Characterization of C-alkyl amidines as bioavailable covalent reversible inhibitors of human DDAH-1.
C-Alkyl amidine analogues of asymmetric N(ω),N(ω)-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 (K(d)=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.
Lluis
Matthew
M
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas, Austin, 78712-0165, USA.
Wang
Yun
Y
Monzingo
Arthur F
AF
Fast
Walter
W
Robertus
Jon D
JD
D000578:Amidines / Q000494:pharmacology; D000619:Aminohydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001682:Biological Availability; D002152:Calorimetry, Differential Scanning; D002460:Cell Line; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000578:Amidines; D004791:Enzyme Inhibitors; D009569:Nitric Oxide; C018524:N,N-dimethylarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000619:Aminohydrolases