Structural modifications of (1S,3S)-3-amino-4-difluoromethylenecyclopentanecarboxylic acid, a potent irreversible inhibitor of GABA aminotransferase

Low brain levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) lead to convulsions. Inhibition of GABA aminotransferase increases the concentration of GABA and can terminate the convulsions. Earlier we reported the synthesis of (1S,3S)-3-amino-4-difluoromethylenecyclopentanecarboxylic acid (2), which is 186 times more potent an inactivator of GABA aminotransferase than the epilepsy drug S-vigabatrin. The corresponding dichloromethylene analogue of 2 (compound 3) has been made, but it shows only weak reversible inhibition of GABA aminotransferase. However, the tetrazole isostere of 2 (compound 4) has been found to be a time-dependent inactivator of GABA aminotransferase. Although it is 20 times less potent than carboxylic acid 2, it is 2.5 times more potent than S-vigabatrin. A calculation of the ClogP values indicates that 4 is the most lipophilic of the three, being 69 times more lipophilic than 2 and 55 times more lipophilic than S-vigabatrin, indicating potential for improved bioavailability.     

β-Lactams: A New Class of Conformationally-Rigid Inhibitors of γ-Aminobutyric Acid Aminotransferase

Abstract

A structural similarity of several monobactams (2–4), 3-aminonocardicinic acid (6), 6-aminopenicillanic acid (7), 7-aminocephalosporanic acid (8), and 7-aminodesacetoxycephalosporanic acids (9, 10) to γ-aminobutyric acid (GABA) and to known inhibitors and substrates of GABA aminotransferase is described. Because of this, the above-mentioned compounds were tested as competitive inhibitors and as inactivators of pig brain GABA aminotransferase. All of the compounds were competitive inhibitors of GABA aminotransferase. On the basis of the inhibitory potency of these conformationally-rigid GABA analogues it is hypothesized that GABA is bound at the active site with its amino and carboxylate groups in a syn orientation. None of the compounds inactivates GABA aminotransferase. These β-lactam analogues represent the first examples of a new class of inhibitors of GABA aminotransferase.     

Degradation of pyrimidines in Saccharomyces kluyveri: transamination of beta-alanine

Beta-alanine is an intermediate in the reductive degradation of uracil. Recently we have identified and characterized the Saccharomyces kluyveri PYD4 gene and the corresponding enzyme beta -alanine aminotransferase ((Sk)Pyd4p), highly homologous to eukaryotic gamma-aminobutyrate aminotransferase (GABA-AT). S. kluyveri has two aminotransferases, GABA aminotransferase ((Sk)Uga1p) with 80% and (Sk)Pyd4p with 55% identity to S. cerevisiae GABA-AT. (Sk)Pyd4p is a typical pyridoxal phosphate-dependent aminotransferase, specific for alpha-ketoglutarate (alpha KG), beta-alanine (BAL) and gamma-aminobutyrate (GABA), showing a ping-pong kinetic mechanism involving two half-reactions and substrate inhibition. (Sk)Uga1p accepts only alpha KG and GABA but not BAL, thus only (Sk)Pydy4p belongs to the uracil degradative pathway.     

Effect of gabaculine on metabolism and release of γ-aminobutyric acid in synaptosomes

Synaptosomes isolated from mouse brain were incubated with [¹⁴C]glutamate and [³H]-aminobutyric acid ([³H]GABA), and then [¹⁴C]GABA (newly synthesized GABA) and [³H]GABA (newly captured GABA) in the synaptosomes were analysed. (1) the [³H]GABA was rapidly degraded in the synaptosomes, (2) when the synaptosomes were treated with gabaculine (a potent inhibitor of GABA aminotransferase), the degradation of [³H]GABA was strongly inhibited, (3) the gabaculine treatment brough about a significant increase in Ca²⁺-independent release of [³H]GABA with no effect on Ca²⁺-dependent release, (4) no effects of gabaculine on degradation and release of [¹⁴C]GABA were observed. The results indicate that there are at least two pools of GABA in synaptosomes and support the possibilities that GABA taken up into a pool which is under the influence of GABA aminotransferase is released Ca²⁺-independently and that GABA synthesized in another pool which is not under the influence of GABA aminotransferase is released Ca²⁺-dependently.     

