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1.
Eur Heart J ; 43(36): 3477-3489, 2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-35728000

RESUMO

AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.


Assuntos
Cardiomiopatia Dilatada , Terapia de Alvo Molecular , Miócitos Cardíacos , Inibidores de Proteínas Quinases , Serina , Troponina T , Fator 4 Ativador da Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Cardiomiopatia Dilatada/tratamento farmacológico , Cardiomiopatia Dilatada/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Glucose/metabolismo , Glicina/biossíntese , Glicina/genética , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Células-Tronco Pluripotentes Induzidas/fisiologia , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Fosfoglicerato Desidrogenase/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Serina/antagonistas & inibidores , Serina/biossíntese , Serina/genética , Troponina T/genética , Troponina T/metabolismo
2.
Lab Invest ; 102(2): 194-203, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34625658

RESUMO

Glioblastoma (GBM) is the most malignant primary tumor in the central nervous system of adults. Temozolomide (TMZ), an alkylating agent, is the first-line chemotherapeutic agent for GBM patients. However, its efficacy is often limited by innate or acquired chemoresistance. Cancer cells can rewire their metabolic programming to support rapid growth and sustain cell survival against chemotherapies. An example is the de novo serine synthesis pathway (SSP), one of the main branches from glycolysis that is highly activated in multiple cancers in promoting cancer progression and inducing chemotherapy resistance. However, the roles of SSP in TMZ therapy for GBM patients remain unexplored. In this study, we employed NCT503, a highly selective inhibitor of phosphoglycerate dehydrogenase (PHGDH, the first rate-limiting enzyme of SSP), to study whether inhibition of SSP may enhance TMZ efficacy in MGMT-positive GBMs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flowcytometry and colony formation assays demonstrated that NCT503 worked synergistically with TMZ in suppressing GBM cell growth and inducing apoptosis in T98G and U118 cells in vitro. U118 and patient-derived GBM subcutaneous xenograft models showed that combined NCT503 and TMZ treatment inhibited GBM growth and promoted apoptosis more significantly than would each treatment alone in vivo. Mechanistically, we found that NCT503 treatment decreased MGMT expression possibly by modulating the Wnt/ß-catenin pathway. Moreover, intracellular levels of reactive oxygen species were elevated especially when NCT503 and TMZ treatments were combined, and the synergistic effects could be partially negated by NAC, a classic scavenger of reactive oxygen species. Taken together, these results suggest that NCT503 may be a promising agent for augmenting TMZ efficacy in the treatment of GBM, especially in TMZ-resistant GBMs with high expression of MGMT.


Assuntos
Neoplasias Encefálicas/metabolismo , Dano ao DNA , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Glioblastoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina/biossíntese , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Sinergismo Farmacológico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Piperazinas/farmacologia , Piridinas/farmacologia , Serina/antagonistas & inibidores , Tioamidas/farmacologia , Carga Tumoral/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
3.
Trends Cancer ; 7(8): 668-670, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34219053

RESUMO

Several recent preclinical studies have demonstrated that simultaneously blocking exogenous and endogenous sources of serine in malignant cells mediates superior anticancer effects as compared with limiting either source alone. Here, we critically summarize key developments in targeting serine to treat cancer and discuss persisting challenges for implementing such a therapeutic approach in patients.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Dieta com Restrição de Proteínas , Neoplasias/terapia , Serina/antagonistas & inibidores , Antimetabólitos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Terapia Combinada/métodos , Proteínas Alimentares/efeitos adversos , Proteínas Alimentares/metabolismo , Humanos , Neoplasias/metabolismo , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Serina/biossíntese , Transaminases/antagonistas & inibidores , Transaminases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Med Chem ; 64(13): 9496-9512, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34143627

