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Identificationnand Optimization of Novel Small-MoleculenCas9 Inhibitors by Cell-Based High-Throughput Screening 基于细胞高通量筛选的新型小分子nCas9抑制剂的鉴定与优化

Figshare Pub Date : 1753-01-01 DOI: 10.1021/acs.jmedchem.1c01834.s002

Sang-Woo Lee (441256), Kim Tai Tran (12071447), Ruben Vazquez-Uribe (8539584), Charlotte Held Gotfredsen (8350599), Mads Hartvig Clausen (9667778), Blanca Lopez Mendez (12071450), Guillermo Montoya (22728), Anders Bach (412488), Morten Otto Alexander Sommer (2632267)

{"title":"Identificationnand Optimization of Novel Small-MoleculenCas9 Inhibitors by Cell-Based High-Throughput Screening","authors":"Sang-Woo Lee (441256), Kim Tai Tran (12071447), Ruben Vazquez-Uribe (8539584), Charlotte Held Gotfredsen (8350599), Mads Hartvig Clausen (9667778), Blanca Lopez Mendez (12071450), Guillermo Montoya (22728), Anders Bach (412488), Morten Otto Alexander Sommer (2632267)","doi":"10.1021/acs.jmedchem.1c01834.s002","DOIUrl":"https://doi.org/10.1021/acs.jmedchem.1c01834.s002","url":null,"abstract":"CRISPR/Cas9 has revolutionizednseveral areas of life science; however,nmethods to control the Cas9 activity are needed for both scientificnand therapeutic applications. Anti-CRISPR proteins are known to inhibitnthe CRISPR/Cas adaptive immunity; however, <i>in vivo</i> delivery of such proteins is problematic. Instead, small-moleculenCas9 inhibitors could serve as useful tools due to their permeable,nproteolytically stable, and non-immunogenic nature. Here, we identifiedna small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughputnscreening utilizing an Escherichia coli selection system. Extensive structure–activity relationshipnstudies, which involved a deconstruction–reconstruction strategy,nresulted in a range of analogues with significant improvements innthe inhibitory activity. Based on NMR and electrophoretic mobilitynshift assays, we propose that the inhibitory action of these compoundsnlikely results from direct binding to apo-Cas9, preventing Cas9:gRNAncomplex formation. These molecules may find use as Cas9 modulatorsnin various applications.","PeriodicalId":522727,"journal":{"name":"Figshare","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"1753-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147902300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

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