Abstract 3847: Target identification of a multi-pass transmembrane G protein coupled receptor using limited-proteolysis coupled mass spectrometry (LiP-MS)

In drug discovery, target identification (ID) after phenotypic screens is a resource-intensive endeavor aimed to understand compound’s mechanism of action. Target ID for membrane proteins is particularly challenging due to hurdles such as poor protein solubility, instability and low expression levels. Addressing these hurdles, recent proteomics-based strategies allow to analyze proteins in their native environment, and do not require compound modification or genetic manipulation of target cell lines. Limited proteolysis coupled to mass spectrometry (LiP-MS) is a peptide-centric strategy that exploits structural protein alterations and steric hindrances induced by drug to detect drug-protein interactions, estimate potency (EC50) and predict binding sites across the proteome. Our previous reports (AACR 2020 and 2021) showed the applicability of LiP-MS on cytosolic proteins such as kinases and phosphatases. Here, LiP-MS workflow was adapted for multi-pass membrane proteins which are often underrepresented in global, unbiased target ID approaches. For protein targets in the cytosol or intracellular organelles, LiP-MS deploys a dose-response (DR) analysis recorded by incubating cell lysates with a given compound. Here, to monitor proteins in plasma membrane, live cells were treated with the compound in a DR curve for a short period of time before lysis and subsequent LiP-MS analysis. Live cell treatment allows to achieve target ID by 1) locking the receptor in its native conformation to detect the structural difference between the drug-bound and unbound forms and; 2) detecting structural changes in downstream signaling cascades resulting from changes in protein-protein interactions, post-translational modifications or other mechanisms. We evaluated the performance of this workflow using a tool compound (IAX16840) targeting specific G-protein coupled receptors. Our unbiased LiP scoring identified atypical chemokine receptor 3 (ACKR3), the primary known compound target, among the top 3 hits. Additional 82 proteins were perturbed in drug-treated samples based on the LiP scores. Mapping the altered peptides on the GPCR signaling network showed enrichment of perturbations in ACKR3 downstream pathways, providing additional evidence of ACKR3 binding. Taken together, we demonstrate that the live cell LiP-MS is applicable for target ID of multi-pass membrane proteins. The approach also provides a system-level view of protein and pathway perturbations downstream of a signaling protein (e.g. GPCR), revealing possible mechanisms of its endogenous action. Further optimization of LiP-MS protocol is sought to accommodate different classes of receptors. Citation Format: Martin Soste, Dominique Kamber, Nadine Dobberstein, Mirjam Zimmermann, Aurelien Rizk, Yuehan Feng, Yaroslav Nikolaev, Nigel Beaton. Target identification of a multi-pass transmembrane G protein coupled receptor using limited-proteolysis coupled mass spectrometry (LiP-MS). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3847.