TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS  

作　　者：Danielle Cicka Qiankun Niu Min Qui Kun Qian Eric Miller Dacheng Fan Xiulei Mo Andrey AIvanov Stefan GSarafianos Yuhong Du Haian Fu 

机构地区：[1]Department of Pharmacology and Chemical Biology,Emory University School of Medicine,Atlanta,GA 30322,USA [2]Graduate Program in Molecular and Systems Pharmacology,Laney Graduate School of Emory University,Atlanta,GA 30322,USA [3]Emory Chemical Biology Discovery Center,Emory University School of Medicine,Atlanta,GA 30322,USA [4]Winship Cancer Institute of Emory University,Atlanta,GA 30322,USA [5]Laboratory of Biochemical Pharmacology,Department of Pediatrics,Emory University School of Medicine,Atlanta,GA 30322,USA

出　　处：《Journal of Molecular Cell Biology》2023年第3期32-43,共12页分子细胞生物学报（英文版）

基　　金：supported in part by the Emory School of Medicine COVID Catalyst-I3 award(H.F.and S.G.S.);the NCI Emory Lung Cancer SPORE(P50CA217691)Career Enhancement Program(A.A.I.);Emory Initiative on Biological Discovery through Chemical Innovation(A.A.I.);R01AI167356(S.G.S.).

摘　　要：SARS-CoV-2,the coronavirus that causes the disease COVID-19,has claimed millions of lives over the past 2 years.This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle.The interaction between host transmembrane serine protease 2(TMPRSS2)and viral SPIKE protein is an important initial step in SARS-CoV-2 infection,offering an opportunity for therapeutic development of viral entry inhibitors.Here,we report the development of a time-resolved fluorescence/Förster resonance energy transfer(TR-FRET)assay for monitoring the TMPRSS2–SPIKE interaction in lysate from cells co-expressing these proteins.The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance.To enable large-scale compound screening,we further miniaturized the assay into 1536-well ultrahigh-throughput screening(uHTS)format.A pilot screen demonstrated the utilization of the assay for uHTS.Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein–protein interaction.

关 键 词：SARS-CoV-2 COVID-19 protein-protein interaction TMPRSS2 SPIKE high-throughput screening TR-FRET 

分 类 号：Q2[生物学—细胞生物学]