{"doi":"10.1093/jmcb/mjad068","title":"A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting the SMAD4–SMAD3–DNA complex","abstract":"The transforming growth factor-beta (TGFβ) signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFβ agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFβ 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 TGFβ pathway, would offer an alternative approach with significant therapeutic potential for anti-TGFβ 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-TGFβ signaling agents.","journal":"Journal of Molecular Cell Biology","year":2023,"id":347065,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":12,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.957,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1089014,"name":"Qianjin Li","orcid":null,"position":1,"is_corresponding":false},{"id":271940,"name":"Qiankun Niu","orcid":"0000-0003-1781-5436","position":2,"is_corresponding":false},{"id":273150,"name":"Min Qui","orcid":null,"position":3,"is_corresponding":false},{"id":254877,"name":"Haian Fu","orcid":"0000-0002-2362-7979","position":4,"is_corresponding":false},{"id":273149,"name":"Yuhong Du","orcid":null,"position":5,"is_corresponding":false},{"id":271941,"name":"Xiulei Mo","orcid":"0000-0002-7558-3453","position":6,"is_corresponding":false},{"id":1089013,"name":"Wukun Ouyang","orcid":null,"position":0,"is_corresponding":true}],"reference_count":37,"raw_metadata":null,"created_at":"2026-07-19T01:11:57.720122Z","pmid":"37968137","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}