INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Nitric oxide synthase (NOS)¹ catalyzes the oxidation of L-arginine to nitric oxide (NO) and citrulline; NADPH and O₂ are cosubstrates (1-3). Three major isoforms of NOS have been identified to date. The neuronal (nNOS) and endothelial (eNOS) isoforms are constitutively expressed and are regulated by Ca²⁺/calmodulin, whereas the activity of the inducible isoform (iNOS) is controlled transcriptionally and is not affected by changes in intracellular Ca²⁺. Although amino acid sequence homology among the isoforms is limited (~50%) (3), all are comprised of a C-terminal reductase domain that binds NADPH and the cofactors FAD and FMN and a N-terminal oxygenase domain that binds L-arginine and the heme and tetrahydrobiopterin (BH₄) cofactors (1-3). The reductase domain is related in function and amino acid sequence to cytochrome P450 reductase (4), whereas the oxygenase domain is related in function (but not sequence) to the cytochromes P450. Binding of Ca²⁺/calmodulin to a region between the domains permits electron flow from the reductase domain to the oxygenase domain and also stimulates electron flow within the reductase domain (5). The resulting reduction of the heme cofactor allows O₂ to be activated, permitting the cytochrome P450-like oxidation of L-arginine to N-hydroxy-L-arginine and the subsequent further oxidation of that tightly bound intermediate to citrulline and NO (1, 2, 6).

Nitric oxide synthase-derived NO is important in many physiological processes including blood pressure homeostasis (7-9), neurotransmission (10, 11), and immune function (12), but overproduction of NO can have pathological consequences (13). For example, excess NO resulting from overexpression of iNOS in response to endotoxin or inflammatory cytokines is a major contributor to the vascular disregulation seen in septic shock (14, 15) and in patients receiving interleukin-2-based immunotherapy (16, 17). Inappropriate activation of nNOS is implicated in chronic visceral pain (18, 19), in migraine headache (20), and in several neurodegenerative diseases (e.g. Parkinson's disease (21, 22)), and is thought to contribute to post-ischemic reperfusion injury in stroke (23).

Appreciation of the pathological roles of NOS-derived NO has stimulated interest in the design and synthesis of NOS inhibitors for possible therapeutic use in disorders associated with overproduction of NO (24, 25). To be pharmacologically useful, compounds should be well transported into the target tissue, strongly inhibitory, NOS isoform-selective, and chemically stable under biological conditions. In attempting to meet these goals, L-arginine analogs are particularly attractive inhibitor candidates because they are effectively transported by the ubiquitous system y⁺ amino acid transporter (26, 27) and thus show good activity in vivo. N-Methyl-L-arginine (L-NMA), the prototypic NOS inhibitor (28), has been shown, for example, to reverse the hypotension of septic shock (14, 15, 29) and cytokine-induced shock (17, 30, 31) in animal studies and in early clinical trials. Unfortunately, NMA and several other L-arginine analogs including N-amino-L-arginine (32-34) and N-(1-iminoethyl)-L-ornithine (L-NIO) (35, 36) show little isoform selectivity; their pharmacological use may thus cause undesirable inhibition of physiological processes controlled by nontargeted NOS isoforms. We (37, 38) and others (39) have shown that the S-alkyl-L-thiocitrullines show modest selectivity (up to 50-fold) for nNOS over eNOS and iNOS and have proposed that these compounds may be of use in treating disorders involving overstimulation of nNOS (e.g. stroke). Improved potency and isoform selectivity would, however, be highly desirable.

