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1.
Antioxidants (Basel) ; 9(2)2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31972975

RESUMO

: A specific light trigger for activating endothelial Nitric Oxide-Synthase (eNOS) in real time would be of unique value to decipher cellular events associated with eNOS activation or to generate on demand cytotoxic levels of NO at specific sites for cancer research. We previously developed novel tools called nanotriggers (NT), which recognized constitutive NO-synthase, eNOS or neuronal NOS (nNOS), mainly via their 2' phosphate group which is also present in NADPH in its binding site. Laser excitation of NT1 bound to eNOS triggered recombinant NOS activity and released NO. We recently generated new NTs carrying a 2' or 3' carboxylate group or two 2' and 3' carboxylate moieties replacing the 2' phosphate group of NADPH. Among these new NT, only the 3' carboxylate derivative released NO from endothelial cells upon laser activation. Here, Molecular Dynamics (MD) simulations showed that the 3' carboxylate NT formed a folded structure with a hydrophobic hub, inducing a good stacking on FAD that likely drove efficient activation of nNOS. This NT also carried an additional small charged group which increased binding to e/nNOS; fluorescence measurements determined a 20-fold improved affinity upon binding to nNOS as compared to NT1 affinity. To gain in specificity for eNOS, we augmented a previous NT with a "hook" targeting variable residues in the NADPH site of eNOS. We discuss the potential of exploiting the chemical diversity within the NADPH site of eNOS for reversal of endothelial dysfunction in cells and for controlled generation of cytotoxic NO-derived species in cancer tissues.

2.
Free Radic Biol Med ; 110: 261-269, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28666850

RESUMO

Previously published studies strongly suggested that insulin- and exercise-induced skeletal muscle glucose uptake require nitric oxide (NO) production. However, the signal transduction mechanisms by which insulin and contraction regulated NO production and subsequent glucose transport are not known. In the present study, we utilized the myotube cell lines treated with insulin or hydrogen peroxide, the latter to mimic contraction-induced oxidative stress, to characterize these mechanisms. We found that insulin stimulation of neuronal nitric oxide synthase (nNOS) phosphorylation, NO production, and GLUT4 translocation were all significantly reduced by inhibition of either nNOS or Akt2. Hydrogen peroxide (H2O2) induced phosphorylation of nNOS at the same residue as did insulin, and also stimulated NO production and GLUT4 translocation. nNOS inhibition prevented H2O2-induced GLUT4 translocation. AMP activated protein kinase (AMPK) inhibition prevented H2O2 activation and phosphorylation of nNOS, leading to reduced NO production and significantly attenuated GLUT4 translocation. We conclude that nNOS phosphorylation and subsequently increased NO production are required for both insulin- and H2O2-stimulated glucose transport. Although the two stimuli result in phosphorylation of the same residue on nNOS, they do so through distinct protein kinases. Thus, insulin and H2O2-activated signaling pathways converge on nNOS, which is a common mediator of glucose uptake in both pathways. However, the fact that different kinases are utilized provides a basis for the use of exercise to activate glucose transport in the face of insulin resistance.


Assuntos
Glucose/metabolismo , Peróxido de Hidrogênio/farmacologia , Insulina/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo I/metabolismo , Estresse Oxidativo , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
4.
J Med Chem ; 58(21): 8694-712, 2015 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-26469213

RESUMO

Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding.


Assuntos
Aminoquinolinas/química , Aminoquinolinas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Éteres Fenílicos/química , Éteres Fenílicos/farmacologia , Aminoquinolinas/farmacocinética , Células CACO-2 , Cristalografia por Raios X , Inibidores Enzimáticos/farmacocinética , Humanos , Modelos Moleculares , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Éteres Fenílicos/farmacocinética , Relação Estrutura-Atividade
5.
Biochem Biophys Res Commun ; 459(3): 393-7, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25732085

RESUMO

Neuronal nitric oxide synthase (nNOS) plays a critical role in regulating cardiomyocyte function. nNOS was reported to decrease superoxide production in the myocardium by inhibiting the function of xanthine oxidoreductase. However, the effect of oxidative stress on nNOS in cardiomyocytes has not been determined. We report here that brief exposure of HL-1 cardiomyocytes to hydrogen peroxide (H2O2) induces phosphorylation of nNOS at serine 1412. This increase in phosphorylation was concomitant with increased nitric oxide (NO) production. Prolonged exposure to the oxidant, however, resulted in decreased expression of the protein. H2O2 treatment for short periods also stimulated phosphorylation of AKT and AMPK. H2O2-induced phosphorylation of nNOS was reduced when AMPK activity was inhibited by compound C, suggesting that AMPK is a mediator of oxidative stress-induced phosphorylation of nNOS. However, inhibition of AKT activity by the pan AKT inhibitor, AKTi, had no effect on nNOS phosphorylation caused by H2O2. These data demonstrate the novel regulation of nNOS phosphorylation and expression by oxidative stress.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Linhagem Celular , Peróxido de Hidrogênio/farmacologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores
6.
J Med Chem ; 57(3): 686-700, 2014 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-24447275

