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1.
Tissue Cell ; 61: 72-78, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31759410

RESUMO

Pyridoxal-5'-phosphate, the active form of vitamin B6, is associated with activities of several enzymes and the treatment of various neurological disorders. Here, we investigated the effects of pyridoxine on the immunoreactivity and protein levels of γ-aminobutyric acid (GABA)-synthesizing and degradation enzymes such as glutamic acid decarboxylase (GAD), GABA transaminase (GABA-T), and succinic semialdehyde dehydrogenase (SSADH), in the hippocampus of mice. The mice intraperitonially received physiological saline and 350 mg/kg pyridoxine, twice a day for 21 days, and were euthanized 2 h after the final dose. In the vehicle-treated group, we observed GAD67 immunoreactivity in the stratum pyramidale of the CA1 and CA3 region, Schaffer collateral, polymorphic layer, and outer granule cell layer of the dentate gyrus. Pyridoxine administration significantly increased GAD67 immunoreactivity, while significantly decreasing GABA-T immunoreactivity in pyridoxine-treated mouse hippocampi (CA1 region and dentate gyrus). In the stratum lacunosum-moleculare of CA1 region, GABA-T immunoreactivity was significantly increased in the pyridoxine-treated group compared to that in the vehicle-treated group, although GAD67 immunoreactivity was similarly observed in these groups. Alternatively, there were no significant differences in SSADH immunoreactivity in any regions of the hippocampus between the vehicle- and pyridoxine-treated groups. Western blot analysis showed significant increases in GAD67 and GABA-T protein levels in the pyridoxine-treated group compared with those in the vehicle-treated group. Therefore, pyridoxine administration facilitates GABA turnover in mouse hippocampus by modulating the GABA-synthesizing and degradation enzymes.


Assuntos
Hipocampo/metabolismo , Piridoxina/metabolismo , Ácido gama-Aminobutírico/biossíntese , 4-Aminobutirato Transaminase/metabolismo , Animais , Glutamato Descarboxilase/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Succinato-Semialdeído Desidrogenase/metabolismo
2.
J Basic Microbiol ; 59(11): 1134-1142, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31549433

RESUMO

Oenococcus oeni is the main bacteria extensively used in malolactic fermentation due to its high tolerance against stress factors in wine production. Among these, ethanol is one of the main challenges to O. oeni, and its ethanol tolerance mechanism remains unclear. In this study, the puuE gene related to ethanol tolerance from O. oeni SD-2a was heterologously expressed in Lactobacillus plantarum WCFS1. Results showed that the recombinant strain (W-pMG36epuuE) exhibited better growth performance and survival rate compared to the control strain (W-pMG36e) under ethanol-stress conditions. In addition, it was found that the activities of superoxide dismutase and the concentration of glutathione of W-pMG36epuuE were significantly higher than those of W-pMG36e. This resulted in the decrease of intracellular reactive oxygen species (ROS) accumulation (10.34% lower than control). Moreover, heterologous expression of puuE in WCFS1 exhibited improved activities of two ATPases in membrane, increasing the cell membrane integrity (37.67% higher than control). These results revealed the role of the puuE gene in improving ethanol tolerance in O. oeni by decreasing ROS accumulation and enhancing cell membrane integrity.


Assuntos
4-Aminobutirato Transaminase/genética , Proteínas de Bactérias/genética , Etanol/metabolismo , Lactobacillus plantarum/metabolismo , Oenococcus/enzimologia , 4-Aminobutirato Transaminase/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Fermentação , Lactobacillus plantarum/genética , Lactobacillus plantarum/crescimento & desenvolvimento , Oenococcus/genética , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Vinho/microbiologia
3.
Oncol Rep ; 42(2): 509-520, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173260

