A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting the SMAD4–SMAD3–DNA complex  

作　　者：Wukun Ouyang Qianjin Li Qiankun Niu Min Qui Haian Fu Yuhong Du Xiulei Mo 

机构地区：[1]Department of Pharmacology and Chemical Biology,Emory University School of Medicine,Atlanta,GA 30322,USA [2]Emory Chemical Biology Discovery Center,Emory University School of Medicine,Atlanta,GA 30322,USA [3]Department of Hematology and Medical Oncology and Winship Cancer Institute,Emory University,Atlanta,GA 30322,USA

出　　处：《Journal of Molecular Cell Biology》2023年第11期21-32,共12页分子细胞生物学报（英文版）

基　　金：supported by the National Cancer Institute(NCI)MERIT Award(R37CA255459 to X.M.);NCI Emory Lung Cancer SPORE(P50CA217691 to H.F.)Career Enhancement Program(P50CA217691 to X.M.);NCI Emory Lung Cancer P01(P01CA257906 to H.F.);NCI Office of Cancer Genomics Cancer Target Discovery and Development(CTD2)initiative network(U01CA217875 to H.F.);Winship Cancer Institute(NIH 5P30CA138292).

摘　　要：The transforming growth factor-beta(TGFβ)signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment,making anti-TGFβagents a significant area of interest in cancer immunotherapy.However,the clinical translation of current anti-TGFβagents that target upstream cytokines and receptors remains challenging.Therefore,the development of small-molecule inhibitors specifically targeting SMAD4,the downstream master regulator of the TGFβpathway,would offer an alternative approach with significant therapeutic potential for anti-TGFβsignaling.In this study,we present the development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer(TR-FRET)assay in an ultrahigh-throughput screening(uHTS)1536-well plate format.This assay enables simultaneous monitoring of the protein–protein interaction between SMAD4 and SMAD3,as well as the protein–DNA interaction between SMADs and their consensus DNA-binding motif.The multiplexed TR-FRET assay exhibits high sensitivity,allowing the dynamic analysis of the SMAD4–SMAD3–DNA complex at single-amino acid resolution.Moreover,the multiplexed uHTS assay demonstrates robustness for screening small-molecule inhibitors.Through a pilot screening of an FDA-approved bioactive compound library,we identified gambogic acid and gambogenic acid as potential hit compounds.These proof-of-concept findings underscore the utility of our optimized multiplexed TR-FRET platform for large-scale screening to discover small-molecule inhibitors that target the SMAD4–SMAD3–DNA complex as novel antiTGFβsignaling agents.

关 键 词：TGFβ/SMAD4 signaling high-throughput screening TR-FRET 

分 类 号：O657.3[理学—分析化学]