Characterization of C-Alkyl Amidines as Bioavailable Covalent Reversible Inhibitors of Human DDAH-1

Abstract

C-Alkyl amidine analogues of asymmetric Nω,Nω-dimethyl-L-arginine are dual-targeted inhibitors of both human DDAH-1 and nitric oxide (NO) synthase, and provide a promising scaffold for the development of therapeutics to control NO overproduction in a variety of pathologies including septic shock and some cancers. Using a two-part click-chemistry-mediated activity probe, a homologated series of C-alkyl amidines were ranked for their ability to inhibit DDAH-1 within cultured HEK 293T cells. N5-(1-Iminopentyl)-L-ornithine was determined to be the most potent compound in-vitro (Kd=7-μM) as well as in cultured cells, and the binding conformation and covalent reversible mode of inhibition was investigated by comparison of interactions made with DDAH-1 and a catalytically inactive C274S variant, as gauged by X-ray crystallography and isothermal titration calorimetry. By interrupting the ability of the inhibitor to form a covalent bond, the contribution of this interaction could be estimated. These results suggest that further stabilization of the covalent adduct is a promising strategy for lead optimization in the design of effective reagents to block NO synthesis.Inhibition, interrupted: A series of dual-targeted DDAH-1/NO synthase inhibitors were ranked for potency within cells, and the most potent compound was characterized in detail. X-ray crystallography and isothermal titration calorimetry were used to compare binding to wild-type and mutant DDAH-1 to dissect the contribution of reversible covalent bond formation to the potency of these C-alkyl amidine inhibitors. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.