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1.
J Pharmacol Sci ; 140(2): 178-186, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31279581

RESUMO

The role of type I diacylglycerol kinases (DGKs) in the regulation of insulin secretion was investigated in MIN6 ß-cells. In intracellular Ca2+ concentration ([Ca2+]i) measurement experiments, 1 µM R59949, a type I DGK inhibitor, and 10 µM DiC8, a diacylglycerol (DAG) analog, amplified 22.2 mM glucose-induced [Ca2+]i oscillations in a protein kinase C (PKC)-dependent manner, whereas 10 µM R59949 and 100 µM DiC8 decreased [Ca2+]i independent of PKC. High concentrations of R59949 and DiC8 attenuated voltage-dependent Ca2+ channel currents. According to these results, 22.2 mM glucose-stimulated insulin secretion (GSIS) was potentiated by 1 µM R59949 but suppressed by 10 µM of the same. The DGKα inhibitor R59022 showed a similar dual effect. Conversely, DiC8 at 10 and 100 µM potentiated GSIS, although 100 µM DiC8 decreased [Ca2+]i. These results suggest that DAG accumulated through declined type I DGK activity shows a dual effect on insulin secretion depending on the degree of accumulation; a mild DAG accumulation induces a PKC-dependent stimulatory effect on insulin secretion, whereas an excessive DAG accumulation suppresses it in a PKC-independent manner, possibly via attenuation of VDCC activity.


Assuntos
Diacilglicerol Quinase/fisiologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células Cultivadas , Diacilglicerol Quinase/antagonistas & inibidores , Diglicerídeos/metabolismo , Relação Dose-Resposta a Droga , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Camundongos , Piperidinas/farmacologia , Proteína Quinase C/fisiologia , Quinazolinonas/farmacologia
2.
Monoclon Antib Immunodiagn Immunother ; 38(4): 175-178, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31355694

RESUMO

The diacylglycerol kinases (DGKs) catalyze the phosphorylation of the cell membrane lipid diacylglycerol (DG), which is important in lipid biochemistry and signal transduction into phosphatidic acid. DG-mediated signal transduction downstream of the T cell receptor has been reported to be terminated by DGKζ, 1 of 10 DGK isoforms in most cases. We previously established an anti-DGKζ monoclonal antibody (mAb) DzMab-1 (rat IgG1, kappa), which reacts with both mouse DGKζ and human DGKζ (hDGKζ). In this study, we characterized the binding epitope of DzMab-1 using Western blotting, and found that Met1 and Pro3 residues of hDGKζ are important for facilitating DzMab-1 binding to hDGKζ. Furthermore, DzMab-1 was shown to be useful for immunohistochemical analyses for formalin-fixed paraffin-embedded HeLa cells. These findings could be applied for the production of more functional anti-hDGKζ mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Diacilglicerol Quinase/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Imuno-Histoquímica/métodos , Anticorpos Monoclonais/genética , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/genética , Células HeLa , Humanos
3.
Viruses ; 11(3)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832223

RESUMO

Filoviruses, such as Ebola virus (EBOV) and Marburg virus, are causative agents of unpredictable outbreaks of severe hemorrhagic fevers in humans and non-human primates. For infection, filoviral particles need to be internalized and delivered to intracellular vesicles containing cathepsin proteases and the viral receptor Niemann-Pick C1. Previous studies have shown that EBOV triggers macropinocytosis of the viral particles in a glycoprotein (GP)-dependent manner, but the molecular events required for filovirus internalization remain mostly unknown. Here we report that the diacylglycerol kinase inhibitor, R-59-022, blocks EBOV GP-mediated entry into Vero cells and bone marrow-derived macrophages. Investigation of the mode of action of the inhibitor revealed that it blocked an early step in entry, more specifically, the internalization of the viral particles via macropinocytosis. Finally, R-59-022 blocked viral entry mediated by a panel of pathogenic filovirus GPs and inhibited growth of replicative Ebola virus. Taken together, our studies suggest that R-59-022 could be used as a tool to investigate macropinocytic uptake of filoviruses and could be a starting point for the development of pan-filoviral therapeutics.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Filoviridae/efeitos dos fármacos , Filoviridae/fisiologia , Pirimidinonas/farmacologia , Tiazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Ebolavirus/fisiologia , Células HEK293 , Humanos , Macrófagos/virologia , Marburgvirus/fisiologia , Pinocitose/efeitos dos fármacos , Receptores Virais , Células Vero , Replicação Viral/efeitos dos fármacos
4.
J Biochem ; 165(6): 517-522, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30715374

