Structural Characterization and Kinetics of Nitric-oxide Synthase Inhibition by Novel N5-(Iminoalkyl)- and N5-(Iminoalkenyl)-ornithines

Abstract

Isoform-specific nitric-oxide synthase (NOS) inhibitors may prove clinically useful in reducing the pathophysiological effects associated with increased neuronal NOS (nNOS) or inducible NOS (iNOS) activity in a variety of neurological and inflammatory disorders. Analogs of the NOS substrate L-arginine are pharmacologically attractive inhibitors because of their stability, reliable cell uptake, and good selectivity for NOS over other heme proteins. Some inhibitory arginine analogs show significant isoform selectivity although the structural or mechanistic basis of such selectivity is generally poorly understood. In the present studies, we determined by x-ray crystallography the binding interactions between rat nNOS and N 5 -(1-imino-3-butenyl)-L-ornithine (L-VNIO), a previously identified mechanismbased, irreversible inactivator with moderate nNOS selectivity. We have also synthesized and mechanistically characterized several L-VNIO analogs and find, surprisingly, that even relatively minor structural changes produce inhibitors that are either iNOS-selective or nonselective. Furthermore, derivatives having a methyl group added to the butenyl moiety of L-VNIO and L-VNIO derivatives that are analogs of homoarginine rather than arginine display slow-on, slow-off kinetics rather than irreversible inactivation. These results elucidate some of the structural requirements for isoform-selective inhibition by L-VNIO and its related alkyl-and alkenyl-imino ornithine and lysine derivatives and may provide information useful in the ongoing rational design of isoform-selective inhibitors. Nitric oxide (NO)1 is a freely diffusible, moderately reactive free radical with diverse biological actions. Three distinct nitric-oxide synthase (NOS) isoforms catalyze the NADPH-and O 2 -dependent conversion of L-arginine to L-citrulline and NO in a wide range of mammalian tissues. Each isoform is associated mainly, but not exclusively, with a specific physiological role, such as neurotransmission or neuromodulation (nNOS) (1, 2), immune response (iNOS) (3), or blood pressure regulation (eNOS) (4). For each isoform, the monomers of the NOS homodimer are comprised of a heme-and tetrahydrobiopterin (BH 4 )-containing oxygenase domain, which binds the substrate arginine, and an FAD-and FMN-containing reductase domain, which binds the substrate NADPH. A calmodulin (CaM)-binding region connects the oxygenase and reductase domains. For nNOS and eNOS, which are constitutively expressed, increases in cellular Ca 2ϩ levels cause Ca 2ϩ -CaM to bind to NOS and activate NO production. In contrast, iNOS is fully active at basal intracellular Ca 2ϩ concentrations and is instead regulated mainly by transcriptional control of enzyme expression.Overproduction of NO or uncoupled reduction of O 2 to superoxide by NOS has been implicated in the morbidity or mortality of cytokine-induced hypotension (5), cardiogenic and septic shock (6 -8), chronic inflammation (9), migraine headache (10 -12), several neurodegenerative diseases (13-15), and the reperfusion inj...

Homoarginine Levels Are Regulated by l -Arginine:Glycine Amidinotransferase and Affect Stroke Outcome

“…08/08 and 10/110). As previously described, creatine deficiency in AGAT -/-and guanidinoacetate N-methyltransferase (GAMT)-deficient mice resulted in reduced body weight, less relative body fat mass, decreased muscle strength and volume [1][2][3] . In addition, cholesterol levels were reduced and glucose tolerance was (ischemia) and after removal of filament (reperfusion).…”

Homoarginine and Cardiovascular Outcome in the Population-Based Dallas Heart Study

“…Of particular note, this phenotype was reversible by homoarginine supplementation, suggesting a causal link between homoarginine and stroke pathogenesis. 10 Although the underlying mechanism linking homoarginine with cardiovascular risk remains poorly understood, involvement in NO generation is plausible 18 because homoarginine is both a substrate for NO synthase 1,19 and a weak inhibitor of arginase activity. 19 By inhibiting arginase, homoarginine can increase the availability of the NO precursor l-arginine.…”

“…The study design, participant selection and phenotypical characterization have been described previously. 1 The study protocol was approved by the University of Texas Southwestern Medical Center Institutional Review Board, and written informed consent was provided by all study participants.…”

“…Homoarginine has been postulated to be an antiatherosclerotic factor because it is a weak substrate for nitric oxide (NO) synthase and can, therefore, be converted to NO. 1 Experimental studies have shown that exogenous homoarginine exerts antihypertensive and antithrombotic effects. 2,3 Recently, clinical data suggested a role of low endogenous homoarginine as a risk marker for cardiovascular and cerebrovascular events.…”

Low Homoarginine Levels in the Prognosis of Patients With Acute Chest Pain

“…Because of a structural similarity with l ‐arginine, homoarginine is supposed to interfere with the nitric oxide (NO) pathway. Homoarginine is a weak alternative NO synthase substrate, competes with l ‐arginine for cellular influx and efflux transporters, and improves l ‐arginine availability, inhibiting its metabolism by arginases 4. In line with these mechanistic insights, low homoarginine concentrations have been linked to phenotypes of (sub)clinical atherosclerosis, that is, brachial intima‐media and aortic wall thickness 5, 6.…”