RESUMO

IncRNAs play an important role in the regulation of gene expression. The present study profiled differentially expressed lncRNAs (DELs) and mRNAs (DEMs) in myelodysplastic syndrome (MDS) to construct a 4­aminobutyrate aminotransferase (ABAT)­DEL­DEM co­expression network in MDS development using the Agilent human BeadChips and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and network analyses. Compared with controls, there were 543 DELs and 2,705 DEMs in MDS patients, among which 285 (52.5%) DELs were downregulated and 258 (47.5%) DELs were upregulated, whereas 1,521 (56.2%) DEMs were downregulated and 1,184 (43.70%) DEMs were upregulated in MDS patients. The ABAT­DEL­DEM co­expression network contained six DELs that were co­expressed with ABAT in MDS. The GO analysis revealed that the co­expression network mainly participated in response to organic cyclic compound, cell proliferation, cell part morphogenesis, regulation of cell proliferation and enzyme­linked receptor protein signaling pathways, while the KEGG database showed that the co­expression network was involved in various pathways, such as phagosome and metabolic pathways. Furthermore, the expression of a selected DEL (lncENST00000444102) and ABAT was shown to be significantly downregulated in MDS patients, and in SKM­1 and THP­1 cells. The selected lncENST00000444102 was then overexpressed and ABAT expression was knocked down in the MDS cell lines using lentiviral transfection. In addition, lncENST00000444102 overexpression reduced the viability and increased the apoptosis of MDS cells, ABAT expression was upregulated by lncENST00000444102.


Assuntos
4-Aminobutirato Transaminase/metabolismo , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Síndromes Mielodisplásicas/patologia , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , 4-Aminobutirato Transaminase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , RNA Mensageiro/genética , Células Tumorais Cultivadas , Adulto Jovem
4.
Neurochem Int ; 125: 151-162, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30822440

RESUMO

The anticonvulsant vigabatrin (VGB; SabrilR) irreversibly inhibits GABA transaminase to increase neural GABA, yet its mechanism of retinal toxicity remains unclear. VGB is suggested to alter several amino acids, including homocarnosine, ß-alanine, ornithine, glycine, taurine, and 2-aminoadipic acid (AADA), the latter a homologue of glutamic acid. Here, we evaluate the effect of VGB on amino acid concentrations in mice, employing a continuous VGB infusion (subcutaneously implanted osmotic minipumps), dose-escalation paradigm (35-140 mg/kg/d, 12 days), and amino acid quantitation in eye, visual and prefrontal cortex, total brain, liver and plasma. We hypothesized that continuous VGB dosing would reveal numerous hitherto undescribed amino acid disturbances. Consistent amino acid elevations across tissues included GABA, ß-alanine, carnosine, ornithine and AADA, as well as neuroactive aspartic and glutamic acids, serine and glycine. Maximal increase of AADA in eye occurred at 35 mg/kg/d (41 ±â€¯2 nmol/g (n = 21, vehicle) to 60 ±â€¯8.5 (n = 8)), and at 70 mg/kg/d for brain (97 ±â€¯6 (n = 21) to 145 ±â€¯6 (n = 6)), visual cortex (128 ±â€¯6 to 215 ±â€¯19) and prefrontal cortex (124 ±â€¯11 to 200 ±â€¯13; mean ±â€¯SEM; p < 0.05), the first demonstration of tissue AADA accumulation with VGB in mammal. VGB effects on basic amino acids, including guanidino-species, suggested the capacity of VGB to alter urea cycle function and nitrogen disposal. The known toxicity of AADA in retinal glial cells highlights new avenues for assessing VGB retinal toxicity and other off-target effects.


Assuntos
4-Aminobutirato Transaminase/metabolismo , Aminoácidos/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Vigabatrina/farmacologia , 4-Aminobutirato Transaminase/antagonistas & inibidores , Aminoácidos/sangue , Aminoácidos/genética , Animais , Anticonvulsivantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Retina/efeitos dos fármacos , Retina/metabolismo
5.
Plant Physiol Biochem ; 136: 188-195, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30685698

RESUMO

This work examines the effect of a treatment with 1 mM of γ-aminobutyric acid (GABA) on zucchini fruit during postharvest cold storage. Specifically, the effect of GABA on postharvest quality was measured, as well as its implication in the GABA shunt and other related metabolic pathways. The treatments were performed in Sinatra, a variety of zucchini highly sensitive to low-temperature storage. The application of GABA improved the quality of zucchini fruit stored at 4 °C, with a reduction of chilling-injury index, weight loss, and cell death, as well as a lower rate of electrolyte leakage. GABA content was significantly higher in the treated fruit than in the control fruit at all times analyzed. At the end of the storage period, GABA-treated fruit had higher contents of both proline and putrescine. The catabolism of this polyamine was not affected by exogenous GABA. Also, over the long term, the treatment induced the GABA shunt by increasing the activities of the enzymes GABA transaminase (GABA-T) and glutamate decarboxylase (GAD). GABA-treated fruit contained higher levels of fumarate and malate than did non-treated fruit, as well as higher ATP and NADH contents. These results imply that the GABA shunt is involved in providing metabolites to produce energy, reduce power, and help the fruit to cope with cold stress over the long term.