RESUMO

Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol (DG) into phosphatidic acid (PA). DG and PA function as lipid messengers contributing to various signalling pathways. Thus, DGK plays a pivotal role in the signalling pathways by maintaining DG and PA levels. For example, DGKδ is involved in diabetes and DGKß is important for higher brain function including memory and emotion. Recently, we also revealed that the activation of DGKα ameliorated diabetic nephropathy (DN) in mice, suggesting that DGK can be therapeutic target. However, there is no commercially available DGK subtype-specific inhibitors or activators. Therefore, in a series of experiment to find DGK subtype-specific inhibitors or activators, we tried to screen novel DGKα activators from 9,600 randomly selected compounds by using high-throughput screening we had recently developed. Finally, we obtained two lead compounds for DGKα activators, KU-8 and KU-10. Focusing KU-8, we assessed the effect of KU-8 on all mammalian DGKs activities. Thus, KU-8 activates not only DGKα but also DGKθ by approximately 20%, and strongly inhibited DGKκ. In conclusion, KU-8 would be a good lead compound for DGKα and DGKθ activators, and useful as a DGKκ inhibitor.


Assuntos
Ciclopropanos/farmacologia , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/metabolismo , Dioxinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Xilenos/farmacologia , Animais , Células COS , Células Cultivadas , Ciclopropanos/química , Dioxinas/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Xilenos/química
5.
Eur J Med Chem ; 164: 378-390, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30611057

RESUMO

As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used an in-silico approach based on chemical homology with the two commercially available DGKα inhibitors R59022 and R59949. Ritanserin and compound AMB639752 emerged from the screening of 127 compounds, showing an inhibitory activity superior to the two commercial inhibitors, being furthermore specific for the alpha isoform of diacylglycerol kinase. Interestingly, AMB639752 was also devoid of serotoninergic activity. The ability of both ritanserin and AMB639752, by inhibiting DGKα in intact cells, to restore restimulation induced cell death (RICD) in SAP deficient lymphocytes was also tested. Both compounds restored RICD at concentrations lower than the two previously available inhibitors, indicating their potential use for the treatment of X-linked lymphoproliferative disease 1 (XLP-1), a rare genetic disorder in which DGKα activity is deregulated.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Transtornos Linfoproliferativos/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Morte Celular/efeitos dos fármacos , Simulação por Computador , Humanos , Piperidinas , Pirimidinonas , Quinazolinonas , Ritanserina , Tiazóis
6.
Artigo em Inglês | MEDLINE | ID: mdl-30648914

RESUMO

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol (DG) to phosphatidic acid (PA). Both DG and PA serve as signaling molecules; therefore, DGK functions as a key enzyme between DG- and PA-mediated signaling. DGKα, one of the 10 DGK isozymes, is involved in T cell function and has been shown to localize in the cytoplasm and nucleus. Furthermore, DGKα translocates to the plasma membrane in response to T cell receptor stimulation. Recently, we developed a specific monoclonal antibody (mAb), DaMab-2 (mouse IgG1, kappa), against DGKα. DaMab-2 is very useful in immunocytochemical analysis using HeLa cells. In this study, we characterized the binding epitope of DaMab-2 using Western blot and revealed that Cys246, Lys249, Pro252, and Cys253 of DGKα are important for DaMab-2 binding to the DGKα protein. Our findings can be applied for the production of more functional anti-DGKα mAbs.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Diacilglicerol Quinase/imunologia , Epitopos/imunologia , Aminoácidos/imunologia , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/uso terapêutico , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/química , Diacilglicerol Quinase/genética , Mapeamento de Epitopos/métodos , Epitopos/química , Células HeLa , Humanos , Ácidos Fosfatídicos/química , Ácidos Fosfatídicos/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/imunologia
7.
Biochem Biophys Res Commun ; 496(2): 309-315, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29326040

