Novel inhibitors of neuronal nitric oxide synthase

Selective inhibitors of neuronal nitric oxide synthase (nNOS), which are devoid of any effect on the endothelial isoform (eNOS), may be required for the treatment of some neurological disorders. In our search for novel nNOS inhibitors, we recently described some 1-[(Aryloxy)ethyl]-1H-imidazoles as interesting molecules for their selectivity for nNOS against eNOS. This work reports a new series of 1-[(Aryloxy)alkyl]-1H-imidazoles in which a longer methylene chain is present between the imidazole and the phenol part of molecule. Some of these molecules were found to be more potent nNOS inhibitors than the parent ethylenic compounds, although this increase in potency resulted in a partial loss of selectivity. The most interesting compound was investigated to establish its mechanism of action and was found to interact with the tetrahydrobiopterin (BH(4)) binding site of nNOS, without interference with any other cofactors or substrate binding sites.     

Thermodynamic analysis of L-arginine and N omega-hydroxy-L-arginine binding to nitric oxide synthase

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS(oxy)) in the presence of the cofactor tetrahydrobiopterin. The intermediate N(omega)-hydroxy-L-arginine (NHA) has a larger affinity than L-Arginine (L-Arg) for nNOS(oxy), with K(d)=0.4+/-0.1 microM and 1.7+/-0.3 microM at 25 degrees C, respectively. nNOS(oxy) binds NHA and L-Arg with DeltaH -4.1+/-0.2 and -1.0+/-0.1 kcal/mol and DeltaS=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to L-Arg. The changes in heat capacity (DeltaC(p)) are relatively small for binding of both NHA and L-Arg (-53+/-18 and -95+/-23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.     

Ligand-protein interactions in nitric oxide synthase

Nitric oxide synthases (NOSs) are heme proteins that catalyze the formation of nitric oxide (NO) from L-arginine and oxygen in a sequential two-step process. Three structurally similar isoforms have been identified that deliver NO to different tissues for specific functions. An understanding of the interactions of ligands with the protein is essential to determine the mechanism of catalysis, the design of inhibitors and the differential auto-inhibitory regulation of the enzymatic activity of the isoforms due to the binding of NO to the heme. Ligand-protein interactions in the three isoforms revealed by resonance Raman scattering studies are reviewed in this article. The CO-related modes in the CO-bound ferrous enzyme are sensitive to the presence of substrate, either L-arginine or N-hydroxy-L-arginine, in the distal pocket, but insensitive to the presence of the tetrahydrobiopterin (H4B) cofactor. In contrast, when NO is coordinated to the ferric heme, the NO is sensitive to the substrate only when H4B is present. Furthermore, in the NO-bound ferric enzyme, the addition of H4B induces a large heme distortion that may modulate heme reduction and thereby regulate the NO auto-inhibitory process. In the metastable O2-bound enzyme, L-arginine binding causes the appearance of a shoulder on the O-O stretching mode, suggesting a specific interaction of the heme-bound dioxygen with the bound-substrate that may be crucial for the oxygenation reaction of the substrate during the catalytic turn-over. It is postulated that spectroscopic differences in the oxy-complex are a consequence of the degree of protonation of the proximal cysteine ligand on the heme. Resonance Raman studies of NOSs expand our understanding of the mechanistic features of this important family of enzymes.     

Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase

Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chem. Soc. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.     

Stopped-flow analysis of substrate binding to neuronal nitric oxide synthase.

The kinetics of binding L-arginine and three alternative substrates (homoarginine, N-methylarginine, and N-hydroxyarginine) to neuronal nitric oxide synthase (nNOS) were characterized by conventional and stopped-flow spectroscopy. Because binding these substrates has only a small effect on the light absorbance spectrum of tetrahydrobiopterin-saturated nNOS, their binding was monitored by following displacement of imidazole, which displays a significant change in Soret absorbance from 427 to 398 nm. Rates of spectral change upon mixing Im-nNOS with increasing amounts of substrates were obtained and found to be monophasic in all cases. For each substrate, a plot of the apparent rate versus substrate concentration showed saturation at the higher concentrations. K(-)(1), k(2), k(-)(2), and the apparent dissociation constant were derived for each substrate from the kinetic data. The dissociation constants mostly agreed with those calculated from equilibrium spectral data obtained by titrating Im-nNOS with each substrate. We conclude that nNOS follows a two-step, reversible mechanism of substrate binding in which there is a rapid equilibrium between Im-nNOS and the substrate S followed by a slower isomerization process to generate nNOS'-S: Im-nNOS + S if Im-nNOS-S if nNOS'-S + Im. All four substrates followed this general mechanism, but differences in their kinetic values were significant and may contribute to their varying capacities to support NO synthesis.     