Assuntos
Cucurbita/efeitos dos fármacos , Armazenamento de Alimentos , Frutas/efeitos dos fármacos , 4-Aminobutirato Transaminase/metabolismo , Trifosfato de Adenosina/metabolismo , Alanina/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Morte Celular/efeitos dos fármacos , Temperatura Baixa , Cucurbita/metabolismo , Armazenamento de Alimentos/métodos , Frutas/metabolismo , Fumaratos/metabolismo , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Malatos/metabolismo , NAD/metabolismo , Prolina/metabolismo , Putrescina/metabolismo , Ácido gama-Aminobutírico/farmacologia
6.
Theranostics ; 9(1): 34-47, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30662552

RESUMO

Basal-like breast cancer (BLBC) is the most aggressive subtype with a poor clinical outcome; however, the molecular mechanisms underlying aggressiveness in BLBC remain poorly understood. Methods: The effects of gamma-aminobutyrate aminotransferase (ABAT) on GABA receptors, Ca2+-NFAT1 axis, and cancer cell behavior were assessed by Ca2+ imaging, Western blotting, immunostaining, colony formation, and migration and invasion assays. We elucidated the relationship between ABAT and Snail by luciferase reporter and ChIP assays. The effect of ABAT expression on BLBC cells was determined by in vitro and in vivo tumorigenesis and a lung metastasis mouse model. Results: We showed that, compared to other subtypes, ABAT was considerably decreased in BLBC. Mechanistically, ABAT expression was downregulated due to Snail-mediated repression leading to increased GABA production. GABA then elevated intracellular Ca2+ concentration by activating GABA-A receptor (GABAA), which contributed to the efficient activation of NFAT1 in BLBC cells. ABAT expression resulted in inhibition of tumorigenicity, both in vitro and in vivo, and metastasis of BLBC cells. Thus, loss of ABAT contributed to BLBC aggressiveness by activating the Ca2+-NFAT1 axis. In breast cancer patients, loss of ABAT expression was strongly correlated with large tumor size, high grade and metastatic tendency, poor survival, and chemotherapy resistance. Conclusions: Our findings have provided underlying molecular details for the aggressive behavior of BLBC. The Snail-mediated downregulation of ABAT expression in BLBC provides tumorigenic and metastatic advantages by activating GABA-mediated Ca2+-NFAT1 axis. Thus, our results have identified potential prognostic indicators and therapeutic targets for this challenging disease.


Assuntos
4-Aminobutirato Transaminase/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Cálcio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Animais , Cátions Bivalentes/metabolismo , Linhagem Celular Tumoral , Neurônios GABAérgicos , Humanos , Camundongos , Modelos Teóricos
7.
FASEB J ; 33(1): 557-571, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30001166

RESUMO

Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1mut), resulting in metabolic stress. Although IDHmut gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDHmut gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1R132H mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1mut- and IDHwt-glioma xenografts. Gene set enrichment analyses of clinical IDH1mut gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDHwt gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDHmut glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDHmut gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Ácido Glutâmico/metabolismo , Isocitrato Desidrogenase/genética , Ácido Láctico/metabolismo , Mutação , Estresse Fisiológico , 4-Aminobutirato Transaminase/genética , 4-Aminobutirato Transaminase/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Isocitrato Desidrogenase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismo , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Plant Cell Rep ; 38(1): 75-84, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30328507