RESUMO

Migration of surviving kidney tubule cells after sub-lethal injury, for example ischemia/reperfusion (I/R), plays a critical role in recovery. Exocytosis is known to be involved in cell migration, and a key component in exocytosis is the highly-conserved eight-protein exocyst complex. We investigated the expression of a central exocyst complex member, Sec10, in kidneys following I/R injury, as well as the role of Sec10 in wound healing following scratch injury of cultured Madin-Darby canine kidney (MDCK) cells. Sec10 overexpression and knockdown (KD) in MDCK cells were used to investigate the speed of wound healing and the mechanisms underlying recovery. In mice, Sec10 decreased after I/R injury, and increased during the recovery period. In cell culture, Sec10 OE inhibited ruffle formation and wound healing, while Sec10 KD accelerated it. Sec10 OE cells had higher amounts of diacylglycerol kinase (DGK) gamma at the leading edge than did control cells. A DGK inhibitor reversed the inhibition of wound healing and ruffle formation in Sec10 OE cells. Conclusively, downregulation of Sec10 following I/R injury appears to accelerate recovery of kidney tubule cells through activated ruffle formation and enhanced cell migration.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Túbulos Renais/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Proteínas de Transporte Vesicular/genética , Animais , Bioensaio , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Cães , Inibidores Enzimáticos/farmacologia , Exocitose , Regulação da Expressão Gênica , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas/farmacologia , Quinazolinonas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteínas de Transporte Vesicular/agonistas , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/metabolismo , Cicatrização/fisiologia
8.
Biochemistry ; 57(2): 231-236, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29155586

RESUMO

Diacylglycerol kinases (DGKs) regulate lipid metabolism and cell signaling through ATP-dependent phosphorylation of diacylglycerol to biosynthesize phosphatidic acid. Selective chemical probes for studying DGKs are currently lacking and are needed to annotate isoform-specific functions of these elusive lipid kinases. Previously, we explored fragment-based approaches to discover a core fragment of DGK-α (DGKα) inhibitors responsible for selective binding to the DGKα active site. Here, we utilize quantitative chemical proteomics to deconstruct widely used DGKα inhibitors to identify structural regions mediating off-target activity. We tested the activity of a fragment (RLM001) derived from a nucleotide-like region found in the DGKα inhibitors R59022 and ritanserin and discovered that RLM001 mimics ATP in its ability to broadly compete at ATP-binding sites of DGKα as well as >60 native ATP-binding proteins (kinases and ATPases) detected in cell proteomes. Equipotent inhibition of activity-based probe labeling by RLM001 supports a contiguous ligand-binding site composed of C1, DAGKc, and DAGKa domains in the DGKα active site. Given the lack of available crystal structures of DGKs, our studies highlight the utility of chemical proteomics in revealing active-site features of lipid kinases to enable development of inhibitors with enhanced selectivity against the human proteome.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteômica/métodos , Ritanserina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Diacilglicerol Quinase/metabolismo , Relação Dose-Resposta a Droga , Desenho de Drogas , Estrutura Molecular , Proteínas Recombinantes/metabolismo , Ritanserina/química , Ritanserina/metabolismo , Ritanserina/farmacologia , Relação Estrutura-Atividade
9.
Neuro Oncol ; 20(2): 192-202, 2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29048560

RESUMO

Background: The mesenchymal phenotype in glioblastoma (GBM) and other cancers drives aggressiveness and treatment resistance, leading to therapeutic failure and recurrence of disease. Currently, there is no successful treatment option available against the mesenchymal phenotype. Methods: We classified patient-derived GBM stem cell lines into 3 subtypes: proneural, mesenchymal, and other/classical. Each subtype's response to the inhibition of diacylglycerol kinase alpha (DGKα) was compared both in vitro and in vivo. RhoA activation, liposome binding, immunoblot, and kinase assays were utilized to elucidate the novel link between DGKα and geranylgeranyltransferase I (GGTase I). Results: Here we show that inhibition of DGKα with a small-molecule inhibitor, ritanserin, or RNA interference preferentially targets the mesenchymal subtype of GBM. We show that the mesenchymal phenotype creates the sensitivity to DGKα inhibition; shifting GBM cells from the proneural to the mesenchymal subtype increases ritanserin activity, with similar effects in epithelial-mesenchymal transition models of lung and pancreatic carcinoma. This enhanced sensitivity of mesenchymal cancer cells to ritanserin is through inhibition of GGTase I and downstream mediators previously associated with the mesenchymal cancer phenotype, including RhoA and nuclear factor-kappaB. DGKα inhibition is synergistic with both radiation and imatinib, a drug preferentially affecting proneural GBM. Conclusions: Our findings demonstrate that a DGKα-GGTase I pathway can be targeted to combat the treatment-resistant mesenchymal cancer phenotype. Combining therapies with greater activity against each GBM subtype may represent a viable therapeutic option against GBM.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Diacilglicerol Quinase/antagonistas & inibidores , Glioblastoma/patologia , Ritanserina/farmacologia , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Diacilglicerol Quinase/genética , Feminino , Humanos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
J Cell Physiol ; 232(9): 2550-2557, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27731506

