Establishment And Application Of Cell-based SARS-CoV-2 3CL Protease Activity Detection Assay

The coronavirus disease 2019 pandemic caused by the severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）has become a global catastrophe.Despite the great efforts made to control the pandemic,the continued emergence of SARS-CoV-2variants,particularly the emergence and spread of the Omicron variant with higher transmissibility,has brought great challenges to epidemic control.There are still few specific inhibitors for SARS-CoV-2,due to the long development cycle of new targeted inhibitors.The 3-chymotrypsin-like protease（3CLpro）is a main protease of SARS-CoV-2,and it has become a hot topic for its pivotal role in viral life cycle,high conservation and low mutation rate.Recently,some assays have been reported for 3CLpro assay and inhibitors screening.However,there are still some problems of susceptibility to promiscuous compounds and incompatibility to high throughput screening.Besides,concerns have also been raised about the potential for 3CLpro inhibitor resistance caused by the continuous emerging 3CLpro mutation in circulating SARS-CoV-2 variants.Therefore,it is urgent to develop a more reliable,sensitive,and facile 3CLpro assay,used for identification and analysis of 3CLpro inhibitors or inhibitors susceptibilities of3CLpro mutants.In this study,based on the finding that the 3CLpro-mediated cytotoxicity and reporter expression suppression can be reversed by an inactivating mutation or treatment with a specific inhibitor,a novel cell-based assay for measuring 3CLpro activity was established,which can quantitatively and sensitively detect the intracellular 3CLpro activity by using the gain-of-signal assay of orthogonal double reporter.Using this assay,positive inhibitors GC376 and PF-00835231 showed commendable dose-dependent inhibitory activity,while non-3CLpro-specific apoptosis or pyroptosis pathway inhibitors did not show inhibition on 3CLpro activity,indicating that the host cell death pathway inhibitors would not interference for assay.Furthermore,we performed high-throughput screening（HTS）of a total of 3785 compounds,including mini scaffold compound library,natural product library and protease inhibitor library,and three hits were identified,which have been reported as 3CLpro inhibitors.Among them S-217622 has the highest potency,whereas Boceprevir and Z-FA-FMK have only weak to moderate potency.In the 45compounds that have been reported to inhibit SARS-CoV-2 3CLpro,except for PF-07321332,GC376 and PF-00835231 and three hits identified in HTS,the other 39compounds,including Ebselen and Masitinib,show no inhibition of 3CLpro,suggesting that they may not be specific inhibitors of 3CLpro.In addition,seven prevalent 3CLpro mutants in circulating SARS-CoV-2 variants were also investigated for their activity and inhibitor susceptibility.It turns out that compared to the wild-type 3CLpro,the Omicron P132H mutant protease showed a substantial decrease in activity about 31%,and all investigated mutants showed IC₅₀ values comparable or lower than that against the wild-type 3CLpro for GC376,PF-00835231,S-217622 and PF-07321332.However,three mutations were identified to result in a decline in the slope of the dose-response curve for PF-07321332（P132H）or S-217622（G15S and T21I）,indicating some decrease in response at high concentrations and possible drug susceptibility alteration.In summary,an orthogonal dual reporter-based gain-of-signal assay was established for measuring 3CLpro activity in living cells.Compared with other reported assays,this assay is more reliable sensitive and convenient,and it can avoid nonspecific off-target effects of the compounds and signal interference from the compounds themselves.Using this method,we performed HTS and identified 3CLpro inhibitors,and investigated the influence of 3CLpro mutation on their activities and inhibitor sensitivities.Considering its utility and versatility,this assay will be conducive to further understanding of the function of 3CLpro and the research of novel 3CLpro targeted inhibitors.