Abstract 6374: COuPLrs, a new class of cysteine-reactive compounds for modulating protein-protein interaction

Abstract Molecular glues have revolutionized the targeting of proteins by disrupting protein complexes, showing immense therapeutic potential. However, the discovery of suitable targets for these agents remains largely serendipitous. Unlike traditional monovalent molecular glues, we have developed a novel class of dual-warhead compounds, chemically distinct cysteine-reactive molecules termed COuPLrs (COvalent Protein Ligators). These compounds are specifically designed to dimerize or disrupt protein complexes, thereby modulating protein function. To systematically identify COuPLrs targets and delineate their associated protein-protein interactions, we employed a combination of systematic fractionation assays and cysteine reactivity mapping. This approach allowed us to uncover targetable interactions with high confidence. Additionally, we conducted a proteomic-based screen across 13 cancer cell lines representing diverse tissue origins and cancer types. This analysis resulted in the creation of a comprehensive map of COuPLrs target proteins, revealing both common and unique oncogenic targets. Notably, several of these targets were previously considered undruggable, and some exhibit mutation-specific engagement. Our investigation highlighted COuPLrs' ability to covalently stabilize members of known protein complexes and revealed their potential to uncover novel interactions. In a non-small cell lung cancer (NSCLC) cell line, we identified COuPLrs binding to the oncogenic fusion protein EML4-ALK, specifically engaging the EML4 domain rather than the ALK kinase domain, the traditional binding site of approved inhibitors. This interaction disrupted ALK signaling and triggered proteasome-dependent degradation of the fusion protein. To enhance compound specificity, we conducted a Structure-Activity Relationship (SAR) analysis, which provided valuable insights for optimizing binding properties. Collectively, our findings introduce a new class of small-molecule tools capable of simultaneously engaging two proteins to disrupt their interaction, structure, and dynamics. These dual-warhead compounds hold significant promise for advancing therapeutic strategies and targeting previously intractable protein interactions. Citation Format: Diane Yang, Stefan Andrew Harry, Harrison Byron Chong, Edwin Zhang, Natalie Shannon Nordenfelt, Nicholas Chen, Elizabeth Codd, Samay Trivedi, Magdy Gohar, Dawn Mitchell, Maolin Ge, Chengzhuo Gao, Zavontae Holmes, Wenxin Yang, Abigail Elizabeth Smith, Alexander Daniel Carlin, Matthew Jacob Lazarov, Neha Khandelwal, Mariko Takahashi, Siwen Zhang, Zhen-Yu Jim Sun, Toshio Fujino, Aaron N Hata, Maristela Onozato, Mohammed Mahamdeh, Roy Soberman, Stefan Kaluziak, Brian B. Liau, A. John Iafrate, Liron Bar-Peled. COuPLrs, a new class of cysteine-reactive compounds for modulating protein-protein interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6374.