RESUMO
The SPRY domain-containing SOCS box proteins SPSB1, SPSB2, and SPSB4 utilize their SPRY/B30.2 domain to interact with a short region in the N-terminus of inducible nitric oxide synthase (iNOS), and recruit an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in the proteasomal degradation of iNOS. Inhibitors that can disrupt the endogenous SPSB-iNOS interactions could be used to augment cellular NO production, and may have antimicrobial and anticancer activities. We previously reported the rational design of a cyclic peptide inhibitor, cR8, cyclo(RGDINNNV), which bound to SPSB2 with moderate affinity. We, therefore, sought to develop SPSB inhibitors with higher affinity. Here, we show that cyclic peptides cR7, cyclo(RGDINNN), and cR9, cyclo(RGDINNNVE), have ~6.5-fold and ~2-fold, respectively, higher SPSB2-bindng affinities than cR8. We determined high-resolution crystal structures of the SPSB2-cR7 and SPSB2-cR9 complexes, which enabled a good understanding of the structure-activity relationships for these cyclic peptide inhibitors. Moreover, we show that these cyclic peptides displace full-length iNOS from SPSB2, SPSB1, and SPSB4, and that their inhibitory potencies correlate well with their SPSB2-binding affinities. The strongest inhibition was observed for cR7 against all three iNOS-binding SPSB proteins.
Assuntos
Peptídeos Cíclicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Humanos , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
In the current study, we utilized molecular modeling and simulation approaches to define putative potential molecular targets for Burdock Inulin, including inflammatory proteins such as iNOS, COX-2, TNF-alpha, IL-6, and IL-1ß. Molecular docking results revealed potential interactions and good binding affinity for these targets; however, IL-1ß, COX-2, and iNOS were identified as the best targets for Inulin. Molecular simulation-based stability assessment demonstrated that inulin could primarily target iNOS and may also supplementarily target COX-2 and IL-1ß during DSS-induced colitis to reduce the role of these inflammatory mechanisms. Furthermore, residual flexibility, hydrogen bonding, and structural packing were reported with uniform trajectories, showing no significant perturbation throughout the simulation. The protein motions within the simulation trajectories were clustered using principal component analysis (PCA). The IL-1ß-Inulin complex, approximately 70% of the total motion was attributed to the first three eigenvectors, while the remaining motion was contributed by the remaining eigenvectors. In contrast, for the COX2-Inulin complex, 75% of the total motion was attributed to the eigenvectors. Furthermore, in the iNOS-Inulin complex, the first three eigenvectors contributed to 60% of the total motion. Furthermore, the iNOS-Inulin complex contributed 60% to the total motion through the first three eigenvectors. To explore thermodynamically favorable changes upon mutation, motion mode analysis was carried out. The Free Energy Landscape (FEL) results demonstrated that the IL-1ß-Inulin achieved a single conformation with the lowest energy, while COX2-Inulin and iNOS-Inulin exhibited two lowest-energy conformations each. IL-1ß-Inulin and COX2-Inulin displayed total binding free energies of - 27.76 kcal/mol and - 37.78 kcal/mol, respectively, while iNOS-Inulin demonstrated the best binding free energy results at - 45.89 kcal/mol. This indicates a stronger pharmacological potential of iNOS than the other two complexes. Thus, further experiments are needed to use inulin to target iNOS and reduce DSS-induced colitis and other autoimmune diseases.
Assuntos
Ciclo-Oxigenase 2 , Interleucina-1beta , Inulina , Simulação de Acoplamento Molecular , Óxido Nítrico Sintase Tipo II , Inulina/química , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/química , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/química , Interleucina-1beta/metabolismo , Animais , Simulação de Dinâmica Molecular , Colite/induzido quimicamente , Colite/metabolismo , Colite/prevenção & controle , Ligação Proteica , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.
