High-Throughput Virtual Screening And In Vitro Evaluation Of Inhibitors Against SARS-CoV-2 Multi-Target Protease

The rapidly evolving Coronavirus Disease 2019（COVID-19）pandemic that was caused by the severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）,has spread worldwide with thousands of deaths and infected cases.For the identification of effective treatments against this disease,the main protease(M^pro)of SARS-CoV-2（PDB id:6LU7）and the papain-like protease(PL^pro)of SARS-CoV-2（PDB id:6WX4）were found to be an attractive drug target,as it played a central role in viral replication and transcription.In this study,natural product library was establish including 1,045,468 ligand structures.These ligands were screened by high-throughput molecular docking to find the potential inhibitors of SARS-CoV-2 M^pro and PL^pro.Then,SARS-CoV-2 M^pro-ligand and SARS-CoV-2 PL^pro-ligand complex were constructed and analyzed their characteristics of the combination.The molecular docking results of the ligand and M^pro showed that 465 promising candidates were obtained with high binding affinities,compared with the comparison drug（conivaptan）.The 465 candidates could be divided into 11 classes by their scaffolds,including biflavones,anthraquinones,flavonoids,polyphenols,phenylpropanoids,terpenoids,stilbenes,isoflavonoids,xanthones,flavonolignans,and alkaloids.Then,the binding characteristics of each class and active pocket of SARS-CoV-2 were analyzed in this work.Pharmacokinetic prediction using ADMET（absorption,distribution,metabolism,excretion and toxicity）and drug-like properties were used to screen the ligands with good docking affinity to obtain the potential inhibitors with good bioavailability.Molecular Dynamics（MD）simulation was an efficient method to predict the stability of the inhibitor-protein complex.Basing on these simulation results,23 kinds of Chinese herbal extracts were employed to study their inhibitory activity for M^pro of SARS-CoV-2.Plants extracts from Forsythiae Fructus,Radix Puerariae,Radix astragali,Anemarrhenae Rhizoma showed acceptable inhibitory efficiencies,which were over 70%.The best candidate was Anemarrhenae Rhizoma,reaching 87.3%.Then,18 promising candidates were selected with the higher binding affinities than reference drug（doxycycline）by molecular docking between ligands and PL^pro of SARS-CoV-2.The results analyzed the key amino acids with in the active pocket of SARS-CoV-2 PL^pro related to the high activity and selectivity of ligand compounds,which were beneficial to design inhibitors against PL^pro.Further,these ligands have been analyzed ADMET and Lipinski’rule to evaluate their drug likeliness.The results implied that magnolol and its derivatives have good Caco-2 and human oral bioavailability values,which means that this compound can be well absorbed in the body.The results of this study can provide a theoretical foundation for the design and modification of multitarget drug of SARS-CoV-2 proteases(M^pro and PL^pro).For example,bisflavonoids had good docking affinity for both M^pro and PL^pro,which indicated that the structure of bisflavonoids can effectively contact the active pocket.It can modify molecular compounds to improve biological activity.At the same time,the enzyme inhibitory activity of plant extracts can screen the effective potential antiviral drugs,health foods or dietary supplements,which provided references for subsequent experiments and clinical applications.