Received for publication August 30, 2007.
Revised December 10, 2007.
Accepted for publication January 4, 2008.

BYK191023 Is A NADPH- And Time-Dependent Irreversible Inhibitor Of Inducible Nitric-Oxide Synthase

Abstract

Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric oxide synthase (iNOS) and BYK191023 (2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine) was found to have very high selectivity in enzymatic and cellular models (Strub et al., 2006). Here we show that BYK191023 irreversibly inactivates murine iNOS in a NADPH- and time-dependent manner, while it acts only as a reversible L-arginine competitive inhibitor in the absence of NADPH or during anaerobic pre-incubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or human iNOS-overexpressing HEK293 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-labeled BYK191023 was recovered quantitatively from irreversibly inactivated iNOS and no inhibitor metabolite was detected by HPLC. Size exclusion chromatography revealed only about 20% iNOS dissociation into monomers. Further 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 (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.

Key words: Nitric oxide, Nitric oxide synthases, Cytochrome P450, Enzymology