Abstract 5754: An LC-MS toolbox for identifying and guiding the development of cysteine-targeted small molecule covalent inhibitors

Covalent inhibitors are a powerful class of therapeutic agents, and have gained particular traction with oncogenic targets. FDA-approved covalent inhibitors have been introduced for treatment of a range of cancers, including mantle cell lymphoma (ibrutinib), non-small cell lung cancer (osimertinib, sotorasib, afatinib), and breast cancer (neratinib). Notably, the recent surge in preclinical covalent inhibitor pipelines across biotech and pharma highlights the methodological and technological advancements in covalent inhibitor research and development. Owing to the mechanism of action of this compound class, mass spectrometry is uniquely positioned as an ideal analysis tool for identification, optimization, and mechanistic characterization of covalent inhibitors across the entire developmental cycle of these therapeutics from library screening to clinical evaluation. IQ Proteomics leverages mass spectrometry to empower biotechnology and pharmaceutical companies as a contract research organization, and has developed a comprehensive, turn-key LC-MS based pipeline for supporting the preclinical development of covalent inhibitors. We present a case study exemplifying this LC-MS pipeline centered on the clinically approved KRAS inhibitor sotorasib. We first describe a high-throughput, proteome-wide cysteine ligandability assay ideally suited for initial identification of library hits and SAR evaluation of preliminary lead compounds, and demonstrate the complimentary utility of multiomics characterization (proteome expression profiling, PTM profiling, etc) to global cysteine profiling data. We further present an optimized targeted LC-MS (PRM) workflow for evaluating target-engagement both indirectly (disappearance of unmodified cysteine peptide) and directly (appearance of adduct peptide) for characterization of preclinical lead compounds over large numbers of in vitro and in vivo samples. We finally evaluate an isobaric multiplexed assay for rapid target engagement characterization of covalent inhibitors in complex matrices. Together, this suite of template assays represents a set of powerful tools for covalent inhibitor discovery, optimization, and therapeutic evaluation that empowers the oncology community as exciting covalent modalities continue to emerge with the potential to treat an increasing number of diseases. Citation Format: Kurt Baltier, Alison Erickson, Craig R. Braun, Ryan Kunz, Brian Erickson. An LC-MS toolbox for identifying and guiding the development of cysteine-targeted small molecule covalent inhibitors [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 5754.