Pharmacophore-based Virtual Screening And Biological Evaluation Of Potent SARS-CoV-2 Main Protease Inhibitors

An outbreak of COVID-19 is a disease caused by the severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）,has posed an extraordinary threat to global public health since December 2019.There are currently some potential drugs that could be used to treat infections caused by SARS-CoV-2,such as Paxlovid,remdesivir,and others.SARS-CoV-2is a member of the Bβcoronavirus,with the viral RNA genome containing 10 open reading frames（ORFs）that encodes for structural proteins.In the lifecycle of the virus,the M^prois essential for processing the polyproteins translated from the viral RNA,and it is one of the two crucial hydrolytic enzymes（pp1a and pp1b）of the virus,which can inhibit the activity of M^proto blocks the Replication of virus.Therefore,M^proprotease plays a key role in mediating the replication and transcription of viral genes.Furthermore,M^prohas become an attractive target for antiviral drug design due to SARS-CoV-2 M^proprotein lack of closely related homologues in humans.Method:Taking SARS-CoV-2 M^proas the research target,we screened the potential SARS-CoV-2 M^proinhibitors from the natural compound library through virtual screening of pharmacophore model,molecular docking and molecular overlap.The compounds with potential inhibitory activity are further evaluated based on the fluorescence resonance energy transfer（FRET）assay.Content:Initially,Preliminary screening of the natural product library of natural compounds by using Lipinski of Five and Veber rules.Then,based a series of active compounds with SARS-CoV-2 obtained from the literature,generate the pharmacophore hypotheses using Hypo Gen algorithm of DS,which has the ability to quantitatively predict activity.The pharmacophore models were evaluated by relevant parameters,training set and test set,molecular superimposition results of GC-376,Fisher’s verification and so on.the Hypo1 pharmacophore model was determined as the optimal model,and the natural compound library was virtually screened using Hypo1.The crystal structure of the SARS-CoV-2 M^pro/GC-376 complex（PDB code:7D1M）was used as the target,and molecular docking was performed using the CDOCKER algorithm in the DS software to analyze the binding mode of compounds with the active site of 7D1M.The conformations of compounds aligned with Hypo1 were superimposed with their docked conformations and the crystallographic active conformation of the known SARS-CoV-2 M^proinhibitor GC-376.Potential SARS-CoV-2 M^proinhibitors were screened based on the superimposition,and their enzymatic activities were evaluated.The docking results of the compounds with good activity were compared with the binding mode of the original ligand GC-376.Result:The results are analyzed through a series of pharmacophore based virtual screening,molecular docking,and molecular superposition techniques.Finally,Seven lead compounds were selected.The activity evaluation results showed that the IC50 of compounds W-1,W-7,and W-6 were 63.31μM,74.59μM,and 253.7μM,respectively.Further analysis of the molecular docking results of compounds W-1,W-6,and W-7,and comparison with the binding mode of GC-376 and SARS-CoV-2 M^pro,showed that,these compounds formed a p-πconjugate with the imidazole ring of the active site His41 similar to GC-376,and formed hydrogen bonds with the amino acid residues Gly143,Cys145,His164,Glu166,and Gln189of SARS-CoV-2 M^pro,which are believed to play a key role in the inhibitory activity of SARS-CoV-2 M^proinhibitors.Conclusion:This study constructed a pharmacophore model based on the SARS-CoV-2M^proinhibitors,and further screened 7 lead compounds through molecular docking and molecular superimposition.Among them,compounds W-1,W-7 and W-6 showed IC50values of 63.31μM,74.59μM and 253.7μM,respectively.Compared with the known SARS-CoV-2 M^proinhibitor GC-376,these three compounds exhibited similar binding modes and binding sites with the active site of SARS-CoV-2 M^pro.Therefore,they are potential novel SARS-CoV-2 M^proinhibitors.