Assuntos
Calmodulina , Espectrofotometria Infravermelho , Calmodulina/química , Calmodulina/metabolismo , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Ligação Proteica , Simulação de Acoplamento MolecularRESUMO
Nitric oxide synthase (NOS) is a homodimeric flavohemoprotein responsible for catalyzing the oxidation of l-arginine (l-Arg) to citrulline and nitric oxide. Electrons are supplied for the reaction via interdomain electron transfer between an N-terminal heme-containing oxygenase domain and a FMN-containing (sub)domain of a C-terminal reductase domain. Extensive attention has focused on elucidating how conformational dynamics regulate electron transfer between the domains. Here we investigate the impact of the interdomain FMN-heme interaction on the heme active site dynamics of inducible NOS (iNOS). Steady state linear and time-resolved two-dimensional infrared (2D IR) spectroscopy was applied to probe a CO ligand at the heme within the oxygenase domain for full-length and truncated or mutated constructs of human iNOS. Whereas the linear IR spectra of the CO ligand were identical among the constructs, 2D IR spectroscopy revealed variation in the frequency dynamics. The wild-type constructs that can properly form the FMN/oxygenase docked state due to the presence of both the FMN and oxygenase domains showed slower dynamics than the oxygenase domain alone. Introduction of the mutation (E546N) predicted to perturb electrostatic interactions between the domains resulted in measured dynamics intermediate between those for the full-length and individual oxygenase domain, consistent with perturbation to the docked/undocked equilibrium. These results indicate that docking of the FMN domain to the oxygenase domain not only brings the FMN cofactor within electron transfer distance of the heme domain but also modulates the dynamics sensed by the CO ligand within the active site in a way expected to promote efficient electron transfer.
Assuntos
Mononucleotídeo de Flavina , Heme , Domínio Catalítico , Transporte de Elétrons , Mononucleotídeo de Flavina/química , Heme/química , Heme Oxigenase (Desciclizante) , Humanos , Ligantes , Óxido Nítrico/química , Óxido Nítrico Sintase Tipo II/químicaRESUMO
Cervical cancer is the second most common cause of cancer deaths in women worldwide and remains the main reason of mortality among women of reproductive age in developing countries. Nitric oxide is involved in several physiological functions inclusive of inflammatory and immune responses. However, the function of NO in tumor biology is debatable. The inducible NOS (iNOS/NOS2) isoform is the one responsible to maintain the levels of NO, and it exhibits pleotropic effects in various cancers with concentration-dependent pro- and anti-tumor effects. iNOS triggers angiogenesis and endothelial cell migration in tumors by regulating the levels of vascular endothelial growth factor (VEGF). In drug discovery, drug repurposing involves investigations of approved drug candidates to treat various other diseases. In this study, we used anti-cancer drugs and small molecules to target iNOS and identify a potential selective iNOS inhibitor. The structures of ligands were geometrically optimized and energy minimized using Hyperchem software. Molecular docking was performed using Molegro virtual docker, and ligands were selected based on MolDock score, Rerank score, and H-bonding energy. In the study shown, venetoclax compound demonstrated excellent binding affinity to iNOS protein. This compound exhibited the lowest MolDock score and Rerank score with better H-bonding energy to iNOS. The binding efficacy of venetoclax was analyzed by performing molecular docking and molecular dynamic simulations. Multiple parameters were used to analyze the simulation trajectory, like root mean square deviation (RMSD), radius of gyration (Rg), and hydrogen bond interactions. Based on the results, venetoclax emerges to be a promising potential iNOS inhibitor to curtail cervical cancer progression.
Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Neoplasias do Colo do Útero/enzimologia , Antineoplásicos/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
<b>Background and Objective:</b> Inflammation occurs <i>via</i> several mechanisms, one of which includes the production of Nitric Oxide (NO) catalyzed by inducible nitric oxide synthase (iNOS), which is inhibited selectively by isothioureas. <i>Ageratum conyzoides</i> L. has shown activity in reducing pain and inflammation, although the molecular mechanism had not been undertaken. The objectives of this work were (1) to study the mechanism of anti-inflammatory activity of <i>A. conyzoides</i> through inhibition of iNOS, (2) to correlate the iNOS inhibitory activity of the plant with the total flavonoid content of the plants and (3) to identify the flavonol synthase (FLS), an enzyme that catalyzes the production of quercetin. <b>Materials and Methods:</b> The inhibitory activity against iNOS was assayed by <i>in vitro</i> method. The total flavonoids (calculated as quercetin) of <i>A. conyzoides</i> were determined by fluorometry. The protein extraction of the leaves was carried out by employing Laing and Christeller's (2004) method, followed with SDS-PAGE. <b>Results:</b> The inhibitory activity (IC<sub>50</sub>) of ethanol extract and ethyl acetate fraction of <i>A. conyzoides</i> against iNOS was 92.05 and 4.78 µg mL<sup></sup><sup>1</sup>, respectively. Pearson correlation analysis resulted in 0.548 (ethanol extract) and 0.696 (ethyl acetate fraction). The total flavonoids (calculated as quercetin) contained in the ethanol extract and ethyl acetate fraction of <i>A. conyzoides</i> were 0.71 and 7.65%, respectively. The FLS in <i>A. conyzoides</i> leaves was identified at 31 kDa. <b>Conclusion:</b> <i>A. </i>c<i>onyzoides</i> L. is potential in inhibiting iNOS due to quercetin contained in the leaves. This report will add a scientific insight of <i>A. conyzoides</i> for biological sciences.
Assuntos
Ageratum/crescimento & desenvolvimento , Ageratum/metabolismo , Óxido Nítrico Sintase/metabolismo , Anti-Inflamatórios , Etanol/química , Flavonoides/química , Indonésia , Concentração Inibidora 50 , Óxido Nítrico/química , Óxido Nítrico Sintase Tipo II/química , Oxirredutases/química , Fenol/química , Extratos Vegetais , Folhas de Planta/efeitos dos fármacos , Proteínas de Plantas/química , Quercetina/farmacologia , Raios UltravioletaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia chebula (TC), a well-known Indian Ayurvedic medicine introduced into China in the Sui and Tang Dynasties, has been recorded and used medicinally as Fructus Chebulae, together with its variety tomentella (TCT) in the Chinese Pharmacopoeia. They have been also used commonly for the treatment of diabetes mellitus by Tibetan medicine. AIM OF THE STUDY: To investigate the main bioactive and therapeutic principles in the fruits of TCT, based on the extensive evaluation of their anti-inflammatory and hypoglycemic activities. MATERIALS AND METHODS: The TCT fresh fruits were analyzed by HPLC and separated further by column chromatography and preparative HPLC. The isolated compounds were identified by extensive spectroscopic analyses, including 1D/2D NMR, MS, UV, IR and ECD. Anti-inflammatory activity was evaluated by inhibition of NO production in RAW264.7 cells. The specific iNOS (PDB ID: 3E7G) structure was prepared by Discovery Studio 4.0, and the molecular docking simulation was performed on GOLD (version 5.2.2). Hypoglycemic activity was measured using the substrate solution of 4-nitrophenyl-α-d-glucopyranoside enzyme and buffer solution. RESULTS: The HPLC analysis method of polyphenols in the fruits of TCT was established, and 13 main chromatographic peaks were identified, including six hydrolyzable tannins (2, 4-7, 10-11), three simple phenols (12-14), and one oleanane pentacyclic triterpene, arjungenin. Extensive chromatographic separation of TCT fresh fruits yielded 14 compounds, including one new natural hydrolyzable tannin, 2,3-(S)-HHDP-6-O-galloyl-d-glucose (1). The known compounds were identified as 10 hydrolyzable tannins (2-11) and three simple phenols (12-14). Compounds 10 (IC50 = 36.43 ± 0.21 µM), 11 (IC50 = 42.28 ± 0.09 µM) displayed stronger NO inhibitory activity than the positive control L-NMMA (IC50 = 42.34 ± 0.66 µM), while 2, 4, and 9 showed moderate inhibitory activity against NO production. Further molecular docking simulation of specific iNOS on 10 and 11, as well as five previously isolated lignans 15-19 showed that there were no obvious rules between docking results and the in vitro NO inhibitory activity for hydrolyzable tannins (10 and 11), while the mechanism of anti-inflammatory activity for lignans was related to the substitution of conjugated aldehyde groups. Moreover, most of the hydrolyzable tannins (1-2, 4-5, 9-11) and simple phenol (12) displayed stronger inhibitory effects on α-glucosidase than the positive control, quercetin (IC50 = 6.118 ± 0.071 µM), with IC50 values ranging from 0.079 to 16.494 µM. Among these bioactive isolates, the hydrolyzable tannins 2, 4-5, and 9-11, and simple phenol 12 are major chemical components in TCT fruit. CONCLUSIONS: The results showed that lignans and hydrolyzed tannins are the main active ingredients of TCT fruits, responsible for the traditional treatment of sore throat and cough. Moreover, hydrolyzed tannins and simple phenolic compounds with potential hypoglycemic activity are closely related to the ethno-pharmacological uses of TCT fruits on diabetes in Tibetan medicine.