In the present studies, we have examined several novel L-arginine antagonists and find that vinyl-L-NIO (N⁵-(1-imino-3-butenyl)-L-ornithine, L-VNIO) (Fig. 1) is a potent, mechanism-based inhibitor that attacks the heme cofactor of NOS. It shows a marked selectivity for nNOS. Abstracts describing this work have been published (40, 41).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Structures of vinyl-L-NIO and related compounds.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials

Most biochemicals and reagents for organic syntheses were obtained from Sigma and Aldrich, respectively. N-Methyl-L-arginine and BH₄ were purchased from Chemical Dynamics (Plainfield, NJ) and Alexis (La Jolla, CA), respectively. L-[¹⁴C]Arginine was from NEN Life Science Products. N-Alkyl-L-arginines (42) and L-NIO (43) were prepared by the general methods indicated. Rat nNOS was isolated from stably transfected kidney 293 cells (44) as described previously (45). Bovine eNOS (46) and mouse iNOS expressed in Escherichia coli² were generous gifts from Dr. Kirkwood Pritchard (Department of Pathology, Medical College of Wisconsin, Milwaukee, WI) and Dr. Bettie S. S. Masters (Department of Biochemistry, University of Texas Health Sciences Center, San Antonio, TX), respectively. ¹H and ¹³C NMR spectra were obtained using a Bruker AC 300 MHz spectrometer. FAB mass spectral analyses were generously carried out by Dr. Frank Laib at the Department of Chemistry, University of Wisconsin, Milwaukee.

General Procedure for Preparation of N⁵-(1-Iminoalkyl)ornithines and N⁵-(1-Iminoalkenyl)ornithines

D- or L-Ornithine HCl (1.68 g, 10 mmol) and cupric acetate (0.5 g, 10 mmol) were dissolved in water (20 ml) and stirred for 10 min at room temperature. The solution was then filtered to remove minor impurities and cooled to 0 °C, and the pH was adjusted to 9.5 by addition of cold 10% NaOH. Alkyl or alkenyl imidate (15 mmol), prepared separately from the corresponding nitrile and HCl_(g) (43) was then added, and the mixture was allowed to stir at pH 9.0-9.5 for 1 h at 0 °C and for 2 h at room temperature. The pH was then adjusted to 7.4 with cold dilute HCl, and the mixture was stirred at room temperature overnight. Hydrogen sulfide gas (caution: toxic) was bubbled through the solution, and the resulting copper sulfide precipitate was removed by filtration through charcoal. The filtrate was passed through Chelex to remove any residual Cu²⁺, and the clear solution was evaporated to dryness by rotary evaporation under reduced pressure. The residue was washed with ethyl acetate, and the product was crystallized from ethanol to give pure N⁵-(1-iminoalkyl)ornithine or N⁵-(1-iminoalkenyl)ornithine.

N⁵-(1-Imino-3-butenyl)-L-ornithine (Vinyl-L-NIO, L-VNIO)-- m.p. 162 °C (dec); ¹H NMR (D₂O):  1.65-2.1 (m, 4H), 3.3 (d, 2H), 3.4(t, 2H), 3.8 (t, 1H), 5.4 (m, 2H), and 5.95 (m, 1H); ¹³C NMR (D₂O):  25.43, 30.35, 39.37, 44.28, 56.93, 124.16, 131.55, 168.72, and 176.98; FABMS: m/e 200 (M + H).

N⁵-(1-Imino-3-butenyl)-D-ornithine (Vinyl-D-NIO; D-VNIO)-- m.p. 170 °C (dec), ¹H NMR (D₂O):  1.6-2.1 (m, 4H), 3.3 (d, 2H), 3.4(t, 2H), 3.8 (t, 1H), 5.4 (m, 2H), and 5.89 (m, 1H); ¹³C NMR (D₂O):  25.43, 30.35, 39.37, 44.27, 56.93, 124.13, 131.55, 168.72, and 176.98; FABMS: m/e 200 (M + H).

N⁵-(1-Iminopropyl)-L-ornithine (Methyl-L-NIO)-- m.p. 150 °C. (dec), ¹H NMR (D₂O):  1.29 (t, 3H), 1.6-1.9 (m, 4H), 2.54 (q, 2H), 3.38 (t, 2H), and 3.84 (t, 1H); ¹³C NMR (D₂O):  13.37, 25.43, 28.10, 30.35, 44.09, 56.94, 171.97, and 177.03; FABMS: m/e 188 (M + H).