PIN inhibits nitric oxide and superoxide production from purified neuronal nitric oxide synthase

A protein inhibitor of neuronal nitric oxide synthase (nNOS) was identified and designated as PIN. PIN was reported to inhibit nNOS activity in cell lysates through disruption of enzyme dimerization. However, there has been lack of direct characterization of the effect of PIN on NO production from purified nNOS. Furthermore, nNOS also generates superoxide (.O(2)(-)) at low levels of L-arginine. It is unknown whether PIN affects .O(2)(-) generation from nNOS. Therefore, we performed direct measurements of the effects of PIN on NO and .O(2)(-) generation from purified nNOS using electron paramagnetic resonance spin trapping techniques. nNOS was isolated by affinity chromatography and a fusion protein CBP-PIN was used to probe the effect of PIN. While the tag CBP did not affect nNOS activity, CBP-PIN caused a dose-dependent inhibition on both NO and L-citrulline production. In the absence of L-arginine, strong .O(2)(-) generation was observed from nNOS, and this was blocked by CBP-PIN in a dose-dependent manner. With low-temperature polyacrylamide gel electrophoresis, neither CBP nor CBP-PIN was found to affect nNOS dimerization. Thus, these results suggested that PIN not only inhibits NO but also .O(2)(-) production from nNOS, and this is through a mechanism other than decomposition of nNOS dimers.     

Hydroxyl-terminated peptidomimetic inhibitors of neuronal nitric oxide synthase

The X-ray structure of previously studied dipeptidomimetic inhibitors bound in the active site of neuronal nitric oxide synthase (nNOS) presented a possibility for optimizing the strength of enzyme-inhibitor interactions as well as for enhancing bioavailability. These desirable properties may be attainable by replacement of the terminal amino group of the parent compounds (1-6) with a hydroxyl group (11-13, and 18-20). The hypothesized effect would be twofold: first, a change from a positively charged amino group to a neutral hydroxyl group might afford more drug-like character and blood-brain barrier permeability to the inhibitors; second, as suggested by docking studies, the incorporated hydroxyl group might displace an active site water molecule with which the terminal amino group of the original compounds indirectly hydrogen bonds. In vitro activity assays of the hydroxyl-terminated analogs (11-13 and 18-20) showed greater than an order of magnitude increase in K(i) values (decreased potency) relative to the amino-terminated compounds. These experimental data support the importance to enzyme binding of a potential electrostatic interaction relative to a hydrogen bonding interaction.     

Cyclopropyl- and methyl-containing inhibitors of neuronal nitric oxide synthase

Inhibitors of neuronal nitric oxide synthase have been proposed as therapeutics for the treatment of different types of neurological disorders. On the basis of a cis-3,4-pyrrolidine scaffold, a series of trans-cyclopropyl- and methyl-containing nNOS inhibitors have been synthesized. The insertion of a rigid electron-withdrawing cyclopropyl ring decreases the basicity of the adjacent amino group, which resulted in decreased inhibitory activity of these inhibitors compared to the parent compound. Nonetheless, three of them exhibited double-digit nanomolar inhibition with high nNOS selectivity on the basis of in vitro enzyme assays. Crystal structures of nNOS and eNOS with these inhibitors bound provide a basis for detailed structure–activity relationship (SAR) studies. The conclusions from these studies will be used as a guide in the future development of selective NOS inhibitors.     

Selective L-nitroargininylaminopyrrolidine and L-nitroargininylaminopiperidine neuronal nitric oxide synthase inhibitors

Selective inhibition of the localized excess production of NO by neuronal nitric oxide synthase (nNOS) has been targeted as a potential means of treating various neurological disorders. Based on observations from the X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-amino)ethylamino]pentyl}-N'-nitroguanidine (L-Arg(NO2)-L-Dbu-NH2 (1) and 4-N-(Nomega-nitro-L-argininyl)-trans-4-amino-L-proline amide (2), a series of descarboxamide analogues was designed and synthesized (3-7). The most potent compound was aminopyrrolidine analogue 3, which exhibited better potency and selectivity for nNOS than parent compound 2. In addition, 3 provided higher lipophilicity and a lower molecular weight than 2, therefore having better physicochemical properties. Nalpha-Methylated analogues (8-11) also were prepared for increased lipophilicity of the inhibitors, but they had 4- to 5-fold weaker binding affinity compared to their parent compounds.     