Assuntos
Anti-Inflamatórios/farmacologia , Hipoglicemiantes/farmacologia , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Terminalia/química , Animais , Anti-Inflamatórios/análise , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Frutas/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Taninos Hidrolisáveis/análise , Taninos Hidrolisáveis/química , Taninos Hidrolisáveis/farmacologia , Hipoglicemiantes/análise , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Técnicas In Vitro , Lignanas/análise , Lignanas/química , Lignanas/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Fenóis/análise , Fenóis/química , Fenóis/isolamento & purificação , Extratos Vegetais/análise , Extratos Vegetais/química , Células RAW 264.7 , Triterpenos/análise , Triterpenos/química , Triterpenos/farmacologia , alfa-Glucosidases/metabolismoRESUMO
Relatively high concentration of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in response to a variety of stimuli is a source of reactive nitrogen species, an important weapon of host innate immune defense. The SPRY domain-containing SOCS box protein 2 (SPSB2) is an E3 ubiquitin ligase that regulates the lifetime of iNOS. SPSB2 interacts with the N-terminal region of iNOS via a binding site on the SPRY domain of SPSB2, and recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, leading to its proteasomal degradation. Although critical residues for the SPSB2-iNOS interaction have been identified, structural basis for the interaction remains to be explicitly determined. In this study, we have determined a crystal structure of the N-terminal region of iNOS in complex with the SPRY domain of SPSB2 at 1.24 Å resolution. We have resolved the roles of some flanking residues, whose contribution to the SPSB2-iNOS interaction was structurally unclear previously. Furthermore, we have evaluated the effects of SPSB2 inhibitors on NO production using transient transfection and cell-penetrating peptide approaches, and found that such inhibitors can elevate NO production in RAW264.7 macrophages. These results thus provide a useful basis for the development of potent SPSB2 inhibitors as well as recruiting ligands for proteolysis targeting chimera (PROTAC) design.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Domínio B30.2-SPRY/efeitos dos fármacos , Cristalografia por Raios X , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Modelos Moleculares , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/química , Peptídeos/farmacologia , Células RAW 264.7 , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/químicaRESUMO
In an effort to discover new agents with high anti-inflammatory activity, 22 new 4-sulfonyloxy/alkoxy benzoxazolone derivatives were synthesized, characterized, and evaluated for their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production and TNF-α expression in RAW 264.7 cells in vitro. Most of these compounds displayed greater inhibitory ability against NO production than the lead compound 4-o-methyl-benzenesulfonyl benzoxazolone, and the most active compound 2h exhibited the strongest inhibitory activity against NO, IL-1ß, and IL-6 production with IC50 values 17.67, 20.07, and 8.61 µΜ, respectively. The effects of 2h were comparable or stronger than those of the positive control celecoxib. Compound 2h also displayed higher activity in vivo than celecoxib in a mouse model of xylene-induced ear edema, based on their inhibitory rates of 42.69% and 30.87%, respectively. Further molecular analysis revealed that compound 2h significantly reduced the iNOS levels in cell supernatant and suppressed the protein expression of iNOS, p-p38, p-ERK, and nuclear NF-κB. The results indicated that the anti-inflammatory effect of 2h might be realized through the regulation of ERK- and p38-mediated mitogen-activated protein kinase (MAPK)-NF-κB/iNOS signaling, thereby reducing the excessive release of NO, IL-1ß, and IL-6. Our findings demonstrated that compound 2h, a new benzoxazolone derivative, could inhibit activation of the MAPK-NF-κB/iNOS pathway, supporting its potential as a novel anti-inflammatory agent.