N⁵-(1-Iminobutyl)-L-ornithine (Ethyl-L-NIO)-- m.p. 145 °C (dec), ¹H NMR (D₂O):  1.01 (t, 3H), 1.65-2.1 (m, 6H), 2.5 (t, 2H), 3.4(t, 2H), and 3.8 (t, 1H); ¹³C NMR (D₂O):  15.06, 22.55, 25.50, 30.44, 37.16, 44.12, 56.97, 170.78, and 177.02; FABMS: m/e 202 (M + H).

Methods

K_i Determination-- Activity of NOS was determined by monitoring the conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline. Reaction mixtures contained in a final volume of 50 µl, 50 mM Na⁺ Hepes buffer, pH 7.4, 100 µM EDTA, 0.2 mM CaCl₂, 10 µg/ml calmodulin, 100 µM dithiothreitol, 50 µM BH₄, 1.0 µM FAD, 1.0 µM FMN, 100 µg/ml bovine serum albumin, 500 µM NADPH, L-[¹⁴C]arginine as indicated, L-VNIO as indicated, and nNOS or eNOS. Reaction mixtures for iNOS were similar, but CaCl₂ and calmodulin were omitted. Reaction was initiated by the addition of enzyme, and the solutions were maintained at 25 °C for 4 min. Reaction mixtures were quenched by the addition of 200 µl of stop buffer containing 100 mM Na⁺ Hepes buffer, pH 5.5, and 5 mM EGTA. Those samples were heated in a boiling water bath for 1 min, chilled and centrifuged. A portion (225 µl) of the supernatant was applied to small Dowex 50 columns (Na⁺ form, 1 ml resin), and the product L-[¹⁴C]citrulline was eluted with 2 ml of water and quantitated by liquid scintillation counting.

Optical Difference Spectroscopy-- Studies were carried out on a Shimadzu model 2501 dual beam UV-visible spectrophotometer using either nNOS as isolated (~20% low spin heme) or nNOS pretreated with imidazole (100% low spin heme). In the former case, 0.5 ml of nNOS as isolated (432 µg) in 50 mM Tris-HCl buffer, pH 7.5, 10% glycerol, and 0.1 mM EDTA was placed in the sample and reference cuvettes at 15 °C, and the base-line spectrum was adjusted to zero. Sequential samples of buffer and L-VNIO in buffer were added to the reference and sample cuvettes, respectively, and optical difference spectra at increasing concentrations of L-VNIO were obtained. Similar studies using imidazole in place of L-VNIO were carried out to determine the K_s for that ligand (K_s^Imid = 86.2 µM, data not shown). The effect of L-VNIO on imidazole liganded nNOS was then determined by initially adding 1.0 mM imidazole to the cuvettes, setting the base line to zero, and then adding sequential samples of buffer and L-VNIO in buffer to the reference and sample cuvettes, respectively, as described above. For these studies K_s^L-VNIO was calculated from the relationship K_s(app)^L-VNIO = K_s^L-VNIO (1 + [Imid]/K_s^Imid), where K_s^L-VNIO is the true binding constant for L-VNIO, K_s(app)^L-VNIO is the apparent binding constant for L-VNIO determined in the presence of imidazole, [Imid] is the concentration of imidazole (1.0 mM) and K_s^Imid is the binding constant for imidazole as determined in the preliminary study mentioned (i.e. 86.2 µM).

