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.     

Crystallographic studies on endothelial nitric oxide synthase complexed with nitric oxide and mechanism-based inhibitors

The crystal structure of the endothelial nitric oxide synthase (NOS) heme domain complexed with NO reveals close hydrogen bonding interactions between NO and the terminal guanidino nitrogen of the substrate, L-arginine. Dioxygen is expected to bind in a similar mode which will facilitate proton abstraction from L-Arg to dioxygen, a required step for O-O bond cleavage. Structures of mechanism-based NOS inhibitors, N(5)-(1-iminoethyl)-L-ornithine and N-(3-(aminomethyl)benzyl)acetamidine, provide clues on how this class of compounds operate as suicide substrate inhibitors leading to heme oxidation.     

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.     

Differential effects of substrate-analogue inhibitors on nitric oxide synthase dimerization

Cigarette smoking has been linked to almost all major types of cancer. Emerging evidence suggests that smoking initiates transformed cell growth and migration by disrupting cell-cell interactions in the polarized mucosal epithelium. Together with other adherens junction proteins, p120-catenin (p120ctn) maintains cell-cell adhesion through its direct interaction with E-cadherin (E-cad). Mislocalization and/or loss of p120ctn have been reported in all lung cancer subtypes and are related to poor prognosis. Here, we showed that p120ctn modulates smoke-induced cell migration via the EGFR/Src-P pathway. Chemical blockade of EGFR/Src signaling inhibited smoke-induced activation of cofilin (an actin severing protein) and promoted cell migration in the presence of p120ctn but had little effect on blocking migration in the absence of p120ctn. These data suggested that smoke-induced cell migration was mediated via an EGFR/Src-dependent signaling pathway in cells that expressed p120ctn, but upon loss of p120ctn, migration continued to occur via an alternative, EGFR/Src-independent pathway. Thus, gradual loss of membrane p120ctn with lung cancer progression may contribute to reduced effectiveness of conventional chemotherapies, such as those directed against EGFR.     

Differences in the effects of treatment of uncompacted and compacted mouse embryos with phorbol esters on pre- and postimplantation development

Abstract. Differences are described in the effects of treatment of preimplantation mouse embryos with low levels (0.01–1 n M ) of phorbol myristate acetate (PMA), during three different periods of a 48-h culture from the 2-cell stage, on pre- and postimplantation development. Treatment of embryos with PMA for 48 h (first group) or 24 h (second group) from the 2-cell stage caused premature cavitation (prior to the 16-cell stage) and it also reduced the size and alkaline phosphatase (ALPase) activity of inner cell masses (ICMs), as well as the numbers of cells in blastocysts, in a dose-dependent manner. Treatment of early morulae with PMA for 24 h (third group) did not have the abovementioned effects on embryos but inhibited the formation and subsequent enlargement of the blastocoel. The blastocysts that were allowed to develop in the three treatment groups were examined for postimplantation development. Implantation was unaffected in all groups. The survival rate after implantation was low in the first and second groups but relatively high in the third group. The results indicate that an embryo exposed to PMA for 24 h from the 2-cell stage forms a premature blastocoel, and, in such an embryo, quantitative and qualitative differentiation into the ICM is blocked but qualitative differentiation into trophectoderm is uninhibited. Consequently, the embryo can implant but does not survive for a long time. When embryos were exposed to PMA for 24 h from the early morula stage, the formation and enlargement of the blastocoel were inhibited even though the treatment had a minimal effect on other developmental events. It is suggested that the effects of PMA on early mouse development are specific to each period at which the drug is applied.     

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.     

Influence of nitric oxide synthase inhibitors on the vasopressin-induced pituitary-adrenocortical activity and hypothalamic catecholamine levels.

In the present study the role of endogenous nitric oxide (NO) in the vasopressin-induced ACTH and corticosterone secretion was investigated in conscious rats. Vasopressin (AVP 5 microg/kg i.p.) considerably augmented ACTH and corticosterone secretion. L-arginine (120 and 300 mg/kg i.p.) did not significantly alter the AVP-induced secretion of those hormones. Nitric oxide synthase (NOS) blockers N(omega)-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) given i.p. 15 min before AVP markedly increased the AVP-induced ACTH secretion. L-NNA (2 mg/kg) more potently and significantly increased the AVP-induced ACTH secretion, whereas L-NAME elicited a weaker and not significant effect. Both those NOS antagonists intensified significantly and to a similar extent the AVP-induced corticosterone secretion. L-arginine (120 mg/kg i.p.) reversed the L-NNA-induced rise in the AVP-stimulated ACTH secretion and substantially diminished the accompanying corticosterone secretion. Neither vasopressin alone nor in combination with L-arginine and L-NAME evoked any significant alterations in the hypothalamic noradrenaline and dopamine levels. L-NNA (2 and 10 mg/kg i.p.) elicited a dose dependent and significant decrease in the hypothalamic noradrenaline level. The hypothalamic dopamine level was not significantly altered by any treatment. These results indicate that in conscious rats endogenous NO has an inhibitory influence on the AVP-induced increase in ACTH and corticosterone secretion. L-NNA is significantly more potent than L-NAME in increasing the AVP-induced ACTH secretion. This may be connected with a considerable increase by L-NNA of hypothalamic noradrenergic system activation which stimulates the pituitary-adrenal axis in addition to specific inhibition of NOS.     