RESUMO

Coadministration of ß-lactam and ß-lactamase inhibitor (BLI) is one of the well-established therapeutic measures for bacterial infections caused by ß-lactam-resistant Gram-negative bacteria, whereas we have only two options for orally active BLI, clavulanic acid and sulbactam. Furthermore, these BLIs are losing their clinical usefulness because of the spread of new ß-lactamases, including extended-spectrum ß-lactamases (ESBLs) belonging to class A ß-lactamases, class C and D ß-lactamases, and carbapenemases, which are hardly or not inhibited by these classical BLIs. From the viewpoints of medical cost and burden of healthcare personnel, oral therapy offers many advantages. In our search for novel diazabicyclooctane (DBO) BLIs possessing a thio-functional group at the C2 position, we discovered a 2-sulfinyl-DBO derivative (2), which restores the antibacterial activities of an orally available third-generation cephalosporin, ceftibuten (CTB), against various serine ß-lactamase-producing strains including carbapenem-resistant Enterobacteriaceae (CRE). It can be orally absorbed via the ester prodrug modification and exhibits in vivo efficacy in a combination with CTB.


Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Ciclo-Octanos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Compostos Azabicíclicos/síntese química , Compostos Azabicíclicos/química , Ciclo-Octanos/síntese química , Ciclo-Octanos/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Enterobacteriaceae/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Serina/antagonistas & inibidores , Serina/metabolismo , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/química
5.
Chem Res Toxicol ; 34(6): 1556-1571, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33900070

RESUMO

Chlorpyrifos (CPF) is an organophosphate (OP) pesticide that causes acute toxicity by inhibiting acetylcholinesterase (AChE) in the nervous system. However, endocannabinoid (eCB) metabolizing enzymes in brain of neonatal rats are more sensitive than AChE to inhibition by CPF, leading to increased levels of eCBs. Because eCBs are immunomodulatory molecules, we investigated the association between eCB metabolism, lipid mediators, and immune function in adult and neonatal mice exposed to CPF. We focused on lung effects because epidemiologic studies have linked pesticide exposures to respiratory diseases. CPF was hypothesized to disrupt lung eCB metabolism and alter lung immune responses to lipopolysaccharide (LPS), and these effects would be more pronounced in neonatal mice due to an immature immune system. We first assessed the biochemical effects of CPF in adult mice (≥8 weeks old) and neonatal mice after administering CPF (2.5 mg/kg, oral) or vehicle for 7 days. Tissues were harvested 4 h after the last CPF treatment and lung microsomes from both age groups demonstrated CPF-dependent inhibition of carboxylesterases (Ces), a family of xenobiotic and lipid metabolizing enzymes, whereas AChE activity was inhibited in adult lungs only. Activity-based protein profiling (ABPP)-mass spectrometry of lung microsomes identified 31 and 32 individual serine hydrolases in neonatal lung and adult lung, respectively. Of these, Ces1c/Ces1d/Ces1b isoforms were partially inactivated by CPF in neonatal lung, whereas Ces1c/Ces1b and Ces1c/BChE were partially inactivated in adult female and male lungs, respectively, suggesting age- and sex-related differences in their sensitivity to CPF. Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) activities in lung were unaffected by CPF. When LPS (1.25 mg/kg, i.p.) was administered following the 7-day CPF dosing period, little to no differences in lung immune responses (cytokines and immunophenotyping) were noted between the CPF and vehicle groups. However, a CPF-dependent increase in the amounts of dendritic cells and certain lipid mediators in female lung following LPS challenge was observed. Experiments in neonatal and adult Ces1d-/- mice yielded similar results as wild type mice (WT) following CPF treatment, except that CPF augmented LPS-induced Tnfa mRNA in adult Ces1d-/- mouse lungs. This effect was associated with decreased expression of Ces1c mRNA in Ces1d-/- mice versus WT mice in the setting of LPS exposure. We conclude that CPF exposure inactivates several Ces isoforms in mouse lung and, during an inflammatory response, increases certain lipid mediators in a female-dependent manner. However, it did not cause widespread altered lung immune effects in response to an LPS challenge.


