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1.
J Transl Med ; 19(1): 340, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34372885

RESUMO

BACKGROUND: To examine the effects of BI 1029539 (GS-248), a novel selective human microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor, in experimental models of acute lung injury (ALI) and sepsis in transgenic mice constitutively expressing the mPGES1 (Ptges) humanized allele. METHODS: Series 1: Lipopolysaccharide (LPS)-induced ALI. Mice were randomized to receive vehicle, BI 1029539, or celecoxib. Series 2: Cecal ligation and puncture-induced sepsis. Mice were randomized to receive vehicle or BI 1029539. RESULTS: Series 1: BI 1029539 or celecoxib reduced LPS-induced lung injury, with reduction in neutrophil influx, protein content, TNF-ɑ, IL-1ß and PGE2 levels in bronchoalveolar lavage (BAL), myeloperoxidase activity, expression of mPGES-1, cyclooxygenase (COX)-2 and intracellular adhesion molecule in lung tissue compared with vehicle-treated mice. Notably, prostacyclin (PGI2) BAL concentration was only lowered in celecoxib-treated mice. Series 2: BI 1029539 significantly reduced sepsis-induced BAL inflammatory cell recruitment, lung injury score and lung expression of mPGES-1 and inducible nitric oxide synthase. Treatment with BI 1029539 also significantly prolonged survival of mice with severe sepsis. Anti-inflammatory and anti-migratory effect of BI 1029539 was confirmed in peripheral blood leukocytes from healthy volunteers. CONCLUSIONS: BI 1029539 ameliorates leukocyte infiltration and lung injury resulting from both endotoxin-induced and sepsis-induced lung injury.


Assuntos
Lesão Pulmonar Aguda , Sepse , Lesão Pulmonar Aguda/tratamento farmacológico , Animais , Ciclo-Oxigenase 2/metabolismo , Dinoprostona , Modelos Animais de Doenças , Humanos , Pulmão/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II , Prostaglandina-E Sintases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
Molecules ; 26(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34443500

RESUMO

Prostaglandins are a group of important cell-signaling molecules involved in the regulation of ovarian maturation, oocyte development, egg laying and associated behaviors in invertebrates. However, the presence of prostaglandin E2 (PGE2), the key enzymes for PGE2 biosynthesis and its interference by drugs were not investigated previously in the ovary of ticks. The present study was undertaken to assess the modulation of the PGE2-mediated pathway in the eclosion blocking effect of flumethrin and terpenoid subfraction isolated from Artemisia nilagirica in Rhipicephalus annulatus ticks. The acaricidal activities and chemical profiling of the terpenoid subfraction were performed. The localization of the cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) enzymes and the quantification of PGE2 in the ovaries of the ticks treated with methanol (control), flumethrin and terpenoid subfraction were also undertaken. In addition, the vitellogenin concentration in hemolymph was also assayed. Both flumethrin and the terpenoid subfraction of A. nilagirica elicited a concentration-dependent inhibition of fecundity and blocking of hatching of the eggs. The COX1 could not be detected in the ovaries of treated and control ticks, while there was no significant difference observed in the concentration of vitellogenin (Vg) in them. The presence of PGES in the oocytes of control ticks was confirmed while the immunoreactivities against PGES were absent in the vitellogenic oocytes of ticks treated with flumethrin and terpenoid subfraction. The levels of PGE2 were below the detection limit in the ovaries of the flumethrin-treated ticks, while it was significantly lower in the ovaries of the terpenoid subfraction-treated ticks. Hence, the prostaglandin E synthase and PGE2 were identified as very important mediators for the signaling pathway for ovarian maturation and oviposition in ticks. In addition, the key enzyme for prostaglandin biosynthesis, PGES and the receptors for PGE2 can be exploited as potential drug targets for tick control. The detection of PGES by immunohistochemistry and quantification of PGE2 by LC-MSMS can be employed as valuable tools for screening newer compounds for their eclosion blocking acaricidal effects.