RESUMO

Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Núcleo Celular/enzimologia , Proliferação de Células , Diacilglicerol Quinase/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Leucemia Eritroblástica Aguda/enzimologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Proliferação de Células/efeitos dos fármacos , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/genética , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Isoenzimas , Células K562 , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção
11.
Biochem Pharmacol ; 123: 29-39, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27974147

RESUMO

Diacylglycerol kinase alpha (DGKα) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Recently, DGKα was identified as a therapeutic target in various cancers, as well as in immunotherapy. Application of small-molecule DGK inhibitors, R59022 and R59949, induces cancer cell death in vitro and in vivo. The pharmacokinetics of these compounds in mice, however, are poor. Thus, there is a need to discover additional DGK inhibitors not only to validate these enzymes as targets in oncology, but also to achieve a better understanding of their biology. In the present study, we investigate the activity of ritanserin, a compound structurally similar to R59022, against DGKα. Ritanserin, originally characterized as a serotonin (5-HT) receptor (5-HTR) antagonist, underwent clinical trials as a potential medicine for the treatment of schizophrenia and substance dependence. We document herein that ritanserin attenuates DGKα kinase activity while increasing the enzyme's affinity for ATP in vitro. In addition, R59022 and ritanserin function as DGKα inhibitors in cultured cells and activate protein kinase C (PKC). While recognizing that ritanserin attenuates DGK activity, we also find that R59022 and R59949 are 5-HTR antagonists. In conclusion, ritanserin, R59022 and R59949 are combined pharmacological inhibitors of DGKα and 5-HTRs in vitro.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirimidinonas/farmacologia , Ritanserina/farmacologia , Tiazóis/farmacologia , Trifosfato de Adenosina/metabolismo , Diacilglicerol Quinase/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Células HeLa , Humanos , Cinética
12.
Naunyn Schmiedebergs Arch Pharmacol ; 390(2): 207-214, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27909743

RESUMO

Although diacylglycerol kinase (DGK) is known to be expressed in vascular smooth muscle cell, its functional significance remains to be clarified. We hypothesized that DGK is involved in the pathway of cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells. The purpose of this study was to investigate the effects of R59949, a diacylglycerol kinase inhibitor, on inducible nitric oxide production in vascular smooth muscle cell. Cultured rat aortic smooth muscle cells (RASMCs) were used to elucidate the effects of R59949 on basal and interleukin-1ß (IL-1ß)-induced NO production. The effects of R59949 on protein and mRNA expression of induced nitric oxide synthase (iNOS) and on transplasmalemmal L-arginine uptake were also evaluated using RASMCs. Treatment of RASMCs with R59949 (10 µM) inhibited IL-1ß (10 ng/ml)-induced NO production but not basal NO production. Neither protein nor mRNA expression level of iNOS after stimulation with IL-1ß was significantly affected by R59949. Estimated enzymatic activities of iNOS in RASMCs were comparable in the absence and presence of R59949. Stimulation of RASMCs with IL-1ß caused a marked increase in transplasmalemmal L-arginine uptake into RASMCs. L-Arginine uptake in the presence of IL-1ß was markedly inhibited by R59949, while basal L-arginine uptake was not significantly affected by R59949. Both IL-1ß-induced NO production and L-arginine uptake were abolished in the presence of cycloheximide (1 µM). The results indicate that R59949 inhibits inducible NO production through decreasing transplasmalemmal L-arginine uptake. DGK is suggested to be involved in cytokine-stimulated L-arginine transport and regulate its intracellular concentration in vascular smooth muscle cell.


Assuntos
Arginina/metabolismo , Membrana Celular/efeitos dos fármacos , Diacilglicerol Quinase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Animais , Transporte Biológico , Membrana Celular/enzimologia , Células Cultivadas , Cicloeximida/farmacologia , Diacilglicerol Quinase/metabolismo , Relação Dose-Resposta a Droga , Interleucina-1beta/farmacologia , Masculino , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Óxido Nítrico Sintase Tipo II/genética , Ratos Wistar
13.
Nat Commun ; 7: 10893, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26964756

RESUMO

Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy.