Assuntos
Anti-Inflamatórios/farmacologia , Benzoxazóis/química , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/uso terapêutico , Benzoxazóis/farmacologia , Benzoxazóis/uso terapêutico , Sítios de Ligação , Regulação para Baixo/efeitos dos fármacos , Edema/induzido quimicamente , Edema/tratamento farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Eight new sesquiterpene derivatives (2, 4-6 and 10-13), along with five known analogues were isolated from the mangrove endophytic fungus Phomopsis sp. SYSU-QYP-23. Their structures of new compounds were established by spectroscopic methods, and the absolute configurations were confirmed by single-crystal X-ray diffraction analysis and comparison of the experimental ECD spectra. The absolute configuration of the side chain in 1 was first defined by modified Mosher's method. Compounds 1-7 showed potent inhibitory activities against nitric oxide (NO) production in lipopolysaccharides (LPS) induced RAW 264.7 cells with IC50 values ranging from 8.6 to 14.5 µM. The molecular docking results implied that the bioactive sesquiterpenes may directly bind with targeting residues in the active cavity of iNOS protein.
Assuntos
Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Phomopsis/química , Sesquiterpenos/farmacologia , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Humanos , Ligação de Hidrogênio , Camundongos , Simulação de Acoplamento Molecular , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Ligação Proteica , Células RAW 264.7 , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/metabolismoRESUMO
Inducible nitric oxide synthase (iNOS) plays critical roles in the inflammatory response and host defense. Previous research on iNOS regulation mainly focused on its gene expression level, and much less is known about the regulation of iNOS function by N-glycosylation. In this study, we report for the first time that iNOS is N-glycosylated in vitro and in vivo. Mass spectrometry studies identified Asn695 as an N-glycosylation site of murine iNOS. Mutating Asn695 to Gln695 yields an iNOS that exhibits greater enzyme activity. The essence of nitric oxide synthase catalytic reaction is electron transfer process, which involves a series of conformational changes, and the linker between the flavin mononucleotide-binding domain and the flavin adenine dinucleotide-binding domain plays vital roles in the conformational changes. Asn695 is part of the linker, so we speculated that attachment of N-glycan to the Asn695 residue might inhibit activity by disturbing electron transfer. Indeed, our NADPH consumption results demonstrated that N-glycosylated iNOS consumes NADPH more slowly. Taken together, our results indicate that iNOS is N-glycosylated at its Asn695 residue and N-glycosylation of Asn695 might suppress iNOS activity by disturbing electron transfer.
Assuntos
Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Polissacarídeos/química , Animais , Asparagina/química , Catálise , Biologia Computacional , Transporte de Elétrons , Retículo Endoplasmático/metabolismo , Ensaios Enzimáticos , Glicosilação , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADP/química , NADP/metabolismo , Polissacarídeos/análise , Células RAW 264.7RESUMO
Intraprotein interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme centers is an obligatory step in nitric oxide synthase (NOS) enzymes. An isoform-specific pivotal region near Leu406 in the heme domain of human inducible NOS (iNOS) was proposed to mediate the FMN-heme domain-domain alignment (J Inorg Biochem 153:186-196, 2015). The FMN-heme IET rate is a measure of the interdomain FMN/heme complex formation. In this work, the FMN-heme IET kinetics in the wild type (wt) human iNOS oxygenase/FMN (oxyFMN) construct were directly measured by laser flash photolysis with added synthetic peptide related to the pivotal region, in comparison with the wt construct alone. The IET rates were decreased by the iNOS HKL peptide in a dose-saturable fashion, and the inhibitory effect was abolished by a single L406 â E mutation in the peptide. A similar trend in change of the NO synthesis activity of wt iNOS holoenzyme by the peptides was observed. These data, along with the kinetics and modeling results for the L406T and L406F mutant oxyFMN proteins, indicated that the Leu406 residue modulates the FMN-heme IET through hydrophobic interactions. Moreover, the IET rates were analyzed for the wt iNOS oxyFMN protein in the presence of nNOS or eNOS-derived peptide related to the equivalent pivotal heme domain site. These results together indicate that the isoform-specific pivotal region at the heme domain specifically interacts with the conserved FMN domain surface, to facilitate proper interdomain docking for the FMN-heme IET in NOS.
