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Experimental Biology and Medicine 228:486-490 (2003)
© 2003 Society for Experimental Biology and Medicine

HEME OXYGENASE

Novel Inhibitors of Neuronal Nitric Oxide Synthase

C. Di Giacomo*,1, V. Sorrenti*, L. Salerno{dagger}, V. Cardile{ddagger}, F. Guerrera{dagger}, M.A. Siracusa{dagger}, M. Avitabile* and A. Vanella*

* Department of Biochemistry, Medical Chemistry and Molecular Biology,
{dagger} Department of Pharmaceutical Sciences,
{ddagger} Department of Physiological Sciences, University of Catania, Catania, Italy

Abstract

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 (BH4) binding site of nNOS, without interference with any other cofactors or substrate binding sites.

Key Words: imidazole derivatives • nNOS • selective inhibitors • BH4

Nitric oxide (NO) is a molecular messenger involved in a number of physiological processes in mammalians (1–4). It is synthesized by nitric oxide synthase (NOS) from L-arginine and molecular oxygen. The catalytic action of NOS requires several cofactors including NADPH, FAD, FMN, Ca2+/calmodulin, BH4, and heme prostetic group. Three distinct NOS enzymes have been identified and characterized: neuronal (nNOS) and endothelial (eNOS), which are constitutively expressed, and inducible (iNOS)(5). Although NO mediates several physiological functions, its overproduction by nNOS has been reported in a number of clinical disorders, including acute (stroke) and chronic (schizophrenia, Alzheimer, Parkinson, and AIDS dementia) neurodegenerative diseases, convulsions, and pain. Overproduction of NO by iNOS has been implicated in various pathological processes, including septic shock, tissue damage after inflammation, and rheumatoid arthritis (6). On the contrary, NO produced by eNOS has mainly physiological roles; in fact, it has been shown to be antiatherogenic and critical for angiogenesis. Accordingly, selective inhibition of nNOS or iNOS may provide a novel therapeutic approach to various diseases, whereas inhibition of eNOS is undesiderable because of its role in the maintenance of vascular tone homeostasis. For these reasons, over the last few years, the design of selective NOS inhibitors has received much attention. To date, many compounds have been shown to inhibit NOS, including mono- or di-substituted arginines, guanidines, isothioureas, amidines, thiazines, benzoxazoles, pyridines, pteridines, indazoles, and imidazoles (7–10). The majority of inhibitors described are nonselective or iNOS-selective, whereas only a few compounds, among which 7-nitro-indazole (11) and l-(2-trifluoromethyl-phenyl)-imidazole (TRIM)(12) and, more recently described, some aromatic amidines (13) and some amino acid derivatives (14), are able to selectively inhibit nNOS. It is therefore conceivable that selective nNOS inhibitors could be developed.

Because the imidazole nucleus possesses a pharmacophore role for NOS inhibitory activity (15, 16), we recently described the synthesis and biological profile as NOS inhibitors of two series of N-1-substituted imidazoles: N-(aryloxy)alkyl-imidazoles (17) and N-phenacyl-imidazoles (18). Some compounds of both series showed a good activity against nNOS together with selectivity over eNOS.

In the present study we synthesized and tested the analogues of compounds 1-[2-(4-bromophenoxy)ethyl]-1H-imidazole (I) and 1-[2-(4-trifluoromethylphenoxy)ethyl]-1H-imidazole (J) in which the imidazole and substituted phenol parts of the molecules were separated by alkyl chains containing from three to six methylenes, instead of two. The aim of this modification was both to further increase the hydrophobicity of these molecules and to determine whether a difference in the distance between these two potential pharmacophores (imidazole and substituted phenol rings) could discriminate between the active sites (arginine, heme, and BH4) of the two investigated isoforms of NOS (nNOS and eNOS). In addition, the action mechanism of a selected compound (B) was analyzed in detail, also to compare it to that of N-(4-nitrophenacyl)imidazole previous described (19).