Assuntos
Neoplasias da Mama/radioterapia , Mama/patologia , Metilação de DNA/genética , Diacilglicerol Quinase/genética , Epigênese Genética/genética , Fibroblastos/patologia , RNA Mensageiro/metabolismo , Lesões por Radiação/genética , Adulto , Idoso , Western Blotting , Estudos de Casos e Controles , Imunoprecipitação da Cromatina , Cromatografia Líquida , Diacilglicerol Quinase/antagonistas & inibidores , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Fibrose/etiologia , Fibrose/genética , Células HCT116 , Células HEK293 , Humanos , Espectrometria de Massas , Pessoa de Meia-Idade , Radioterapia/efeitos adversos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Transcriptoma
14.
J Lipid Res ; 57(3): 368-79, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26768655

RESUMO

Diacylglycerol kinase (DGK) consists of 10 isozymes. The α-isozyme enhances the proliferation of cancer cells. However, DGKα facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGKα enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGKα activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGKα with an IC50 value of 0.6 µM. CU-3 targeted the catalytic region, but not the regulatory region, of DGKα. CU-3 competitively reduced the affinity of DGKα for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGKα and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Diacilglicerol Quinase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Rodanina/análogos & derivados , Sulfonamidas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Tiazóis/farmacologia , Animais , Células COS , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Concentração Inibidora 50 , Interleucina-2/biossíntese , Isoenzimas/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Rodanina/farmacologia , Especificidade por Substrato , Linfócitos T/metabolismo
15.
Sci Transl Med ; 8(321): 321ra7, 2016 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-26764158

RESUMO

X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8(+) T cells after Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase α (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ (protein kinase Cθ). We show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the proapoptotic proteins NUR77 and NOR1. Pharmacological inhibition of DGKα prevents the excessive CD8(+) T cell expansion and interferon-γ production that occur in SAP-deficient mice after lymphocytic choriomeningitis virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Morte Celular/efeitos dos fármacos , Citocinas/biossíntese , Diacilglicerol Quinase/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/metabolismo , Ativação Linfocitária , Contagem de Linfócitos , Transtornos Linfoproliferativos/tratamento farmacológico , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/deficiência , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/metabolismo , Tiazóis/farmacologia , Proteínas ras/metabolismo
16.
Clin Cancer Res ; 21(22): 5008-12, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26420856

RESUMO

Lipid kinases have largely been neglected as targets in cancer, and an increasing number of reports suggest diacylglycerol kinase alpha (DGKα) may be one with promising therapeutic potential. DGKα is one of 10 DGK family members that convert diacylglycerol (DAG) to phosphatidic acid (PA), and both DAG and PA are critical lipid second messengers in the plasma membrane. A host of important oncogenic proteins and pathways affect cancer cells in part through DGKα, including the c-Met and VEGF receptors. Others partially mediate the effects of DGKα inhibition in cancer, such as mTOR and HIF-1α. DGKα inhibition can directly impair cancer cell viability, inhibits angiogenesis, and notably may also boost T-cell activation and enhance cancer immunotherapies. Although two structurally similar inhibitors of DGKα were established decades ago, they have seen minimal in vivo usage, and it is unlikely that either of these older DGKα inhibitors will have utility for cancer. An abandoned compound that also inhibits serotonin receptors may have more translational potential as a DGKα inhibitor, but more potent and specific DGKα inhibitors are sorely needed. Other DGK family members may also provide therapeutic targets in cancer, but require further investigation.


Assuntos
Diacilglicerol Quinase/genética , Inibidores Enzimáticos/uso terapêutico , Imunoterapia , Neoplasias/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/química , Inibidores Enzimáticos/química , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Neoplasias/patologia , Neoplasias/terapia , Ácidos Fosfatídicos/genética , Proteínas Proto-Oncogênicas c-met/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética
17.
Pharm Pat Anal ; 4(1): 17-27, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25565157

RESUMO

The epithelial Na(+) channel, ENaC, is a key regulator of the volume of airway surface liquid in the human airway epithelium. In cystic fibrosis (CF), Na(+) hyperabsorption through ENaC in the absence of CFTR-mediated anion secretion results in the dehydration of respiratory secretions and the impairment of mucociliary clearance. The hypothesis of utilizing an ENaC-blocking molecule to facilitate restoration of the airway surface liquid volume sufficiently to allow normal mucociliary clearance is of interest in the management of lung disease in CF patients. This article summarizes the published patent applications from 2010 that claim approaches to inhibit the function of ENaC for utility in the treatment of CF. Patents were located though SciFinder(®), using "ENaC" as the keyword from 2010 onwards; documents not relevant to CF were then manually removed.