RESUMO

KEY MESSAGE: The new transient protein expression system using the pBYR2HS vector is applicable to several tomato cultivars and wild species with high level of protein expression. Innovation and improvement of effective tools for transient protein expression in plant cells is critical for the development of plant biotechnology. We have created the new transient protein expression system using the pBYR2HS vector that led to about 4 mg/g fresh weight of protein expression in Nicotiana benthamiana. In this study, we validated the adaptability of this transient protein expression system by agroinfiltration to leaves and fruits of several tomato cultivars and wild species. Although the GFP protein was transiently expressed in the leaves and fruits of all tomato cultivars and wild species, we observed species-specific differences in protein expression. In particular, GFP protein expression was higher in the leaves and fruits of Micro-Tom, Solanum pimpinellifolium (0043) and S. pimpinellifolium (0049-w1) than in those of cultivars and wild species. Furthermore, Agrobacterium with GABA transaminase enhanced transient expression in tomato fruits of Micro-Tom. Taken together with these results, our system is applicable to several tomato cultivars and species as well as a model tomato, even though characteristics are often different among tomato cultivars or species. Thus, the system is an effective, simple, and valuable tool to achieve rapid transgene expression to examine gene function in tomato plant cells.


Assuntos
Lycopersicon esculentum/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , 4-Aminobutirato Transaminase/genética , 4-Aminobutirato Transaminase/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética
9.
Biochem Biophys Res Commun ; 506(4): 891-894, 2018 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-30392904

RESUMO

BACKGROUND: Gamma aminobutyric acid (GABA), an inhibitory neurotransmitter, is produced via decarboxylation of l-glutamate through the glutamic acid decarboxylase (GAD) enzyme. The synchronic action of GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) enzymes convert the GABA metabolite into succinate. Given this background, our research was aimed at probing the effect of Magainin II, on the activity of GABA shunt metabolizing enzymes. METHODS: Male NIH mice were administered peripherally by Magainin II (50 µg/kg body weight) and saline solution (%0.9 (w/v)) as the control vehicle. At different time intervals, the mice were sacrificed to evaluate the effect of Magainin II injection on the GABA shunt pathway. The activity of hypothalamic GAD, GABA-T and SSADH enzymes were determined using relevant enzyme assays. RESULTS: Magainin II effectively enhanced the activity of GAD, by %90, 24 h after injection, while quenching the activities of GABA-T and SSADH by %43 and %71, respectively. In vitro models also revealed the direct but reversible interaction between the peptide and each of the individual enzymes of GABA shunt pathway. CONCLUSION: This study confirms the probable role of Magainin II in increasing the GABA content of the mouse hypothalamus. This property might candidate the peptide as a novel agent for improving the symptoms of many GABA dependent psychiatric disorders.


Assuntos
Magaininas/farmacologia , Ácido gama-Aminobutírico/metabolismo , 4-Aminobutirato Transaminase/metabolismo , Animais , Glutamato Descarboxilase/metabolismo , Magaininas/metabolismo , Masculino , Camundongos , Succinato-Semialdeído Desidrogenase/metabolismo
10.
Enzyme Microb Technol ; 118: 57-65, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30143200

RESUMO

Glutaric acid is one of the promising C5 platform compounds in the biochemical industry. It can be produced chemically, through the ring-opening of butyrolactone followed by hydrolysis. Alternatively, glutaric acid can be produced via lysine degradation pathways by microorganisms. In microorganisms, the overexpression of enzymes involved in this pathway from E. coli and C. glutamicum has resulted in high accumulation of 5-aminovaleric acid. However, the conversion from 5-aminovaleric acid to glutaric acid has resulted in a relatively low conversion yield for unknown reasons. In this study, as a solution to improve the production of glutaric acid, we introduced gabTD genes from B. subtilis to E. coli for a whole cell biocatalytic approach. This approach enabled us to determine the effect of co-factors on reaction and to achieve a high conversion yield from 5-aminovaleric acid at the optimized reaction condition. Optimization of whole cell reaction by different plasmids, pH, temperature, substrate concentration, and cofactor concentration achieved full conversion with 100 mM of 5-aminovaleric acid to glutaric acid. Nicotinamide adenine dinucleotide phosphate (NAD(P)+) and α-ketoglutaric acid were found to be critical factors in the enhancement of conversion in selected conditions. Whole cell reaction with a higher concentration of substrates gave 141 mM of glutaric acid from 300 mM 5-aminovaleric acid, 150 mM α-ketoglutaric acid, and 60 mM NAD+ at 30 °C, with a pH of 8.5 within 24 h (47.1% and 94.2% of conversion based on 5-aminovaleric acid and α-ketoglutaric acid, respectively). The whole cell biocatalyst was recycled 5 times with the addition of substrates; this enabled the accumulation of extra glutaric acid.