Assuntos
Artemisia/química , Dinoprostona/metabolismo , Piretrinas/farmacologia , Rhipicephalus/efeitos dos fármacos , Terpenos/isolamento & purificação , Terpenos/farmacologia , Animais , Anticorpos/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Hemolinfa/metabolismo , Imersão , Ovário/efeitos dos fármacos , Ovário/enzimologia , Peroxidase/metabolismo , Prostaglandina-E Sintases/metabolismo , Vitelogeninas/metabolismo
3.
J Clin Invest ; 131(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34101620

RESUMO

Inhibitors of microsomal prostaglandin E synthase 1 (mPGES-1) are in the early phase of clinical development. Deletion of mPges-1 in mice confers analgesia, restrains atherogenesis, and fails to accelerate thrombogenesis, while suppressing prostaglandin E2 (PGE2), but increasing the biosynthesis of prostacyclin (PGI2). In low-density lipoprotein receptor-deficient (Ldlr-/-) mice, this last effect represents the dominant mechanism by which mPges-1 deletion restrains thrombogenesis, while suppression of PGE2 accounts for its antiatherogenic effect. However, the effect of mPges-1 depletion on blood pressure (BP) in this setting remains unknown. Here, we show that mPges-1 depletion significantly increased the BP response to salt loading in male Ldlr-/- mice, whereas, despite the direct vasodilator properties of PGI2, deletion of the I prostanoid receptor (Ipr) suppressed this response. Furthermore, combined deletion of the Ipr abrogated the exaggerated BP response in male mPges-1-/- mice. Interestingly, these unexpected BP phenotypes were not observed in female mice fed a high-salt diet (HSD). This is attributable to the protective effect of estrogen in Ldlr-/- mice and in Ipr-/- Ldlr-/- mice. Thus, estrogen compensates for a deficiency in PGI2 to maintain BP homeostasis in response to high salt in hyperlipidemic female mice. In male mice, by contrast, the augmented formation of atrial natriuretic peptide (ANP) plays a similar compensatory role, restraining hypertension and oxidant stress in the setting of Ipr depletion. Hence, men with hyperlipidemia on a HSD might be at risk of a hypertensive response to mPGES-1 inhibitors.


Assuntos
Pressão Sanguínea , Homeostase , Receptores de Epoprostenol/deficiência , Caracteres Sexuais , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Receptores de Epoprostenol/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo
4.
Bioorg Chem ; 112: 104861, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33826984

RESUMO

Microsomal prostaglandin E2 synthase-1 (mPGES-1), 5-lipoxygenase (5-LO) and 5- lipoxygenase-activating protein (FLAP) are key for biosynthesis of proinflammatory lipid mediators and pharmacologically relevant drug targets. In the present study, we made an attempt to explore the role of small heteroaromatic fragments on the 4,5-diarylisoxazol-3-carboxylic acid scaffold, which are selected to interact with focused regions in the active sites of mPGES-1, 5-LO and FLAP. We report that the simple structural variations on the benzyloxyaryl side-arm of the scaffold significantly influence the selectivity against mPGES-1, 5-LO and FLAP, enabling to produce multi-target inhibitors of these protein targets, exemplified by compound 18 (IC50 mPGES-1 = 0.16 µM; IC50 5-LO = 0.39 µM) with in vivo efficacy in animal model of inflammation. The computationally modeled binding structures of these new inhibitors for three targets provide clues for rational design of modified structures as multi-target inhibitors. In conclusion, the simple synthetic procedure, and the possibility of enhancing the potency of this class of inhibitors through structural modifications pave the way for further development of new multi-target inhibitors against mPGES-1, 5-LO and FLAP, with potential application as anti-inflammatory agents.


Assuntos
Androstenóis/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Carboxílicos/farmacologia , Inibidores Enzimáticos/farmacologia , Prostaglandina-E Sintases/antagonistas & inibidores , Adolescente , Adulto , Idoso , Androstenóis/síntese química , Androstenóis/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Estrutura Molecular , Prostaglandina-E Sintases/metabolismo , Relação Estrutura-Atividade , Adulto Jovem
5.
Parasit Vectors ; 14(1): 206, 2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33874988

RESUMO

BACKGROUND: The salmon louse (Lepeophtheirus salmonis) is a parasite of salmonid fish. Atlantic salmon (Salmo salar) exhibit only a limited and ineffective immune response when infested with this parasite. Prostaglandins (PGs) have many biological functions in both invertebrates and vertebrates, one of which is the regulation of immune responses. This has led to the suggestion that prostaglandin E2 (PGE2) is important in the salmon louse host-parasite interaction, although studies of a salmon louse prostaglandin E2 synthase (PGES) 2 gene have not enabled conformation of this hypothesis. The aim of the present study was, therefore, to characterize two additional PGES-like genes. METHODS: Lepeophtheirus salmonis microsomal glutathione S-transferase 1 like (LsMGST1L) and LsPGES3L were investigated by sequencing, phylogenetics, transcript localization and expression studies. Moreover, the function of these putative PGES genes in addition to the previously identified LsPGES2 gene was analyzed in double stranded (ds) RNA-mediated knockdown (KD) salmon louse. RESULTS: Analysis of the three putative LsPGES genes showed a rather constitutive transcript level throughout development from nauplius to the adult stages, and in a range of tissues, with the highest levels in the ovaries or gut. DsRNA-mediated KD of these transcripts did not produce any characteristic changes in phenotype, and KD animals displayed a normal reproductive output. The ability of the parasite to infect or modulate the immune response of the host fish was also not affected by KD. CONCLUSIONS: Salmon louse prostaglandins may play endogenous roles in the management of reproduction and oxidative stress and may be a product of salmon louse blood digestions.