Assuntos
Clorpirifos/farmacologia , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Metabolismo dos Lipídeos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Serina/antagonistas & inibidores , Animais , Clorpirifos/química , Inibidores Enzimáticos/química , Hidrolases/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Serina/imunologia
6.
FEBS Open Bio ; 10(7): 1316-1325, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32421926

RESUMO

Aging is a major risk factor for hypertension and atherosclerosis, and vascular smooth muscle cell (VSMC) senescence can promote aging-related vascular diseases. Sirtuin-1 (SIRT1) and AMP-activated protein kinase (AMPK) were previously reported to modulate vascular senescence; however, its effects have not been well characterized. To determine the nature of the interaction between SIRT1 and AMPK in VSMC senescence, we investigated the effects of SRT1720 on its downstream targets of SIRT1 and the phosphorylation of AMPKα at Ser485. During Adriamycin-induced VSMC senescence, SRT1720 increased the activity of SIRT1 and AMPKα phosphorylation at Ser485 via the cAMP-protein kinase A (PKA) pathway. Telomere length and telomerase reverse transcriptase expression were increased by SIRT1 activation with SRT1720. Taken together, these data show that activation of the SIRT1/cAMP-PKA/p-AMPKα (Ser485) pathway may be an effective antisenescence mechanism for VSMCs.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Serina/antagonistas & inibidores , Sirtuína 1/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Serina/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-31838993

RESUMO

BACKGROUND: Human Immunodeficiency Virus 1 (HIV-1) is a lentivirus, which causes various HIV-associated infections. The HIV-1 core dissociation is essential for viral cDNA synthesis and phosphorylation of HIV-1 capsid protein (HIV-1 CA) plays an important role in it. OBJECTIVE: The aim of this study was to explicate the role of three phosphoserine sites i.e. Ser109, Ser149 and Ser178 in the structural stability of HIV-1 CA, and it's binding with GS-CA1, a novel potent inhibitor. METHODS: Eight complexes were analyzed and Molecular Dynamics (MD) simulations were performed to observe the stability of HIV-1 CA in the presence and absence of phosphorylation of serine residues at four different temperatures i.e. 300K, 325K, 340K and 350K, along with molecular docking and DFT analysis. RESULTS: The structures showed maximum stability in the presence of phosphorylated serine residue. However, GS-CA1 docked most strongly with the native structure of HIV-1 CA i.e. binding affinity was -8.5 kcal/mol (Ki = 0.579 µM). CONCLUSION: These results suggest that the phosphorylation of these three serine residues weakens the binding of GS-CA1 with CA and casts derogatory effect on inhibition potential of this inhibitor, but it supports the stability of HIV-1 CA structure that can enhance regulation and replication of HIV-1 in host cells.


Assuntos
Proteínas do Capsídeo/química , Teoria da Densidade Funcional , HIV-1/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Serina/química , Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , HIV-1/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Serina/antagonistas & inibidores , Serina/metabolismo , Temperatura
8.
Cell Commun Signal ; 17(1): 100, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429764

RESUMO

BACKGROUND: Androgen receptor (AR) plays important role in the development, progression, and metastasis of prostate cancer (PCa). Caffeic acid phenethyl ester (CAPE) is the main component of honey bee propolis. We determined if CAPE affects the signaling and stability of AR in PCa cells. METHODS: Effects of CAPE on AR transcriptional activity and localization were determined by reporter gene assay and immunofluorescent microscopy. Western blotting, fluorescent polarization, computer simulation, and animal experiment were performed to investigate the molecular mechanism how CAPE reduces the stability of AR. RESULTS: CAPE treatment dose-dependently suppressed the transcriptional activity of AR as well as the protein levels of AR and its target gene PSA. Cyclohexamide treatment revealed that androgen stabilized AR protein, but AR stability was diminished by CAPE. Fluorescence microscopy demonstrated that androgen promoted the nucleus translocation of AR in PCa cells, while treatment with CAPE reduced protein level of AR in both nucleus and cytoplasm. CAPE treatment suppressed the phosphorylation of Ser81 and Ser213 on AR, which regulates the stability of AR. CDK1 and AKT are the kinases phosphorylating Ser81 and Ser213 on AR, respectively. CAPE treatment significantly reduced the protein level and activity of CDK1 and AKT in PCa cells. Overexpression of CDK1 or AKT rescued the AR protein level under CAPE treatment. CONCLUSIONS: Our results suggested that CAPE treatment reduced AR stability and AR transcriptional activity in PCa cells, implying the possibility of using CAPE as a treatment for advanced PCa.