Assuntos
4-Aminobutirato Transaminase/metabolismo , Aminoácidos Neutros/metabolismo , Bacillus subtilis/enzimologia , Escherichia coli/metabolismo , Glutaratos/metabolismo , Succinato-Semialdeído Desidrogenase/metabolismo , 4-Aminobutirato Transaminase/genética , Bacillus subtilis/genética , Biocatálise , Escherichia coli/genética , Succinato-Semialdeído Desidrogenase/genética
11.
Luminescence ; 33(4): 722-730, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29653023

RESUMO

γ-Aminobutyric acid (GABA) is a well-known neurotransmitter that regulates inhibitory neurotransmission in the mammalian central nervous system and participates in several processes outside the brain. A reliable quantification method is needed to determine its role in different physiological and pathological conditions. However, GABA measurements have several challenges because GABA is neither fluorescent nor electroactive, and it is difficult to detect using enzymatic reactions because no oxidases or dehydrogenases have been identified. Several methods have been developed to quantify GABA concentrations based on the instrumentation available, the sensitivity required, and the volume of samples analyzed. Most of these methods use high-performance liquid chromatography (HPLC). Here, we describe a method for quantifying GABA concentrations in small volume samples using enzymatically-induced electrochemiluminescence with the well-known GABAse complex, which produces glutamate for use in a luminescent reaction with glutamate oxidase and luminol in an electrochemiluminescence cell. The luminescence obtained was proportional to the GABA concentrations in the micromolar range (1-1000), with linear r2 values > 0.95. GABA standards were treated with the enzymatic reactors to generate glutamate (Glu), which was measured simultaneously with an HPLC technique, to validate this new procedure. The assay was further used to determine GABA concentrations in hippocampal extracts. This alternative may be used to quantify GABA levels in fluid samples, such as microdialysates, other perfusates and tissue extracts. Thus, the method presented here is a good alternative for monitoring GABA levels with good sensitivity compared with the traditional methods that are still in use.


Assuntos
4-Aminobutirato Transaminase/metabolismo , Aldeído Oxirredutases/metabolismo , Técnicas Eletroquímicas , Luminescência , Ácido gama-Aminobutírico/análise , 4-Aminobutirato Transaminase/química , Aldeído Oxirredutases/química , Animais , Masculino , Ratos , Ratos Wistar , Análise de Regressão , Ácido gama-Aminobutírico/metabolismo
12.
Chem Rev ; 118(7): 4037-4070, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29569907

RESUMO

When the brain concentration of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) diminishes below a threshold level, the excess neuronal excitation can lead to convulsions. This imbalance in neurotransmission can be corrected by inhibition of the enzyme γ-aminobutyric acid aminotransferase (GABA-AT), which catalyzes the conversion of GABA to the excitatory neurotransmitter l-glutamic acid. It also has been found that raising GABA levels can antagonize the rapid elevation and release of dopamine in the nucleus accumbens, which is responsible for the reward response in addiction. Therefore, the design of new inhibitors of GABA-AT, which increases brain GABA levels, is an important approach to new treatments for epilepsy and addiction. This review summarizes findings over the last 40 or so years of mechanism-based inactivators (unreactive compounds that require the target enzyme to catalyze their conversion to the inactivating species, which inactivate the enzyme prior to their release) of GABA-AT with emphasis on their catalytic mechanisms of inactivation, presented according to organic chemical mechanism, with minimal pharmacology, except where important for activity in epilepsy and addiction. Patents, abstracts, and conference proceedings are not covered in this review. The inactivation mechanisms described here can be applied to the inactivations of a wide variety of unrelated enzymes.


Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Inibidores Enzimáticos/química , Epilepsia/tratamento farmacológico , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , 4-Aminobutirato Transaminase/metabolismo , Animais , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares
13.
J Am Chem Soc ; 140(6): 2151-2164, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29381352

RESUMO

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. Inhibition of GABA aminotransferase (GABA-AT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, has been established as a possible strategy for the treatment of substance abuse. The raised GABA levels that occur as a consequence of this inhibition have been found to antagonize the rapid release of dopamine in the ventral striatum (nucleus accumbens) that follows an acute challenge by an addictive substance. In addition, increased GABA levels are also known to elicit an anticonvulsant effect in patients with epilepsy. We previously designed the mechanism-based inactivator (1S,3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (2), now called CPP-115, that is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved drug that is an inactivator of GABA-AT. CPP-115 was found to have high therapeutic potential for the treatment of cocaine addiction and for a variety of epilepsies, has successfully completed a Phase I safety clinical trial, and was found to be effective in the treatment of infantile spasms (West syndrome). Herein we report the design, using molecular dynamics simulations, synthesis, and biological evaluation of a new mechanism-based inactivator, (S)-3-amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic acid (5), which was found to be almost 10 times more efficient as an inactivator of GABA-AT than CPP-115. We also present the unexpected crystal structure of 5 bound to GABA-AT, as well as computational analyses used to assist the structure elucidation process. Furthermore, 5 was found to have favorable pharmacokinetic properties and low off-target activities. In vivo studies in freely moving rats showed that 5 was dramatically superior to CPP-115 in suppressing the release of dopamine in the corpus striatum, which occurs subsequent to either an acute cocaine or nicotine challenge. Compound 5 also attenuated increased metabolic demands (neuronal glucose metabolism) in the hippocampus, a brain region that encodes spatial information concerning the environment in which an animal receives a reinforcing or aversive drug. This multidisciplinary computational design to preclinical efficacy approach should be applicable to the design and improvement of mechanism-based inhibitors of other enzymes whose crystal structures and inactivation mechanisms are known.


Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Prolina/análogos & derivados , 4-Aminobutirato Transaminase/química , 4-Aminobutirato Transaminase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Dopamina/metabolismo , Antagonistas de Dopamina/química , Antagonistas de Dopamina/farmacocinética , Antagonistas de Dopamina/farmacologia , Inibidores Enzimáticos/farmacocinética , Glucose/metabolismo , Humanos , Masculino , Modelos Moleculares , Prolina/química , Prolina/farmacocinética , Prolina/farmacologia , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismo
14.
Bioorg Chem ; 77: 56-67, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29331765

RESUMO

Even after considerable advances in the field of epilepsy treatment, convulsions are inefficiently controlled by standard drug therapy. Herein, a series of pyrimidine-carbothioamide derivatives 4(a-t) was designed as anticonvulsant agents by doing some important structural modifications in well-known anticonvulsant drugs. Two classical animal models were used for the in vivo anticonvulsant screening, maximum electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) models; followed by motor impairment study by rotarod method. The most active compound 4g effectively suppressed seizure effect in both the animal models with median doses of 15.6 mg/kg (MES ED50), 278.4 mg/kg (scPTZ ED50) and 534.4 mg/kg (TD50) with no sign of neurotoxicity. Furthermore, in vitro GABA-AT enzyme activity assay of 4g showed inhibitory potency (IC50) of 12.23 µM. The docking study also favored the animal studies.


Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Anticonvulsivantes/farmacologia , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Convulsões/tratamento farmacológico , Tioamidas/farmacologia , 4-Aminobutirato Transaminase/metabolismo , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Eletrochoque , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Masculino , Camundongos , Modelos Moleculares , Estrutura Molecular , Pentilenotetrazol , Pirimidinas/síntese química , Pirimidinas/química , Convulsões/induzido quimicamente , Relação Estrutura-Atividade , Tioamidas/síntese química , Tioamidas/química
15.
Biochem J ; 475(4): 749-758, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29339464