Materials and Methods

Chemistry.
Synthetic pathways for the synthesis of title compounds are in agreement with standard protocols (17). In brief, N-(Aryloxy)alkyl-imidazoles A–H were prepared by alkylation of imidazole with the corresponding (aryloxy)alkyl bromide in dry DMF and sodium hydride. Yields were between 40 and 60%. (Aryloxy)alkyl bromides were prepared by a Williamson’s reaction on substituted phenols with appropriate dibromoalkane carried out in acetone and K2CO3. Imidazole and phenols were purchased from Aldrich. Purification of all synthesized compounds was usually performed with flash chromatography. Purity and structures of all synthesized compounds were confirmed by elemental analyses and 1H NMR spectral data. Analytical and 1H NMR data of compound B are reported as a representative compound:

1-[4-(4-bromophenoxy)butyl]-1H-imidazole (B).
Yield: 45%; mp59.5–99.9°C; 1H NMR (CDCl3): 1.73–2.03 (m, 4H, CH2CH2); 3.9–4.06 (m, 4H, CH2O, and NCH2); 6.70–6.79 (m, 2H, Ar); 6.91 (bs, 1H, H5 Im); 7.08 (bs, 1H, H4 Im); 7.32–7.39 (m, 2H, Ar); 7.52 (bs, 1H, H2 Im). Anal. C13H15BrN2O.

Biology.
Enzymatic assay.
NOS isoenzymes.
Neuronal rat recombinant nitric oxide synthase isolated from a Baculovirus overexpression system in SF9 cells, was purchased from ALEXIS. Endothelial nitric oxide synthase was prepared by a transformed endothelial cell (EC) line from the heart of C57BL/6 mice (H5V). EC (passage 5) were cultured in flasks (Falcon, Becton Dickinson) until confluent in Dulbecco’s modified Eagle’s medium (Life Technologies), with 10% fetal calf serum, 1mM glutamine and antibiotics and incubated at 37°C in a humidified 5% CO2 atmosphere. The medium was changed every 2 days and subcultures were performed every 4-5 days following treatment with trypsin-EDTA.

ECs were scraped and washed in phosphate-buffered saline. Approximately 1 x 109 ECs were suspended in 1.5 ml of 50 mM Tris-HCl pH 7.4 containing 10 mM EDTA, 5 mM glucose, 1.15% w/v KCl, 0.1 mM DTT, 2 mg/l leupeptin, 2 mg/l pepstatin, and 44 mg/l phenylmethylsulfonyl fluoride. The cell suspensions were homogenized by sonication twice for 5 sec with a Soniprep and then centrifuged at 0°C (20,000 x g for 20 min). The supernatant was then used for the enzymatic assay.

The assay for NO synthase activities was carried out measuring the conversion rate of oxyhemoglobin to methemoglobin at {lambda}= 401 nm according to Hevel and Marletta (20). Because compound B was the most interesting because of its potency against nNOS together with its selectivity with respect to eNOS (Ki ratio = 19.53), its mechanism of action was further investigated. Absorbance spectra of recombinant nNOS and effects of varying L-arginine and BH4 concentrations in the presence or absence of compound B were assayed as previously reported (19).

Results

In the present study, the ability of 1-substituted imidazoles A–H to inhibit nNOS and eNOS was determined by monitoring the conversion of oxyhemoglobin to methemoglobin. Results reported in Table IGo represent the Ki values in the micromolar range for the inhibition of nNOS and eNOS activities. TRIM, 1-phenyl-imidazole and L-nitroarginine were always used as reference substances.


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  		Table I. Inhibitory Activities of NOS of Compounds A–J
 
From Table 1Go, it is clear that, increasing hydrophobicity by an elongation of the carbon chain between the imidazole and the phenol rings, improved nNOS inhibitory activity only among the 4-Br substituted compounds, in particular when four methylenes were present (the order of activity was I = A < B > C).

Because compound B was the most potent nNOS inhibitor among this group of 1-[(Aryloxy)alkyl]-1H-imidazoles and maintained a discreet selectivity over eNOS (Ki eNOS/nNOS ratio = 19.53), it was selected to study its mechanism of action on the NOS neuronal isoform and compare it to that of the previously studied N-phenacyl-imidazoles (19).

In this work, we verified whether compound B interferes with the binding tetrahydrobiopterin or with other sites of the nNOS enzyme. In the presence of 12 µM BH4, compound B appeared to be a less potent inhibitor of nNOS. In fact, the absence of BH4 in the incubation medium, increased its inhibitory effect (Fig. 1Go). As shown in Figure 2Go, compound B did not compete with L-arginine. In fact, variations in the concentration of L-arginine (1.25 µM or 50 µM) produced no significant change in the inhibitory capacity of this compound.



