MOLECULAR DOCKING STUDY TO IDENTIFY POTENTIAL INHIBITOR OF COVID-19 MAIN PROTEASE ENZYME: AN IN-SILICO APPROACH
Coronavirus became pandemic very soon and is a potential threat to human lives across the globe. No approved drug is currently available therefore an urgent need has been developed for any antiviral therapy for COVID-19. For the molecular docking study, ten herbal molecules have been included in the current study. The three-dimensional chemical structures of molecules were prepared through ChemSketch 2015 freeware. Molecular docking study was performed using AutoDock 4.2 simulator and Discovery studio 4.5 was employed to predict the active site of target enzyme. Result indicated that all-natural molecules found in the active site of enzyme after molecular docking. Oxyacanthine and Hypericin (-10.990 and -9.05 and kcal/mol respectively) have shown good binding efficacy among others but Oxyacanthine was the only natural product which made some of necessary interactions with residues in the enzyme require for target inhibition. Therefore Oxyacanthine may be considered to be potential inhibitor of main protease enzyme of virus but need to be explored for further drug development process.
<!>Material and Methods<!>Results and Discussion<!>CONCLUSION
<p>However, information about intermediary reservoir is still unknown [1]. Coronaviruses (CoVs) are the enveloped positive stranded RNA viruses, supposed to be possessing largest viral RNA genome known till date [2,3]. As per the recent situational report released by World Health Organization (WHO) on March 28, 2020, 571678 COVID-19 cases have been confirmed globally, including 62514 new cases and total 26494 deaths [4]. SARS-CoV-2 a highly infectious and pathogenic coronavirus has become global concern [5].</p><p>Recently sequence analysis confirmed that the SARS-CoV-2 belongs to the class of β-coronaviruses including SARS-CoV and MERS-CoV. [6)]. Since, SARS-CoV-2 is novel pathogenic coronavirus, till date no effective antiviral therapy is available. Preventive and supportive therapies are main approach for management of COVID-19. Around the globe researchers are endeavoring for the development of effective prevention and treatment strategies for COVID-19. Preliminary studies have suggested potential repurposing of available anti-viral drugs like protease inhibitors lopinavir/ritonavir, nucleoside analogues, neuraminidase inhibitors, remdesivir, umifenovir (arbidol), tenofovir disoproxil (TDF), and lamivudine (3TC), etc. for the treatment of COVID-19 infected patients [7]. In another study Camostat mesylate, a clinically approved TMPRSS2 inhibitor was found to block SARS-CoV-2 entry to human cells, indicating its therapeutic potential as a drug against COVID-19 [8]. Xu et al. (2020) in another investigation screened 1903 approved drugs by homology modeling, molecular docking and binding free energy calculation and suggested nelfinavir as potential inhibitor against SARS CoV-2 [9].</p><p>A sizeable body of data demonstrates anti-coronaviral effect from several natural products and herbal medicines. In separate investigations Phenolic compounds of Isatisindigotica, Amentoflavone isolated from Torreya nucifera and Houttuyniacordata water extract have shown SARS-CoV 3CL protease enzyme inhibitory potential against SARS-CoV. (10)(11)(12) Since the Main Protease M pro or Chemotrypsin-like-protease (3CL) which is highly conservable among SARS CoV and SARS-CoV-2 proved by sequential analysis; is suggested to be potential target to fight SARS-CoV-2 [6]. Recently, Khaerunnisa et al, reported M pro Inhibitory potential of kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, and epicatechin-gallate against COVID-19 [13]. Prompted by this, in present study we investigated Caffeine, Capsaicin, Hypericin, Gossypol, Luteolin, Berberine, Embelin,Oxyacanthine, Sanguinarine and Emodin as potential inhibitor candidates for SARS-CoV-2 M pro with the help of molecular docking study.</p><!><p>The crystal structure of the molecular target, protease enzyme (COVID-19 3cl pro /M pro (PDB ID: 6LU7), was retrieved from RCSB protein data bank (https://www.rcsb.org/) [14]. Target needs to be prepared before starting the molecular docking process, which involves removal of the water molecules and native ligand attached with target and other heteroatoms which may provide hindrance in the simulation. Besides, hydrogen atoms were added into target. These all processes were carried out in the Auto Dock window execution file. Investigational ligands were designed using ChemSketch (ACD 2012) and optimized for energy minimization using MM2 force field and saved in.mol format subsequently converted into .pdb format by Open Bable -2.3.2 software. The investigation ligand was