RESUMO

Propionic acidemia is the accumulation of propionate in blood due to dysfunction of propionyl-CoA carboxylase. The condition causes lethargy and striatal degeneration with motor impairment in humans. How propionate exerts its toxic effect is unclear. Here, we show that intravenous administration of propionate causes dose-dependent propionate accumulation in the brain and transient lethargy in mice. Propionate, an inhibitor of histone deacetylase, entered GABAergic neurons, as could be seen from increased neuronal histone H4 acetylation in the striatum and neocortex. Propionate caused an increase in GABA (γ-amino butyric acid) levels in the brain, suggesting inhibition of GABA breakdown. In vitro propionate inhibited GABA transaminase with a Ki of ∼1 mmol/l. In isolated nerve endings, propionate caused increased release of GABA to the extracellular fluid. In vivo, propionate reduced cerebral glucose metabolism in both striatum and neocortex. We conclude that propionate-induced inhibition of GABA transaminase causes accumulation of GABA in the brain, leading to increased extracellular GABA concentration, which inhibits neuronal activity and causes lethargy. Propionate-mediated inhibition of neuronal GABA transaminase, an enzyme of the inner mitochondrial membrane, indicates entry of propionate into neuronal mitochondria. However, previous work has shown that neurons are unable to metabolize propionate oxidatively, leading us to conclude that propionyl-CoA synthetase is probably absent from neuronal mitochondria. Propionate-induced inhibition of energy metabolism in GABAergic neurons may render the striatum, in which >90% of the neurons are GABAergic, particularly vulnerable to degeneration in propionic acidemia.


Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Neurônios GABAérgicos/efeitos dos fármacos , Letargia/metabolismo , Propionatos/administração & dosagem , Acidemia Propiônica/metabolismo , 4-Aminobutirato Transaminase/metabolismo , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Neurônios GABAérgicos/metabolismo , Glucose/metabolismo , Inibidores de Histona Desacetilases/administração & dosagem , Histona Desacetilases , Humanos , Letargia/induzido quimicamente , Letargia/fisiopatologia , Metilmalonil-CoA Descarboxilase/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , Neocórtex/patologia , Acidemia Propiônica/induzido quimicamente , Acidemia Propiônica/fisiopatologia , Ácido gama-Aminobutírico/metabolismo
16.
Acta Pharm ; 68(3): 349-359, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31259693

RESUMO

In an attempt to clarify the controversial role of nitric oxide (NO) in seizures, the effects of NO on brain GABA transaminase (GABA-T) activity and GABA levels were investigated. To this aim, the effects of the substrate (l-arginine) and inhibitors (Nω-nitro-l-arginine methyl ester, 7-nitroindazole) of NO synthase (NOS) on GABA-T activity and GABA levels in vitro and ex vivo were analyzed. In vitro NO diminished GABA-T activity and increased GABA. Ex vivo NO modified GABA-T activity and GABA levels biphasically. Inhibition of endothelial and neuronal NOS (eNOS and nNOS) had opposite effects on GABA-T activity and GABA levels, even during seizures induced by pentylenetetrazole. Different effects of NO on GABA-T activity and on GABA levels, depending on the NOS isoform involved, may explain its contradictory role in seizures, the endothelial NOS acting as an anticonvulsant and the neuronal NOS as a proconvulsant. nNOS inhibitors may represent a new generation of antiepileptics.


Assuntos
4-Aminobutirato Transaminase/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Convulsões/fisiopatologia , Ácido gama-Aminobutírico/metabolismo , Animais , Anticonvulsivantes/farmacologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Pentilenotetrazol/farmacologia , Convulsões/enzimologia
17.
Cell Rep ; 21(11): 3040-3048, 2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29241534

RESUMO

White adipocytes are specialized for energy storage, whereas brown adipocytes are specialized for energy expenditure. Explicating this difference can help identify therapeutic targets for obesity. A common tool to assess metabolic differences between such cells is the Seahorse Extracellular Flux (XF) Analyzer, which measures oxygen consumption and media acidification in the presence of different substrates and perturbagens. Here, we integrate the Analyzer's metabolic profile from human white and brown adipocytes with a genome-scale metabolic model to predict flux differences across the metabolic map. Predictions matched experimental data for the metabolite 4-aminobutyrate, the protein ABAT, and the fluxes for glucose, glutamine, and palmitate. We also uncovered a difference in how adipocytes dispose of nitrogenous waste, with brown adipocytes secreting less ammonia and more urea than white adipocytes. Thus, the method and software we developed allow for broader metabolic phenotyping and provide a distinct approach to uncovering metabolic differences.


