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1.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 6): 412-418, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204687

RESUMO

The SPRY domain-containing SOCS box protein 2 (SPSB2) is one of four mammalian SPSB proteins that are characterized by a C-terminal SOCS box and a central SPRY/B30.2 domain. SPSB2 interacts with inducible nitric oxide synthase (iNOS) via the SPRY domain and polyubiquitinates iNOS, resulting in its proteasomal degradation. Inhibitors that can disrupt SPSB2-iNOS interaction and augment NO production may serve as novel anti-infective and anticancer agents. The previously determined murine SPSB2 structure may not reflect the true apo conformation of the iNOS-binding site. Here, the crystal structure of human SPSB2 SPRY domain in the apo state is reported at a resolution of 1.9 Å. Comparison of the apo and ligand-bound structures reveals that the iNOS-binding site is highly preformed and that major conformational changes do not occur upon ligand binding. Moreover, the C-terminal His6 tag of the recombinant protein binds to a shallow pocket adjacent to the iNOS-binding site on a crystallographically related SPSB2 molecule. These findings may help in structure-based and fragment-based SPSB2 inhibitor design in the future.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Modelos Moleculares , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Sequência de Aminoácidos , Domínio B30.2-SPRY , Cristalografia por Raios X , Humanos , Óxido Nítrico Sintase Tipo II/química , Conformação Proteica
2.
Environ Sci Pollut Res Int ; 26(13): 13539-13550, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30915694

RESUMO

In the current report, we examined the potential beneficial role of soursop fruit extract (SSFE) on liver injury induced by a single paracetamol (APAP) overdose (2000 mg/kg). Thirty-five Wistar albino rats were randomly divided into five groups as follows: control, SSFE, APAP, SSFE+APAP, and silymarin (SIL)+APAP. APAP intoxication was found to elevate alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin levels. Moreover, it increased the levels of malondialdehyde, nitrites, and nitrates and depleted glutathione, superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase. APAP intoxication inactivated the nuclear factor erythroid 2-related factor 2 (Nrf2) defense pathway and upregulated the expression of heme oxygenase-1 (HO-1). APAP administration enhanced the activation of nuclear factor-kappa B (NF-κB), the elevation of tumor necrosis factor-alpha and interleukin 1-beta levels, and the upregulation of inducible nitric oxide synthase mRNA expression. In addition, APAP activated the overexpression of Bax protein, increased release of cytochrome c, and the downregulation of Bcl-2 protein. Finally, APAP-induced overexpression of transforming growth factor-beta (TGF-ß) further suggested enhanced liver damage. On the other hand, SSFE pretreatment attenuated these biochemical, molecular, and histopathological alterations in the liver, which might be partially due to the regulation of hepatic Nrf2/HO-1 and downregulation of NF-κB and TGF-ß.


Assuntos
Acetaminofen/metabolismo , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Aspartato Aminotransferases/metabolismo , Catalase/metabolismo , Frutas/metabolismo , Glutationa/metabolismo , Fígado/metabolismo , Malondialdeído/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Superóxido Dismutase/metabolismo , Acetaminofen/química , Alanina Transaminase/metabolismo , Animais , Annona , Anti-Inflamatórios/química , Antioxidantes/química , Apoptose , Aspartato Aminotransferases/química , Fator 2 Relacionado a NF-E2/química , Óxido Nítrico Sintase Tipo II/química , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo , Verduras
3.
Molecules ; 24(5)2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841642

RESUMO

: Kudzu (Pueraria thunbergiana Benth.) has long been used as a food and medicine for many centuries. The root is the most commonly used portion of the plant, but the aerial parts are occasionally used as well. In this study, we investigated the constituent compounds and biological activities of the aerial parts, leaves, stems, and sprouts, and compared their constituents and activities with those of roots. Leaf extract showed a significantly higher TPC level at 59 ± 1.6 mg/g and lower free radical scavenging (FRS) values under 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and NO inhibition at 437 ± 11, 121 ± 6.6 µg/mL and 107 ± 4.9 µg/mL, respectively, than those of sprout, stem, and root extract. Leaf extract also significantly suppressed lipopolysaccharide (LPS)-mediated gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The main components of leaf extract were found to be genistin and daidzin. This study suggests that the leaves of kudzu are a good source of biological activities and isoflavones that can be used in functional or medicinal foods and cosmetics for the prevention or treatment of diseases related to inflammation and oxidative stress.


