Developing COUPLrs, dual-warhead cysteine-reactive compounds, to target oncoproteins through interaction modulation and degradation

COUPLrs (COvalent Protein Ligators) are cysteine-reactive small molecules designed to dimerize or disrupt protein complexes, thereby modulating protein function. In our previous work, we identified COUPLrs that bind to the oncogenic fusion protein EML4-ALK in non-small cell lung cancer (NSCLC) cells. These compounds engaged the EML4 rather than the ALK kinase domain targeted by clinical inhibitors. This unconventional binding disrupted ALK signaling and induced proteasome-dependent degradation of the fusion protein, establishing proof of concept that COUPLrs can engage two protein partners simultaneously, altering their interactions and stability. These findings define COUPLrs as a new mechanistic class of molecules capable of bridging or disrupting protein complexes and driving selective degradation, offering opportunities to target proteins lacking conventional binding pockets.To systematically characterize COUPLrs' targets and interaction networks, we employed a two-pronged workflow combining Cysteine Druggability Mapping to identify reactive and accessible cysteine residues across oncogenic proteins, while SDS-PAGE and fractionation assays monitored changes in molecular weight, revealing COUPLr-involved protein complex formation. We synthesized a 32-member library of cysteine-reactive compounds with diverse scaffolds, warheads, and reactivities. Library characterization revealed proteome-wide COUPLr targets, including inter- and intramolecular protein coupling.To expand this analysis, we conducted a proteomic screen across 13 cancer cell lines representing diverse tissue origins, generating a comprehensive atlas of COUPLrs-targetable proteins. This screen revealed both shared and lineage-specific targets, including several proteins previously considered undruggable, such as transcription factors. Notably, some protein showed mutation-specific engagement by COUPLrs, suggesting potential to selectively target oncogenic variants resistant to existing therapies.Collectively, our work establishes COUPLrs as powerful tools for modulating protein-protein interactions. By integrating covalent chemistry with proteomic profiling, we delineated protein interactions stabilized or disrupted by COUPLrs. These findings open new avenues for therapeutic development against previously inaccessible targets in cancer biology.