Global Assessment of Drug Target Engagement and Selectivity of Covalent Cysteine-Reactive Inhibitors Using Alkyne-Functionalized Probes

Elisabeth M Rothweiler; Kilian V M Huber

doi:10.1007/978-1-0716-3397-7_14

Global Assessment of Drug Target Engagement and Selectivity of Covalent Cysteine-Reactive Inhibitors Using Alkyne-Functionalized Probes

Methods Mol Biol. 2023:2706:191-200. doi: 10.1007/978-1-0716-3397-7_14.

Authors

Elisabeth M Rothweiler^{1

2}, Kilian V M Huber^{3

4}

Affiliations

¹ Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
² Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
³ Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, UK. kilian.huber@cmd.ox.ac.uk.
⁴ Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK. kilian.huber@cmd.ox.ac.uk.

PMID: 37558950
DOI: 10.1007/978-1-0716-3397-7_14

Abstract

Covalent inhibitors are emerging as a promising therapeutic means for efficient and sustained targeting of key disease-driving proteins. As for classic non-covalent inhibitors, understanding target engagement and selectivity is essential for determining optimal dosing and limiting potential on- or off-target toxicity. Here, we present a complementary activity-based protein profiling (ABPP) strategy for unbiased proteome-wide profiling of cysteine-reactive inhibitors based on two orthogonal approaches. We illustrate the use of clickable alkyne probes for in-gel fluorescence and mass spectrometry studies using a series of therapeutic XPO1 inhibitors as an example.

Keywords: Activity-based protein profiling; Chemoproteomics; Covalent inhibitors; Cysteine; Target engagement.

MeSH terms

Alkynes* / chemistry
Cysteine* / chemistry
Mass Spectrometry

Substances

Cysteine
Alkynes