Integrated bioinformatics–cheminformatics approach toward locating pseudo‐potential antiviral marine alkaloids against SARS‐CoV‐2‐Mpro

Abstract The emergence of the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) with the most contagious variants, alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and Omicron (B.1.1.529) has continuously added a higher number of morbidity and mortality, globally. The present integrated bioinformatics–cheminformatics approach was employed to locate potent antiviral marine alkaloids that could be used against SARS‐CoV‐2. Initially, 57 antiviral marine alkaloids and two repurposing drugs were selected from an extensive literature review. Then, the putative target enzyme SARS‐CoV‐2 main protease (SARS‐CoV‐2‐Mpro) was retrieved from the protein data bank and carried out a virtual screening‐cum‐molecular docking study with all candidates using PyRx 0.8 and AutoDock 4.2 software. Further, the molecular dynamics (MD) simulation of the two most potential alkaloids and a drug docking complex at 100 ns (with two ligand topology files from PRODRG and ATB server, separately), the molecular mechanics/Poisson‐Boltzmann surface area (MM/PBSA) free energy, and contributions of entropy were investigated. Then, the physicochemical‐toxicity‐pharmacokinetics‐drug‐likeness profiles, the frontier molecular orbitals energies (highest occupied molecular orbital, lowest unoccupied molecular orbital, and ΔE), and structural–activity relationship were assessed and analyzed. Based on binding energy, 8‐hydroxymanzamine (−10.5 kcal/mol) and manzamine A (−10.1 kcal/mol) from all alkaloids with darunavir (−7.9 kcal/mol) and lopinavir (−7.4 kcal/mol) against SARS‐CoV‐2‐Mpro were recorded. The MD simulation (RMSD, RMSF, Rg, H‐bond, MM/PBSA binding energy) illustrated that the 8‐hydroxymanzamine exhibits a static thermodynamic feature than the other two complexes. The predicted physicochemical, toxicity, pharmacokinetics, and drug‐likeness profiles also revealed that the 8‐hydroxymanzamine could be used as a potential lead candidate individually and/or synergistically with darunavir or lopinavir to combat SARS‐CoV‐2 infection after some pharmacological validation.


| INTRODUCTION
have been posing more challenges for the applied or proposed drugs and vaccines. [2][3][4] The discriminating infection and re-infection (second and third waves of SARS-CoV-2) without confirmative symptoms and unexpected morbidity-mortality rates in certain regions and communities without any potential therapy by the Food and Drug Administration (FDA), "added fuel to the fire" at this health emerging situation. 3,4 At present, re-purposing anti-viral, anti-inflammatory, immune-modulating, antiparasitic regimens, including natural/herbal regimens is a frontline approach to moderately control SARS-CoV-2 pathogenesis. 5,6 However, the efficacy of applied re-purposing drugs and vaccines varies from patient to region. 7,8 Although, proposed drugs and vaccine candidates got permission for emergency use and few drugs were also recommended by FDA to control the pandemic situation. But we still need more potential drugs with higher efficacy and fewer side effects. 7,8 Therefore, the current situation directly indicated exploring and developing more therapeutic candidates at a priority basis for the ongoing drug development module to save the human community from highly pathogenic SARS-CoV-2 strains. 5,9 Therefore, locating potential natural products from existing resources is considered as ideal research to accelerate and/or offer other antiviral drug development research groups or pharmaceutical companies to take over the preliminary findings for further pharmacological investigation against SARS-CoV-2. [9][10][11][12] Prima facie, several natural secondary metabolites isolated from eukaryotic and prokaryotic origins are continuously investigated against SARS-CoV-2 experimental (in vitro) and computational (in silico) methods. Opportunistically some of the regimens are also entered into different stages of clinical trials. Thus, in an emergency, the computer-aided drug design (CADD) platform can trace several potential leads and proceed with such recognized potent candidates for an experimental validation expected to be the cost-effective or resource-saving drug development strategy against SARS-CoV-2. 6,[13][14][15][16][17] From the history of natural product-based drug discovery, marine diversity is one of the most substantial contributors of potentially unique metabolites with a wide range of mainstream therapeutic applications, mainly against cancer and infectious diseases. 6,[18][19][20]  and so on from previous experimental reports (see Table S1). Overall, the alkaloid class of compounds has a higher success rate than other natural products. Thus, selecting such broad-spectrum antiviral marine alkaloids from previous experimental and using those alkaloids against SARS-CoV-2 could be a rational approach in the current drug development module.
Currently, the massive effort of worldwide scientific groups, crystallographic protein structures of newly emerged SARS-CoV-2 main protease or 3-chymotrypsin-like cysteine protease (Mpro or 3CLpro), papine-like protease (PLpro), spike glycoprotein (S-protein) and RNAdependent RNA polymerase (RdRp) provided structural insights for novel drug development. 21,22 Significantly, the Mpro is one of the most putative drug targets for antiviral drug development associated with viral-host interaction for assembly, formation, and replication of virus genomes in the host cell. [9][10][11][12]21 This work examined 57 antiviral marine alkaloids (see Table S1) followed by an advanced computational platform targeting SARS-CoV-2-Mpro. Further, advanced molecular dynamics simulation (MD simulation) at 100 ns with comparative analysis two different topology files generated by the PRO-DRG and the automated force field topology builder (ATB) server, the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) binding energy calculation with drug-ability-toxicity-pharmacokinetics prediction, structural activity relationship (SAR), and frontier molecular orbitals (FMOs) in a combined "biophysics-quantum chemistry" approach using various bioinformatics and cheminformatics tools to locate the potential "lead" against SARS-CoV-2-Mpro (see Figure 1).