Assuntos
Isoflavonas/química , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Raízes de Plantas/química , Pueraria/química , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Camundongos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/química , Estresse Oxidativo , Fenóis/química , Células RAW 264.7
4.
J Biol Chem ; 294(19): 7904-7916, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30926606

RESUMO

Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (kr ), of dissociation of NO from the ferric heme-NO complex (kd ), and of oxidation of the ferrous heme-NO complex (k ox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outer-sphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound FeIII-N(O)OO- intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidine-coordinated heme.


Assuntos
Modelos Químicos , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico/química , Substituição de Aminoácidos , Animais , Heme , Camundongos , Mutação de Sentido Incorreto , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Oxirredução , Ratos
5.
Mol Cancer Ther ; 18(1): 62-74, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30297361

RESUMO

Oleanolic acid exhibits extensive pharmacologic activities and takes significant antitumor effects. Its pharmacologic mechanism, however, still remained to be further clarified. In this study, we demonstrated that oleanolic acid attenuated the migration and invasion abilities, resulting in the suppression of the epithelial-mesenchymal transition (EMT) process in liver cancer cells, and inhibited the tumor growth of the peritoneal lymphocytes-bearing mice. We further proved that inducible nitric oxide synthase (iNOS) may be the potential target of oleanolic acid. We confirmed that oleanolic acid could promote the dimerization of iNOS, activating it, and subsequently increasing the production of nitric oxide. Further experiments indicated that oleanolic acid promoted the nitration of specific proteins and consequently suppressed their EMT-related biological functions. Furthermore, it has been confirmed that oleanolic acid enhanced the antitumor effects of regorafenib in liver cancer treatment. These results deepened our understanding of the pharmacologic mechanism of the antitumor effect oleanolic acid, and the importance of nitric oxide synthetase as a therapeutic target for liver cancer treatment.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/química , Ácido Oleanólico/administração & dosagem , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Invasividade Neoplásica , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Multimerização Proteica/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cell Physiol Biochem ; 51(2): 746-762, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30463066

RESUMO

BACKGROUND/AIMS: This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). METHODS: We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO3) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3',5' monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. RESULTS: Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. CONCLUSION: These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.


Assuntos
Diferenciação Celular , Nanopartículas/química , Óxido Nítrico Sintase Tipo II/química , Osteogênese , Fosfatase Alcalina/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , GMP Cíclico/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Redes Reguladoras de Genes , Cinética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
7.
Protein Pept Lett ; 25(12): 1101-1107, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30381058

RESUMO

BACKGROUND: Inducible Nitric Oxide Synthase (iNOS or NOS2) produces Nitric Oxide (NO) and related reactive nitrogen species, which are critical effectors of the host innate response and play key roles in the intracellular killing of bacterial and parasitic pathogens. The SPRY domain- containing SOCS box proteins SPSB1 and SPSB2 are key physiological regulators of this important enzyme. Disrupting the endogenous SPSB-iNOS interaction should prolong the intracellular lifetime of iNOS and enhance the production of NO, and therefore be beneficial in treating chronic and persistent infections such as tuberculosis. By using structure-based design, potent peptide inhibitors of this interaction have been developed. CONCLUSION: Inhibitors of the SPSB-iNOS interaction have therapeutic potential as a novel class of anti-infective agents. Various strategies are being pursued to target these peptide inhibitors to macrophages and deliver them to the cytoplasm of these cells. It will then be possible to assess the efficacy of such inhibitors in boosting the capacity of macrophages to destroy infectious pathogens.