Assuntos
Ácidos Cafeicos/farmacologia , Álcool Feniletílico/análogos & derivados , Receptores Androgênicos/metabolismo , Serina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Álcool Feniletílico/farmacologia , Fosforilação/efeitos dos fármacos , Receptores Androgênicos/genética , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Células Tumorais Cultivadas
9.
J Med Chem ; 62(17): 7976-7997, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31365252

RESUMO

Phosphoglycerate dehydrogenase (PHGDH) is known to be the rate-limiting enzyme in the serine synthesis pathway in humans. It converts glycolysis-derived 3-phosphoglycerate to 3-phosphopyruvate in a co-factor-dependent oxidation reaction. Herein, we report the discovery of BI-4916, a prodrug of the co-factor nicotinamide adenine dinucleotide (NADH/NAD+)-competitive PHGDH inhibitor BI-4924, which has shown high selectivity against the majority of other dehydrogenase targets. Starting with a fragment-based screening, a subsequent hit optimization using structure-based drug design was conducted to deliver a single-digit nanomolar lead series and to improve potency by 6 orders of magnitude. To this end, an intracellular ester cleavage mechanism of the ester prodrug was utilized to achieve intracellular enrichment of the actual carboxylic acid based drug and thus overcome high cytosolic levels of the competitive cofactors NADH/NAD+.


Assuntos
Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Serina/antagonistas & inibidores , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indóis/síntese química , Indóis/química , Modelos Moleculares , Estrutura Molecular , Fosfoglicerato Desidrogenase/metabolismo , Serina/biossíntese , Relação Estrutura-Atividade
10.
Bioorg Med Chem Lett ; 28(9): 1615-1620, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29588215

RESUMO

Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel therapeutic treatment for Parkinson's disease. Herein we disclose the discovery of a 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors identified through a kinase-focused set screening. Optimization of the physicochemical properties and kinase selectivity led to the discovery of compound 7, which exhibited potent in vitro inhibition of LRRK2 kinase activity, good physicochemical properties and kinase selectivity across the kinome. Moreover, compound 7 was able to penetrate into the CNS, and in vivo pharmacology studies revealed significant inhibition of Ser935 phosphorylation in the brain of both rats (30 and 100 mg/kg) and mice (45 mg/kg) following oral administration.


Assuntos
Descoberta de Drogas , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Ratos , Serina/antagonistas & inibidores , Serina/metabolismo , Relação Estrutura-Atividade
11.
Mol Nutr Food Res ; 61(11)2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28759161

RESUMO

SCOPE: Serine lies at the central node linking biosynthetic flux from glycolysis to glutathione synthesis and one-carbon metabolic cycle which are closely related to antioxidant capacity. The present study was conducted to determine the effects of serine supplementation on oxidative stress and its relative mechanisms. METHODS AND RESULTS: Diquat treatment was performed to induce oxidative stress in mice and primary hepatocytes. The results showed that hepatic glutathione anti-oxidant systems were impaired and reactive oxygen species and homocysteine were increased in diquat-induced mice and hepatocytes, while such disadvantageous changes were diminished by serine supplementation both in vivo and in vitro. However, when cystathionine ß-synthase expression was inhibited by interference RNA in hepatocytes, the effects of serine supplementation on the improvement of glutathione synthesis and the alleviation of oxidative stress were diminished. Moreover, when hepatocytes were treated with cycloleucine, an inhibitor of methionine adenosyltransferase, the effects of serine supplementation on the improvement of methionine cycle and the alleviation of DNA hypomethylation and oxidative stress were also diminished. CONCLUSION: Our results indicated that serine supplementation alleviated oxidative stress via supporting glutathione synthesis and methionine cycle, mostly by condensing with homocysteine to synthesize cysteine and providing one-carbon units for homocysteine remethylation.


