Abstract
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.
Footnotes
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This work was supported by ALTANA Pharma AG.
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ABBREVIATIONS: NOS, nitric-oxide synthase(s); iNOS, inducible nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; CaM, calmodulin; H4B, (6R)-5,6,7,8-tetrahydro-l-biopterin; Arg, l-arginine; BYK191023, 2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine; 1400W, N-(3-(aminomethyl)benzyl)acetamidine; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; BSA, bovine serum albumin; SOD, superoxide dismutase; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; IFN, interferon; LPS, lipopolysaccharide; HPLC, high-performance liquid chromatography; PVDF, polyvinylidene difluoride; P450, cytochrome P450; P420, cytochrome P420; ROS, reactive oxygen species.
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↵1 Current affiliation: Vascular Medicine Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
- Received August 29, 2007.
- Accepted January 4, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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