Assuntos
Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Anti-Infecciosos/química , Sistemas de Liberação de Medicamentos , Desenho de Drogas , Humanos , Imunidade Inata/efeitos dos fármacos , Modelos Moleculares , Óxido Nítrico Sintase Tipo II/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Supressoras da Sinalização de Citocina/química
8.
BMC Complement Altern Med ; 18(1): 271, 2018 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-30285710

RESUMO

BACKGROUND: The extracts of the ten selected Sri Lankan medicinal plants have been traditionally used in the treatment of inflammatory mediated diseases. The extracts were investigated for anti-inflammatory and anti-oxidant potential in vitro to identify bio-active extracts for further chemical characterization. METHODS: In vitro anti-inflammatory activities of total ethanol extracts were investigated measuring the inhibitory activities of four pro-inflammatory enzymes, arachidonate-5- lipoxygenase (A5-LOX), hyaluronidase (HYL), xanthine oxidase (XO) and inducible nitric oxide (iNO) synthase. Cytotoxicity of extracts were determined by MTT assay. Oxidative burst inhibition (OBI) on human whole blood (WB) and isolated polymorphoneutrophils (PMNs) was carried out for a selected bio-active extract. Anti- oxidant activities of the extracts were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, ferric reducing antioxidant power (FRAP), ferrous ion chelation (FIC) and oxygen radical absorbance capacity (ORAC) assays. Total polyphenol and total Flavonoid contents of the extracts were also determined. The most active plant extract was analysed using Gas chromatography-Mass spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC). RESULTS: The ethanol bark extract of Flacourtia indica showed the highest A5-LOX (IC50: 22.75 ± 1.94 g/mL), XO (70.46 ± 0.18%; 250 µg/mL) and iNOs inhibitory activities on LPS- activated raw 264.7 macrophage cells (38.07 ± 0.93%; 500 µg/mL) with promising OBI both on WB (IC50: 47.64 2.32 µg/mL) and PMNs (IC50: 5.02 0.38 µg/mL). The highest HYL inhibitory activity was showed by the leaf extracts of Barathranthus nodiflorus (42.31 ± 2.00%; 500 µg/mL) and Diospyros ebenum (41.60 ± 1.18%; 500 µg/mL). The bark and leaf extracts of Callophyllum innophyllum (IC50: 6.99 ± 0.02 µg/mL) and Symplocus cochinchinesis (IC50: 9.85 ± 0.28 µg/mL) showed promising DPPH free radical scavenging activities. The GC-MS analysis of ethanol bark extract of F. indica showed the presence of two major bio-active compounds linoleic acid ethyl ester and hexadecanoic acid, ethyl ester (> 2% peak area). The HPLC analysis showed the presence polyphenolic compounds. CONCLUSION: The ethanol bark extract of F. indica can be identified as a potential candidate for the development of anti-inflammatory agents, which deserves further investigations. The bio-active plant extracts may be effectively used in the applications of cosmetic and health care industry.


Assuntos
Anti-Inflamatórios/química , Antioxidantes/química , Inibidores Enzimáticos/química , Extratos Vegetais/química , Plantas Medicinais/química , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hialuronoglucosaminidase/antagonistas & inibidores , Hialuronoglucosaminidase/química , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Extratos Vegetais/farmacologia , Células RAW 264.7 , Explosão Respiratória/efeitos dos fármacos , Sri Lanka , Xantina Oxidase/antagonistas & inibidores , Xantina Oxidase/química
9.
J Phys Chem B ; 122(49): 11048-11057, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-29965771

RESUMO

Nitric oxide synthases (NOSs) are heme enzymes that generate highly reactive nitric oxide from l-arginine (l-Arg) in a complex mechanism that is still only partially understood. We have studied carbon monoxide (CO) binding to the oxygenase domain of murine inducible NOS (iNOS) by using flash photolysis. The P420 and P450 forms of the enzyme, assigned to a protonated and unprotonated proximal cysteine, through which the heme is anchored to the protein, show markedly different CO rebinding properties. The data suggest that P420 has a widely open distal pocket that admits water. CO rebinding to the P450 form strongly depends on the presence of the substrate l-Arg, the intermediate Nω-hydroxy-l-arginine, and the cofactor tetrahydrobiopterin. After adding these small molecules to the enzyme solution, the CO kinetics change slowly over the hours. This process can be described as a relaxation from a fast rebinding, metastable species to a slowly rebinding, thermodynamically stable species, which we associate with the enzymatically active form. Our results allow us to determine kinetic parameters of l-Arg binding to the ferrous deoxy iNOS protein for the first time and also provide clues regarding the nature of structural differences between the two interconverting species.