Irreversible Inactivation of NOS-- Time- and concentration-dependent inactivation kinetics for nNOS and eNOS treated with various inhibitors were determined at 25 °C in reaction mixtures (final volume = 0.15 ml) containing 50 mM Na⁺ Hepes buffer, pH 7.4, 0.1 mM EDTA, 50 µM BH₄, 2.0 mM glutathione, 1.0 µM FAD, 1.0 µM FMN, 1 mg/ml bovine serum albumin, 100 units of superoxide dismutase, 0.2 mM CaCl₂, 10 µg/ml calmodulin, 1.0 mM NADPH, L-arginine or inhibitor as indicated, and ~40 µg NOS. Residual activity was determined after various time intervals by adding a 25-µl aliquot of the reaction mixture to a cuvette containing, in a final volume of 0.5 ml, 50 mM Hepes buffer, pH 7.4, 0.1 mM EDTA, 50 µM BH₄, 10 µg/ml calmodulin, 0.2 mM CaCl₂, 0.1 mM GSH, 1.0 µM FAD, 1.0 µM FMN, 1 mg/ml bovine serum albumin, 0.5 mM NADPH, and 0.25 mM of L-arginine and 5 µM bovine oxyhemoglobin (prepared by reduction with sodium dithionite followed by gel filtration). Reaction mixtures for iNOS were similar but lacked CaCl₂ and calmodulin. Nitric oxide-mediated oxidation of oxyhemoglobin was monitored at 401 nm ( = 0.038 µM¹) (47); the reference cuvette contained a similar mixture without enzyme. The rate of NO formation was determined and used to calculate the residual activity.

Determination of Heme Loss-- The ability of carbon monoxide (CO) to bind to the reduced heme cofactor of NOS and elicit a characteristic absorption maxima at 443 nm was used to determine the loss of heme cofactor after inactivation of nNOS by L-VNIO. The incubation conditions were similar to those used to determine irreversible inactivation except the final volume was 1.8 ml. After specific time intervals (0, 10, and 20 min), an aliquot (200 µl) of the inactivation reaction mixture was added to both sample and reference cuvettes containing 50 mM Na⁺ Hepes buffer, pH 7.4, and 10% glycerol to give final volume of 0.5 ml. The buffer in the sample cuvette was then saturated with CO, and the difference spectrum between 400 and 500 nm was determined (48).

Determination of NOS-mediated Cytochrome c Reduction-- Reaction mixtures of 500 µl contained 50 mM Na⁺ Hepes buffer, pH 7.4, 100 µM EDTA, 50 µM NADPH, 50 µM bovine heart cytochrome c, and nNOS. NADPH-dependent reduction of cytochrome c was monitored at 550 nm ( = 0.021 µM¹). Where indicated 10 µM L-VNIO, 0.2 mM CaCl₂, 10 µg/ml calmodulin, and/or 800 units/ml superoxide dismutase were added to the reaction mixtures.

Determination of NOS-mediated NADPH Oxidation-- The rate of NADPH oxidation by NOS was determined spectrophotometrically by monitoring the decrease in absorbance at 340 nm with time ( = 6.22 mM¹). The reaction mixtures contained in a final volume of 0.5 ml 50 mM Na⁺ Hepes buffer, pH 7.4, 0.1 mM EDTA, 50 µM BH₄, 2.0 mM GSH, 1.0 µM FAD, 1.0 µM FMN, 1 mg/ml bovine serum albumin, 0.2 mM CaCl₂, 10 µg/ml calmodulin, 0.25 mM NADPH, where indicated 10 µM L-VNIO, and nNOS. NADPH oxidation was initiated by addition of enzyme.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Inhibition of NOS Isoforms by L-VNIO and Related Compounds-- All NOS isoforms are inhibited by a variety of L-arginine analogs that compete with L-arginine for the amino acid binding site; we have reported previously that N-alkyl-L-arginines with n-alkyl substituents up to 4 carbons and the isosteric N⁵-(1-iminoalkyl)-L-ornithines are good to excellent inhibitors (24). Consistent with these findings, in initial rate studies L-VNIO was found to be a potent inhibitor of nNOS, and its binding was competitive with L-arginine (Fig. 2). A replot of the data shows that K_i^L-VNIO is ~100 nM (Fig. 2, inset); this value is substantially lower than the K_m for L-arginine (1.4 µM). Similar studies showed L-VNIO also inhibits eNOS and iNOS competitively with respect to L-arginine, but the K_i values are much higher (i.e. 12.0 µM for eNOS and 60 µM for iNOS) (Table I). Note that the K_i^L-VNIO/K_m^L-Arg ratios for nNOS, eNOS, and iNOS are 0.07, 3.33 and 4.80, respectively, indicating that L-VNIO competes for the L-arginine binding site of nNOS particularly well (Table I). D-Arginine is neither a substrate nor an inhibitor of NOS (1), and none of the NOS isoforms was inhibited by D-VNIO when tested at 100 µM in the presence of 20 µM L-arginine (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Kinetic analysis of nNOS inhibition by L-VNIO. Product formation was determined using the L-[14C]arginine assay as described under "Methods." The inset shows a replot of the data indicating that the Ki for L-VNIO is 100 nM.