Assuntos
Fibrose Cística/tratamento farmacológico , Bloqueadores do Canal de Sódio Epitelial/uso terapêutico , Canais Epiteliais de Sódio/metabolismo , Animais , Fibrose Cística/metabolismo , Diacilglicerol Quinase/antagonistas & inibidores , Indústria Farmacêutica , Humanos , Patentes como Assunto
18.
J Mol Neurosci ; 56(1): 78-88, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25451596

RESUMO

Hypoxia-inducible factors (HIF) play a fundamental role in retinal neovascularization (NV) induced by low oxygen tension. In the presence of oxygen, the HIF-α subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases (PHD), which triggers HIF-α for degradation. In our present study, we examined the effect of R59949, the diacylglycerol kinase (DGK) inhibitor II, on the retinal NV and its potential mechanism in an oxygen-induced retinopathy (OIR) model. OIR model was induced by exposure of hyperoxia (75 % oxygen) to C57BL/6J mice from postnatal day 7 (P7) to P12 and then returned to room air. By intraperitoneal injection once a day (10 µg/g/day) from P12 to P17, R59949 not only effectively prevented pathologic NV but also preserved the astrocyte morphology. Furthermore, the expression of PHD-2 was upregulated and HIF-1α and vascular endothelial growth factor (VEGF) were downregulated in the retina of OIR mice following R59949 treatment. These findings suggested a potential possibility that R59949 suppressed retinal neovascular pathophysiology via PHD2/HIF-1α/VEGF pathway.


Assuntos
Astrócitos/efeitos dos fármacos , Diacilglicerol Quinase/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Piperidinas/uso terapêutico , Quinazolinonas/uso terapêutico , Retinopatia da Prematuridade/tratamento farmacológico , Animais , Astrócitos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Retina/efeitos dos fármacos , Retina/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
Oncotarget ; 5(20): 9710-26, 2014 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-25339152

RESUMO

Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.


Assuntos
Diacilglicerol Quinase/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Quinases da Família src/metabolismo , Animais , Células CACO-2 , Processos de Crescimento Celular/fisiologia , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/biossíntese , Diacilglicerol Quinase/genética , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinonas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidores
20.
BMC Res Notes ; 7: 391, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24962347

RESUMO

BACKGROUND: Elevated fatty acids contribute to the development of type 2 diabetes and affect skeletal muscle insulin sensitivity. Since elevated intramuscular lipids and insulin resistance is strongly correlated, aberrant lipid storage or lipid intermediates may be involved in diabetes pathogenesis. The aim of this study was to develop a method to determine the dynamic metabolic fate of lipids in primary human skeletal muscle cells and in intact mouse skeletal muscle. We report a simple and fast method to characterize lipid profiles in skeletal muscle using thin layer chromatography. FINDINGS: The described method was specifically developed to assess lipid utilization in cultured and intact skeletal muscle. We determined the effect of a pan-diacylglycerol kinase (DGK) class I inhibitor (R59949) on lipid metabolism to validate the method. In human skeletal muscle cells, DGK inhibition impaired diacylglycerol (DAG) conversion to phosphatidic acid and increased triglyceride synthesis. In intact glycolytic mouse skeletal muscle, DGK inhibition triggered the accumulation of DAG species. Conversely, the DGK inhibitor did not affect DAG content in oxidative muscle. CONCLUSION: This simple assay detects rapid changes in the lipid species composition of skeletal muscle with high sensitivity and specificity. Determination of lipid metabolism in skeletal muscle may further elucidate the mechanisms contributing to the pathogenesis of insulin resistance in type 2 diabetes or obesity.


Assuntos
Bioensaio , Diacilglicerol Quinase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Diacilglicerol Quinase/antagonistas & inibidores , Diglicerídeos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Ácidos Fosfatídicos/metabolismo , Piperidinas/farmacologia , Cultura Primária de Células , Quinazolinonas/farmacologia , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Triglicerídeos/biossíntese
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