Assuntos
Óxido Nítrico Sintase Tipo II/química , Animais , Sítios de Ligação , Cinética , Ligantes , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Conformação Proteica , Termodinâmica
10.
Br J Pharmacol ; 175(17): 3470-3485, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29888783

RESUMO

BACKGROUND AND PURPOSE: Beta cell apoptosis is a major feature of type 1 diabetes, and pro-inflammatory cytokines are key drivers of the deterioration of beta cell mass through induction of apoptosis. Mitochondrial stress plays a critical role in mediating apoptosis by releasing cytochrome C into the cytoplasm, directly activating caspase-9 and its downstream signalling cascade. We aimed to identify new compounds that protect beta cells from cytokine-induced activation of the intrinsic (mitochondrial) pathway of apoptosis. EXPERIMENTAL APPROACH: Diabetogenic media, composed of IL-1ß, IFN-γ and high glucose, were used to induce mitochondrial stress in rat insulin-producing INS1E cells, and a high-content image-based screen of small molecule modulators of Casp9 pathway was performed. KEY RESULTS: A novel small molecule, ATV399, was identified from a high-content image-based screen for compounds that inhibit cleaved caspase-9 activation and subsequent beta cell apoptosis induced by a combination of IL-1ß, IFN-γ and high glucose, which together mimic the pathogenic diabetic milieu. Through medicinal chemistry optimization, potency was markedly improved (6-30 fold), with reduced inhibitory effects on CYP3A4. Improved analogues, such as CAT639, improved beta cell viability and insulin secretion in cytokine-treated rat insulin-producing INS1E cells and primary dispersed islet cells. Mechanistically, CAT639 reduced the production of NO by allosterically inhibiting dimerization of inducible NOS (iNOS) without affecting its mRNA levels. CONCLUSION AND IMPLICATIONS: Taken together, these studies demonstrate a successful phenotypic screening campaign resulting in identification of an inhibitor of iNOS dimerization that protects beta cell viability and function through modulation of mitochondrial stress induced by cytokines.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Animais , Caspase 9/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Dimerização , Ativação Enzimática , Glucose/farmacologia , Células Secretoras de Insulina/citologia , Óxido Nítrico Sintase Tipo II/química , Ratos , Transdução de Sinais
11.
Chem Biodivers ; 15(9): e1800203, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29933520

RESUMO

Calvatia species, generally known as puffball mushrooms, are used both as sources of food and as traditional medicine. Among the Calvatia genus, Calvatia nipponica (Agaricaceae) is one of the rarest species. Using bioassay-guided fractionation based on anti-inflammatory effects, five alkaloids (1 - 5), two phenolics (6 and 7), and a fatty acid methyl ester (8) were isolated from the fruiting bodies of C. nipponica. Compound 8 was identified from C. nipponica for the first time, and all isolates (1 - 8) were tested for inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Compound 7 showed mild inhibition while compound 8 significantly inhibited NO production with an IC50 value of 27.50 ± 0.08 µm. The mechanism of NO inhibition of compound 7 was simulated by molecular docking analysis against nitric oxide synthase (iNOS), which revealed the interactions of 7 with the key amino acid residue and the heme in the active site. With the most potent inhibition against LPS-induced inflammation, compound 8 was further investigated with respect to its mechanism of action, and the activity was found to be mediated through the inhibition of iNOS and COX-2 expression.


