SUMMARY Tumor suppressor genes represent a major class of oncogenic “drivers” and offer robust window for therapeutic intervention. However, direct targeting loss-of-function tumor suppressor genes remains challenging, because that majority of tumor suppressors do not have enzymatic activity and exert their normal function through protein- protein interaction (PPI). Noteworthy, a unique class of tumor suppressor mutations are missense mutations encoding single amino acid substitutions that impair the normal PPI. These tumor suppressor mutations are defined as “loss-of-interaction” mutation. We aim to directly target the “loss-of-interaction” tumor suppressor mutations through discovery of small molecule PPI inducers to restore their anticancer functions. SMAD4 is such a tumor suppressor with “loss-of-interaction” mutations in cancer that disrupt its normal PPI with SMAD3. Using SMAD4 as a proof-of-concept study, we propose to utilize our newly developed TR-FRET SMAD4-SMAD3 PPI screening platform to reveal novel small molecule mutant SMAD4-PPI inducer (MuSMADid) that can induce the mutant SMAD4 PPI with SMAD3 and restore the pathway and cellular response to the tumor suppressive TGF-b signaling. Preliminary studies showed that the SMAD4-SMAD3 TR-FRET assay is robust and scalable in 1536-well uHTS format and is sensitive to monitor the SMAD4-SMAD3 PPI dynamic at single amino acid resolution. From a bioactive chemical library, Ro-31-8220, a bisindolylmaleimide derivative, was identified as potential MuSMADid that induced the mutant SMAD4 PPI with SMAD3 and restored the responsiveness of SMAD4 mutant colon cancer cells to the TGF-b anti-proliferation signaling. Identification of Ro-31-8220 as a potential MuSMADid provides strong evidence for direct targeting “loss-of-interaction” SMAD4 mutations. Together, this preliminary data supports our central premise that novel chemical probes can be discovered as potential MuSMADid by leveraging the established uHTS TR-FRET assay to screen structurally diverse chemical libraries. Based on the stages of discovery research, our proposal will focus on Aim 1 “Primary Screen Implementation” to identify MuSMADid hits with new chemical scaffolds, followed by verification with orthogonal PPI assays, and on Aim 2 “Functional Validation” to prioritize a list of validated novel small molecule anti-tumor MuSMADid for future hit-to-lead optimization phase. We will use the uHTS TR-FRET platform to rapidly identify primary hits and validated hits followed by characterization of their PPI induction and cellular activities in restoring the TGF-b tumor suppressive signaling. Accomplishing the goals of the proposed study is anticipated to generate a list of prioritized and confirmed small molecule MuSMADid compounds that show potent biochemical and biological activities in inducing the mutant SMAD4-SMAD3 PPI and restoring the TGF-b anti-proliferation signaling pathways. Top ranked anti-tumor MuSMADid with the strongest structura...