Hydroxyethylene isosteres of selective neuronal nitric oxide synthase inhibitors

Nitric oxide (NO) is an important second messenger molecule for blood pressure homeostasis, as a neurotransmitter, and in the immune defense system. Excessive NO can lead to neurodegeneration and connective tissue damage. Three different isozymes of the enzyme nitric oxide synthase regulate NO production in endothelial (eNOS), neuronal (nNOS), and macrophage (iNOS) cells. Whereas creating a lower level of NO in some cells could be beneficial, it also could be detrimental to the protective effects that NO has on other cells. Therefore, it is essential that therapeutic NOS inhibitors be made that are subtype selective. Previously, we reported a series of nitroarginine-containing dipeptide amides as potent and selective nNOS inhibitors. Here we synthesize peptidomimetic hydroxyethylene isosteres of these dipeptide amides for potential increased bioavailability. None of the compounds is as potent or selective as the dipeptide amides, but they exhibit good inhibition and selectivity. When the terminal amino group was converted to a hydroxyl group, potency and selectivity greatly diminished, supporting the importance of the terminal amino group for binding.     

Substrate-ligand interactions in Geobacillus stearothermophilus nitric oxide synthase

Nitric oxide synthase (NOS) generates NO via a sequential two-step reaction [l-arginine (l-Arg) --> N-hydroxy-l-arginine (NOHA) --> l-citrulline + NO]. Each step of the reaction follows a distinct mechanism defined by the chemical environment introduced by each substrate bound to the heme active site. The dioxygen complex of the NOS enzyme from a thermophilic bacterium, Geobacillus stearothermophilus (gsNOS), is unusually stable; hence, it provides a unique model for the studies of the mechanistic differences between the two steps of the NOS reaction. By using CO as a structural probe, we found that gsNOS exhibits two conformations in the absence of substrate, as indicated by the presence of two sets of nu(Fe-CO)/nu(C-O) modes in the resonance Raman spectra. In the nu(Fe-CO) versus nu(C-O) inverse correlation plot, one set of data falls on the correlation line characterized by mammalian NOSs (mNOS), whereas the other set of data lies on a new correlation line defined by a bacterial NOS from Bacillus subtilis (bsNOS), reflecting a difference in the proximal Fe-Cys bond strength in the two conformers of gsNOS. The addition of l-Arg stabilizes the conformer associated with the mNOS correlation line, whereas NOHA stabilizes the conformer associated with the bsNOS correlation line, although both substrates introduce a positive electrostatic potential into the distal heme pocket. To assess how substrate binding affects Fe-Cys bond strength, the frequency of the Fe-Cys stretching mode of gsNOS was monitored by resonance Raman spectroscopy with 363.8 nm excitation. In the substrate-free form, the Fe-Cys stretching mode was detected at 342.5 cm(-1), similar to that of bsNOS. The binding of l-Arg and NOHA brings about a small decrease and increase in the Fe-Cys stretching frequency, respectively. The implication of these unique structural features with respect to the oxygen chemistry of NOS is discussed.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

Relationship between structure and inhibitory effect of arginine analogues on neuronal nitric oxide synthase activity

Since nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) froml-arginine (Arg) which has an amidino group in its molecule, we, examined the effect of 29 kinds of Arg analogues on neuronal NOS (nNOS) activity in the rat brain. None of the Arg analogues acted as a substrate for nNOS. Diamidinocystamine, hirudonine, and guanethidine inhibited nNOS activity to 67.3%, 64.2% and 74.1%, respectively, but their inhibitory efficiency was lower than N^G-monomethyl-l-arginine (to 36.5%) which is a well known NOS inhibitor. Dimethylguanidine and N-benzoylguanidine also significantly inhibited nNOS activity to 88.0% and 90.7%, respectively. Whereas almost all of the NOS inhibitors previously reported were synthesizdd by substituting the amidino nitrogen of Arg, none of these new inhibitors were substituted at this position. Furthermore, hirudonine, which is a naturally occurring compound, was thought to act as an agonist at polyamine binding site of the N-methyl-d-aspartate type of glutamate receptor complex. It is also interesting that guanethidine, an antihypertensive agent, inhibit nNOS activity. These new drugs are useful for the investigation not only of the chemical nature of nNOS but also of the physiologic function of NO.     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K_m for arginine 2.8, 2.0 and 2.3 μM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

Inhibition of neuronal nitric oxide synthase by N-phenacyl imidazoles.

