AUTHOR=Jones Hannah B. L. , Heilig Raphael , Fischer Roman , Kessler Benedikt M. , Pinto-Fernández Adán TITLE=ABPP-HT - High-Throughput Activity-Based Profiling of Deubiquitylating Enzyme Inhibitors in a Cellular Context JOURNAL=Frontiers in Chemistry VOLUME=9 YEAR=2021 URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2021.640105 DOI=10.3389/fchem.2021.640105 ISSN=2296-2646 ABSTRACT=

The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.