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.
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas, Austin, 78712-0165, USA.
Matthew
M
Lluis
Yun
Y
Wang
Arthur F
AF
Monzingo
Walter
W
Fast
Jon D
JD
Robertus
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