Assuntos
Proteínas de Artrópodes/metabolismo , Copépodes/enzimologia , Doenças dos Peixes/parasitologia , Prostaglandina-E Sintases/metabolismo , Animais , Proteínas de Artrópodes/genética , Copépodes/classificação , Copépodes/genética , Copépodes/crescimento & desenvolvimento , Feminino , Interações Hospedeiro-Parasita , Masculino , Filogenia , Prostaglandina-E Sintases/genética , Prostaglandinas/metabolismo , Salmo salar/parasitologia
6.
Toxins (Basel) ; 13(4)2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33916734

RESUMO

Tetraponera rufonigra (Arboreal Bicoloured Ant) venom induces pain, inflammation, and anaphylaxis in people and has an increased incident in Southeast Asia regions. The bioactive components and mechanism of action of the ant venom are still limited. The aim of this research was to identify the protein composition and inflammatory process of the ant venom by using RAW 264.7 macrophage cells. The major venom proteins are composed of 5' nucleotidase, prolyl endopeptidase-like, aminopeptidase N, trypsin-3, venom protein, and phospholipase A2 (PLA2). The venom showed PLA2 activity and represented 0.46 µg of PLA2 bee venom equivalent/µg crude venom protein. The venom induced cytotoxic in a dose- and time-dependent manner with IC20 approximately at 4.01 µg/mL. The increased levels of COX-2 and PGE2 were observed after 1 h of treatment correlating with an upregulation of COX-2 expression. Moreover, the level of mPGES-1 expression was obviously increased after 12 h of venom induction. Hence, our results suggested that the induction of COX-2/mPGEs-1 pathway could be a direct pathway for the ant venom-induced inflammation.


Assuntos
Venenos de Formiga/toxicidade , Formigas , Mediadores da Inflamação/metabolismo , Inflamação/induzido quimicamente , Proteínas de Insetos/toxicidade , Macrófagos/efeitos dos fármacos , Animais , Venenos de Formiga/enzimologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Proteínas de Insetos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Células RAW 264.7 , Fatores de Tempo , Regulação para Cima
7.
PLoS One ; 16(4): e0250638, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33891661

RESUMO

Prostaglandin E2 (PGE2) is known to have important roles in labor, but the detailed mechanism underlying the spontaneous human labor remains unknown. Here, we examined the involvement of prostaglandin biosynthetic enzymes and transporter in the accumulation of PGE2 in amniotic fluid in human labor. PGE2 and its metabolites were abundant in amniotic fluid in deliveries at term in labor (TLB), but not at term not in labor (TNL). In fetal-membrane Transwell assays, levels of PGE2 production in both maternal and fetal compartments were significantly higher in the TLB group than the TNL group. In fetal-membrane, the mRNA level of PTGES3, which encodes cytosolic prostaglandin E synthase (cPGES), was significantly higher in TLB than in TNL, but the mRNA levels of the other PGE2-synthase genes were not affected by labor. Moreover, the mRNA level of PTGS2, which encodes cyclooxygenase-2 (COX-2) in the amnion was significantly higher in TLB than in TNL. Western blot analyses revealed that the levels of COX-1 and COX-2 were comparable between the two groups, however, the level of cPGES was relatively higher in TLB than in TNL. COXs, cPGES, and prostaglandin transporter (SLCO2A1) proteins were all expressed in both chorionic trophoblasts and amniotic epithelium. These findings suggest that COXs, cPGES and SLCO2A1 contribute to PGE2 production from fetal-membrane in labor.


