Hypomorph mutation-directed small-molecule protein-protein interaction inducers to restore mutant SMAD4-suppressed TGF-β signaling

Tumor suppressor genes represent a major class of oncogenic drivers. However, direct targeting of loss-offunction 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-beta 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 SMAD4(R361H) with SMAD3 and identified Ro-31-8220, a bisindolylmaleimide derivative, as a SMAD4(R361H)/ SMAD3 interaction inducer. Ro-31-8220 reactivated the dormant SMAD4(R361H)-mediated transcriptional activity and restored TGF-b-induced tumor suppression activity in SMAD4 mutant cancer cells. Thus, demonstration of Ro-31-8220 as a SMAD4(R361H)/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.

Automated summary

A variant-directed chemical biology approach was explored to reverse the lost function of tumor suppressors, using SMAD4 mutation as an example, and Ro-31-8220 was identified as a compound that could induce interactions between SMAD4(R361H) and SMAD3, reinstating tumor suppression activity in mutant cancer cells.