Development Of A Simple Sandwich-Like Fuorescence Polarization Assay For Rapid Screening Of SARS-CoV-2 Main Protease Inhibitors

Objective:SARS-Co V-2 main protease（Mpro）is a highly conserved coronaviral en-zyme,which plays a pivotal role in viral replication and host immune evasion,making it an attractive target for the development of broad-spectrum anti-coronaviral agents.In this study,we first combined the fluorescence polarization（FP）technique with biotin-avidin system（BAS）to develop a simple sandwich-like FP assay for rapid screening of novel Mpro inhibitors from a natural product library.Methods:A codon-optimized SARS-Co V-2 Mpro gene was ligated into a p ET-28a vec-tor to construct a recombinant plasmid termed by p ET-21a-Mpro.After transformation into E.coli Rosetta（DE3）competent cells,the soluble Mpro was expressed under a low tempreture condition and further tested by the SDS-PAGE assay.Subsequently,the sol-uble Mpro was purified by a His Trap^TM column,and the enzymatic activity and mich-aelis constant（K_m）value of the pure Mpro were determined by a fluorescence resonance energy transfer（FRET）assay.The pure Mpro was used as immunogen to inoculate rats.The titer,selectivity,and sensitivity of polyclonal antibody against Mpro were analyzed using the enzyme-linked immunosorbent assay（ELISA）and Western blot assay.Ac-cording to the FP principle,a fluorescein isothiocyanate（FITC）and biotin dual-labeled peptide termed FITC-Substrate-Biotin was used as a tracer in Mpro enzyme reaction.The tracer,avidin,and Mpro working concentration were optimized,and the reaction conditions（incubation temperature,time,and DMSO tolerance）have been investigated for hit compounds screening from a natural product library.In addition,an in vitro in-hibition effect of hit compounds on Mpro enzyme was determined by the surface plasmon resonance（SPR）,dimerization-dependent red fluorescent protein（dd RFP）,FRET,and molecular docking assays,respectively.Results:The DNA sequence alignment and double digestion assay results showed that the recombinant plasmid p ET-21a-Mpro is successfully constructed.The SDS-PAGE assay showed that the soluble Mpro is highly expressed in E.coli cells,and the purity of purified Mpro is more than 90%.Importantly,the FRET assay showed that the pure Mpro exhibits a desirable enzymatic activity（40 000 U/mg）,and the K_m value was19.78μmol/L.After three immunization cycles,the ELISA and Western blot assay demonstrated that the highly sensitive polyclonal antibody against Mpro could specially recognize Mpro protein,and the titer could reach 1:256 000.Through the systematic optimizations,a sandwich-like FP screening assay was performed using 20 nmol/L tracer,0.2μmol/L Mpro,and 50 nmol/L avidin,and a high Z′factor of 0.85 was achieved.With this screening assay,dieckol was identified as a novel competitive Mpro inhibitor in vitro.Additionally,dieckol exhibited a high affinity with Mpro using SPR assay,and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model.Conclusion:Through the codon-optimization strategy,the soluble Mpro is successfully expressed in E.coli cells,which exhibits an ideal enzymatic activity.This simple,ro-bust,and reliable sandwich-like FP screening assay enables the rapid discovery of an-tiviral agents targeting viral protease.A natural product dieckol provides an excellent starting point to generate more potent antiviral agents targeting Mpro.