Assuntos
Antioxidantes/uso terapêutico , Suplementos Nutricionais , Glutationa/metabolismo , Hepatócitos/metabolismo , Metionina/metabolismo , Estresse Oxidativo , Serina/uso terapêutico , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Cicloleucina/farmacologia , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Metilação de DNA/efeitos dos fármacos , Desfolhantes Químicos/antagonistas & inibidores , Desfolhantes Químicos/toxicidade , Diquat/antagonistas & inibidores , Diquat/toxicidade , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Homocisteína/metabolismo , Masculino , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/metabolismo , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Interferência de RNA , Distribuição Aleatória , Serina/antagonistas & inibidores , Serina/metabolismo , Organismos Livres de Patógenos Específicos
12.
Microbes Infect ; 17(6): 395-401, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25749709

RESUMO

Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVß3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVß3, and that a mutant lacking SdrG failed to bind. Blocking αVß3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Integrina alfaVbeta3/metabolismo , Osteoblastos/metabolismo , Staphylococcus epidermidis/crescimento & desenvolvimento , Proteínas de Transporte/imunologia , Humanos , Osteomielite/etiologia , Osteomielite/imunologia , Osteomielite/terapia , Ligação Proteica/imunologia , Serina/antagonistas & inibidores , Serina/imunologia , Staphylococcus epidermidis/imunologia
13.
Pharmacol Biochem Behav ; 130: 46-52, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25579325

RESUMO

Glycinergic transmission has an important role in regulating nociception in the spinal cord. The glycine transporter-2 (GlyT2) is localized at presynaptic terminals of glycinergic neurons and eliminates glycine from the synaptic cleft to terminate glycinergic transmission. Systemic and intrathecal administration of GlyT2 inhibitors alleviate various types of pain. Although the GlyT2s and glycine receptors are widely distributed in the central nervous system, little is known about the role of glycinergic transmission in pain perception at supraspinal regions. The present study examined the antinociceptive effect of intracerebroventricular (i.c.v.) administration of the selective GlyT2 inhibitor ALX1393 on inflammatory and neuropathic pain in experimental models. For i.c.v. administration, a guide cannula was implanted into the right lateral ventricle of male Sprague-Dawley rats. Normal rats were used to assess inflammatory nociception using the formalin test and motor function using the rotarod test. Chronic constriction injury (CCI) to the sciatic nerve was induced in the rats. The CCI rats were then used to assess mechanical, cold, and thermal hyperalgesia using the electronic von Frey test, cold plate test, and the plantar test, respectively. ALX1393 (25, 50, and 100 µg) was administered i.c.v. to examine its effects on supraspinal antinociception. Supraspinal ALX1393 in normal rats suppressed the late-phase response in the formalin test but did not affect motor performance. In the CCI rats, ALX1393 inhibited mechanical and cold hyperalgesia in a dose-dependent manner. The antihyperalgesic effects of ALX1393 (100 µg) were reversed completely by i.c.v. pretreatment with a glycine receptor antagonist strychnine (10 µg). These results suggest that GlyT2 contributes to nociceptive transmission at supraspinal level and that the selective GlyT2 inhibitor is a promising candidate for the treatment of inflammatory and neuropathic pain without causing motor dysfunction.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Inflamação/tratamento farmacológico , Neuralgia/tratamento farmacológico , Dor/complicações , Dor/tratamento farmacológico , Serina/análogos & derivados , Analgésicos/administração & dosagem , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hiperalgesia/complicações , Hiperalgesia/tratamento farmacológico , Inflamação/complicações , Infusões Intraventriculares , Masculino , Medição da Dor , Ratos , Teste de Desempenho do Rota-Rod , Serina/administração & dosagem , Serina/antagonistas & inibidores , Serina/farmacologia , Serina/uso terapêutico , Estricnina/farmacologia
14.
Bioorg Med Chem Lett ; 24(19): 4763-4767, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25205188