RESUMO

Selective inhibitors of neuronal nitric oxide synthase (nNOS) are regarded as valuable and powerful agents with therapeutic potential for the treatment of chronic neurodegenerative pathologies and human melanoma. Here, we describe a novel hybrid strategy that combines the pharmacokinetically promising thiophene-2-carboximidamide fragment and structural features of our previously reported potent and selective aminopyridine inhibitors. Two inhibitors, 13 and 14, show low nanomolar inhibitory potency (Ki = 5 nM for nNOS) and good isoform selectivities (nNOS over eNOS [440- and 540-fold, respectively] and over iNOS [260- and 340-fold, respectively]). The crystal structures of these nNOS-inhibitor complexes reveal a new hot spot that explains the selectivity of 14 and why converting the secondary to tertiary amine leads to enhanced selectivity. More importantly, these compounds are the first highly potent and selective nNOS inhibitory agents that exhibit excellent in vitro efficacy in melanoma cell lines.


Assuntos
Amidinas/síntese química , Compostos de Anilina/síntese química , Antineoplásicos/síntese química , Melanoma/tratamento farmacológico , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Tiofenos/síntese química , Amidinas/química , Amidinas/farmacologia , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Bovinos , Linhagem Celular Tumoral/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoenzimas/antagonistas & inibidores , Camundongos , Modelos Moleculares , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Ratos , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologia
7.
PLoS One ; 8(9): e75638, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086598

RESUMO

NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for cytochromes P450, dehydrocholesterol reductase, heme oxygenase, and squalene monooxygenase. Human patients with specific mutations in POR exhibit severe developmental malformations including disordered steroidogenesis, sexual ambiguities and various bone defects, similar to those seen in patients with Antley-Bixler syndrome (ABS). To probe the role of POR during bone development, we generated a conditional knockout mouse (CKO) by cross breeding Por (lox/lox) and Dermo1 Cre mice. CKO mice were smaller than their littermate controls and exhibited significant craniofacial and long bone abnormalities. Differential staining of the CKO mice skull bases shows premature fusion of the sphenooccipital and basioccipital-exoccipital synchondroses. Class III malocclusion was noted in adult knockout mice with an unusual overgrowth of the lower incisors. Shorter long bones were observed along with a reduction in the bone volume fraction, measured by microCT, in the Por-deleted mice compared to age- and sex-matched littermate controls. Concerted up- or down-regulation of proteins in the FGF signaling pathway observed by immunohistochemistry in the tibia samples of CKO mice compared to wild type controls shows a decrease in the FGF signaling pathway. To our knowledge, this is the first report of a mouse model that recapitulates both skull and long bone defects upon Por deletion, offering an approach to study the sequelae of POR mutations. This unique model demonstrates that P450 metabolism in bone itself is potentially important for proper bone development, and that an apparent link exists between the POR and FGF signaling pathways, begging the question of how an oxidation-reduction flavoprotein affects developmental and cellular signaling processes.


Assuntos
Desenvolvimento Ósseo/genética , Desenvolvimento Ósseo/fisiologia , NADPH-Ferri-Hemoproteína Redutase/genética , Deleção de Sequência/genética , Células-Tronco/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/fisiopatologia , Animais , Fenótipo de Síndrome de Antley-Bixler/genética , Fenótipo de Síndrome de Antley-Bixler/metabolismo , Fenótipo de Síndrome de Antley-Bixler/fisiopatologia , Regulação para Baixo/genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Knockout , Mutação/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Crânio/crescimento & desenvolvimento , Crânio/metabolismo , Células-Tronco/fisiologia , Tíbia/crescimento & desenvolvimento , Tíbia/metabolismo , Regulação para Cima/genética
8.
J Am Chem Soc ; 132(2): 798-806, 2010 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-20014790

RESUMO

The heme-thioether ligand interaction often occurs between heme iron and native methionine ligands, but thioether-based heme-coordinating (type II) inhibitors are uncommon due to the difficulty in stabilizing the Fe-S bond. Here, a thioether-based inhibitor (3) of neuronal nitric oxide synthase (nNOS) was designed, and its binding was characterized by spectrophotometry and crystallography. A crystal structure of inhibitor 3 coordinated to heme iron was obtained, representing, to our knowledge, the first crystal structure of a thioether inhibitor complexed to any heme enzyme. A series of related potential inhibitors (4-8) also were evaluated. Compounds 4-8 were all found to be type I (non-heme-coordinating) inhibitors of ferric nNOS, but 4 and 6-8 were found to switch to type II upon heme reduction to the ferrous state, reflecting the higher affinity of thioethers for ferrous heme than for ferric heme. Contrary to what has been widely thought, thioether-heme ligation was found not to increase inhibitor potency, illustrating the intrinsic weakness of the thioether-ferric heme linkage. Subtle changes in the alkyl groups attached to the thioether sulfur caused drastic changes in the binding conformation, indicating that hydrophobic contacts play a crucial role in stabilizing the thioether-heme coordination.


