition, Geraniin is also shown to elicit conventional hydrogen bond interactions with Asn691, Asn760, and Asp623 while a carbon-hydrogen bond interaction was formed with Lys621 in deeper regions of the inhibitor binding site.These interactions could have accounted for its favourable docking orientation within the SARS-CoV-2 RdRp inhibitor binding pocket relative to the other phytochemical compounds as established in the molecular docking simulation.A comparison of the docking score of Geraniin (-10.4kcal/mol) with that of the docking score of Remdesivir (-5.9kcal/mol) [124], within the same grid box dimensions, revealed that Geraniin showed a relatively higher docking score than Remdemsivir.This relatively higher docking score of Geraniin, in addition to its strong intermolecular interactions engaged with binding pocket residues of SARS-CoV-2 RdRp conforms with a possible inhibitory activity.In addition to its previously antiviral activity [80,82], these revealed atomistic binding insights against SARS-CoV-2 RdRp suggests that Geraniin could be further investigated as a possible inhibitor against SARS-CoV-2.

SARS-CoV-2 Receptor Binding Domain-Geraniin complexes
As shown in table 2 Geraniin also exhibited the highest docking score toward the RBD of SARS-CoV-2 viral Sprotein.By examining its residue interaction pro le with the RBD, we explored its possible binding mechanism.A successful blockage of the RBD of viral S-protein by Geranin could prevent the binding of RBD of viral S-protein and SARS-CoV-2.As shown in Figure 3, Geraniin is engaged in a wide network of interactions, notably, conventional hydrogen bond interactions were formed with Arg403, Tyr495, Tyr453, Ser494, Gln493, Gln498 and Tyr505 while a carbon-hydrogen interaction is observed with Gln498.These strong conventional hydrogen interactions could anchor Geraniin within the binding pocket to ensure its stability for favourable binding and consequential interruption of the activity of RBD of the viral S-protein.
The interacting residues were also consistent with prominent residues reported by several studies in which novel SARS-CoV-2 inhibitors have been predicting thus further establishing the potential inhibitory prowess of Geraniin [125].In addition to the previously reported antiviral activity of Geraniin [80,82], the atomistic and structural insights provided in this study warrants further exploration of Geraniin as a possible binder of the receptor-binding domain of viral S-protein towards COVID-19 therapy.

Identi ed Hits exhibit favorable binding free energy towards SARS-CoV-2 3CL pro , RdRp and RBD of viral S protein
Inhibitor stability within the binding pocket is very crucial in determining biological processes with consequential pharmaceutical implications.Therefore, to establish the stability of the identi ed hits within the respective SARS-CoV-2 target, we assessed their binding free energy over the simulation period using the MMPB-SA approach since binding a nities from molecular docking are inconclusive.The MMPB-SA calculations also allowed for a quantitative determination of absolute binding a nities of the identi ed hits [126].The calculated binding free energies allow for a thorough understanding of the mechanism by which the respective SARS-CoV-2 targets recognizes the identi ed hits which in turn in uences the inhibitory potential of the hits against those targets [127].Favourable binding free energies of hit compounds indicate stability within the pocket which could in turn favour binding site interactions [126].As shown in table 3 the binding free energy of Geraniin towards RdRp and RBD of viral S protein was calculated to be -25.87kcal/moland -21.74kcal/mol respectively while the binding free energy of 2-O-Caffeoyl-(+)allohydroxycitric acid was estimated as -32.00kcal/mol.Overall, all three compounds bound exhibited strong binding a nity towards their respective target corroborating with the strong interaction bonds elicited binding pockets as revealed in the interaction dynamics.2-O-Caffeoyl-(+)-allohydroxycitric acid exhibited almost similar binding free energy with Ritonavir which exhibited a total binding free energy of -32.34kcal/mol.Also, a comparison of the binding free energy of Geraniin to the known SARS-CoV-2 RdRp inhibitor, Remdesivir, showed that Geraniin exhibited a relatively lower binding free energy with Remdesivir demonstrating binding free energy of -33.34kcal/mol.This further highlighted the therapeutic potential of these hits and hence warrants their further experimental investigations.ΔE ele = electrostatic energy; ΔE vdW = van der Waals energy; ΔG bind = total binding free energy; ΔG sol = solvation free energy ΔG gas = gas phase free energy.