Assuntos
Agaricales/química , Anti-Inflamatórios/farmacologia , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Óleos Voláteis/química , Terpenos/isolamento & purificação , Animais , Anti-Inflamatórios/química , Península Balcânica , Clima , Análise Discriminante , Concentração Inibidora 50 , Camundongos , Simulação de Acoplamento Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Células RAW 264.7 , Terpenos/farmacologia
12.
FEBS Lett ; 592(14): 2425-2431, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29904908

RESUMO

The interface between calmodulin (CaM) and the NO synthase (NOS) heme domain is the least characterized interprotein interface that the NOS isoforms must traverse through during catalysis. Our previous molecular dynamics simulations predicted a salt bridge between K497 in human inducible NOS (iNOS) heme domain and D118(CaM). Herein, the FMN - heme interdomain electron transfer (IET) rate was found to be notably decreased by charge-reversal mutation, while the IET in the iNOS K497D mutant is significantly restored by the CaM D118K mutation. The results of wild-type protein with added synthetic peptides further demonstrate the critical nature of K497 relative to the rest of the peptide sequence in modulating the IET. These data provide definitive evidence supporting the regulatory role of the isoform-specific K497 residue.


Assuntos
Calmodulina/metabolismo , Mononucleotídeo de Flavina/metabolismo , Heme/metabolismo , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Calmodulina/química , Calmodulina/genética , Códon sem Sentido , Transporte de Elétrons/fisiologia , Heme/química , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Óxido Nítrico Sintase Tipo II/genética , Oxirredução , Domínios e Motivos de Interação entre Proteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Estrutura Secundária de Proteína
13.
Toxicol Lett ; 294: 20-26, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29751043

RESUMO

Gentamycin is one of the most clinically used aminoglycoside antibiotics which induce intrinsic apoptosis of hair cells. Tauroursodeoxycholic acid (TUDCA) is known as safe cell-protective agent in disorders associated with apoptosis. We aimed to investigate the protective effects of TUDCA against gentamicin-induced ototoxicity. House Ear Institute-Organ of Corti 1(HEI-OC1) cells and explanted cochlear tissue were treated with gentamicin and TUDCA, followed by serial analyses including cell viability assay, hair cell staining, qPCR, ELISA and western blotting to determine the cell damage by the parameters relevant to cell apoptosis and endoplasmic reticulum stress. TUDCA significantly attenuated gentamicin-induced cell damage in cultured HEI-OC1 cells and explanted cochlear hair cells. TUDCA alleviated gentamicin-induced cell apoptosis, supported by the decreased Bax/Bcl2 ratio compared with that of gentamicin treated alone. TUDCA decreased gentamicin-induced nitric oxide production and protein nitration in both models. In addition, TUDCA suppressed gentamicin-induced endoplasmic reticulum stress as reflected by inversing the expression levels of Binding immunoglobulin protein (Bip), CCAAT/-enhancer-binding protein homologous protein (CHOP) and Caspase 3. TUDCA attenuated gentamicin-induced hair cell death by inhibiting protein nitration activation and ER stress, providing new insights into the new potential therapies for sensorineural deafness.


Assuntos
Antibacterianos/química , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Gentamicinas/antagonistas & inibidores , Células Ciliadas Auditivas/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Antibacterianos/efeitos adversos , Biomarcadores/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Cóclea/patologia , Cóclea/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Gentamicinas/efeitos adversos , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas/ultraestrutura , Perda Auditiva Neurossensorial/induzido quimicamente , Perda Auditiva Neurossensorial/etiologia , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/prevenção & controle , Humanos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidores da Síntese de Proteínas/efeitos adversos , Inibidores da Síntese de Proteínas/química , Técnicas de Cultura de Tecidos , Tirosina/análogos & derivados , Tirosina/metabolismo
14.
Molecules ; 23(5)2018 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-29710774

RESUMO

The synthesis and anti-inflammatory effects of certain pyrazolo[4,3-c]quinoline derivatives 2a⁻2r are described. The anti-inflammatory activities of these derivatives were evaluated by means of inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Among them, 3-amino-4-(4-hydroxyphenylamino)-1H-pyrazolo[4,3-c]-quinoline (2i) and 4-(3-amino-1H-pyrazolo[4,3-c]quinolin-4-ylamino)benzoic acid (2m) exhibited significant inhibition of LPS-stimulated NO production with a potency approximately equal to that of the positive control, 1400 W. Important structure features were analyzed by quantitative structure⁻activity relationship (QSAR) analysis to give better insights into the structure determinants for predicting the inhibitory effects on the accumulation of nitric oxide for RAW 264.7 cells in response to LPS. In addition, our results indicated that their anti-inflammatory effects involve the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expression. Further studies on the structural optimization are ongoing.


