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.
© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.

The signaling pathway of transforming growth factor-beta (TGFβ) 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 TGFβ 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 (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-TGFβ signaling agents.

Protein-protein interactions (PPIs) have emerged as promising yet challenging therapeutic targets. A robust bioassay is required for rapid PPI modulator discovery. Here, we present a time-resolved Förster's (fluorescence) resonance energy transfer assay protocol for PPI modulator screening in a 1536-well plate format. We use hypomorph SMAD4R361H-SMAD3 PPI as an example to illustrate the application of the protocol for screening of variant-directed PPI inducers. This platform can be readily adapted for the discovery of both small-molecule PPI inducers and inhibitors. For complete details on the use and execution of this protocol, please refer to Tang et al. (2020).
© 2021.

Tumor suppressor genes represent a major class of oncogenic drivers. However, direct targeting of loss-of-function tumor suppressors remains challenging. To address this gap, we explored a variant-directed chemical biology approach to reverse the lost function of tumor suppressors using SMAD4 as an example. SMAD4, a central mediator of the TGF-β pathway, is recurrently mutated in many tumors. Here, we report the development of a TR-FRET technology that recapitulated the dynamic differential interaction of SMAD4 and SMAD4R361H with SMAD3 and identified Ro-31-8220, a bisindolylmaleimide derivative, as a SMAD4R361H/SMAD3 interaction inducer. Ro-31-8220 reactivated the dormant SMAD4R361H-mediated transcriptional activity and restored TGF-β-induced tumor suppression activity in SMAD4 mutant cancer cells. Thus, demonstration of Ro-31-8220 as a SMAD4R361H/SMAD3 interaction inducer illustrates a general strategy to reverse the lost function of tumor suppressors with hypomorph mutations and supports a systematic approach to develop small-molecule protein-protein interaction (PPI) molecular glues for biological insights and therapeutic discovery.
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