Assessing the structural and conformational changes of SARS-CoV-2 therapeutic targets upon binding of Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid
As a reliable and widely employed computational technique, molecular dynamics simulations were used to conduct a time-dependent prediction of the structural and conformational motions that occur on the SARS-CoV-2 therapeutic targets upon the binding of the identi ed bioactive compounds [128][129][130].Any observed structural changes on these enzymes could provide essential conformational insights that could contribute to the potential inhibitory activity of the compounds.MD simulations also allowed for an assessment of the conformational stability of the targets upon binding of the compounds since these could provide valuable insights into the potential inhibitory prowess of the compounds.Also, since molecular docking calculations were done using the rigid X-ray crystal structures of the target SARS-CoV-2 enzymes, MD simulation allowed for an assessment of target receptor and corresponding bound inhibitor interactions in the dynamic behaviour of both the enzymes and the identi ed bioactive compounds.With an adequate 200ns MD simulation period, we calculated the root mean square deviation (RMSD) [131] and root mean square uctuation (RMSF) [130,132] to assess conformational stability and residue exibility.The structural and conformational alterations that were unraveled suggest a possible inhibitory mechanism of the identi ed antiviral phytochemical compounds since these changes could interfere with enzyme functions.

2-O-Caffeoyl-(+)-allohydroxycitric acid-binding perturbs 3CL pro
Several recent reports have investigated the conformational dynamics of unliganded of SARS-CoV-2 3CL pro including a recent molecular dynamics simulations study by Suarez and Diaz, (2020) [133] where they revealed that the domain III of 3CL pro is generally unstable while the presence of peptide substrate, Ace-Ala-Val-Leu-Gln∼Ser-Nme, induces a stable interdomain arrangement in the monomeric conformation of the protease.These conformational changes of enzymes are the hallmarks of their dynamics that correlate with the overall functions of these enzymes [133].Therefore, when induced by potential inhibitors, these conformational changes could interfere with their known functions.By calculating the RMSD of the Ca atoms of 3CL pro over the 200ns simulation period, the impact of the binding of 2-O-Caffeoyl-(+)allohydroxycitric acid on the stability of 3CL pro was assessed.The stability of an enzyme structure is crucial in the maintenance of its function [134].As shown in the Figure 4A and 4B, both unbound and bound simulated models of 3CL pro converged around 75ns after an initial jump in deviation due to the expansion of atoms.On average, the unbound conformation of 3CL pro exhibited a relatively higher RMSD of 2.53Å while the inhibitor bound conformation exhibited a lower RMSD of 2.43Å.This suggested that the binding mechanism of 2-O-Caffeoyl-(+)-allohydroxycitric acid involved a consequential increase in the stability of 3CL pro consistent with the earlier reports by Suarez and Natalia in which substrate binding was shown to induce a stable interdomain conformation.The stabilized conformation of 3CL pro upon the binding of 2-O-Caffeoyl-(+)-allohydroxycitric acid could further facilitate a favorable interaction with crucial active site residues to impede the functions of 3CL pro .Root Mean Square Fluctuation (RMSF) which provided atomistic insights on the exibility of each of the 306 amino acid residues of 3CL pro was estimated as presented in gure 4. Comparatively, the unbound simulated model of 3CL pro exhibited an average RMSF of 9.90Å while the 2-O-Caffeoyl-(+)-allohydroxycitric acid bound model showed a relatively lower average RMSF of 9.16Å.This suggested that the binding of 2-O-Caffeoyl-(+)-allohydroxycitric acid impeded the exibility of individual amino acids of 3CL pro , consistent with the relatively lower average RMSD of the bound conformation as observed.This impeded residue exibility could intend to interfere with the essential residue mobility required for the function of 3CL pro .From the RMSF and RMSD calculations, it could therefore be inferred that the binding of 2-O-Caffeoyl-(+)-allohydroxycitric acid is characterised by a stabilized structural conformation and an impeded residue exibility which in turn interfere with essential enzyme mobility.