Assuntos
Anti-Inflamatórios/síntese química , Macrófagos/citologia , Pirazóis/síntese química , Quinolinas/síntese química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Modelos Moleculares , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Relação Quantitativa Estrutura-Atividade , Quinolinas/química , Quinolinas/farmacologia , Células RAW 264.7
15.
Int J Mol Sci ; 19(2)2018 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-29401674

RESUMO

The iridoids of Hedyotis diffusa Willd play an important role in the anti-inflammatory process, but the specific iridoid with anti-inflammatory effect and its mechanism has not be thoroughly studied. An iridoid compound named scandoside (SCA) was isolated from H. diffusa and its anti-inflammatory effect was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Its anti-inflammatory mechanism was confirmed by in intro experiments and molecular docking analyses. As results, SCA significantly decreased the productions of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and inhibited the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 messenger RNA (mRNA) expression in LPS-induced RAW 264.7 macrophages. SCA treatment suppressed the phosphorylation of inhibitor of nuclear transcription factor kappa-B alpaha (IκB-α), p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The docking data suggested that SCA had great binding abilities to COX-2, iNOS and IκB. Taken together, the results indicated that the anti-inflammatory effect of SCA is due to inhibition of pro-inflammatory cytokines and mediators via suppressing the nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, which provided useful information for its application and development.


Assuntos
Anti-Inflamatórios/farmacologia , Hedyotis/química , Iridoides/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/química , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Iridoides/química , Iridoides/isolamento & purificação , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/química , Células RAW 264.7 , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
J Inorg Biochem ; 181: 28-40, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29407906

RESUMO

Nitric oxide (NO·) is a messenger molecule with diverse physiological roles including host defense, neurotransmission and vascular function. The synthesis of NO· from l-arginine is catalyzed by NO-synthases and occurs in two steps through the intermediary Nω-hydroxy-l-arginine (NHA). In both steps the P450-like reaction cycle is coupled with the redox cycle of the cofactor tetrahydrobiopterin (H4B). The mechanism of the second step is studied by Density Functional Theory calculations to ascertain the canonical sequence of proton and electron transfer (PT and ET) events. The proposed mechanism is controlled by the interplay of two electron donors, H4B and NHA. Consistent with experimental data, the catalytic cycle proceeds through the ferric-hydroperoxide complex (Cpd 0) and the following aqua-ferriheme resting state, and involves interim partial oxidation of H4B. The mechanism starts with formation of Cpd 0 from the ferrous-dioxy reactant complex by PT from the C-ring heme propionate coupled with hole transfer to H4B through the highest occupied π-orbital of NHA as a bridge. This enables PT from NHA+· to the proximal oxygen leading to the shallow ferriheme-H2O2 oxidant. Subsequent Fenton-like peroxide bond cleavage triggered by ET from the NHA-derived iminoxy-radical leads to the protonated Cpd II diradicaloid singlet stabilized by spin delocalization in H4B, and the closed-shell coordination complex of HO- with iminoxy-cation. The complex is converted to the transient C-adduct, which releases intended products upon PT to the ferriheme-HO- complex coupled with ET to the H4B+·. Deferred ET from the substrate or undue ET from/to the cofactor leads to side products.


Assuntos
Arginina/análogos & derivados , Biopterina/análogos & derivados , Modelos Moleculares , NADP/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Arginina/química , Arginina/metabolismo , Biocatálise , Biopterina/química , Biopterina/metabolismo , Domínio Catalítico , Citrulina/química , Citrulina/metabolismo , Sequência Conservada , Bases de Dados de Proteínas , Transporte de Elétrons , Humanos , Ligações de Hidrogênio , NADP/química , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/química , Oxirredução , Prótons , Teoria Quântica , Termodinâmica
17.
Bioorg Chem ; 76: 53-60, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29128707

RESUMO

A phytochemical investigation to obtain new NO inhibitors resulted in the isolation of five new spiro diterpenoids (1 -5) from the aerial parts of Scutellaria formosana. The structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of these compounds were established via comparison of experimental and calculated electronic circular dichroism (ECD) spectra. The nitric oxide (NO) inhibitory effects were evaluated and all of the compounds showed inhibitory effects on lipopolysaccharide-induced NO production in murine microglial BV-2 cells. The possible mechanism of NO inhibition of bioactive compounds was also investigated using molecular docking, which revealed the interactions of bioactive compounds with the iNOS protein.


