Abstract 4499: Drug target ID and binding site mapping in complex cellular environments using LiP-MS

Abstract Identifying drug targets within cellular contexts is critical to advancing both target-based and phenotypic drug discovery, particularly for small molecule degraders, such as molecular glues (MG). In most common cases of MG, target identification involves two key mechanism of actions (MoA): determining specific protein target(s) that are engaged by the E3 ligase when a drug binds the E3 ligase, and, conversely, identifying the E3 ligase (adaptor) that is recruited to form the ternary complex when the drug directly targets the protein itself. Limited proteolysis combined with mass spectrometry (LiP-MS) has emerged as a robust, label-free method for deconvoluting small molecule and peptide targets directly within cell lysates, avoiding the need for compound modification or cell line manipulation. This approach leverages a non-specific protease under controlled conditions to detect conformational changes or steric hindrance induced by drug-protein interactions or protein-protein interactions. Using quantitative MS, LiP monitors over 250, 000 peptides, representing more than 8, 000 proteins in a mammalian cell line, allowing for proteome-wide profiling of drug-target interactions. A comprehensive concentration-response study spanning seven concentrations, combined with machine learning-based LiP-scoring, provides a precise ranking of targets and predicts binding sites with peptide-level resolution. Beyond global target identification, we also established a high-throughput HR-LiP workflow that offers detailed peptide-level insights into small molecule-protein interactions directly in cell lysates from cell lines overexpressing target proteins or complexes. This workflow bypasses the need for protein purification, mitigating risks of protein truncation or misfolding. To evaluate LiP-MS’ performance on molecular glue MoAs, we performed global target ID experiments using two well-characterized models: the cyclin K degraders CR8 and SR-4835 and the GSPT1 degraders MRT-2359 and Eragidomide. While the cyclin K degraders bind directly to CDK12, inducing its interaction with DDB1 and leading to the degradation of cyclin K within the CDK12 complex, the GSPT1 compounds engage CRBN, which in turn recruits and degrades GSPT1 as a neosubstrate. Applying HR-LiP, we mapped the binding site of the BRD4 inhibitor JQ1 on full-length BRD4, a large protein challenging to study via standard methods, using a mammalian transient overexpression system. Similarly, we located binding sites for EGFR inhibitors gefitinib and afatinib on the membrane-bound EGFR protein. Additionally, we pinpointed the SMER28 binding site on the hexameric ATPase VCP, an autophagy enhancer, demonstrating the method's applicability to large protein complexes. In conclusion, LiP-MS provides a versatile toolbox for identifying drug targets and mapping binding sites within complex cellular environments. Citation Format: Martin Soste, Polina Shichkova, Roland Bruderer, Matevz Stefancic, Daniel Redfern, Francesca Cavallo, Kuhulika Bhalla, Helen Burston, Prasamit S. Baruah, Roland Hjerpe, Stuart Thomson, Allan Jordan, Yuehan Feng. Drug target ID and binding site mapping in complex cellular environments using LiP-MS [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 4499.

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