RESUMO

Natural compounds are regarded as a rich source for potential anti-inflammatory and anti-carcinogenic agents. Increasing evidence indicates that histone phosphorylation at Ser10 is a marker for cell cycle progression during the mitosis and the induction of immediate pro-inflammatory genes during the interphase. In the present study, we have screened our in-house natural compounds to find out new chemical inhibitor(s) of histone H3 phosphorylation at Ser10. As a result, we observed that α-amyrin, oleanolic acid, marliolide, and 4'-O-ß-D-glucosyl-5-O-methylvisamminol decreased the levels of histone H3 phosphorylation at Ser10 and c-Jun. In particular, we observed that 4'-O-ß-D-glucosyl-5-O-methylvisamminol suppressed the direct interaction of histone H3 with 14-3-3ε, inhibited the aurora B kinase activity and delayed the mitotic cell cycle progression. We reports 4'-O-ß-D-glucosyl-5-O-methylvisamminol as the first epigenetic natural chemical inhibitor that can abrogates the mitotic cell cycle progression and immediate pro-inflammatory gene expressions via suppression of histone H3 phosphorylation at Ser10 and its interaction with 14-3-3ε.


Assuntos
Proteínas 14-3-3/antagonistas & inibidores , Cromonas/farmacologia , Epigênese Genética/efeitos dos fármacos , Glucosídeos/farmacologia , Histonas/antagonistas & inibidores , Serina/antagonistas & inibidores , Proteínas 14-3-3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromonas/síntese química , Cromonas/química , Glucosídeos/síntese química , Glucosídeos/química , Células HT29 , Histonas/metabolismo , Humanos , Conformação Molecular , Fosforilação/efeitos dos fármacos , Serina/metabolismo , Relação Estrutura-Atividade
15.
J Clin Psychiatry ; 75 Suppl 1: 21-6, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24581451

RESUMO

Current treatments for schizophrenia, although effective for positive symptoms, have not proven as effective for negative symptoms and cognitive dysfunction. Additional strategies, such as combining antipsychotics or adding adjunctive agents to antipsychotics, have also yielded disappointing results in both negative and cognitive symptom domains. However, the N-methyl-d-aspartate (NMDA) receptor hypofunction hypothesis, with its focus on the glutamate system's effect on dopamine, can explain the positive, negative, and cognitive symptoms in schizophrenia. Therapeutic targets are being explored that focus on NMDA receptors (eg, glycine, d-serine), glycine reuptake inhibition (such as sarcosine and bitopertin), and, through a different pathway, α-7 nicotinic acetylcholine receptor agonism (eg, encenicline).


Assuntos
Antipsicóticos , Terapia de Alvo Molecular/métodos , Esquizofrenia/tratamento farmacológico , Antipsicóticos/uso terapêutico , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/metabolismo , Glicina/antagonistas & inibidores , Glicina/fisiologia , Humanos , Receptores de Glutamato/metabolismo , Receptores de Glutamato/fisiologia , Esquizofrenia/metabolismo , Serina/antagonistas & inibidores , Serina/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas
16.
PLoS One ; 8(6): e67044, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23825613

RESUMO

To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF). Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1) reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2) improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3) increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca(2+)-activated K(+) channels on the cerebral blood vessel endothelium.


Assuntos
Circulação Cerebrovascular/efeitos dos fármacos , Infarto da Artéria Cerebral Média/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Serina/farmacologia , Animais , Apamina/farmacologia , Charibdotoxina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Endotélio Vascular/efeitos dos fármacos , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos , Ratos Sprague-Dawley , Serina/antagonistas & inibidores , Fatores de Tempo
17.
J Dent Res ; 92(9): 808-13, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23881719

