A Novel and Potent Inhibitor of Dimethylarginine Dimethylaminohydrolase: A Modulator of Cardiovascular Nitric Oxide

Yohannes T. Ghebremariam; Daniel A. Erlanson; John P. Cooke

doi:10.1124/jpet.113.206847

Abstract

PD 404182 [6H-6-imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine], a heterocyclic iminobenzothiazine derivative, is a member of the Library of Pharmacologically Active Compounds (LOPAC) that is reported to possess antimicrobial and anti-inflammatory properties. In this study, we used biochemical assays to screen LOPAC against human dimethylarginine dimethylaminohydrolase isoform 1 (DDAH1), an enzyme that physiologically metabolizes asymmetric dimethylarginine (ADMA), an endogenous and competitive inhibitor of nitric oxide (NO) synthase. We discovered that PD 404182 directly and dose-dependently inhibits DDAH. Moreover, PD 404182 significantly increased intracellular levels of ADMA in cultured primary human vascular endothelial cells (ECs) and reduced lipopolysaccharide-induced NO production in these cells, suggesting its therapeutic potential in septic shock–induced vascular collapse. In addition, PD 404182 abrogated the formation of tube-like structures by ECs in an in vitro angiogenesis assay, indicating its antiangiogenic potential in diseases characterized by pathologically excessive angiogenesis. Furthermore, we investigated the potential mechanism of inhibition of DDAH by this small molecule and found that PD 404182, which has striking structural similarity to ADMA, could be competed by a DDAH substrate, suggesting that it is a competitive inhibitor. Finally, our enzyme kinetics assay showed time-dependent inhibition, and our inhibitor dilution assay showed that the enzymatic activity of DDAH did not recover significantly after dilution, suggesting that PD 404182 might be a tightly bound, covalent, or an irreversible inhibitor of human DDAH1. This proposal is supported by mass spectrometry studies with PD 404182 and glutathione.

Footnotes

Received May 28, 2013.
Accepted October 17, 2013.

This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants 1U01HL100397 and K12HL087746] (to J.P.C.); the American Heart Association [Grant 11IRG5180026]; Stanford SPARK Translational Research Program (to Y.T.G.); the National Institutes of Health National Heart, Lung, and Blood Institute [Grant 1K01HL118683-01] (to Y.T.G.); and the Tobacco-Related Disease Research Program of the University of California [Grant 18XT-0098]. Y.T.G. was a recipient of the Stanford School of Medicine Dean’s fellowship [Grant 1049528-149-KAVFB] and the Tobacco-Related Disease Research Program of the University of California [Grant 20FT-0090].
Conflict of interest: Y.T.G. and J.P.C. are inventors on patents, owned by Stanford University, that protect the use of agents that modulate the NOS/DDAH pathway for therapeutic application. Ghebremariam YT and Cooke JP (2012) inventors; Stanford University, assignee. Dimethylarginine dimethylaminohydrolase inhibitors and methods of use thereof. U.S. patent WO 2013123033A1. Application Pending.
dx.doi.org/10.1124/jpet.113.206847.
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