Effect of L-VNIO Binding on the Heme Spectrum of nNOS-- A variety of studies indicate that the reactive guanidinium nitrogen of substrate L-arginine is bound near the iron of the NOS heme cofactor; N-hydroxy-L-arginine is then formed when that guanidinium nitrogen is oxidized by O₂, which has been bound and activated as a sixth, axial heme iron ligand (1, 2). Although L-arginine does not bind near enough to heme iron to act as a sixth axial ligand (49), other inhibitors such as L-thiocitrulline do covalently interact with heme iron (24, 50, 51). Such interactions can be revealed by optical difference spectroscopy in which perturbations of the heme spectrum caused by substrates or inhibitors are determined (49). As shown in Fig. 3A, increasing concentrations of L-VNIO, when added to solutions of native nNOS, cause a type I difference spectrum. This result, which is similar to that seen with L-arginine, indicates that L-VNIO does not interact covalently with heme iron but does bind sufficiently close to its sixth axial position to displace an endogenous heme ligand (the identity of the ligand displaced is presently unknown). The displacement of the endogenous ligand, which is present in ~20% of nNOS as isolated (49), is responsible for the spectral change shown in Fig. 3A. Fig. 3B is a replot of the data in Fig. 3A showing that the L-VNIO dissociation constant, K_s^L-VNIO, is 1.1 µM; the previously reported value for K_s^L-Arg is 2.5 µM (49). We also determined K_s^L-VNIO using imidazole-saturated nNOS. These studies confirmed that L-VNIO gives a type I optical difference spectrum and provide a potentially more accurate estimate of K_s^L-VNIO since the signal is larger (i.e. heme iron is initially 100% low spin) and the amounts of L-VNIO added are larger, making it unnecessary to correct for L-VNIO bound to nNOS. With imidazole saturated nNOS, K_s^L-VNIO, calculated as described under "Methods," was 1.4 µM (data not shown). Note that K_s^L-VNIO is a simple binding constant measured in the absence of NADPH whereas K_i^L-VNIO (Table I) is determined under conditions of substrate turnover. The two values need not agree (52).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Binding spectrum of nNOS with L-VNIO. Panel A, the sample of nNOS (432 µg) (prepared in 50 mM Tris-HCl buffer, pH 7.4, 10% glycerol, and 0.1 mM EDTA) was titrated by adding L-VNIO to final concentrations of 1.2 (a), 2.2 (b), 3.2 (c), 4.2 (d), and 5.2 (e) µM. Panel B, double-reciprocal plot of absorbance difference (390-420 nm) versus L-VNIO concentration. A spectral dissociation constant (Ks) of 1.1 µM was established.

