Abstract LB195: Activity based protein profiling on kinase PROTACs leads to the discovery of selective APT1/2 degrader

Abstract Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent protein degradation. Most success has been achieved by designing selective degraders starting from known binders of a protein of interest (POI). Chemo-proteomics analysis of these degraders can be used to confirm their selectivity for target proteins but often results in the serendipitous discovery of additional degradation targets. While this has provided a means to the discovery of unanticipated degraders, the relatively low throughput of proteomics combined with the idiosyncrasy of discovery has prevented this approach being systematically applied. To improve the throughput of this approach, we explored using activity-based protein profiling (ABPP) to prescreen a degrader library to select for compounds that could potentially inhibit or degrade targets of interest before performing chemo-proteomics profiling. Here, we demonstrate the application of this combined ABPP-chemo-proteomics workflow using a library of heterobifunctional kinase degraders which allowed us to efficiently discover acyl-protein thioesterase APT1/2 as off-targets of a compound originally developed as a FGFR1/2 hetero-bivalent degrader. APT1/2 is responsible for removing S-palmitoylation, a post-translational modification that regulates oncoprotein function and localization in cancer cells. Further optimization and molecular engineering results in the development of a potent APT1/2 degrader TWQ-02-083 that no longer induces FGFR1/2 degradation. We show that TWQ-02-083 induces a strong ternary complex between VHL and APT1/2 and has high cooperativity comparable to classical molecular glues. Our work highlights the potential utility of ABPP strategies for rapid discovery of heterobifunctional degraders on a subset of proteins and delivers a robust and scalable workflow for identifying novel unanticipated degraders that could be valuable in cancer research. Citation Format: Tian Qiu, Samuel Maiwald, Brendan G. Dwyer, Leyna Duong, Katherine A. Donovan, Woong Sub Byun, Hannah M. Jones, Eric S. Fischer, Stephen M. Hinshaw, Tinghu Zhang, Brenda A. Schulman, Nathanael S. Gray. Activity based protein profiling on kinase PROTACs leads to the discovery of selective APT1/2 degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB195.