Screening And Mechanism Study Of Natural Small Molecule Compounds Against SARS-CoV-2

In late 2019,SARS-CoV-2 suddenly struck and subsequently the virus became pandemic in various parts of the world.To date,many people have lost their lives in this epidemic worldwide,which seriously affects the global economy and public health and safety.SARS-CoV-2 infestation of hosts is dependent on a variety of host factors and viral protein that may be key targets for the prevention and treatment of viral infections.Natural compounds have been an important reserve for drug development and have been considered as a source of new lead compounds for the treatment of various diseases,including antiviral therapy,with great research and development value.The research involves the screening of a small molecule compound of plant origin with the aim of obtaining compounds of natural origin with anti-SARS-CoV-2,with the following main results:Ⅰ.Screening and mechanistic study of natural compounds that inhibit SARS-CoV-2 entryThe binding of viral spike protein to ACE2 protein is the first step of SARS-CoV-2invasion,followed by the entry of the virus into the cell in the form of membrane fusion mediated by the host membrane surface protease and in the form of cytokinesis not mediated by the membrane surface protease.SARS-CoV-2 invasion has been a key target for drug development as an important part of the viral life cycle.In this study,we performed a drug screen on a library of plant-derived compounds,which resulted in the identification of two compounds,Miyabenol C and trans-ε-Viniferin,with the ability to inhibit SARS-CoV-2 invasion.First,we screened the plant-derived compound library using HIV-1-based SARS-CoV-2 pseudovirus infection assay and dual split protein(DSP)assay and found that Miyabenol C and trans-ε-Viniferin specifically inhibited SARS-CoV-2 entry.Secondly,both resveratrol oligomers exhibited inhibition of histone protease CatL activity in pseudovirus infection of different cell lines assay and in vitro fluorescence kinetics assay.SARS-CoV-2 mutant pseudovirus infection assay revealed that Miyabenol C and trans-ε-Viniferin exhibited broad spectrum inhibition of mutant pseudovirus infection.In addition,molecular docking experiments showed that two resveratrol oligomers could occupy the active cavity of CatL to exert inhibitory activity.Surface plasmon resonance(SPR)experiments,revealed that the equilibrium dissociation constants(K_D)of Miyabenol C-CatL and trans-ε-Viniferin-CatL were 5.54μmol/L and8.54μmol/L,respectively.This new finding clarifies that Miyabenol C and trans-ε-Viniferin inhibit SARS infection by targeting CatL to block the function of SARS-CoV-2 entry.Ⅱ.Inhibition of SARS-CoV-2 3CL protease compound screeningSARS-CoV-2 3CLpro is translated from the SARS-CoV-2 nsp5 gene.The cis-cleavage of the viral polyprotein by 3CLpro,which forms a single active protein,is essential for the SARS-CoV-2 viral genome to be able to complete subsequent processes after entering the cell,and inhibition of it’s activity can be effectively inhibit the proliferation of the virus eventually.In the present study,we performed an inhibitor screening of SARS-CoV-2 3CLpro based on the FRET principle,established a 3CLpro prokaryotic expression system and successfully expressed and purified the active 3CLpro for inhibitor screening.Meanwhile,the pc DNA3.1-N5-S-Luc plasmid was constructed in this study for simulating intracellular cis-cleavage of 3CLpro.Based on the FRET principle screening method,our preliminary screening of the plant compound library did not find any compounds that could inhibit3CLpro activity and the 3CLpro intracellular cis-cleavage model was not sensitive and needed subsequent optimization.Through the established screening system,we have identified some compounds from the small molecule library that can treat SARS-CoV-2 invasion and clarify their mechanism of action,which is of significance for the research and treatment of SARS-CoV-2.