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A Nucleophilic Activity-Based Probe Enables Profiling of PLP-Dependent Enzymes.

Scott I BrodyJoseph A BuonomoMoyosore O OrimoloyeZiyi JiaSachin SharmaChristopher D BrownAnthony D BaughnCourtney C Aldrich
Published in: Chembiochem : a European journal of chemical biology (2023)
PLP-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.
Keyphrases
  • mycobacterium tuberculosis
  • living cells
  • mass spectrometry
  • quantum dots
  • emergency department
  • mesenchymal stem cells
  • bone marrow
  • cell therapy
  • hiv infected
  • hyaluronic acid
  • gram negative
  • fluorescence imaging