TY - JOUR T1 - BYK191023 (2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3<em>H</em>-imidazo[4,5-<em>b</em>]pyridine) Is an NADPH- and Time-Dependent Irreversible Inhibitor of Inducible Nitric-Oxide Synthase JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1244 LP - 1253 DO - 10.1124/mol.107.041319 VL - 73 IS - 4 AU - Mauro Tiso AU - Andreas Strub AU - Christian Hesslinger AU - Claire T. Kenney AU - Rainer Boer AU - Dennis J. Stuehr Y1 - 2008/04/01 UR - http://molpharm.aspetjournals.org/content/73/4/1244.abstract N2 - Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric-oxide synthase (iNOS), and 2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) was found to have very high selectivity in enzymatic and cellular models ( Mol Pharmacol:-337, 2006 ). Here, we show that BYK191023 irreversibly inactivates murine iNOS in an NADPH- and time-dependent manner, whereas it acts only as a reversible l-arginine-competitive inhibitor in the absence of NADPH or during anaerobic preincubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or iNOS-overexpressing human embryonic kidney 293 cell lines. The mechanism of BYK191023 inhibition in the presence of NADPH was studied using spectral, kinetic, chromatographic, and radioligand binding methods. BYK191023-bound iNOS was spectrally indistinguishable from l-arginine-bound iNOS, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. [3H]BYK191023 was recovered quantitatively from irreversibly inactivated iNOS, and no inhibitor metabolite was detected by high-performance liquid chromatography (HPLC). Size exclusion chromatography revealed only about 20% iNOS dissociation into monomers. Furthermore, HPLC and spectrophotometric analysis showed that the irreversible inhibition was associated with loss of heme from iNOS and a reduced ability to form the distinctive ferrous heme-CO complex (cytochrome P450). Thus, enzyme inactivation is mainly caused by heme loss, and it occurs in the inhibitor-bound enzyme in the presence of electron flux from NADPH. The American Society for Pharmacology and Experimental Therapeutics ER -