| MM/PBSA and binding free energy per residue analysis
To distinguish the dynamic behavior of ligand interaction in the form of the binding energy of two separately generated topology files for simulated protein-ligand docking complexes, the widely used MM/PBSA method for binding free energy (ΔG bind ) with the g_mmpbsav5.12 suite in the GROMACS platform was performed. 16,17,22 The following equations was used to calculate Gibb's binding free energy (ΔG bind ): where ΔG = free energy; ΔG complex = free energy of protein-ligand complex, ΔG protein = free energy of protein, and ΔG ligand free energy of ligand in the solvent. TΔS = entropic contribution in vacuum for free energy, T = temperature, and S = entropy. ΔG solv = solvation free energy, G polar = electrostatic, and G nonpolar = nonelectrostatic contributions to the solvation free energy. ΔE MM is the vacuum potential energy of both bonded and nonbonded interactions.

| Physicochemical and toxicity profiles prediction
Using generated simplified molecular-input line-entry system

| Pharmacokinetics and overall drug-likeness profiles prediction
Using the same SMILES code, assessed the absorption, distribution, metabolism, excretion, and toxicity (ADME/T) with bioavailability overall known as pharmacokinetics profiles of ligands structure using the Swiss-ADME tool. Finally, the drug-likeness score for each alkaloid along with two anti-HIV drugs was predicated using the tool,

| FMOs and SAR analyses
We have analyzed the intermolecular reaction or kinetic outlines in the form of the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO) plots, and energy gap (ΔE) of two potential marine alkaloids with two standard drugs using software, Avogadro-ORCA 1.2.0 platform. 27 The Universal Force Field (UFF) with steepest descent algorithms for energy minimization of structure with the restricted Hartree-Fock (RHF) principle was employed with single point calculation method to compute the electron wave function in form of HOMO, LUMO, and ΔE orbital energy.
Simultaneously, the most potential theoretical drug prospective T A B L E 1 Molecular docking score (kcal/mol) against SARS-CoV-2-Mpro with the drug-likeness score, LD 50 value and toxicity profiles of antiviral marine alkaloids and two anti-HIV standard drugs  (Table 1)