Assuntos
Âmnio/metabolismo , Dinoprostona/metabolismo , Membranas Extraembrionárias/metabolismo , Trabalho de Parto/metabolismo , Prostaglandina-E Sintases/metabolismo , Líquido Amniótico/metabolismo , Cromatografia Líquida de Alta Pressão , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/análise , Membranas Extraembrionárias/patologia , Feminino , Humanos , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Gravidez , Prostaglandina-E Sintases/genética , RNA Mensageiro/metabolismo , Espectrometria de Massas em Tandem , Regulação para Cima
8.
Bioorg Med Chem Lett ; 41: 127992, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33775835

RESUMO

Our previous research showed that N-carboxy-phenylsulfonyl hydrazide (scaffold A) could reduce LPS-stimulated PGE2 levels in RAW 264.7 macrophage cells by an inhibition of mPGES-1 enzyme. However, a number of scaffold A derivatives showed the drawbacks such as the formation of regioisomers and poor liver metabolic stability. In order to overcome these synthetic and metabolic problems, therefore, we decided to replace N-carboxy-phenylsulfonyl hydrazide (scaffold A) with N-carboxy-phenylsulfonamide (scaffold B) or N-amido-phenylsulfonamide frameworks (scaffold C) as a bioisosteric replacement. Among them, MPO-0186 (scaffold C) inhibited the production of PGE2 (IC50: 0.24 µM) in A549 cells via inhibition of mPGES-1 (IC50: 0.49 µM in a cell-free assay) and was found to be approximately 9- and 8-fold more potent than MK-886 as a reference inhibitor, respectively. A molecular docking study theoretically suggests that MPO-0186 could inhibit PGE2 production by blocking the PGH2 binding site of mPGES-1 enzyme. Furthermore, MPO-0186 demonstrated good liver metabolic stability and no significant inhibition observed in clinically relevant CYP isoforms except CYP2C19. This result provides a potential starting point for the development of selective and potent mPGES-1 inhibitor with a novel scaffold.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Prostaglandina-E Sintases/antagonistas & inibidores , Sulfonamidas/farmacologia , Células A549 , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Fígado/química , Fígado/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Prostaglandina-E Sintases/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
9.
Cell Death Dis ; 12(2): 135, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33542207

RESUMO

Despite the progress to understand inflammatory reactions, mechanisms causing their resolution remain poorly understood. Prostanoids, especially prostaglandin E2 (PGE2), are well-characterized mediators of inflammation. PGE2 is produced in an inducible manner in macrophages (Mϕ) by microsomal PGE2-synthase-1 (mPGES-1), with the notion that it also conveys pro-resolving properties. We aimed to characterize the role of mPGES-1 during resolution of acute, zymosan-induced peritonitis. Experimentally, we applied the mPGES-1 inhibitor compound III (CIII) once the inflammatory response was established and confirmed its potent PGE2-blocking efficacy. mPGES-1 inhibition resulted in an incomplete removal of neutrophils and a concomitant increase in monocytes and Mϕ during the resolution process. The mRNA-seq analysis identified enhanced C-X3-C motif receptor 1 (CX3CR1) expression in resident and infiltrating Mϕ upon mPGES-1 inhibition. Besides elevated Cx3cr1 expression, its ligand CX3CL1 was enriched in the peritoneal lavage of the mice, produced by epithelial cells upon mPGES-1 inhibition. CX3CL1 not only increased adhesion and survival of Mϕ but its neutralization also completely reversed elevated inflammatory cell numbers, thereby normalizing the cellular, peritoneal composition during resolution. Our data suggest that mPGES-1-derived PGE2 contributes to the resolution of inflammation by preventing CX3CL1-mediated retention of activated myeloid cells at sites of injury.


Assuntos
Quimiocina CX3CL1/metabolismo , Dinoprostona/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Peritonite/enzimologia , Prostaglandina-E Sintases/antagonistas & inibidores , Animais , Anticorpos Neutralizantes/farmacologia , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Quimiocina CX3CL1/antagonistas & inibidores , Quimiocina CX3CL1/genética , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Feminino , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Peritonite/genética , Peritonite/imunologia , Fenótipo , Prostaglandina-E Sintases/metabolismo , Regulação para Cima
10.
Biochem Biophys Res Commun ; 546: 124-129, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33582554

