Neo‐Cysteine Molecular Glues for Targeting Mutated SMAD4 Protein

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.