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

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 increased 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

INSTRUMENT(S): timsTOF Pro, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Brain, Cell Culture

SUBMITTER: Raphael A Heilig

LAB HEAD: Roman Fischer

PROVIDER: PXD023036 | Pride | 2021-02-24

REPOSITORIES: Pride

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			Action	DRS
	QE1063_MSQ1553_20181206_RaphaelBravo_HT-IP_G-Hepes.raw	Raw
	QE1063_MSQ1553_20181206_RaphaelBravo_HT-IP_G-TFA_181210041356.raw	Raw
	QE1063_MSQ1553_20181206_RaphaelBravo_HT-IP_adj_txt.zip	Other
	QE1063_MSQ1553_20181206_RaphaelBravo_HT-IP_insol_E.raw	Raw
	QE1063_MSQ1553_20181206_RaphaelBravo_HT-IP_insol_F.raw	Raw

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Project description:Comparative, dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-targets, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of drug selectivity and efficacy. In this study, we further refined the site-specific activity-based protein profiling strategy, PhosID-ABPP, on a timsTOF Pro mass spectrometer. This refinement enables dose-dependent competition of inhibitors within a single cellular proteome. Here, a comparative analysis of two activity-based probes (ABPs), developed to selectively target the epidermal growth factor receptor (EGFR), namely PF-06672131 and PF-6422899, facilitated the simultaneous identification of ABP-specific binding sites at a proteome-wide scale within a cellular proteome. Dose-dependent probe-binding preferences for proteinaceous cysteines, even at low nanomolar ABP concentrations, could be revealed. Notably, while both ABPs showed comparable affinities for the EGFR, PF-06672131 had a broader off-target reactivity profile. In contrast, PF-6422899 exhibited higher affinity for the ERBB2 receptor and bound to catalytic cysteines in several other enzymes, which is likely to disrupt their catalytic activity. Notably, PF-06672131 also effectively labeled ADP/ATP translocase proteins at a concentration of just 1 nanomolar. Additionally, analysis of different binding sites within the EGF receptor and the voltage-dependent anion channel 2 revealed secondary binding sites of both probes and provided insights into the binding poses of inhibitors on these proteins. Insights from the PhosID-ABPP analysis of these two ABPs serve as a valuable resource for understanding drug on- and off-target engagement in a dose- and site-specific manner.

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

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