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

Hannah Jones, Raphael Heilig, Roman Fischer, Benedikt M Kessler, Adan Pinto-Fernandez
doi: https://doi.org/10.1101/2020.12.10.419499
Hannah Jones
1Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom
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Raphael Heilig
1Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom
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Roman Fischer
1Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom
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Benedikt M Kessler
1Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom
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Adan Pinto-Fernandez
1Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, United Kingdom
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  • For correspondence: adan.pintofernandez@ndm.ox.ac.uk
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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.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 10, 2020.
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ABPP-HT - high-throughput activity-based profiling of deubiquitylating enzyme inhibitors in a cellular context
Hannah Jones, Raphael Heilig, Roman Fischer, Benedikt M Kessler, Adan Pinto-Fernandez
bioRxiv 2020.12.10.419499; doi: https://doi.org/10.1101/2020.12.10.419499
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ABPP-HT - high-throughput activity-based profiling of deubiquitylating enzyme inhibitors in a cellular context
Hannah Jones, Raphael Heilig, Roman Fischer, Benedikt M Kessler, Adan Pinto-Fernandez
bioRxiv 2020.12.10.419499; doi: https://doi.org/10.1101/2020.12.10.419499

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