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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

Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio (M.T., C.T.K., D.J.S.); and the Department of Biochemistry, Nycomed, Konstanz, Germany (A.S., C.H., R.B.)

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[Abstract/Free Full Text] ). 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.

Address correspondence to: Dr. Dennis J. Stuehr, Department of Pathobiology, NC-20, The Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195. E-mail: stuehrd{at}ccf.org or Dr. Andreas Strub, Nycomed, Byk-Gulden-Str. 2, 78467, Konstanz, Germany. E-mail: andreas.strub{at}nycomed.com