Abstract 1660: Seeking new glues - Toward an E3 ligase-agnostic molecular glue degrader identification platform

Abstract Targeted Protein Degradation (TPD) is a rapidly growing modality in oncology and is broadly driven by development of bifunctional (PROTAC-type) or monovalent degraders. Whilst the latter group largely consists of molecular glue degraders (MGDs), it also encompasses compounds that act as degraders via allosteric or other mechanisms. Screening for compounds with MGD properties can be undertaken via biophysical methods, where proximity of an effector and target protein is measured, limiting the effect of the compound to a known, selected effector, such as a particular ubiquitin-ligase of interest. Whereas this approach benefits from full control over the target and ligase pairing, cell-based systems offer the advantage of screening for target degradation against the entire complement of expressed ubiquitin ligases, potentially increasing the probability of identifying a hit compound. Conversely, a key challenge with cell-based screening approaches is to understand the mechanism by which the target is degraded, relying on the identification of the ubiquitin ligase or adaptor protein that is mediating the effect of the compound. Here, we demonstrate the utility of our cell-based screening platform by developing degrader assays for two separate targets, Cyclin K and GSPT1, both therapeutically relevant for oncology, and with respective MGDs in clinical trials (CT7439 for cyclin K by Carrick Therapeutics, and MRT-2359 and Eragidomide from Monte Rosa Therapeutics and Cellgene/BMS, respectively). Further, to address the challenge of deconvoluting the mechanistic basis for monovalent degrader hits from cell-based assays, we have utilized limited proteolysis coupled with mass spectrometry (LiP-MS). LiP-MS has become a powerful, label-free technique for identifying small molecule and peptide targets directly in cell lysates. This methodology allows the identification of the proteins involved in mediating degradation of a target, such as a ubiquitin ligase recruited to a target via an MGD. Our approach integrates flexible cell-based assays for degrader screening with a powerful method of mechanistic deconvolution, allowing identification of the specific ligase driving target degradation. This knowledge can then facilitate the development of ligase- and target-specific molecular glue assays to drive hit optimisation and, ultimately, the delivery of novel MGD chemotypes against new oncology targets of choice. Citation Format: Allan M. Jordan, Kuhulika Bhalla, Helen Burston, Francesca Cavallo, Prasamit Baruah, Martin Soste, Polina Scichkova, Roland Bruderer, Daniel Redfern, Matevz Stefancic, Yuehan Feng, Stuart Thomson, Roland Hjerpe. Seeking new glues - Toward an E3 ligase-agnostic molecular glue degrader identification platform [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 1660.