Assuntos
Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Metabolismo Energético/genética , Genoma Humano , Redes e Vias Metabólicas/genética , 4-Aminobutirato Transaminase/metabolismo , Adipócitos Marrons/citologia , Adipócitos Brancos/citologia , Amônia/metabolismo , Linhagem Celular Transformada , Glucose/metabolismo , Glutamina/metabolismo , Homeostase , Humanos , Metaboloma/genética , Especificidade de Órgãos , Consumo de Oxigênio/genética , Ácido Palmítico/metabolismo , Cultura Primária de Células , Software , Ureia/metabolismo , Ácido gama-Aminobutírico/metabolismo
18.
Bioorg Chem ; 74: 166-178, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28806600

RESUMO

Several new 5,6-dihydropyrimidine-2(1H)-thione derivatives have been prepared and investigated for their potencies for anticonvulsant activity against maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) test in mice. The acute neurotoxicity was measured by rotarod test. Compounds 3c and 3l were found active in both of the animal models. Further, in vitro GABA-AT enzyme activity assay was carried out to investigate the possible mechanism of action through GABA-AT inhibition. The most potent compounds 3c and 3l showed inhibitory potency (IC50) of 18.42µM and 19.23µM, respectively. The molecular modeling was performed for all the synthesized compounds. The docking results were found in concordant with the observed animal studies.


Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Anticonvulsivantes/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Convulsões/tratamento farmacológico , Tionas/farmacologia , 4-Aminobutirato Transaminase/metabolismo , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Relação Dose-Resposta a Droga , Eletrochoque , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Pentilenotetrazol , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Tionas/síntese química , Tionas/química
19.
Biochemistry ; 56(37): 4951-4961, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28816437

RESUMO

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.


Assuntos
4-Aminobutirato Transaminase/antagonistas & inibidores , Aspartato Aminotransferases/antagonistas & inibidores , Cicloleucina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Ornitina-Oxo-Ácido Transaminase/antagonistas & inibidores , Fosfato de Piridoxal/metabolismo , 4-Aminobutirato Transaminase/química , 4-Aminobutirato Transaminase/metabolismo , Aspartato Aminotransferases/química , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cicloleucina/química , Cicloleucina/metabolismo , Cicloleucina/farmacologia , Bases de Dados de Compostos Químicos , Bases de Dados de Proteínas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ligantes , Conformação Molecular , Ornitina-Oxo-Ácido Transaminase/química , Ornitina-Oxo-Ácido Transaminase/genética , Ornitina-Oxo-Ácido Transaminase/metabolismo , Conformação Proteica , Fosfato de Piridoxal/química , Piridoxamina/química , Piridoxamina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato
20.
Neurosci Lett ; 653: 283-287, 2017 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-28579483

RESUMO

Breathing oxygen at partial pressures ≥2.5 atmospheres absolute, which can occur in diving and hyperbaric oxygen (HBO2) therapy, can rapidly become toxic to the central nervous system (CNS). This neurotoxicity culminates in generalized EEG epileptiform discharges, tonic-clonic convulsions and ultimately death. Increased production of neuronal nitric oxide (NO) has been implicated in eliciting hyperoxic seizures by altering the equilibrium between glutamatergic and GABAergic synaptic transmission. Inhibition of glutamic acid decarboxylase (GAD) activity in HBO2 promotes this imbalance; however, the mechanisms by which this occurs is unknown. Therefore, we conducted a series of experiments using mice, a species that is highly susceptible to CNS oxygen toxicity, to explore the possibility that NO modulates GABA metabolism. Mice were exposed to 100% oxygen at 4 ATA for various durations, and brain GAD and GABA transaminase (GABA-T) activity, as well as S-nitrosylation of GAD65 and GAD67 were determined. HBO2 inhibited GAD activity by 50% and this was negatively correlated with S-nitrosylation of GAD65, whereas GABA-T activity and S-nitrosylation of GAD67 were unaltered. These results suggest a new mechanism by which NO alters GABA metabolism, leading to neuroexcitation and seizures in HBO2.


Assuntos
4-Aminobutirato Transaminase/metabolismo , Glutamato Descarboxilase/metabolismo , Oxigenação Hiperbárica/efeitos adversos , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Oxigênio/toxicidade , Ácido gama-Aminobutírico/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL
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