The rational design of inhibitors of nitric oxide formation by inducible nitric oxide synthase

A series of compounds was rationally designed as inhibitors of dimer formation of the inducible isoform of nitric oxide synthase, and subsequent nitric oxide production. The conformation of two fragments obtained from a crystal structure was utilized to design a tether connecting those same two fragments. The resulting compounds were potent dimerization inhibitors that bound to the enzyme in a similar conformation as the fragments.     

Desmin and titin expression in early postimplantation mouse embryos

The expression of the intermediate filament (IF) constituents desmin, vimentin and keratin, as well as the striated-muscle-specific marker titin, was studied in mouse embryos of 8.0 to 9.5 days post coitum (d.p.c.), using the indirect immunofluorescence technique in combination with polyclonal and monoclonal antibodies. During the development of the embryo, desmin was first detected at 8.25 d.p.c. in the ectoderm, where it was transiently coexpressed with keratin and vimentin. At later stages, the ectoderm contained only keratin and to a certain extent also vimentin IF. At 8.5 d.p.c., desmin was found exclusively in the heart rudiment, and remained present with increasing intensity in the myocardial cells during later cardiogenesis. Striation of desmin in the heart muscle cells was observed in 9.5 d.p.c. embryos. At these stages (8.5-9.5 d.p.c.), triple expression of the IF proteins desmin, vimentin and keratin was evident in these cells. From 9.0 d.p.c. onwards, desmin could be detected in the myotomes as well. Immunoblotting studies of 9.5 d.p.c. mouse embryos confirmed the immunohistochemical data. Titin was found in the early heart anlage at stage 8.25 d.p.c., when no desmin expression was observed in this tissue. At this stage the titin appeared in a punctate pattern, similar to that observed in cardiac myofibrils of early chicken embryos (Tokuyasu and Maher, 1987; J. Cell Biol. 105, 2781-2793). In 8.5 d.p.c. mouse embryos, this punctate titin staining pattern was still observed, while, at this stage, a filamentous staining reaction could be seen with the desmin antibodies. During further development, cross-striation was detected within myocardial cells using the polyclonal titin antibody from 9.0 d.p.c. onwards, i.e. before such striation could be detected with the desmin antibodies. From these data, we conclude that titin synthesis may anticipate desmin expression in the developing mouse myocard, although the level of expression of the former protein remains low until 9.0 d.p.c.     

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.     

Effects of nitric oxide synthase inhibitors on porcine oocyte meiotic maturation

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p < 0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.     

Circadian variation of nitric oxide synthase activity in mouse tissue

Endogenous nitric oxide (NO) is an important mediator in the processes that control biological clocks and circadian rhythms. The present study was designed to elucidate if NO synthase (NOS) activity in the brain, kidney, testis, aorta, and lungs and plasma NO_x levels in mice are controlled by an endogenous circadian pacemaker. Male BALB/c mice were exposed to two different lighting regimens of either light-dark 14:10 (LD) or continuous lighting (LL). At nine different equidistant time points (commencing at 09:00h) blood samples and tissues were taken from mice. The plasma and tissue homogenates were used to measure the levels of NO₂+ NO₃^- (NO_x) and total protein. The NO_x concentrations were determined by a commercial nitric oxide synthase assay kit, and protein content was assessed in each homogenate tissue sample by the Lowry method. Nitric oxide synthase activity was calculated as pmol/mg protein/h. The resulting patterns were analyzed by the single cosinor method for pre-adjusted periods and by curve-fitting programs to elucidate compound rhythmicity. The NOS activity in kidneys of mice exposed to LD exhibited a circadian rhythm, but no rhythmicity was detected in mice exposed to LL. Aortic NOS activity displayed 24h rhythmicity only in LL. Brain, testis, and lung NOS activity and plasma NO_x levels displayed 24h rhythms both in LD and LL. Acrophase values of NOS activity in brain, kidney, testis, and lungs were at midnight corresponding to their behavioral activities. Compound rhythms were also detected in many of the examined patterns. The findings suggest that NOS activity in mouse brain, aorta, lung, and testis are regulated by an endogenous clock, while in kidney the rhythm in NOS activity is synchronized by the exogenous signals.     