Assuntos
Diterpenos/farmacologia , Óxido Nítrico/antagonistas & inibidores , Scutellaria/química , Animais , Domínio Catalítico , Linhagem Celular , Diterpenos/química , Diterpenos/isolamento & purificação , Camundongos , Microglia/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Óxido Nítrico Sintase Tipo II/química , Componentes Aéreos da Planta/química
18.
Fish Shellfish Immunol ; 74: 94-100, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29277697

RESUMO

Nitric oxide (NO) is an important effector molecule which is involved in a myriad of biological processes, including immune responses against pathogens such as parasites, virus and bacteria. During the inflammatory processes in vertebrates, NO is produced by the inducible nitric oxide synthase (iNOS) enzyme in practically all nucleated cells to suppress or kill intracellular pathogens. The aim of the present study was to characterize the full coding region of the iNOS gene of pacu (Piaractus mesopotamicus), an economically and ecologically important South American fish species, and to analyze mRNA expression levels following intraperitoneal infection with the pathogenic bacterium Aeromonas dhakensis by means of quantitative real time PCR (qPCR). The results showed that the pacu iNOS transcript is 3237 bp in length, encoding a putative protein composed of 1078 amino acid residues. The amino acid sequence showed similarities ranging from 69.03% to 94.34% with other teleost fish and 57.70% with the human iNOS, with all characteristic domains and cofactor binding sites of the enzyme detected. Phylogenetic analysis showed that the iNOS from the red-bellied piranha, another South American characiform, was the closest related sequence to the pacu iNOS. iNOS transcripts were constitutively detected in the liver, spleen and head kidney, and there was a significant upregulation in the liver and spleen at 12, 24 and 48 h after infection with A. dhakensis. No significant variations were observed in the head kidney during the periods analyzed. These results show that iNOS expression was induced by A. dhakensis infection and suggest that this enzyme may be involved in the response to this bacterium in pacu.


Assuntos
Caraciformes/genética , Caraciformes/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Imunidade Adaptativa , Aeromonas/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Infecções por Bactérias Gram-Negativas/imunologia , Óxido Nítrico Sintase Tipo II/química , Filogenia , Distribuição Aleatória , Alinhamento de Sequência/veterinária
19.
Bioorg Chem ; 76: 449-457, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29275263

RESUMO

Our continuous search for new nitric oxide (NO) inhibitory substances as anti-neuroinflammatory agents for AD resulted in the isolation of one new labdane diterpenoid and three new guaiane sesquiterpenoids, as well as ten known compounds from Blumea balsamifera. Their structures were elucidated by NMR spectroscopic data analysis and the time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The anti-neuroinflammatory effects were examined by inhibiting NO release in LPS-induced murine microglial BV-2 cells. The possible mechanism of NO inhibition of some bioactive compounds was also investigated using molecular docking, which revealed the interactions of bioactive compounds with the iNOS protein.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Asteraceae/química , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/antagonistas & inibidores , Terpenos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Linhagem Celular , Camundongos , Microglia/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Óxido Nítrico Sintase Tipo II/química , Componentes Aéreos da Planta/química , Terpenos/química , Terpenos/isolamento & purificação
20.
PLoS Pathog ; 13(12): e1006744, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29220410

RESUMO

Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.


Assuntos
Infecções por Coxsackievirus/virologia , Cisteína Endopeptidases/metabolismo , Enterovirus Humano B/enzimologia , Miocardite/virologia , Miócitos Cardíacos/virologia , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/imunologia , Enterovirus Humano B/fisiologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos A , Mutação , Miocardite/imunologia , Miocardite/metabolismo , Miocardite/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteólise , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Replicação Viral
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