Inactivation of nNOS by L-VNIO-- The rate measurements used to construct Fig. 2 were obtained immediately after initiation of the enzymatic reaction. If the reaction mixtures were monitored for several minutes, the progress curves were clearly concave downward indicating occurrence of a progressive irreversible inhibition of nNOS. Such inactivation was examined directly by incubating nNOS with various concentrations of L-VNIO in the presence of NADPH and monitoring the reaction mixtures for residual nNOS activity at intervals (Fig. 4A). As shown, L-VNIO, but not D-VNIO, caused a first-order inactivation of nNOS. There was no evidence of nNOS reactivation in these studies; product formation was constant over the time of the assay. In separate studies, passage of reaction mixtures containing L-VNIO-inactivated nNOS through small gel-filtration columns did not restore activity (not shown). A replot of the data in Fig. 4A shows that the rate constant for inactivation, k_inact, is 0.078 min¹ and K_I, the equilibrium binding constant of L-VNIO to nNOS prior to inactivation, is 90 nM (Fig. 4B). The latter value is similar to the K_i measured under turnover conditions.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Panel A, time- and concentration-dependent inactivation kinetics of nNOS with L-VNIO at 25 °C. Reactions were carried out as described under "Methods." Panel B, double-reciprocal plot for the determination of kinact of nNOS with L-VNIO. The values for 1/kobs (min1) were calculated from the inactivation experiments shown in panel A.

Inactivation of nNOS by Related N⁵-(1-Iminoalkyl)-L-ornithines and N-Methyl-L-arginine-- Previous studies established that nNOS as well as the other NOS isoforms are inhibited and/or inactivated by L-NMA, the prototypic NOS inhibitor (24, 28), and by L-NIO (24, 35, 36). In Fig. 5, inactivation of nNOS by these compounds is compared with that seen with L-VNIO. As shown, inactivation by 0.5 µM L-VNIO is comparable to that seen with 2.0 µM L-NMA and greater than that seen with 10 µM L-NIO. As was seen with L-VNIO, inhibition by L-NIO is NADPH-dependent; nNOS inactivation by L-NMA has previously been shown to be NADPH-dependent (53, 54). Interestingly, the next higher homolog of L-NIO, methyl-L-NIO, is a much weaker inactivator of nNOS, requiring a 10-fold higher concentration (100 µM) to duplicate the inactivation seen with L-NIO. Further extension of the iminoalkyl group to form the saturated analog of L-VNIO (i.e. ethyl-L-NIO) results in a compound that does not inactivate nNOS under the conditions examined (Fig. 5). Note that ethyl-L-NIO does bind competitively with L-arginine and thereby inhibits nNOS (Table I); such inhibition does not, however, progress to inactivation.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Time-dependent inactivation of nNOS by L-NIO, methyl-L-NIO, ethyl-L-NIO, L-NMA, and L-VNIO. Conditions were similar to those used for the studies shown in Fig. 4.

Effect of L-VNIO on Other Reactions Catalyzed by nNOS-- In addition to NO synthesis, nNOS catalyzes the NADPH-dependent reduction of cytochrome c, an activity attributed to the reductase domain (1, 2). It also catalyzes the O₂- and Ca²⁺/calmodulin-dependent, L-arginine-independent oxidation of NADPH, an activity requiring both the reductase and the oxygenase nNOS domains and resulting in the formation of superoxide (1, 2). As shown in Table III (experiments 1 and 2), L-VNIO has no effect on cytochrome c reduction but potently inhibits NADPH oxidation. That cytochrome c reduction is due to direct transfer of electrons to it from the reductase domain and not to formation of superoxide by the oxygenase domain is evident from experiments 3 and 4 showing that superoxide dismutase has no effect on the rate of cytochrome c reduction in the presence or absence of L-VNIO. In the absence of Ca²⁺/calmodulin the rate of electron flow within the reductase domain is greatly diminished and electrons do not flow from the reductase domain to the heme cofactor of the oxygenase domain (5). As shown in experiments 5-8, omission of Ca²⁺/calmodulin from the reaction mixtures decreases cytochrome c reduction ~ 96% and nearly eliminates NADPH oxidation. There is again no effect of L-VNIO and superoxide dismutase on cytochrome c reduction, confirming that this reductase domain specific reaction is not affected. The minimal residual NADPH oxidase activity seen in the absence of Ca²⁺/calmodulin (experiment 5) is apparently unrelated to that seen in the presence of Ca²⁺/calmodulin (experiment 1) since it is not inhibited by L-VNIO; it may reflect a very slow, normally undetectable formation of superoxide by the flavins of the reductase domain.