RESUMO

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and prostacyclin (PGI2) synthase (PGIS) are PG terminal synthases that work downstream of cyclooxygenase and synthesize PGE2 and PGI2, respectively. Although the involvement of PG receptors in acquired cutaneous immune responses was recently shown, the roles of these PG terminal synthases remain unclear. To identify the pathophysiological roles of mPGES-1 and PGIS in cutaneous immune systems, we applied contact hypersensitivity (CHS) to mPGES-1 and PGIS knockout (KO) mice as a model of acquired immune responses. Mice were treated with 1-fluoro-2,4-dinitrobenzene (DNFB) and evaluated for ear thickness and histopathological features. The results showed that the severity of ear swelling in both gene-deficient mice was much lower than that in wild-type (WT) mice. Histological examination of DNFB-treated ears showed that inflammatory cell infiltration and edema in the dermis were also less apparent in both genotypic mice. LC-MS analysis further showed that the increment in PGE2 levels in DNFB-treated ear tissue was reduced in mPGES-1 KO mice, and that 6-keto PGF1α (a stable metabolite of PGI2) was not detected in PGIS KO mice. Furthermore, we made bone marrow (BM) chimera and found that transplantation of WT mouse-derived BM cells restored the impaired CHS response in mPGES-1 KO mice but did not restore the response in PGIS KO mice. These results indicated that mPGES-1 in BM-derived cells and PGIS in non-BM-derived cells might play critical roles in DNFB-induced CHS. mPGES-1-derived PGE2 and PGIS-derived PGI2 might coordinately promote acquired cutaneous immune responses.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Dermatite de Contato/enzimologia , Oxirredutases Intramoleculares/metabolismo , Prostaglandina-E Sintases/metabolismo , Transferência Adotiva , Animais , Células da Medula Óssea , Sistema Enzimático do Citocromo P-450/deficiência , Sistema Enzimático do Citocromo P-450/genética , Dermatite de Contato/etiologia , Dermatite de Contato/genética , Dinitrofluorbenzeno/efeitos adversos , Orelha/patologia , Feminino , Interferon gama/metabolismo , Interleucinas/metabolismo , Oxirredutases Intramoleculares/deficiência , Oxirredutases Intramoleculares/genética , Camundongos , Camundongos Knockout , Prostaglandina-E Sintases/deficiência , Prostaglandina-E Sintases/genética , Prostaglandinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
Nat Commun ; 12(1): 828, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547294

RESUMO

The co-chaperone p23 is a central part of the Hsp90 machinery. It stabilizes the closed conformation of Hsp90, inhibits its ATPase and is important for client maturation. Yet, how this is achieved has remained enigmatic. Here, we show that a tryptophan residue in the proximal region of the tail decelerates the ATPase by allosterically switching the conformation of the catalytic loop in Hsp90. We further show by NMR spectroscopy that the tail interacts with the Hsp90 client binding site via a conserved helix. This helical motif in the p23 tail also binds to the client protein glucocorticoid receptor (GR) in the free and Hsp90-bound form. In vivo experiments confirm the physiological importance of ATPase modulation and the role of the evolutionary conserved helical motif for GR activation in the cellular context.


Assuntos
Adenilil Imidodifosfato/química , Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Prostaglandina-E Sintases/química , Receptores de Glucocorticoides/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
12.
Sci Rep ; 11(1): 880, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441600

RESUMO

Increased prostaglandin E2 (PGE2) levels were detected in mitochondrial disease patient cells harboring nuclear gene mutations in structural subunits of complex I, using a metabolomics screening approach. The increased levels of this principal inflammation mediator normalized following exposure of KH176m, an active redox-modulator metabolite of sonlicromanol (KH176). We next demonstrated that KH176m selectively inhibited lipopolysaccharide (LPS) or interleukin-1ß (IL-1ß)-induced PGE2 production in control skin fibroblasts. Comparable results were obtained in the mouse macrophage-like cell line RAW264.7. KH176m selectively inhibited mPGES-1 activity, as well as the inflammation-induced expression of mPGES-1. Finally, we showed that the effect of KH176m on mPGES-1 expression is due to the inhibition of a PGE2-driven positive feedback control-loop of mPGES-1 transcriptional regulation. Based on the results obtained we discuss potential new therapeutic applications of KH176m and its clinical stage parent drug candidate sonlicromanol in mitochondrial disease and beyond.


Assuntos
Cromanos/farmacologia , Dinoprostona/biossíntese , Prostaglandina-E Sintases/metabolismo , Animais , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Fibroblastos/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Oxirredutases Intramoleculares/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Microssomos/metabolismo , Doenças Mitocondriais/metabolismo , Oxirredução/efeitos dos fármacos , Células RAW 264.7
13.
Protein J ; 40(1): 63-67, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33403608

RESUMO

Prostaglandin E synthase (PGES) catalyzes the conversion of prostaglandin H2 to prostaglandin E2 in the presence of glutathione (GSH) in mammals. Amid the limited knowledge on prostaglandin and its related enzymes in insects, we recently identified PGES from the silkworm Bombyx mori (bmPGES) and determined its crystal structure complexed with GSH. In the current study, we investigated the substrate-binding site of bmPGES by site-directed mutagenesis and X-ray crystallography. We found that the residues Tyr107, Val155, Met159, and Glu203 are located in the catalytic pockets of bmPGES, and mutagenesis of each residue reduced the bmPGES activity. Our results suggest that these four residues contribute to the catalytic activity of bmPGES. Overall, this structure-function study holds implications in controlling pests by designing rational and efficient pesticides.


