A Nucleophilic Activity‐Based Probe Enables Profiling of PLP‐Dependent Enzymes

Abstract

AbstractPLP‐dependent enzymes represent an important class of highly “druggable” enzymes that perform a wide array of critical reactions to support all organisms. Inhibition of individual members of this family of enzymes has been validated as a therapeutic target for pathologies ranging from infection with Mycobacterium tuberculosis to epilepsy. Given the broad nature of the activities within this family of enzymes, we envisioned a universally acting probe to characterize existing and putative members of the family that also includes the necessary chemical moieties to enable activity‐based protein profiling experiments. Hence, we developed a probe that contains an N‐hydroxyalanine warhead that acts as a covalent inhibitor of PLP‐dependent enzymes, a linear diazirine for UV crosslinking, and an alkyne moiety to enable enrichment of crosslinked proteins. Our molecule was used to study PLP‐dependent enzymes in vitro as well as look at whole‐cell lysates of M. tuberculosis and assess inhibitory activity. The probe was able to enrich and identify LysA, a PLP‐dependent enzyme crucial for lysine biosynthesis, through mass spectrometry. Overall, our study shows the utility of this trifunctional first‐generation probe. We anticipate further optimization of probes for PLP‐dependent enzymes will enable the characterization of rationally designed covalent inhibitors of PLP‐dependent enzymes, which will expedite the preclinical characterization of these important therapeutic targets.