BYK191023 (2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3h-imidazo[4,5-b]pyridine) is an NADPH- and time-dependent irreversible inhibitor of inducible nitric-oxide synthase.
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 69: 328-337, 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. [(3)H]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.
Tiso
Mauro
M
Department of Pathobiology, NC-20, The Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195, USA.
Strub
Andreas
A
Hesslinger
Christian
C
Kenney
Claire T
CT
Boer
Rainer
R
Stuehr
Dennis J
DJ
D000693:Anaerobiosis / Q000187:drug effects; D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006418:Heme / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry* / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009249:NADP / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors*; D010084:Oxidation-Reduction / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism; D011725:Pyridines / Q000737:chemistry* / Q000494:pharmacology; D013997:Time Factors; D014316:Tritium
C507627:2-(2-(4-methoxypyridin-2-yl)ethyl)-3H-imidazo(4,5-b)pyridine; D004791:Enzyme Inhibitors; D007093:Imidazoles; D011725:Pyridines; D014316:Tritium; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D002248:Carbon Monoxide; D007501:Iron; D052247:Nitric Oxide Synthase Type II; D010100:Oxygen