Assuntos
Bombyx/química , Dinoprostona/química , Glutationa/química , Proteínas de Insetos/química , Prostaglandina-E Sintases/química , Motivos de Aminoácidos , Animais , Bombyx/enzimologia , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Dinitroclorobenzeno/química , Dinitroclorobenzeno/metabolismo , Dinoprostona/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
14.
Brain Res ; 1750: 147153, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33049240

RESUMO

Prostaglandin E2 (PGE2) is a lipid mediator which plays a role in the generation of inflammatory and neuropathic pain. In the peripheral nervous system, PGE2 sensitizes nociceptive afferent neurons through E-prostanoid (EP) receptors. In the central nervous system, PGE2 modulates pain sensitivity and contributes to the development of neuropathic pain. However, the distribution of PGE2 and EP receptors in the spinal cord remains unclear. In the present study, we examined the expression of PGE2 synthases (microsomal PGE synthase [mPGES]-1, mPGES-2, and cytosolic PGE synthase [cPGES]) and EP receptors (EP1-4) in a rat model of neuropathic pain. We identified that mPGES-1 mRNA was upregulated in spinal endothelial cells after nerve injury and exhibited co-localization with cyclooxygenase-2 (COX-2). We detected that mPGES-2 mRNA and cPGES mRNA were expressed in spinal neurons and noted that their expression level was not affected by nerve injury. With respect to EP receptors, EP2 mRNA and EP4 mRNA were expressed in spinal neurons in the dorsal horn. EP3 mRNA was expressed in motor neurons, whereas EP1 mRNA was not detected in the spinal cord. Intrathecal injection of tumor necrosis factor alpha (TNFα) upregulated mPGES-1 mRNA in blood vessels in the spinal cord. Intrathecal injection of a TNFα-neutralizing antibody partially inhibited the upregulation of mPGES-1 mRNA after nerve injury. These results indicate that PGE2 is synthesized by COX-2/mPGES-1 in spinal endothelial cells after nerve injury. These results suggest that in neuropathic pain condition, endothelial cell-derived PGE2 may act on EP2 and EP4 receptors on spinal neurons and modulate pain sensitivity.


Assuntos
Neuralgia/fisiopatologia , Prostaglandina-E Sintases/metabolismo , Receptores de Prostaglandina E/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Expressão Gênica/genética , Oxirredutases Intramoleculares/metabolismo , Masculino , Limiar da Dor/efeitos dos fármacos , Prostaglandina-E Sintases/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E/fisiologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Medula Espinal/fisiologia
15.
J Cell Physiol ; 236(6): 4764-4777, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33275302

RESUMO

Primary cilia have been found to function as mechanosensors in low-magnitude high-frequency vibration (LMHFV)-induced osteogenesis. The PGE2 also regulates bone homeostasis and mechanical osteogenesis through its receptor EP4 signaling, but its involvement in LMHFV-induced or in primary cilia-induced osteogenesis has not been investigated. We hypothesized that LMHFV stimulates osteoblast (OB) differentiation by activating the COX2-PGE2-EP pathway in a manner dependent on primary cilia and that primary cilia are also affected by the PGE2 pathway. In this study, through western blot analysis, RNA interference, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and cytochemical staining, we observed that COX2, mPGES-1, and PGE2 levels were markedly elevated in cells treated with LMHFV and were greatly decreased in LMHFV-treated cells following IFT88 silencing. EP4 expression was significantly increased in OBs following LMHFV treatment, but IFT88 silencing significantly blocked this increase. EP4 localized to the bases of primary cilia. LMHFV reduced the length and abundance of primary cilia, but the cells could self-repair their primary cilia after mechanical damage. EP4 antagonism significantly blocked the LMHFV-induced increase in IFT88 expression and blocked the recovery of primary cilia length and the proportion of cells with primary cilia. In addition, COX2 or EP4 antagonism disrupted LMHFV-induced osteogenesis. These results demonstrate the integration of and crosstalk between primary cilia and the COX2-PGE2-EP4 signaling pathway under mechanical stimulation.