Assuntos
Mononucleotídeo de Flavina/metabolismo , Heme/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Transporte de Elétrons , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Mutação , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Domínios ProteicosRESUMO
Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.
Assuntos
Encéfalo/enzimologia , DNA/química , Corantes Fluorescentes/química , Óxido Nítrico Sintase Tipo II , Animais , Encéfalo/metabolismo , Química Encefálica , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Técnicas de Inativação de Genes , Camundongos , Microglia/química , Microglia/enzimologia , Microglia/metabolismo , Microscopia de Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Fagossomos/química , Fagossomos/metabolismo , Peixe-ZebraRESUMO
Bearing in mind that pain and major depressive disorder (MDD) often share biological pathways, this condition is classified as depression-pain syndrome. Mounting evidence suggests that oxidative stress is implicated in the pathophysiology of this syndrome. The development of effective pharmacological interventions for the depression-pain syndrome is of particular importance as clinical treatments for this comorbidity have shown limited efficacy. Therefore, the present study aimed to evaluate whether the 3,5-dimethyl-1-phenyl-4-(phenylselanyl)-1H-pyrazole (SePy) was able to reverse the depression-pain syndrome induced by intracerebroventricular (i.c.v) streptozotocin (STZ) in mice and the possible modulation of oxidative and nitrergic pathways in its effect. The treatment with SePy (1 and 10 mg/kg) administered intragastrically (i.g.) reversed the increased immobility time in the tail suspension test, decreased grooming time in the splash test, latency time to nociceptive response in the hot plate test, and the response frequency of Von Frey hair (VFH) stimulation induced by STZ (0.2 mg/4 µl/per mouse). Additionally, SePy (10 mg/kg, i.g.) reversed STZ-induced alterations in the levels of reactive oxygen species, nitric oxide, and lipid peroxidation and the superoxide dismutase and catalase activities in the prefrontal cortices (PFC) and hippocampi (HC) of mice. Treatment with SePy (10 mg/kg, i.g.) also reversed the STZ-induced increased expression of inducible nitric oxide synthase (iNOS) and glycogen synthase kinase 3 beta (GSK3ß) in the PFC and HC. An additional molecular docking investigation found that SePy binds to the active site of iNOS and GSK3ß. Altogether, these results indicate that the antidepressant-like effect of SePy is accompanied by decreased hyperalgesia and mechanical allodynia, which were associated with its antioxidant effect.