| MD simulation of docked protein-ligand complexes
The RMSD-protein backbone, RMSF-C-alpha and Rg-score plots from    Figures 2B and S1B). From generated RMSF plots, a higher binding affinity with less fluctuation in C-alpha residues of both alkaloids than darunavir complex was observed in PRODRG-derived topology files ( Figures 2C and S1C). RMSF plots of marine-1 and marine-2 complexes deviated throughout 100 ns, but from minute observation marine-1 is more deviated in PRODRG files and both complexes exhibited more relative deviation in MD simulation with ATB server derived ligand topology files (Figures 2C and S1C). Overall, in terms of the ligand RMSD, a substantial difference has been observed where the RMSD of both the marine alkaloids deviated from 0.1 nm to 0.25 nm, whereas using PRODRG topology, the RMSD of the two marine alkaloids seems stable $0.15 nm. The difference has been notably observed due to the charge distribution pattern while generating the topology from the aforementioned servers. Based on the RMSF-analyses, among three ligand complexes exhibited more relative deviations, while marine-2 was comparatively more stable in PRODRG server-generated topology files ( Figures 2C and S1C). The overall compactness and the RMSF remain the same in both the marine-1 and marine-2 topologies. Some minute differences have been observed, which does not impact significantly the overall parametric calculations. Further, Rg-plots displayed the compactness or solidity in marine-2 with more squeezed Rg-score than other complexes in both PRODRG and ATB-derived topology files ( Figures 2D   and S1D).  (Table 2). Parallelly, the van der Waal energy, electrostatic energy, polar solvation energy, and solvent accessible surface area (SASA) energy were too calculated for each complex ( Table 2).
The ATB-server-derived topology file's binding energies of marine-1 and marine-2 were documented, separately. Mainly the atomic charged ATB-files showed their energy in a lower value as À93.070 ± 0.925 kJ/mol binding energy for marine-1 complex and À143.9 ± 0.985 kJ/mol for the marine-2 complex (Table S2). According to another energy was also recorded in a higher range (Table S2). Overall, from both topology file analyses, the 8-hydroxymanzamine was more potential than against SARS-CoV-2-Mpro as per the energy per binding energies and residues calculation in a graph format within 100 ns ( Figure 6). There is a maximum number of common residues with comparatively lower binding energy in marine-2 than marine-1 and darunavir within 100 ns. Thus, the MM/PBSA is widely accepted in the current CADD module to calculate absolute binding affinity per residue during ligand integration with the target molecule. 17,30

| Topology-based comparison and contributions of entropy analyses
As mentioned earlier, the topology-based analysis of the two marine alkaloids has been performed using ATB and compared with PRODRG   In addition, Schlitter's formula and quasi-harmonic analyses were evaluated against the two most potential marine alkaloids that reproduce translational, rotational, and vibrational entropies.

| Physicochemical and toxicity profiles prediction
The physicochemical/RO5 properties of all marine-alkaloids and reference anti-HIV drugs were recorded (Table S3). Briefly, 23 marinealkaloids did not follow the standardized molecular weight (≤500 g/ prediction suggested that most potential alkaloids displayed in a higher range than standardized parameters due to large chemical structure but not that much to disrupt the oral bioavailability (Table S3). Nevertheless, RO5 is a standardized parameter to locate bioactive oral drugs with some ideal metabolism for the current drug development modules. 33 From the predicted toxicity profiles, marine-alkaloids and anti-HIV drugs exhibited comparatively moderate hepatotoxicity, carcinogenicity, and mutagenicity profiles with adverse immunotoxicity profiles tabulated in Table 1 with lopinavir drug were safer candidates with toxicity profiles class-VI (Table 1).

| Pharmacokinetics and overall drug-likeness profile prediction
Possible pharmacokinetic profiles of marine-alkaloids and anti-HIV drugs were recorded (Table S4 and Figure S5). The drug-likeness score for each marine-alkaloid was recorded in  Figure S6). However, the >2 scores are also not good ideal as per the predicted tool. Mainly, the score within 0-2 is the more preferred score for a candidate under the predefined bell-shaped curve for identification of drug or nondrug category by Molsoft. To conclude, drugability is the sum of all drug-parameter scores as per the chemical composition of a candidate. 17,33,34 Thus, the predicted drug-likeness score could be another crucial parameter used to select lead candidates, where marine alkaloids displayed potential drug-ability scores like a mainstream drug.

| FMOs and SAR analyses
In  As per the prediction, inappropriate drug-likeness profiles could be overcome through advanced nano-formulation and structural modification approaches later. [35][36][37][38] Nevertheless, exploring more bioactive compounds from different ethnomedical-natural sources is one of the potential directions in the primary drug discovery module. 11,18,19,39 Simultaneously, the use of advanced artificial intelligence programs accommodating to find out the potential candidates (herein marine alkaloids) against specific diseases (herein SARS-CoV-2 targeting