Assuntos
Diferenciação Celular , Cílios/enzimologia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Mecanotransdução Celular , Osteoblastos/enzimologia , Osteogênese , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Células 3T3 , Animais , Diferenciação Celular/efeitos dos fármacos , Cílios/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Estimulação Física , Antagonistas de Prostaglandina/farmacologia , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Vibração
16.
Biochim Biophys Acta Gen Subj ; 1865(2): 129800, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33246032

RESUMO

BACKGROUND: Due to the importance of both prostaglandins (PGs) and leukotrienes (LTs) as pro-inflammatory mediators, and the potential for eicosanoid shunting in the presence of pathway target inhibitors, we have investigated an approach to inhibiting the formation of both PGs and LTs as part of a multi-targeted drug discovery effort. METHODS: We generated ligand-protein X-ray crystal structures of known inhibitors of microsomal prostaglandin E2 synthase-1 (mPGES-1) and the 5-Lipoxygenase Activating Protein (FLAP), with their respective proteins, to understand the overlapping pharmacophores. We subsequently used molecular modeling and structure-based drug design (SBDD) to identify hybrid structures intended to inhibit both targets. RESULTS: This work enabled the preparation of compounds 4 and 5, which showed potent in vitro inhibition of both targets. SIGNIFICANCE: Our findings enhance the structural understanding of mPGES-1 and FLAP's unique ligand binding pockets and should accelerate the discovery of additional dual inhibitors for these two important integral membrane protein drug targets.


Assuntos
Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Descoberta de Drogas , Eicosanoides/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Prostaglandina-E Sintases/antagonistas & inibidores , Inibidores da Proteína Ativadora de 5-Lipoxigenase/química , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Eicosanoides/metabolismo , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Prostaglandina-E Sintases/metabolismo , Relação Estrutura-Atividade
17.
Int J Mol Sci ; 21(22)2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228217

RESUMO

Dry eye disease (DED) is commonly associated with ocular surface inflammation and pain. In this study, we evaluated the effectiveness of repeated instillations of transient receptor potential melastatin 8 (TRPM8) ion channel antagonist M8-B on a mouse model of severe DED induced by the excision of extra-orbital lacrimal and Harderian glands. M8-B was topically administered twice a day from day 7 until day 21 after surgery. Cold and mechanical corneal sensitivities and spontaneous ocular pain were monitored at day 21. Ongoing and cold-evoked ciliary nerve activities were next evaluated by electrophysiological multi-unit extracellular recording. Corneal inflammation and expression of genes related to neuropathic pain and inflammation were assessed in the trigeminal ganglion. We found that DED mice developed a cold allodynia consistent with higher TRPM8 mRNA expression in the trigeminal ganglion (TG). Chronic M8-B instillations markedly reversed both the corneal mechanical allodynia and spontaneous ocular pain commonly associated with persistent DED. M8-B instillations also diminished the sustained spontaneous and cold-evoked ciliary nerve activities observed in DED mice as well as inflammation in the cornea and TG. Overall, our study provides new insight into the effectiveness of TRPM8 blockade for alleviating corneal pain syndrome associated with severe DED, opening a new avenue for ocular pain management.


Assuntos
Anti-Inflamatórios/farmacologia , Síndromes do Olho Seco/tratamento farmacológico , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Ácidos Nicotínicos/farmacologia , Canais de Cátion TRPM/genética , Administração Oftálmica , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Temperatura Baixa , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/fisiopatologia , Modelos Animais de Doenças , Síndromes do Olho Seco/complicações , Síndromes do Olho Seco/genética , Síndromes do Olho Seco/metabolismo , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Gânglios Parassimpáticos/efeitos dos fármacos , Gânglios Parassimpáticos/metabolismo , Gânglios Parassimpáticos/fisiopatologia , Regulação da Expressão Gênica , Glândula de Harder/cirurgia , Hiperalgesia/etiologia , Hiperalgesia/genética , Hiperalgesia/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Aparelho Lacrimal/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/etiologia , Neuralgia/genética , Neuralgia/metabolismo , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/fisiopatologia
18.
Am J Respir Cell Mol Biol ; 63(6): 819-830, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32926636

