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 ¼ 0.028 mM), and H-Phe-Leu-NHOH discriminated between human and bacterial ortholog of the M49 family.

Dynamic properties of dipeptidyl peptidase III from Bacteroides thetaiotaomicron and the structural basis for its substrate specificity – a computational study

“…36 Tyr286 is 100% conserved within the M49 family and is presumed to be a part of the S1 active subsite. 37 Both experimental 38…”

“…In the experimentally determined 'closed' structure of the Bt.DPP III this distance is 22.2 Å. During the MD simulations resulting with the protein closure Tyr286 forms hydrogen bonds with Lys468, Gly461 and, most notably, Ser472 which is thought to be a part of the S2 subsite 37.…”

Conservation of the conformational dynamics and ligand binding within M49 enzyme family

“…The K m values for hydrolysis of uorogenic substrate Arg 2 -2NA were determined at pH 7.4 (20 mM Tris-HCl buffer) and at 25 C, from the initial reaction rates as previously described. 26 Under the conditions used, the K m values were 6.0 mM, 12 mM and 0.8 mM for human, yeast, and B. thetaiotaomicron DPP III, and 0.3 mM, 15.9 mM and 23.8 mM for Porphyromonas gingivalis, Caldithrix abyssi and Physcomitrella patens enzyme, respectively.…”

“…Detailed expression protocols were described elsewhere. 8,12,25,26,33 Protein purity was conrmed by SDS-PAGE according to Laemmli. 34 Protein concentrations were determined by the Bradford method.…”

Validation of flavonoids as potential dipeptidyl peptidase III inhibitors: Experimental and computational approach

“…Although previous studies have shown that some pentapeptides and heptapeptide spinorphin potently inhibit mammalian DPP III, it has been reported that peptidic inhibitors are degraded in the human body by (amino) peptidases . More recent studies of the effect of dipeptide derivatives of hydroxamic acid on the human and bacterial DPP III have shown that these compounds competitively inhibit the enzyme . We have investigated and reported the inhibitory effect of benzimidazole derivatives against DPP III from human erythrocytes and found the key features responsible for their inhibitory activity .…”