Inactivation of nNOS by L-VNIO Results in Heme Loss-- Brief treatment of nNOS with dithionite or with NADPH in the presence of Ca²⁺/calmodulin reduces the heme cofactor from the Fe³⁺ to the Fe²⁺ state. With heme reduced, nNOS binds CO with a characteristic increase in absorption at 443 nm that is useful in quantitating bound, functional heme (1, 2). As shown in Table IV, treatment of nNOS with L-VNIO, but not D-VNIO, causes loss of the heme cofactor as judged by CO binding studies. Loss of heme closely parallels the loss of overall NOS activity indicating that loss of heme functionality or heme binding to the enzyme can account for virtually all mechanism-based inactivation.

Ability of L-VNIO to Inactivate eNOS and iNOS-- As shown in Fig. 6, L-VNIO is a relatively weak inactivator of eNOS and it does not inactivate iNOS. Inactivation of eNOS is NADPH-dependent and follows first order kinetics, but even with a 20-fold higher concentration of L-VNIO the rate of inactivation of eNOS does not match that seen with nNOS (compare the rate of eNOS inactivation using 10 µM L-VNIO to that seen with nNOS using 0.5 µM L-VNIO). Detectable inactivation of iNOS did not occur even with 100 µM L-VNIO.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Time-dependent inactivation of NOS isoforms by L-VNIO. Conditions were similar to those shown in Fig. 4, except that CaCl2 and calmodulin were omitted in the studies with iNOS.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Studies from this laboratory and others establish that all NOS isoforms bind a variety of L-arginine and L-homoarginine analogs. Initial binding is in all cases competitive with L-arginine, and all of the analogs inhibit all NOS isoforms if added in sufficiently high concentration (1, 24). Three general mechanisms of inhibition and/or inactivation have been distinguished. Some analogs such as L-canavanine (55), N-cyclopropyl-L-arginine (56), N-nitro-L-arginine with iNOS (57) and N⁵-(1-iminobutyl)-L-ornithine with nNOS (this work) bind to and dissociate from NOS rapidly but do not react covalently; they are classic competitive inhibitors and do not cause inactivation. L-Thiocitrulline is also in this class, although it reversibly interacts to a limited degree (~20%) as a covalent sixth axial ligand to the heme cofactor (50). "Slow on-slow off" inhibitors comprise the second type of NOS inhibitors and are exemplified by the slow binding and very slow dissociation of N-nitro-L-arginine from nNOS and eNOS (58, 59). These inhibitors, which include the S-alkyl-L-thiocitrullines (38, 39), are not altered by the enzyme, but their binding is apparently accompanied by a conformational change in NOS that is only slowly reversible.

Mechanism-based inhibitors (k_cat inhibitors) constitute the third type of NOS-inhibiting amino acids. Of these, the best characterized is L-NMA, which has been shown to be hydroxylated by nNOS and iNOS as a pseudosubstrate; that product, N-hydroxy-N-methyl-L-arginine, undergoes further NOS-mediated reaction to form CH₃NO⁺, formaldehyde, NO, and citrulline by complex mechanisms (53, 54). A presently unidentified intermediate in this latter transformation apparently causes damage to and loss of the NOS heme cofactor, but the product formed is not yet identified (60). N-Allyl-L-arginine (56) and N-amino-L-arginine (34) have also been previously shown to cause mechanism-based, irreversible inactivation of NOS, and there is a brief report that L-NIO (35) (this work) causes similar inactivation. Inactivation by these compounds has not yet been mechanistically characterized. Although N-amino-L-arginine causes somewhat more rapid inactivation of nNOS (k_inact(max) = 0.35 min¹) (34), neither it nor the other previously reported mechanism-based arginine analog inactivators shows significant NOS isoform selectivity.