RESUMO

Trigeminal neuropathic pain affects millions of people worldwide. Despite decades of study on the neuronal processing of pain, mechanisms underlying enhanced pain states after injury remain unclear. N-methyl-D-aspartate (NMDA) receptor-dependent changes play a critical role in triggering central sensitization in neuropathic pain. These receptors are regulated at the glycine site through a mandatory endogenous co-agonist D-serine, which is synthesized by astrocytes. Therefore, the present study was carried out to determine whether astrocytes are involved, through D-serine secretion, in dynamic mechanical allodynia (DMA) obtained after chronic constriction of the infraorbital nerve (CCI-IoN) in rats. Two weeks after CCI-IoN, an important reaction of astrocytes was present in the medullary dorsal horn (MDH), as revealed by an up-regulation of glial fibrillary acidic protein (GFAP) in allodynic rats. In parallel, an increase in D-serine synthesis, which co-localized with its synthesis enzyme serine racemase, was strictly observed in astrocytes. Blocking astrocyte metabolism by intracisternal delivery of fluorocitrate alleviated DMA. Furthermore, the administration of D-amino-acid oxidase (DAAO), a D-serine-degrading enzyme, or that of L-serine O-sulfate (LSOS), a serine racemase inhibitor, significantly decreased pain behavior in allodynic rats. These results demonstrate that astrocytes are involved in the modulation of orofacial post-traumatic neuropathic pain via the release of the gliotransmitter D-serine.


Assuntos
Astrócitos/metabolismo , Serina/metabolismo , Neuralgia do Trigêmeo/fisiopatologia , Animais , Astrócitos/efeitos dos fármacos , Cisterna Magna , Citratos/farmacologia , Constrição Patológica/fisiopatologia , D-Aminoácido Oxidase/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Injeções , Isomerismo , Masculino , Proteínas de Membrana/metabolismo , Neurotransmissores/metabolismo , Órbita/inervação , Células do Corno Posterior/metabolismo , Racemases e Epimerases/antagonistas & inibidores , Racemases e Epimerases/metabolismo , Ratos , Ratos Sprague-Dawley , Serina/análogos & derivados , Serina/antagonistas & inibidores , Serina/farmacologia , Regulação para Cima
18.
Curr Neurovasc Res ; 10(3): 208-15, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23713735

RESUMO

Ischemic stroke causes a significant amount of cell damage resulting from an insufficient supply of glucose and oxygen to central nervous system tissue and finding more effective therapeutic neuroprotective agents has become a priority in the treatment of ischemic stroke. Hsp20, one of the small heat shock proteins, has been implicated in multiple physiological and pathophysiological processes and is a potential neuroprotective agents. To investigate whether Hsp20 exerts protective effects on in vitro ischemia-reperfusion injury, mouse neuroblastoma cells were subjected to oxygen-glucose deprivation/reoxygenation (OGDR) insult. The N2a cells transfected with Hsp20 and constitutively phosphorylated Hsp20 (S16D) had significantly less cell loss and less proportion of apoptotic cells compared to N2a cells transfected with pEGFP-N1 after oxygen-glucose deprivation (OGD) 4 h plus 12 and 24 h reperfusion, which showed no difference in N2a cells transfected with nonphosphorylatable Hsp20 (S16A). Meanwhile, transfected with Hsp20 and constitutively phosphorylated Hsp20 (S16D) also significantly attenuated mitochondrial fragmentation and modulated Bcl-2 and Bax expression level after OGD 4 h plus 12 reperfusion, which were not affected in N2a cells transfected with Hsp20 (S16A). In conclusion, our data demonstrated that increased Hsp20 and Hsp20 (S16D) expression in mouse N2A neuroblastoma cells protected against ischemia-reperfusion injury, the neuroprotective mechanism may be related to regulate Bcl-2 and Bax expression. However, blockade of Ser16-Hsp20 phosphorylation attenuated the neuroprotective effects of Hsp20. Therefore, Hsp20 and factors that contribute to regulation of phosphorylation on Ser16 of Hsp20 are potential new therapeutic targets for the treatment of cerebral ischemia-reperfusion injury.


