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Mechanistic studies of inactivation of inducible nitric oxide synthase by amidines.

ABSTRACT: Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide. N(5)-(1-Iminoethyl)-L-ornithine (L-NIO), an amidine-containing molecule, is a natural product known to be an inactivator of inducible NOS (iNOS). Because of the presence of the amidine methyl group in place of the guanidine amino group of substrate L-arginine, the active site heme peroxy intermediate sometimes cannot be protonated, thereby preventing its conversion to the heme oxo intermediate; instead, a heme oxygenase-type mechanism occurs, leading to conversion of the heme to biliverdin. This might be a new and general inactivation mechanism for heme-containing enzymes. In the studies described here, we attempted to provide support for amidines as substrates and inactivators of iNOS by the design and synthesis of amidine analogues of L-NIO having groups other than the amidine methyl group. No nitric oxide- or enzyme-catalyzed products could be detected by incubation of these amidines with iNOS. Although none of the L-NIO analogues acted as substrates, they all inhibited iNOS; increased inhibitory potency correlated with decreased substituent size. Computer modeling and molecular dynamics simulations were run on 10 and 11 to rationalize why these compounds do not act as substrates. Unlike the methyl amidine (L-NIO), the other alkyl groups block binding of O2 at the heme iron. Compounds 8, 9, and 11 were inactivators; however, no heme was lost, and no biliverdin was formed. No kinetic isotope effect on inactivation was observed with perdeuterated ethyl 8. A small amount of dimer disruption occurred with these inactivators, although the amount would not account for complete enzyme inactivation. The L-NIO analogues inactivate iNOS by a yet unknown mechanism; however, it is different from that of L-NIO, and the inactivation mechanism previously reported for L-NIO appears to be unique to methyl amidines.

SUBMITTER: Tang W

PROVIDER: S-EPMC4410366 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Mechanistic studies of inactivation of inducible nitric oxide synthase by amidines.

Tang Wei W Li Huiying H Poulos Thomas L TL Silverman Richard B RB

Biochemistry 20150407 15

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide. N(5)-(1-Iminoethyl)-L-ornithine (L-NIO), an amidine-containing molecule, is a natural product known to be an inactivator of inducible NOS (iNOS). Because of the presence of the amidine methyl group in place of the guanidine amino group of substrate L-arginine, the active site heme peroxy intermediate sometimes cannot be protonated, thereby preventing its conversion to the heme oxo intermediate; i ...[more]

PMID: 25811913

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Project description:UnlabelledInducible nitric oxide synthase (iNOS) activity increases in acute and chronic inflammatory lung diseases. Imaging iNOS expression may be useful as an inflammation biomarker for monitoring lung disease activity. We developed a novel tracer for PET that binds to iNOS in vivo, (18)F-NOS. In this study, we tested whether (18)F-NOS could quantify iNOS expression from endotoxin-induced lung inflammation in healthy volunteers.MethodsHealthy volunteers were screened to exclude cardiopulmonary disease. Qualifying volunteers underwent a baseline, 1-h dynamic (18)F-NOS PET/CT scan. Endotoxin (4 ng/kg) was then instilled bronchoscopically in the right middle lobe. (18)F-NOS imaging was performed again approximately 16 h after endotoxin instillation. Radiolabeled metabolites were determined from blood samples. Cells recovered by bronchoalveolar lavage (BAL) after imaging were stained immunohistochemically for iNOS. (18)F-NOS uptake was quantified as the distribution volume ratio (DVR) determined by Logan plot graphical analysis in volumes of interest placed over the area of endotoxin instillation and in an equivalent lung region on the left. The mean Hounsfield units (HUs) were also computed using the same volumes of interest to measure density changes.ResultsSeven healthy volunteers with normal pulmonary function completed the study with evaluable data. The DVR increased by approximately 30%, from a baseline mean of 0.42 ± 0.07 to 0.54 ± 0.12, and the mean HUs by 11% after endotoxin in 6 volunteers who had positive iNOS staining in BAL cells. The DVR did not change in the left lung after endotoxin. In 1 volunteer with low-level iNOS staining in BAL cells, the mean HUs increased by 7% without an increase in DVR. Metabolism was rapid, with approximately 50% of the parent compound at 5 min and 17% at 60 min after injection.Conclusion(18)F-NOS can be used to image iNOS activity in acute lung inflammation in humans and may be a useful PET tracer for imaging iNOS expression in inflammatory lung disease.

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Mechanistic studies of inactivation of inducible nitric oxide synthase by amidines.

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Mechanistic studies of inactivation of inducible nitric oxide synthase by amidines.

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