| DISCUSSION
The marine ecosystem produces a plethora of bioactive metabolites that are recognized from their previous contribution, namely, streptomycin, rifamycin, vancomycin, and so on. 16,19,43 From the antiviral drug development point of view, previous experimental records and the present computational investigation indicated that the marine sponge is the most suitable source of anti-viral alkaloids (see Table S1). In contrast, plant genera also provide several alkaloid classes of drug namely quinine, morphine, nicotine, vinblastine, vincamine and emetine, and so on, to mainstream medicine. However, the structural composition and complexity have differentiated the uniqueness of marine alkaloid and plant alkaloid metabolites. 18,44,45 Therefore, alkaloids class of metabolites are always a most potent source of mainstream drug development and herein marine alkaloids also showed potential activity for SARS-CoV-2-Mpro, computationally.
In the present scenario, the CADD platform is a time and costsaving method in the ongoing drug discovery module to select potential lead candidates at an early stage. Mainly, the computational screening method locates any bioactive candidate based on lower binding energy known as docking scores represented in kilocalorie per mole unit and recently several natural anti-SARS-CoV-2 products were also highlighted/recognized through the computational program. 13,[15][16][17] Furthermore, before synthesis and expensive experimental validation, primary molecular interaction studies with molecular docking-simulation for biological activity and other drug-likeness predictions directly help to reduce resources and time compared to ongoing traditional drug selection procedures. 13,[15][16][17]22 Thus, the CADD/ artificial intelligence is a promising platform for the rapid selection of most potential anti-CoV candidates from various conventional/ existing sources for further pharmacological/clinical validation. [15][16][17] F I G U R E 7 Frontier molecular orbitals (FMOs) analysis in the form of LUMO, HOMO, and their energy gap (ΔE in kcal/mol) were predicted for two potent candidates along with two repurposing anti-HIV drugs in Avogadro-ORCA planform. The red color spear represents positive electron density and the blue color for negative electron density spear The toxicity profile is a significant obstacle for any therapeutic agent. Accordingly, the maximum number of lead drug candidates are withdrawing in different clinical trial stages due to side effects. In alkaloid cases, nitrogen atoms in the alkaloid moiety possess higher toxicity than other natural chemical classes such as terpenoids and flavonoids, primarily in higher concentrations. 6,11 Nevertheless, most F I G U R E 8 The overall computational prediction associated with structural activity relationships of potential manzamine alkaloid class of derivatives against SARS-CoV-2-Mpro alkaloids showed potential biological activities at a lower concentration. 18,44,45 Drugs such as rifampicin, tigecycline, vancomycin, darunavir and lopinavir have been approved by the FDA despite violations of the RO5. For example, rifampicin, tigecycline, vancomycin, even darunavir, and lopinavir. Sometimes, strict implementation of the RO5 opposes specific bioactive compounds where opportunities exist.
Therefore, a negotiation between the biological activity and physiological profile of any bioactive molecules should be needed during selection or undergoes further formulation toward fulfilling the ideal pharmacokinetics. 46,47 Overall, the cost-effective computational RO5 prediction in the early stage is more helpful for current drug development modules, but selection only through RO5 is challenging. 47,48 Simultaneously, the pharmacokinetics profile also plays a crucial role in the dose formulation of a newer drug, which is associated with physicochemical property or RO5. Briefly, the transport protein present in the GI tract resides is fully responsible for facilitating the transport of nutrition/drugs across the intestine. 6,49,50 Similarly, p-gp is another factor associated with drug distribution across the cell membrane/efflux-membrane transporter, directly linked to optimal drug delivery. 51,52 Overall, the computational prediction has reduced the resource and cost of selecting ideal lead candidates by filtering the essential drug parameters. [52][53][54][55] Concludingly, this cost-effective, resource-saving, and highly efficient computational screening is helpful to locate several repurposing candidates like marine alkaloids toward control of gruesome SARS-CoV in this health emergency.

| CONCLUSIONS
In the present scenario, exploring natural regimens like marine alkaloids based on previous pharmacological reports to combat SARS-CoV-2 is provisionally an ideal approach. Herein, 57 antiviral marinealkaloids were examined through bioinformatics and cheminformatics tools followed by a step-by-step procedure in the CADD platform.
Primarily, based on docking scores, two marine alkaloids manzamine A (À10.2 kcal/mol) and 8-hydroxymanzamine (À10.5 kcal/mol) were the most potential candidates than drug darunavir (À7.9 kcal/mol) and Strategically, CADD could be a promising platform to recognize possible natural "lead" drug candidates against SARS-CoV-2 at a primary level than the traditional hit-and-trial to accelerate anti-CoV drug discovery.