Geraniin binding distorts conformational integrity of SARS-CoV-2 RdRP
A recent comparative molecular dynamics simulations study by Koulgi et al (2020) in which the unbound and Remdesivir-complexed structures of SARS-CoV-2 RdRp showed the blocking of the template entry site upon Remdesivir binding [135].Their report further revealed that Remdesivir binding is characterised by structural instability and increased residue exibility.Therefore, to ascertain the inhibitory potential of Geraniin against RdRp, we also assessed the conformational dynamics of RdRp upon Geraniin binding.In a similar mechanism as Remdesivir, the binding of Geraniin also increased the deviation of c-a atoms of RdRp consistent with structural instability.As shown in Figure 5A, a relatively higher average RMSD of 3.08Å was calculated for the Geraniin bound RdRp while the unbound RdRp exhibited an average RMSD of 2.5Å.Likewise, as shown in Figure 5B the binding of Geraniin also induced prominent residue uctuations as was reported for Remdesivir binding in the study by Koulgi et al.,(2020) [135].An average RMSF of 32.01Å was estimated for the Geraniin bound RdRp while an average RMSF of 21.70Å was calculated for the unbound conformation.Taken together, it could be inferred that the binding of Geraniin distorts the structural integrity of SARS-CoV-2 RdRp in a similar mechanism as Remdesivir.As such, Geraniin could further be investigated as a potential inhibitor of SARS-CoV-2 RdRp.

Geraniin binding in uence the receptor accessibility or inaccessibility of the spike protein
According to a recent report by Gur et al, (2019) [136], the down and up positions of SARS-CoV-2 RBD can interfere with the accessibility of the spike protein by controlling its open (receptor accessible) and closed (receptor inaccessible) positions.Therefore, it is evident that any conformational changes of RBD induced by a bound inhibitor could in uence any intended therapeutic inhibition.A calculation of the RMSD of the simulated RBD models as presented in Figures 6A and 6B revealed that the unbound conformation of RBD showcased an average RMSD of 7.20Å while the Geraniin bound RBD showed an average RMSD of 10.17Å.The signi cantly higher average RMSD of the bound conformation suggests that the binding of Geraniin possibly increased the deviation of c-a atoms and hence subsequently decreased the conformational stability of RBD.The exibility of the individual amino acids of RBD was also accessed to unravel any conformational changes on RBD upon Geraniin binding.As shown in Figure 6, an average RMSF of 12.96Å and 13.08Å was calculated for the unbound and inhibitor bound conformation of RBD respectively.Although the difference in average residue uctuations between the bound and unbound conformations was minimal, the relatively higher average RMSF in the Geraniin bound structure confers with increased residue exibility suggesting that the binding of Geraniin distorted the residue integrity of RBD which subsequently increased the residue motions as observed.This increased residue mobility of RBD upon Geraniin binding could in turn favour a down and up motion of RBD and hence possibly in uence the receptor accessibility or inaccessibility of the spike protein as postulated by Gur et al,(2019) [136].

Assessing the pharmacokinetic properties of Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid
The physicochemical and pharmacokinetic features of drugs are very crucial to their overall therapeutic success.As such, we analysed the physicochemical and pharmacokinetic properties of Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid using the online platform SwissADME [105].An in silico assessment of these properties, notably absorption, distribution, metabolism, and excretion offers insights into the pharmacokinetics of a given small molecular inhibitor in vivo while minimizing the risk of being disapproved during late stages of drug development [137,138].As shown in table 4, the properties as assessed from SwissADME are presented [105].Since natural products are usually not compatible with Lipinski's Rule of 5 [106][107][108], rules propounded from relatively simple small molecules, it suggests Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid warrant further experimental investigation for their potential anti-SARS-CoV-2 properties.Using the Brain Or IntestinaL EstimateD permeation method (BOILED-Egg) concept [139], the SwissADME platform was also used to predict the lipophilicity (log P) and polarity of Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid compound [140,141].By calculating the LogP of the compounds, we predicted their permeability across cellular membranes.According to Lipinski's rule of ve, the Log P of a compound intended for oral administration should not be more than 5 [142].As such, higher logP is usually associated with a compound with minimal potential of permeating the lipid membrane.With a logP of -1.71 and -0.65 for Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid respectively, it suggests both compounds will exhibit high membrane permeability, hence a possibly high bioavailability and absorption.An assessment of the molecular weights (MW) of both compounds was found to be 952.64g/moland 379.27g/mol for Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid.The relatively lower MW of 2-O-Caffeoyl-(+)-allohydroxycitric acid (<500g/mol), suggests that it could possess minimal toxicity tendencies [143].Although Geraniin has a large MW of 952.64g/mol, its synthetic fragmentation into smaller simpler compounds could increase its bioactivity and decrease toxicity [144].

