High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors

The global COVID-19 coronavirus pandemic has infected over 109 million people,leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment.Here,we screened about 1.8 million small molecules against the main protease(M^(pro))and papain like protease(PL^(pro)),two major proteases in severe acute respiratory syndrome-coronavirus 2 genome,and identified 1851M^(pro)inhibitors and 205 PL^(pro)inhibitors with low nmol/l activity of the best hits.Among these inhibitors,eight small molecules showed dual inhibition effects on both M^(pro)and PL^(pro),exhibiting potential as better candidates for COVID-19 treatment.The best inhibitors of each protease were tested in antiviral assay,with over 40%of M^(pro)inhibitors and over 20%of PL^(pro)inhibitors showing high potency in viral inhibition with low cytotoxicity.The X-ray crystal structure of SARS-CoV-2 M^(pro)in complex with its potent inhibitor 4a was determined at 1.8Åresolution.Together with docking assays,our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors

A new coronavirus(SARS-CoV-2)has been identified as the etiologic agent for the COVID-19 outbreak.Currently,effective treatment options remain very limited for this disease;therefore,there is an urgent need to identify new anti-COVID-19 agents.In this study,we screened over 6,000 compounds that included approved drugs,drug candidates in clinical trials,and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease(PLpro).Together with main protease(Mpro),PLpro is responsible for processing the viral replicase polyprotein into functional units.There-fore,it is an attractive target for antiviral drug develop-ment.Here we discovered four compounds,YM155,cryptotanshinone,tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 pmol/L.These compounds also exhibit strong antiviral activities in cell-based assays.YM155,an anti-cancer drug candidate in clinical trials,has the most potent antiviral activity with an EC50 value of 170 nmol/L.In addition,we have determined the crystal structures of this enzyme and its complex with YM155,revealing a unique binding mode.YM155 simultaneously targets three"hot"spots on PLpro,including the substrate-binding pocket,the interferon stimulating gene product 15(ISG15)binding site and zinc finger motif.Our results demonstrate the efficacy of this screening and repur-posing strategy,which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

OsMADS32 interacts with PI-like proteins and regulates rice flower development

OsMADS32 is a monocot specific MIKC^c type MADS-box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clari fied. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1-3 and identi fied its function in controlling rice flower development by bioinformatics and protein-protein interaction analysis. The cfo1-3 mutant produces defective flowers,including loss of lodicule identity, formation of ectopic lodicule or hull-like organs and decreased stamen number,mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis-splicing of OsMADS32 transcripts in the cfo1-3 mutant resulted in an extra eight amino acids in the K-domain of OsMADS32 protein.By yeast two hybrid and bimolecular fluorescence complementation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of Os MADS32 affects the interaction between OsMADS32 with PISTILLATA(PI)-like proteins OsMADS2 and OsMADS4. This work provides new insight into the mechanism by which Os MADS32 regulates rice lodicule and stamen identity, by interaction with two PI-like proteins via its K domain.