Assuntos
Regulação para Baixo/fisiologia , Proteínas de Choque Térmico HSP20/antagonistas & inibidores , Fármacos Neuroprotetores/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Serina/metabolismo , Regulação para Cima/fisiologia , Proteína X Associada a bcl-2/fisiologia , Animais , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Proteínas de Choque Térmico HSP20/metabolismo , Camundongos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Serina/antagonistas & inibidores
19.
Brain ; 136(Pt 4): 1216-30, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23518710

RESUMO

Cocaine seeking behaviour and relapse have been linked to impaired potentiation and depression at excitatory synapses in the nucleus accumbens, but the mechanism underlying this process is poorly understood. We show that, in the rat nucleus accumbens core, D-serine is the endogenous coagonist of N-methyl-D-aspartate receptors, and its presence is essential for N-methyl-D-aspartate receptor-dependent potentiation and depression of synaptic transmission. Nucleus accumbens core slices obtained from cocaine-treated rats after 1 day of abstinence presented significantly reduced D-serine concentrations, increased expression of the D-serine degrading enzyme, D-amino acid oxidase, and downregulated expression of serine racemase, the enzyme responsible for D-serine synthesis. The D-serine deficit was associated with impairment of potentiation and depression of glutamatergic synaptic transmission, which was restored by slice perfusion with exogenous D-serine. Furthermore, in vivo administration of D-serine directly into the nucleus accumbens core blocked behavioural sensitization to cocaine. These results provide evidence for a critical role of D-serine signalling in synaptic plasticity relevant to cocaine addiction.


Assuntos
Cocaína/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Equidae , Masculino , Camundongos , Núcleo Accumbens/patologia , Núcleo Accumbens/ultraestrutura , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Serina/metabolismo , Serina/farmacologia
20.
J Pharmacol Sci ; 120(3): 213-27, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23099320

RESUMO

Polyriboinosinic-polyribocytidilic acid (polyI:C) is a synthetic analog that elicits viral-like immune responses in mammals. We have recently found that polyI:C treatment in neonatal mice induced abnormalities of emotional, cognitive, and sensorimotor gating and dysfunction of glutamatergic neurotransmission in adulthood. In this study, we investigated the effect of the NMDA-receptor co-agonist D-serine on polyI:C-induced behavioral abnormalities in mice. Neonatal ICR mice were repeatedly injected with polyI:C for 5 days from postnatal day 2 to 6. At 10 weeks, sensorimotor gating function was analyzed in the prepulse inhibition (PPI) test. Emotional function was analyzed in open field and social interaction tests. Cognitive function was analyzed by novel object recognition tests. D-Serine dose-dependently improved polyI:C-induced impairment of emotional and cognitive behaviors whereas it had no effect on PPI deficit in adults. The ameliorating effects of D-serine were antagonized by pretreatment with an NMDA-receptor antagonist, MK-801. Although the mRNA level of D-amino acid oxidase (DAAO) was increased in the prefrontal cortex and hippocampus of neonatal polyI:C-treated mice in adulthood, no changes were observed in D-serine content and DAAO enzymatic activity. These results suggest that D-serine ameliorates emotional and cognitive impairments of the polyI:C-treated mice through potentiating NMDA receptor activity.


Assuntos
Sintomas Afetivos/prevenção & controle , Viroses do Sistema Nervoso Central/fisiopatologia , Córtex Cerebral/efeitos dos fármacos , Transtornos Cognitivos/prevenção & controle , Modelos Animais de Doenças , Receptores de N-Metil-D-Aspartato/agonistas , Serina/análogos & derivados , Sintomas Afetivos/etiologia , Animais , Animais Recém-Nascidos , Ansiolíticos/antagonistas & inibidores , Ansiolíticos/metabolismo , Ansiolíticos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Viroses do Sistema Nervoso Central/imunologia , Córtex Cerebral/imunologia , Córtex Cerebral/metabolismo , Transtornos Cognitivos/etiologia , D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Neurônios/metabolismo , Nootrópicos/antagonistas & inibidores , Nootrópicos/metabolismo , Nootrópicos/uso terapêutico , Distribuição Aleatória , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/antagonistas & inibidores , Serina/metabolismo , Serina/uso terapêutico , Organismos Livres de Patógenos Específicos
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