3. 1
Investigation of the possible binding mechanisms of antiviral phytochemical ethanolic leaf extract of S. mombin against SARS-CoV-2 therapeutic targets 3.1.1Molecular Docking of the antiviral phytochemical ethanolic leaf extracts of S. mombin with SARS-CoV-2 RNA dependent RNA polymerase, SARS-CoV-2 3CL pro and RBD of viral S-protein

3. 1 . 2
Exploring the binding mechanisms of identi ed hit bioactive phytochemicals against SARS-CoV-2 therapeutic targetsThe mechanism of binding of inhibitors to biological targets is in uenced by the nature of the interactions engaged between the inhibitor and binding site amino acid residues.These interactions consequentially in uence the conformational stability and binding a nity of inhibitors towards their targets.Therefore, inhibitor-residue interactions are very crucial in the overall therapeutic potential of inhibitors.Using the Discovery Studio[121], we visualized and explored the residue interaction pro le of both Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid upon binding to SARS-CoV-2 3CL pro , SARS-CoV-2 RdRp, and the RBD of viral S-protein.This revealed essential atomistic insights that could have informed the favourable binding orientations as observed in the molecular docking simulation.3.1.3SARS-CoV-2 RNA 3 C-like Main Protease-2-O-Caffeoyl-(+)-allohydroxycitric acid complexAfter exhibiting the highest docking towards SARS-CoV-2 3CL pro amongst all the investigated compounds as shown in Table2, we further explored the possible binding mechanisms of 2-O-Caffeoyl-(+)-allohydroxycitric acid by analysing its interaction pro le with binding site residues.As shown in Figure3an exploration of the binding interactions of 2-O-Caffeoyl-(+)-allohydroxycitric acid towards 3CL pro revealed the formation of strong intermolecular interactions with crucial binding site residues.Notably, strong conventional hydrogen bond interactions were formed with Csy145, Asn142 and His163.Cys145 is also shown to engage in an additional pi-cation interaction with the bound inhibitor emphasizing its cruciality to the binding of 2-O-Caffeoyl-(+)-allohydroxycitric acid.A study byHall et al,(2020) This in silico study identi ed two bioactive compounds; Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid as potential repurposing therapeutic inhibitors against SARS-CoV-2.Using Molecular Mechanics/Poisson-Boltzmann Surface Area (MMPB-SA) approach to calculate binding free energy, Geraniin exhibited binding free energy (ΔG bind ) of -25.87kcal/mol and -21.74kcal/mol respectively towards SARS-CoV-2 RdRp and RBD of viral S protein respectively.2-O-Caffeoyl-(+)-allohydroxycitric acid on the other hand exhibited a ΔG bind of -32kcal/mol towards 3CL pro .The binding of both Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid were characterised strong interactions with respective SARS-CoV-2 therapeutic target suggesting an inhibitory potential and their ability to bind favourably to SARS-CoV-2 RNA-dependent polymerase, 3CL pro and RBD of the viral S-protein.Molecular Dynamics (MD) simulations further revealed crucial structural changes induced Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid which possibly interfered with enzyme functions.Notable structural changes included, increased residue exibility of and a distortion of the structural integrity of SARS-CoV-2 RdRp RBD upon Geraniin binding.The binding of 2-O-Caffeoyl-(+)allohydroxycitric acid also stabilized the structural conformation and impeded residue exibility of 3CL pro .The molecular insights provided with regards to the inhibitory potency of the two phytochemicals warrant an extensive experimental evaluation towards the discovery of novel SARS-CoV-2 therapeutics.To the best of our knowledge, this is the rst time Geraniin and 2-O-Caffeoyl-(+)-allohydroxycitric acid have been reported as potential repurposing SARS-CoV-2 inhibitors.

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Table 1 :
Selected Phytochemicals isolated from ethanolic extracts of the leaves of S. mombin with reported antiviral and anti-malarial properties.

Table 2 :
Docking scores of selected phytochemical compounds against SARS-CoV-2 RdRp, 3CL pro and RBD of viral S-protein.

Table 3 .
MM/PBSA-based binding free energy profile of identified hit compounds against