Abstract 5771: Utilizing a photoreactive chloroalkane capture tag for target deconvolution

Abstract

Abstract Identifying the physiologically relevant targets of bioactive compounds is often the rate limiting step toward understanding their mechanism of action. To this end, we developed a streamlined chemical proteomic strategy that utilizes a novel photoreactive cleavable chloroalkane tag, which can be attached to bioactive compounds to isolate their respective cellular targets for identification by mass spectrometry. The tag does not significantly affect compounds potency and membrane permeability, allowing for binding interactions to be established within intact cells under appropriate physiological conditions. UV-induced covalent photo-crosslinking “freezes” the cellular interactions and prevents their dissociation upon cell lysis. Effective enrichment of the cellular targets is achieved through their selective covalent capture onto HaloTag coated particles and subsequent selective release by tag cleavage. Putative targets identified by mass spectrometry can be readily verified using resonance energy transfer. By exchanging the chloroalkane tag for a fluorophore, direct binding to the targets can be revealed through proximity of the fluorophore to a small NanoLuc luciferase genetically fused to the putative target. This approach was used to identify physiologically relevant targets for bioactive compounds including those with low affinity and/or low abundance as well as insoluble targets with multiple trans membrane domains. Citation Format: Tetsuo Uyeda, Robin Hurst, Sergiy Levin, Mike Rosenblatt, Kris Zimmermann, Rachel F. Ohana. Utilizing a photoreactive chloroalkane capture tag for target deconvolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5771.