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bioRxiv · 10.1101/2020.12.10.419499

ABPP-HT - high-throughput activity-based profiling of deubiquitylating enzyme inhibitors in a cellular context

Jones, H.·Heilig, R.·Fisher, R.·Kessler, B. M.·Pinto-Fernandez, A.
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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 (ABPs) 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.

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Jones, H., Heilig, R., Fisher, R., Kessler, B. M., Pinto-Fernandez, A.. 2020-12-10. ABPP-HT - high-throughput activity-based profiling of deubiquitylating enzyme inhibitors in a cellular context. https://doi.org/10.1101/2020.12.10.419499

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