Assuntos
Inibidores Enzimáticos/farmacologia , Heme/farmacologia , Ferro/química , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Sulfetos/química , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Heme/química , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade
9.
Bioorg Med Chem ; 15(18): 6096-108, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17614291

RESUMO

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.


Assuntos
Amidas/farmacologia , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Etilenos/farmacologia , Mimetismo Molecular , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Animais , Bovinos , Dipeptídeos/síntese química , Dipeptídeos/química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Etilenos/síntese química , Etilenos/química , Macrófagos/enzimologia , Camundongos , Estrutura Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ratos , Estereoisomerismo , Relação Estrutura-Atividade
10.
J Med Chem ; 50(9): 2089-99, 2007 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-17425297

RESUMO

The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N'-nitroguanidine (1) and 4-N-(Nomega-nitro-l-argininyl)-trans-4-amino-l-proline amide (2), led to the discovery of a conserved structural water molecule that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). On the basis of this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of 2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues 3-5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized. X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety (Figures 5 and 6). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1). Rationalizations for the small increase in potency are consistent with other changes in the crystal structures.


Assuntos
Heme/química , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/química , Nitroarginina/análogos & derivados , Nitroarginina/síntese química , Peptídeos/química , Água/química , Biomimética , Cristalografia por Raios X , Desenho de Fármacos , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Nitroarginina/química , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Estereoisomerismo , Relação Estrutura-Atividade , Termodinâmica
11.
Bioorg Med Chem ; 14(11): 3681-90, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16480878

RESUMO

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.


Assuntos
Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Peptídeos/farmacologia , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Peptídeos/síntese química , Peptídeos/química , Estereoisomerismo , Relação Estrutura-Atividade
12.
Biochem Biophys Res Commun ; 338(1): 543-9, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16154533

RESUMO

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.


Assuntos
Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nitroso/química , Óxido Nitroso/metabolismo , Animais , Escherichia coli , Cromatografia Gasosa-Espectrometria de Massas/métodos
13.
J Clin Endocrinol Metab ; 90(2): 1100-5, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15562034

RESUMO

Type 2 diabetes is an insulin-resistant state characterized by hyperinsulinemia and accelerated atherosclerosis. In vitro and in vivo studies in rodents have suggested that nitric oxide generation plays an important role in glucose transport and insulin action. We determined nitric oxide synthase (NOS) activity in skeletal muscle of 10 type 2 diabetic (hemoglobin A(1C) = 6.8 +/- 0.1%) and 11 control subjects under basal conditions and during an 80 mU/m(2).min euglycemic insulin clamp performed with vastus lateralis muscle biopsies before and after 4 h of insulin. In diabetics, insulin-stimulated glucose disposal (Rd) was reduced by 50%, compared with controls (5.4 +/- 0.3 vs. 10.4 +/- 0.5 mg/kg.min, P < 0.01). Basal NOS activity was markedly reduced in the diabetic group (101 +/- 33 vs. 457 +/- 164 pmol/min.mg protein, P < 0.05). In response to insulin, NOS activity increased 2.5-fold in controls after 4 h (934 +/- 282 pmol/min.mg protein, P < 0.05 vs. basal), whereas insulin failed to stimulate NOS activity in diabetics (86 +/- 28 pmol/min.mg protein, P = NS from basal). Basal NOS protein content in muscle was similar in controls and diabetics and did not change following insulin. In controls, insulin-stimulated NOS activity correlated inversely with fasting plasma insulin concentration (r = -0.58, P = 0.05) and positively with Rd (r = 0.71, P = 0.03). In control and diabetic groups collectively, Rd correlated with insulin-stimulated NOS activity (r = 0.52, P = 0.02). We conclude that basal and insulin-stimulated muscle NOS activity is impaired in well-controlled type 2 diabetic subjects, and the defect in insulin-stimulated NOS activity correlates closely with the severity of insulin resistance. These results suggest that impaired NOS activity may play an important role in the insulin resistance in type 2 diabetic individuals.


Assuntos
Diabetes Mellitus Tipo 2/enzimologia , Resistência à Insulina/fisiologia , Músculo Esquelético/enzimologia , Óxido Nítrico Sintase/metabolismo , Biópsia , Glicemia/metabolismo , Índice de Massa Corporal , Ácidos Graxos não Esterificados/sangue , Feminino , Técnica Clamp de Glucose , Humanos , Insulina/sangue , Insulina/farmacologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Valores de Referência , Sístole/fisiologia
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