The present studies establish that L-VNIO is a potent, mechanism-based NOS inactivator with substantial nNOS selectivity. Thus, initial binding of L-VNIO to nNOS is followed by NADPH- and O₂-dependent mechanism-based inactivation of the overall reaction and of the oxygenase domain-dependent NADPH oxidase reactions. The reductase domain-specific cytochrome c reduction activity in the presence or absence of Ca²⁺/calmodulin is not lost. Consistent with damage to the oxygenase domain, inactivation correlates with loss of the heme cofactor as judged by CO difference spectroscopy. Isoform selectivity derives both from differences in initial binding (i.e. the K_i values for eNOS and iNOS are 120- and 600-fold higher than that for nNOS) and from differences in subsequent enzyme-mediated activation of L-VNIO to a reactive derivative (i.e. iNOS is not subject to mechanism-based inactivation).

The precise mechanism by which L-VNIO is transformed by nNOS (and presumably by eNOS, albeit more slowly) into an inactivating intermediate is not yet known. Previous studies establish that NOS-mediated metabolism of L-arginine and L-NMA proceeds via cytochrome P450-like hydroxylation and oxidation reactions (1, 2); in the case of L-NMA, carbon-centered free-radical intermediates are formed (54). We designed L-VNIO with the intent of developing an arginine analog that would place an allyl moiety, known to be highly susceptible to free radical activation, in close proximity to the NOS heme cofactor. The observations that initial binding is competitive with L-arginine and that steady-state K_i values are lower than or comparable to the K_m for L-arginine support the view that L-VNIO binds to nNOS as an L-arginine analog. The observation that a type I difference spectrum accompanies L-VNIO binding to imidazole liganded or native nNOS indicates that a portion of the inhibitor binds closely enough to the heme cofactor to displace (but not replace) imidazole or the endogenous sixth axial heme ligand present in nNOS as isolated. Because the binding site for the non-reactive guanidinium nitrogen of L-arginine (i.e. the nitrogen not near the heme iron and thus not hydroxylated or converted to NO) does not accomodate groups larger than =NH or -NH₂ (1),³ we know further that it is the allyl moiety of L-VNIO that must be near heme. Several specific mechanisms for nNOS-mediated L-VNIO activation thus seem plausible. Activated oxygen bound to heme may epoxidize the allyl moiety, forming N⁵-(1-imino-3,4-epoxybutyl)-L-ornithine; this species could then react with protein or heme nucleophiles in a manner that destroys heme functionality or binding. Alternatively, by a process similar to that described for L-NMA (54), the allyl moiety of L-VNIO may lose H^· to form a reactive allyl radical that damages or derivatizes the heme cofactor. Finally, we have considered the possibility that -electrons of the allyl moiety attack the highly electrophilic perferryl heme species that normally hydroxylates L-arginine to form N-hydroxy-L-arginine (1, 2); this reaction would generate a highly reactive carbocation at the terminus of L-VNIO. Although additional studies are necessary to confirm which of these or related mechanisms is(are) operative, we note that all of the proposed mechanisms depend critically on the unsaturation of the L-VNIO side chain. The mechanistic proposals are thus consistent with our observation that the saturated analog, ethyl-L-NIO, does not inactivate nNOS.

In summary, the present studies establish L-VNIO as the first neuronal isoform selective, mechanism-based amino acid inactivator of NOS. Our studies show further that selectivity can be achieved both in initial binding and in subsequent enzyme-mediated activation. The latter observation is of particular interest. Whereas all NOS isoforms catalyze the same overall reaction for L-arginine, our results indicate the isoforms can differ qualitatively as well as quantitatively in their ability to metabolize and thereby activate L-arginine analogs. There is thus the possibility of designing mechanism-based inhibitors that will have extremely high levels of isoform selectivity.