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

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

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The transforming growth factor-beta(TGF β)signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment,making anti-TGF β agents a significant area of interest in cancer immunotherapy.However,the clin-ical translation of current anti-TGF β agents that target upstream cytokines and receptors remains challenging.Therefore,the development of small-molecule inhibitors specifically targeting SMAD4,the downstream master regulator of the TGF β pathway,would offer an alternative approach with significant therapeutic potential for anti-TGF β signaling.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 anti-TGF β signaling agents.

TGFβ/SMAD4 signalinghigh-throughput screeningTR-FRET

Wukun Ouyang、Qianjin Li、Qiankun Niu、Min Qui、Haian Fu、Yuhong Du、Xiulei Mo

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Department of Pharmacology and Chemical Biology,Emory University School of Medicine,Atlanta,GA 30322,USA

Emory Chemical Biology Discover y Center,Emor y University School of Medicine,Atlanta,GA 30322,USA

Department of Hematology and Medical Oncology and Winship Cancer Institute,Emory University,Atlanta,GA 30322,USA

National Cancer Institute(NCI)MERIT AwardNCI Emory Lung Cancer SPORECareer Enhancement ProgramNCI Emory Lung Cancer P01NCI Office of Cancer Genomics Cancer Target Discovery and Development(CTD2)initiative networkWinship Cancer Institute

R37CA255459P50CA217691P50CA217691P01CA257906U01CA217875NIH 5P30CA138292

2023

10.1093/jmcb/mjad068

分子细胞生物学报(英文版)
中国科学院上海生命科学研究院,生物化学与细胞生物学研究所,中国细胞生物学学会

分子细胞生物学报(英文版)

CSTPCDCSCD
影响因子：0.595
ISSN：1673-520X
年,卷(期)：2023.(11)
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