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

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.