RESUMO

Pathological changes in the biomechanical environment are implicated in the progression of idiopathic pulmonary fibrosis (IPF). Stiffened matrix augments fibroblast proliferation and differentiation and activates TGF-ß1 (transforming growth factor-ß1). Stiffened matrix impairs the synthesis of the antifibrogenic lipid mediator prostaglandin E2 (PGE2) and reduces the expression of the rate-limiting prostanoid biosynthetic enzyme cyclooxygenase-2 (COX-2). We now show that prostaglandin E synthase (PTGES), the final enzyme in the PGE2 biosynthetic pathway, is expressed at lower levels in the lungs of patients with IPF. We also show substantial induction of COX-2, PTGES, prostaglandin E receptor 4 (EP4), and cytosolic phospholipase A2 (cPLA2) expression in human lung fibroblasts cultured in soft collagen hydrogels or in spheroids compared with conventional culture on stiff plastic culture plates. Induction of COX-2, cPLA2, and PTGES expression in spheroid cultures was moderately inhibited by the p38 mitogen-activated protein kinase inhibitor SB203580. The induction of prostanoid biosynthetic enzyme expression was accompanied by an increase in PGE2 levels only in non-IPF-derived fibroblast spheroids. Our study reveals an extensive dysregulation of prostanoid biosynthesis and signaling pathways in IPF-derived fibroblasts, which are only partially abrogated by culture in soft microenvironments.


Assuntos
Microambiente Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Imidazóis/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ciclo-Oxigenase 2/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Prostaglandina-E Sintases/metabolismo
19.
FASEB J ; 34(8): 11101-11114, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32623799

RESUMO

The majority of anti-cancer therapies target the proliferating tumor cells, while the tumor stroma, principally unaffected, survives, and provide a niche for surviving tumor cells. Combining tumor cell and stroma-targeting therapies thus have a potential to improve patient outcome. The neuroblastoma stroma contains cancer-associated fibroblasts expressing microsomal prostaglandin E synthase-1 (mPGES-1). mPGES-1-derived prostaglandin E2 (PGE2 ) is known to promote tumor growth through increased proliferation and survival of tumor cells, immune suppression, angiogenesis, and therapy resistance, and we, therefore, hypothesize that mPGES-1 constitutes an interesting stromal target. Here, we aimed to develop a relevant in vitro model to study combination therapies. Co-culturing of neuroblastoma and fibroblast cells in 3D tumor spheroids mimic neuroblastoma tumors with regard to the cyclooxygenase/mPGES-1/PGE2 pathway. Using the spheroid model, we show that the inhibition of fibroblast-derived mPGES-1 enhanced the cytotoxic effect of doxorubicin and vincristine and significantly reduced tumor cell viability and spheroid growth. Cyclic treatment with vincristine in combination with an mPGES-1 inhibitor abrogated cell repopulation. Moreover, inhibition of mPGES-1 potentiated the cytotoxic effect of vincristine on established neuroblastoma allografts in mice. In conclusion, we established a 3D neuroblastoma model, highlighting the potential of combining stromal targeting of mPGES-1 with tumor cell targeting drugs like vincristine.


Assuntos
Antineoplásicos/farmacologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neuroblastoma/metabolismo , Prostaglandina-E Sintases/metabolismo
20.
Sci Rep ; 10(1): 10187, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576928

RESUMO

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is known as an ideal target for next generation of anti-inflammatory drugs without the side effects of currently available anti-inflammatory drugs. However, there has been no clinically promising mPGES-1 inhibitor identified through traditional drug discovery and development route. Here we report a new approach, called DREAM-in-CDM (Drug Repurposing Effort Applying Integrated Modeling-in vitro/vivo-Clinical Data Mining), to identify an FDA-approved drug suitable for use as an effective analgesic targeting mPGES-1. The DREAM-in-CDM approach consists of three steps: computational screening of FDA-approved drugs; in vitro and/or in vivo assays; and clinical data mining. By using the DREAM-in-CDM approach, lapatinib has been identified as a promising mPGES-1 inhibitor which may have significant anti-inflammatory effects to relieve various forms of pain and possibly treat various inflammation conditions involved in other inflammation-related diseases such as the lung inflammation caused by the newly identified COVID-19. We anticipate that the DREAM-in-CDM approach will be used to repurpose FDA-approved drugs for various new therapeutic indications associated with new targets.


Assuntos
Anti-Inflamatórios/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Lapatinib/farmacologia , Pneumonia Viral/tratamento farmacológico , Prostaglandina-E Sintases/antagonistas & inibidores , Betacoronavirus , COVID-19 , Química Computacional/métodos , Infecções por Coronavirus/patologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Inflamação/tratamento farmacológico , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral/patologia , Prostaglandina-E Sintases/metabolismo , SARS-CoV-2
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