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  		Figure 1. Effect of compound B on nNOS activity in the presence of increasing concentrations of tetrahydrobiopterin. Results represent the mean ± SD of six experiments (SDs lie within dimension of symbols).

 


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  		Figure 2. Dixon plot for the inhibition of nNOS activity by compound B.

 
The optical spectra of recombinant nNOS, in the presence and absence of BH4, or BH4 + compound B, are shown in Figure 3Go. The peak near {lambda} = 380 nm in the SORET region of the absolute spectrum of nNOS, indicates the presence of low-spin-state heme iron in a predominantly high-spin heme iron enzyme preparation. Absolute spectra obtained in the presence of BH4, or BH4 + compound B, characterize the conversion of the low-spin-state heme iron to high-spin heme complex according to reports of other authors about several molecules (21–23). Figure 4Go shows the perturbation difference spectra obtained for BH4, BH4 + compound B with nNOS. These are "type I" difference spectra.



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  		Figure 3. Absolute spectra of recombinant nNOS in absence (a) and in presence of 12 µM BH4 (c), and 12 µM BH4 + 50 µM compound B (b). Optical spectra were obtained from 5 U of nNOS in 100 mM HEPES buffer, pH 7.4.

 


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  		Figure 4. Perturbation difference spectra induced by 12 µM BH4 (B), 12 µM BH4 + 50 µM compound B (C), binding to recombinant nNOS. Five units of nNOS in 100 mM HEPES buffer, pH 7.4 were placed in the sample and reference cuvettes, which were mantained at 25°C in the spectrophotometer; the absorbance difference was adjusted to zero, and the baseline was recorded (A).

 
Discussion

At the biochemical level, NOS catalysis requires various cofactors including NADPH, FAD, FMN and BH4; the last markedly stimulates NOS catalytic activity by a unique mechanism involving enzyme stabilization (24–27). Raman et al. (28) reported that pterin binding is critical for dimer formation and consequently the activation of iNOS and nNOS but not of eNOS. Moreover, Werner et al. (29) reported that novel tetrahydrobiopterin derivatives show high selectivity for nNOS. In view of these considerations, the BH4 site of NOS may represent an ideal target for selective inhibitors of nNOS with respect to eNOS, and then compounds that mainly act at this site may be considered noteworthy.

The results of the present study are in agreement with data obtained in our previous research for N-phenacyl imidazole derivatives (19). Compound B inhibited nNOS activity "noncompetitively" versus arginine, but "competitively" versus BH4. In fact, the removal of exogenous BH4 from the incubation medium significantly influenced the capacity of compound B to inhibit nNOS. Moreover, these data are in agreement with Handy and Moore (30), who reported a similar mechanism of action for the nNOS selective inhibitor TRIM, although TRIM inhibited nNOS "competitively" both versus arginine and tetrahydrobiopterin. Spectral changes observed in both absolute and difference spectra, in the presence of BH4 and compound B, indicate that this inhibitor exerts its effect without interaction with heme iron, in contrast to other imidazole derivatives that selectively inhibit iNOS (16, 31).

In conclusion, although the inhibitory potency of some compounds (B, C, and F) against nNOS increased with the distance between imidazole and phenol in comparison with the ethylenic analogues, selectivity decreased, indicating that this modification is a good approach for improving the inhibitory potency against nNOS but not for discriminating between neuronal and endothelial isoforms. However, compound B was found to be a more potent inhibitor of nNOS than 1-phenyl imidazole, with a similar potency to TRIM, and more selective than L-nitroarginine. Moreover, although the observed structure-activity relationships among previously studied N-phenacyl imidazoles and 1-[(Aryloxy) alkyl]-1H-imidazoles were different, their mechanism of action was the same. In fact, compound B specifically interacts with the BH4 binding site of nNOS and does not interfere with any other cofactors or substrate binding, in agreement with Frohlich et al. (32) who described a group of pteridine-derived compounds as potent inhibitors of the neuronal isoform. This confirms the potentiality of 1-[(Aryloxy)alkyl]-1H-imidazoles as nNOS inhibitors and opens the way for further modifications aimed at improving selectivity.

Footnotes

1 To whom requests for reprints should be addressed at Department of Biochemistry, Medical Chemistry and Molecular Biology, Viale Andrea Doria 6, 95125, Catania, Italy. E-mail: cdigiaco{at}unict.it Back

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