loaded and their torsion salong with rotatable bonds are assigned and the files are saved as ligand. PDBQT. In the current study, identification of binding modes of the herbal molecules with targetwas identified using Auto Dock 4.2 software program. Moreover, to confirm actual binding interaction with targets blind docking was performed and the best conformers were represented with lowest binding energy (-kcal/mol) which might pave the way to disclose the mode of actions of these ligands. The docking parameters were defined as coordinates of the center of binding site with x = 126, y = 126, z = 126 and binding radius = 0.375 Å. All AutoDock output file (.dlg) were then analyzed through Analysis option provided in MGLTools-1.5.6 rc3. Top-scoring molecules in the largest cluster were analyzed.</p><p>Conformers of the ligand were automatically docked to the enzymes and most stable conformer in terms of binding affinity (most negative) was used for post-docking analysis.</p><!><p>Table1 shows the ligands structures and necessary hydrogen bond formation by ligands with targets required for inhibition of target enzyme of covid-19. The rationale of choosing these ligands is only that they have shown efficacy in preclinical experimental models in different diseases. The SARS coronavirus main peptidase (SARS-CoVMpro) plays an essential role in the life cycle of the virus and is a primary target for the development of effective antiviral agents in case of COVID-19 infection. Figure 1 indicates the active site of target to confirm the binding site of investigational ligands if they were interacted with the amino acids in the active site of target or found to be attached any other site inside the target. The active site of this target enzyme comprises The active site of this target enzyme is composed of HIS41, MET 49, PHE140, LUE141, ASN142, GLY143, HIS163, HIS164, MET165, GLU166, LEU167, PRO168, HIS172, GLN189, THR190, ALA191 (Figure 1).</p><p>The best poses of natural ligands and enzyme complexes are described, and the number of hydrogen bonds formed by both proteins with the drug are displayed in figure 2(a-j) as well as in the table1.</p><p>Figure1: Active site of target enzyme.  GLY143, HIS163, HIS164, GLU166, GLU189 and THR190. In the current study, molecular docking analysis reveals that although all-natural ligands interacted in the active site of target enzyme but some of them could not interact through hydrogen bonds with target which are required for enzyme inhibition.Caffeine is a CNS stimulant, exhibited least binding affinity (-4.520 kcal/mol) towards enzyme but could afford to form required some hydrogen bonds with GLY143 and HIS163 amino acids residues of enzyme. Berberine is an alkaloid, showed slightly good binding energy (-7.910 kcal/mol) than that of Caffeine but could interact with enzyme through different amino acid viz. ASP187 and produced different conformational changes in the target enzyme after binding than that of caffeine (see figure2 b and 3b).</p><p>Although Luteolin exhibited moderate binding affinity (-6.890 kcal/mol) to target but could not make mandatory hydrogen bonds with enzyme for demonstrating inhibitory activity and made hydrogen bonds with ASP187 and GLU192 amino acids residues of enzyme and revealed quite similar conformational changes in the target enzyme (see figure 4b).</p><p>Therefore, Berberine and Luteolin could not be used as inhibitors of main protease enzyme.</p><p>Sanguinarine also demonstrated a good binding energy but could not afford any hydrogen bond with virus enzyme. Embelin, Capsaicin, Emodin and Gossypol were found to be in the active site and interacted with enzyme through requisite hydrogen bonds but could not exhibit good binding affinity toward target enzyme (-4.940 kcal/mol, -5.510 kcal/mol, -6.490 kcal/mol and -6.290 kcal/mol respectively). Hypericin is a naphthodianthrone, an anthraquinone derivative expressed better binding affinity i.e. -9.050 kcal/mol but made unsuitable hydrogen bond with ASN142 residue in the active site of target enzyme also made a necessary hydrogen bond with HIS 164 residue required for enzyme inhibition.Oxyacanthine displayed the highest binding affinity those of among other ligands involved in the study i.e. -10.990 kcal/mol and produced in the significant changes in the conformations of main protease enzyme (see figure.11b).</p><!><p>A new strain of coronavirus has now been extended from Wuhan city of China to the other parts of world for which not any approved medications are existed. Therefore, from the current molecular docking study it has been concluded that Oxyacanthine can act as Mpro inhibitor but need to be explored for more experimental work regarding development of anti SARS-Mpro inhibitor.</p>