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

The signaling pathway of transforming growth factor-beta (TGF{beta}) plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGF{beta} agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGF{beta} agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small molecule inhibitors specifically targeting SMAD4, the downstream master regulator of TGF{beta} pathway, would offer an alternative approach with significant therapeutic potential for anti-TGF-{beta} 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 (PPI) between SMAD4 and SMAD3, as well as the protein-DNA interaction (PDI) 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 and 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{beta} signaling agents.