Electrochemistry of pterin cofactors and inhibitors of nitric oxide synthase.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), but its function is not fully understood. Specifically, it is unclear whether BH4 participates directly in electron transfer. We investigated the redox properties of BH4 and several other pteridines with cyclic voltammetry and Osteryoung square wave voltammetry. BH4 was oxidized at a potential of +0.27 V vs normal hydrogen electrode (NHE); the corresponding reductive signal after the reversal of the scan direction was very small. Instead, reduction occurred at a potential of -0.16 V vs NHE; there was no corresponding oxidative signal. These two transitions were interdependent, indicating that the reductive wave at -0.16 V represented the regeneration of BH4 from its product of oxidation at +0.27 V. Similar voltammograms were obtained with tetrahydroneopterin and 6,7-dimethyltetrahydropterin, both of which can substitute for BH4 in NOS catalysis. Completely different voltammograms were obtained with 7,8-dihydrobiopterin, sepiapterin, 2'-deoxysepiapterin, and autoxidized BH4. These 7,8-dihydropterins, which do not sustain NOS catalysis, were oxidized at much higher potentials (+0.82-1.04 V vs NHE), and appreciable reduction did not occur between +1.2 and -0.8 V, in line with the concept of a redox role for BH4 in NOS catalysis. However, the electrochemical properties of the potent pterin-site NOS inhibitor 4-amino-BH4 resembled those of BH4, whereas the active pterin cofactor 5-methyl-BH4 was not re-reduced after oxidation. We conclude that the 2-electron redox cycling of the pterin cofactor between BH4 and quinonoid dihydrobiopterin is not essential for NO synthesis. The data are consistent with 1-electron redox cycling between BH4 and the trihydrobiopterin radical BH3(*).     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats

Endotoxin shock is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors and acute lung edema. A nitric oxide synthase (NOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA) has been shown to be effective in reversing acute lung injury. In the present study, we evaluated the effects of NOS blockade by different mechanisms on the endotoxin-induced changes. In anesthetized rats, lipopolysaccharide (LPS,Klebsiella pneumoniae) was administered intravenously in a dose of 10 mg/kg. LPS caused sustained systemic hypotension accompanied by an eightfold increase of exhaled NO during an observation period of 4 h. After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expressions of inducible NOS (iNOS), interleukin-1 (IL-1) and tumor necrosis factor--(TNF-). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1 and TNF- were absent. Four hours after LPS, the mRNA expressions of iNOS and IL-1 were still significantly enhanced, but TNF- was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial damage and interstitial edema. Various NOS inhibitors were given 1 h after LPS administration. These agents included N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), a constitutive NOS and iNOS inhibitor; S,S-1,4-phenylene-bis-(1,2-ethanedinyl) bis-isothiourea dihydrobromide (1,4-PBIT, 10 mg/kg), a relatively specific iNOS inhibitor, and dexamethasone (3 mg/kg), an inhibitor of iNOS expression. These NOS inhibitors all effectively reversed the systemic hypotension, reduced the exhaled NO concentration and prevented acute lung injury. The LPS-induced mRNA expressions of iNOS and IL-1 were also significantly depressed by these NOS inhibitors. Our results suggest that NO production through the iNOS pathway is responsible for endotoxin-induced lung injury. Certain cytokines such as IL-1 are possibly involved. These changes are minimized by NOS inhibitors through different mechanisms.     

The non specificity of specific nitric oxide synthase inhibitors.

L-NAME (Nw-Nitro-L-arginine methylester) and L-NMMA (NG- Monomethyl-L-arginine, monoacetate) are used widely as nitric oxide (NO) synthase inhibitors. Because of their functional groups (alcohols, amines and carboxylates), it appeared that they could interact with iron in a variety of systems. Using three in vitro models we observed these two compounds had inhibitory effects on cytochrome C reduction by ferrous iron, by ferrous iron accelerated by an unsaturated fatty acid or by epinephrine. This suggests that L-NAME and L-NMMA could have effects in iron containing systems found intracellularly apart from their inhibition of (NO) synthesis.