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.