Nitric oxide (NO) mediates a series of physiological processes, including regulation of vascular tone, macrofage-mediated neurotoxicity, platelet aggregation, learning and long-term potentiation, and neuronal transmission. Although NO mediates several physiological functions, overproduction of NO can be detrimental and play multiple roles in several pathological diseases. Accordingly, more potent inhibitors, more selective for neuronal nitric oxide synthase (nNOS) than endothelial NOS (eNOS) or inducible NOS (iNOS), could be useful in the treatment of cerebral ischemia and other neurodegenerative diseases. We recently described the synthesis of a series of imidazole derivatives. Among them N-(4-nitrophenacyl) imidazole (A) and N-(4-nitrophenacyl)-2-methyl-imidazole (B) were considered selective nNOS inhibitors. In the present study the action mechanism of compounds A and B was analyzed. Spectral changes observed in the presence of compound A indicate that this inhibitor exerts its effect without interaction with heme iron. Moreover compounds A and B, inhibit nNOS "noncompetitively" versus arginine, but "competitively" versus BH(4).     

A cellular model for screening neuronal nitric oxide synthase inhibitors

Nitric oxide synthase (NOS) inhibitors are potential drug candidates because it has been well demonstrated that excessive production of nitric oxide critically contributes to a range of diseases. Most inhibitors have been screened in vitro using recombinant enzymes, leading to the discovery of a variety of potent compounds. To make inhibition studies more physiologically relevant and bridge the gap between the in vitro assay and in vivo studies, we report here a cellular model for screening NOS inhibitors. Stable transformants were generated by overexpressing rat neuronal NOS in HEK 293T cells. The enzyme was activated by introducing calcium ions into cells, and its activity was assayed by determining the amount of nitrite that was formed in culture medium using the Griess reagent. We tested a few NOS inhibitors with this assay and found that the method is sensitive, versatile, and easy to use. The cell-based assay provides more information than in vitro assays regarding the bioavailability of NOS inhibitors, and it is suitable for high-throughput screening.     

Novel 2-aminobenzothiazoles as selective neuronal nitric oxide synthase inhibitors

A series of substituted 2-aminobenzothiazole compounds have been synthesized and evaluated as nitric oxide synthase (NOS) inhibitors. Compound 14 shows activity in the nM range and is selective for the human neuronal NOS isoform. We have also evaluated the compounds against the rat NOS isoforms. For some of the compounds, there are significant differences in NOS inhibitory activities between the human and rat enzymes. For example, compound 10b has nM activity against the rat nNOS while low microM activity against the human nNOS.     

Spin trapping nitric oxide from neuronal nitric oxide synthase: A look at several iron-dithiocarbamate complexes

The free radical, nitric oxide ( radicalNO), is responsible for a myriad of physiological functions. The ability to verify and study radicalNO in vivo is required to provide insight into the events taking place upon its generation and in particular the flux of radicalNO at relevant cellular sites. With this in mind, several iron-chelates (Fe2+(L)2) have been developed, which have provided a useful tool for the study and identification of radicalNO through spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. However, the effectiveness of radicalNO detection is dependent on the Fe2+(L)2 complex. The development of more efficient and stable Fe2+(L)2 chelates may help to better understand the role of radicalNO in vivo. In this paper, we present data comparing several proline derived iron-dithiocarbamate complexes with the more commonly used spin traps for radicalNO, Fe2+-di(N-methyl-D-glutamine-dithiocarbamate) (Fe2+(MGD)2) and Fe2+-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2). We evaluate the apparent rate constant (kapp) for the reaction of radicalNO with these Fe2+(L)2complexes and the stability of the corresponding Fe2+(NO)(L)2 in presence of NOS I.