Novel dipeptidyl hydroxamic acids that inhibit human and bacterial dipeptidyl peptidase III

Abstract

Human dipeptidyl peptidase III (hDPP III), a zinc-metallopeptidase of the family M49, is an activator of the Keap1-Nrf2 cytoprotective pathway involved in defense against oxidative stress. Pathophysiological roles of DPP III have not been elucidated yet, partly due to the lack of specific inhibitors. We showed that substrate analog H-Tyr-Phe-NHOH is a strong competitive inhibitor of hDPP III, while H-Tyr-Gly-NHOH expresses much weaker inhibition. To investigate the effects of amino acid substitutions in inhibitor P1 position, we synthesized three new dipeptidyl hydroxamates and examined their influence on the activity of hDPP III and DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The extent of inhibition of hDPP III, but not of bacterial enzyme, was dependent on the amino acid in P1. H-Phe-Phe-NHOH is recognized as one of the strongest inhibitors of hDPP III (K_i = 0.028 μM), and H-Phe-Leu-NHOH discriminated between human and bacterial ortholog of the M49 family.

Keywords:

• Bacteroides thetaiotaomicron
• binding specificity
• competitive inhibitor
• family M49
• metallopeptidase

Acknowledgements

We are grateful to Dr. Jeffrey I. Gordon and Dr. Janaki L. Guruge for the kind gift of genomic DNA isolated from B. thetaiotaomicron. We thank Marija Kozlović, M.S., for the purification of recombinant human DPP III, Dr. Ivanka Jerić for help with purification of hydroxamic acid inhibitor by HPLC, and Dr. Karlo Wittine and Dr. Uroš Anđelković for HRMS analyses.

Declaration of interest

The authors report that they have no conflicts of interest. We thank the Croatian Ministry of Science, Education and Sport (project 098-1191344-2938) and the Croatian Science Foundation (projects number 7235 “Flexibility, activity and structure correlations in the dipeptidyl peptidase III family”, and IP-11-2013-7387 “Supramolecular Synthesis of Self-Assembled Functional Nanomaterials and Complex Chemical Systems”) for the financial support for this study. We greatly acknowledge the access to equipment in possession of University of Rijeka within the project RISK “Development of University of Rijeka campus laboratory research infrastructure”, financed by European Regional Development Fund (ERDF).

Supplementary material available online.