Neo‐Cysteine Molecular Glues for Targeting Mutated SMAD4 Protein

Abstract Neo‐protein‐protein interactions (neoPPIs), directed by genetic mutation‐encoded neo‐amino acid residues, represent a promising class of precision medicine targets. Small molecules can mimic genetic mutational effects, creating neo‐surfaces and acting as molecular glues to mediate neoPPIs and reprogram biological circuitry. This convergence of genomic alterations and chemical interventions highlights a strategy for targeting disease‐associated mutations using neo‐amino acid residue‐directed molecular glues. Among these, neo‐cysteine at the protein‐protein interaction (PPI) interface represents unique opportunities to develop covalent molecular glues. Despite this promise, identifying neo‐cysteine molecular glues (neoCMGs) remains challenging. Here, we report the discovery of a neoCMG through a systematic chemical screening approach, using SMAD4, a frequently mutated tumor suppressor gene, as a model system. We established a robust PPI biosensor assay for high‐throughput chemical screening, leading to the identification of neoCMG101. Biophysical and biochemical characterization revealed that neoCMG101 selectively and covalently modifies the neo‐C361 residue on SMAD4, enhance SMAD4‐R361C/SMAD3 PPI and restore SMAD‐dependent transcriptional activity. This work establishes the feasibility of leveraging neo‐cysteine‐directed molecular glues to restore mutant PPIs, supporting a generalizable strategy for identifying neoCMG hits through unbiased screening. Such an approach provides a framework for targeting mutation‐disrupted signaling networks in cancer and other diseases.