Assuntos
Depressão/tratamento farmacológico , Estresse Nitrosativo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Dor/tratamento farmacológico , Pirazóis/administração & dosagem , Selênio/administração & dosagem , Animais , Depressão/induzido quimicamente , Depressão/metabolismo , Glicogênio Sintase Quinase 3 beta/química , Glicogênio Sintase Quinase 3 beta/metabolismo , Injeções Intraventriculares , Masculino , Camundongos , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Nitrosativo/fisiologia , Estresse Oxidativo/fisiologia , Dor/induzido quimicamente , Dor/metabolismo , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Estrutura Secundária de Proteína , Estreptozocina/administração & dosagem , Estreptozocina/toxicidadeRESUMO
Joint inflammation is a key player in the pathogenesis of osteoarthritis (OA). Imperatorin, a plant-derived small molecule has been reported to have anti-inflammatory properties; however, its effect on chondrocytes is not known. Here, we investigated the effects of Imperatorin on interleukin-1ß (IL-1ß) induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in primary human OA chondrocytes and cartilage explants culture under pathological conditions and explored the associated signaling pathways. We pretreated chondrocytes or explants with Imperatorin (50 µM) followed by IL-1ß (1 ng/ml), and the culture supernatant was used to determine the levels of nitrite production by Griess assay and chondrocytes were harvested to prepare cell lysate or RNA for gene expression analysis of iNOS by Western blot or qPCR and in explants by immunohistochemistry (IHC). Pretreatment of primary chondrocytes and cartilage explants with Imperatorin suppressed IL-1ß induced expression of iNOS and NO production. Imperatorin blocked the IL-1ß-induced phosphorylation of ERK-MAPK/AP1 signaling pathway to suppress iNOS expression. The role of ERK in the regulation of iNOS expression was verified by using ERK inhibitor. Interestingly, we also found that Imperatorin binds to iNOS protein and inhibits its activity in vitro. Our data demonstrated that Imperatorin possess strong anti-inflammatory activity and may be developed as a therapeutic agent for the management of OA.
Assuntos
Anti-Inflamatórios/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Furocumarinas/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite/prevenção & controle , Fator de Transcrição AP-1/metabolismo , Anti-Inflamatórios/uso terapêutico , Cartilagem/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/ultraestrutura , Furocumarinas/uso terapêutico , Humanos , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/toxicidade , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Nitritos/análise , Cultura Primária de Células , Proteoma/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
"Hot loop" protein segments have variable structure and conformation and contribute crucially to protein-protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and -restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.
Assuntos
Peptídeos Cíclicos/metabolismo , Receptores de Melanocortina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteína Relacionada com Agouti/química , Proteína Relacionada com Agouti/metabolismo , Epitopos , Humanos , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Ligação Proteica , Conformação Proteica , EstereoisomerismoRESUMO
INTRODUCTION: Nitric oxide (NO) is an important mediator in the pathogenesis and control of immune system-related disorders and its levels are modulated by inducible NO synthase (iNOS). Oxidative stress is another pathological indication in majority of autoimmune disorders. The present study aims at the development of coupled molecules via selection of pharmacophores for both immunomodulatory and antioxidant activities through iNOS inhibition. METHODS: Variedly substituted coumarin moieties are coupled with naturally occurring phenols through an amide linkage and were predicted for activities using computer-based program PASS. The compounds predicted to have dual activities were synthesized. Docking studies were carried out against iNOS (PDB 1R35) and compounds having good docking score were evaluated for immunomodulatory and antioxidant activities. RESULTS: The synthesized compounds were found to be pure and were obtained in good yields. Compounds with maximum docking score (YR1a, YR2e, YR2c and YR4e) were selected for evaluation by in vitro models. Compounds YR2e and YR2c markedly inhibited the reduction of NBT dye and showed maximum % iNOS inhibition. In DPPH assay, compound YR4e was observed as the most potent antioxidant (EC50 0.33 µM/mL). Based on these studies, compounds YR2e and YR2c were selected for haemagglutination test. Compound YR2e was observed as the most active immunosuppressant with maximal inhibitory ability of iNOS and NBT reduction and lower HAT value of 3.5. CONCLUSION: Compound YR2e can be utilized as a pharmacological agent in the prevention or treatment of immunomodulatory diseases such as tumors, rheumatoid arthritis, ulcerative colitis, organ transplant and other autoimmune disorders.
Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Imunossupressores/química , Imunossupressores/farmacologia , Produtos Biológicos/síntese química , Produtos Biológicos/metabolismo , Técnicas de Química Sintética , Desenho de Fármacos , Imunossupressores/síntese química , Imunossupressores/metabolismo , Simulação de Acoplamento Molecular , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Conformação ProteicaRESUMO
A considerable number of human diseases have an inflammatory component, and a key mediator of immune activation and inflammation is inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO) from l-arginine. Overexpressed or dysregulated iNOS has been implicated in numerous pathologies including sepsis, cancer, neurodegeneration, and various types of pain. Extensive knowledge has been accumulated about the roles iNOS plays in different tissues and organs. Additionally, X-ray crystal and cryogenic electron microscopy structures have shed new insights on the structure and regulation of this enzyme. Many potent iNOS inhibitors with high selectivity over related NOS isoforms, neuronal NOS, and endothelial NOS, have been discovered, and these drugs have shown promise in animal models of endotoxemia, inflammatory and neuropathic pain, arthritis, and other disorders. A major issue in iNOS inhibitor development is that promising results in animal studies have not translated to humans; there are no iNOS inhibitors approved for human use. In addition to assay limitations, both the dual modalities of iNOS and NO in disease states (ie, protective vs harmful effects) and the different roles and localizations of NOS isoforms create challenges for therapeutic intervention. This review summarizes the structure, function, and regulation of iNOS, with focus on the development of iNOS inhibitors (historical and recent). A better understanding of iNOS' complex functions is necessary before specific drug candidates can be identified for classical indications such as sepsis, heart failure, and pain; however, newer promising indications for iNOS inhibition, such as depression, neurodegenerative disorders, and epilepsy, have been discovered.
Assuntos
Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Animais , Doença , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Biológicos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
(1) Introduction: Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules that play important roles in the progression of inflammatory disorders. The objective of this study was to explore the use of myrtucommuacetalone-1 (MCA-1), as a novel compound of natural origin and a potential anti-inflammatory agent. (2) Methodology: The anti-inflammatory potential of MCA-1, which was isolated from Myrthus communis Linn, was determined by assaying superoxide, hydrogen peroxide, and nitric oxide production in macrophages. Furthermore, the effects of the compound were analyzed via phosphorylation and translocation of the transcription factor NF kappa B, which is a key regulator of iNOS activation. The effect of MCA-1 on the inducible nitric oxide synthase (iNOS) enzyme was also examined using in silico docking studies. The anticancer potential for MCA-1 was evaluated with an MTT cytotoxic assay. (3) Results: In stimulated macrophages, MCA-1 inhibited superoxide production by 48%, hydrogen peroxide by 53%, and nitric oxide (NO) with an IC50 of <1 µg/mL. MCA-1 also showed a very strong binding pattern within the active site of the inducible nitric oxide synthase enzyme. Furthermore, 25 µg/mL of MCA-1 inhibited inducible nitric oxide synthase expression and abolished transcription factor (NFκB) phosphorylation and translocation to the nucleus. Cytotoxicity analyses of MCA-1 on 3T3 mouse fibroblasts, CC1 liver cell line, J774.2, macrophages and MDBK bovine kidney epithelial cell, yielded IC50 values of 6.53 ± 1.2, 4.6 ± 0.7, 5 ± 0.8, and 4.6 ± 0.7, µg/mL, respectively. (4) Conclusion: Our results suggest that MCA-1, a major phloroglucinol-type compound, shows strong anti-inflammatory activity and has a potential to be a leading therapeutic agent in the future.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Macrófagos/efeitos dos fármacos , Myrtus/química , Animais , Anti-Inflamatórios/isolamento & purificação , Linhagem Celular , Humanos , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , NF-kappa B/química , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/imunologia , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A total of 18 diterpenoids, including 10 new analogues (1-10), were isolated from Euphorbia antiquorum. The structures were characterized by spectroscopic techniques, and circular dichroism data analysis was adopted to confirm the absolute configurations of 1-10. Compounds 1-9 were classified as ent-atisane diterpenoids, and 10 was assigned as an ent-kaurane diterpenoid. The biological evaluation of nitric oxide (NO) production inhibition was conducted, and all of these isolates showed the property of inhibiting NO generation in lipopolysaccharide-induced BV-2 cells. Further research on molecular docking disclosed the affinities between the diterpenoids obtained and inducible nitric oxide synthase.