Early changes in GABA and glutamate levels and aminotransferase activity in parts of the rat brain following whole-body gamma irradiation at an absolutely lethal dose

The contents of gamma-aminobutyric acid (GABA) and glutamate (GL) as well as GABA-aspartate- and alanine aminotransferase activities were measured in rat cerebellum, cerebral cortex and truncus cerebri 1, 3, 6, 24 and 48 hr following total-body gamma-irradiation (60Co) with a dose of 30 Gy. All the indices under study changed in a similar way in the cortex and truncus cerebri while in the cerebellum, GABA level increased and GABA-alpha-ketoglutarate aminotransfearse activity decreased 60 min after irradiation. The levels of GABA and GL in the cortex and truncus cerebri decreased immediately and increased 24 hr after irradiation. Activity of aminotransferases changed in a phase manner: changes in aspartate- and alanine aminotransferase activity were more pronounced than those of GABA-alpha-ketoglutarate aminotransferase activity and correlated with the glutamate level changes.     

Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of l-glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.     

Acute metabolic effects of taurine on the enzymes metabolizing glutamate and gaba

100 mg of taurine per kg body weight had been administered intraperitoneally and 30 min after the administration the animals were sacrificed. Glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, glutaminase, glutamine synthetase, glutamate decarboxylase and GABA aminotransferase along with the content of glutamate and GABA in cerebral cortex, cerebellum and brain stem were studied and compared with the same obtained in the rats treated with normal saline in place of taurine. The results indicated a significant decrease in the activity of glutamate dehydrogenase in cerebral cortex and cerebellum and a significant increase in brain stem. Glutaminase and glutamine synthetase were found to increase significantly both in cerebral cortex and cerebellum. The activities of glutamate decarboxylase was found to increase in all the three regions along with a significant decrease in GABA aminotransferase while the content of glutamate showed a decrease in all the three brain regions, the content of GABA was observed to increase significantly. The above effects of taurine on the metabolism of glutamate and GABA are discussed in relation to the functional role of GABA and glutamate. The results indicate that taurine administration would result in a state of inhibition in brain.     

Crystal structure of GABA-aminotransferase, a target for antiepileptic drug therapy.

gamma-Aminobutyrate aminotransferase (GABA-AT), a pyridoxal phosphate-dependent enzyme, is responsible for the degradation of the inhibitory neurotransmitter GABA and is a target for antiepileptic drugs because its selective inhibition raises GABA concentrations in brain. The X-ray structure of pig GABA-AT has been determined to 3.0 A resolution by molecular replacement with the distantly related enzyme ornithine aminotransferase. Both omega-aminotransferases have the same fold, but exhibit side chain replacements in the closely packed binding site that explain their respective specificities. The aldimines of GABA and the antiepileptic drug vinyl-GABA have been modeled into the active site.     

A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast

In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiae UGA1 and Schizosaccharomyces pombe UGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5'-phosphate is needed for enzymatic activity and alpha-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (V(max)/K(m)=0.78 U x mg(-1) x mm(-1)), but can also use GABA (V(max)/K(m)=0.42 U x mg(-1) x mm(-1)), while SkUga1p only uses GABA (V(max)/K(m)=4.01 U x mg(-1) x mm(-1)). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently 'distinguish' between the two reactions in a single cell. It is likely that upon duplication approximately 200 million years ago, a specialized Uga1p evolved into a 'novel' transaminase enzyme with broader substrate specificity.     

The Synthesis and Activity of cis-and trans-2-(Aminomethyl) cyclopropanecarboxylic Acid as Conformationally Restricted Analogues of GABA

Abstract: The synthesis of cis -2-(aminomethyl) cyclopropanecarboxylic acid, a new analogue of GABA in a folded conformation, is described, as is also an improved preparation of trans -2-(aminomethyl) cyclopropanecarboxylic acid. When adminstered microelectrophoretically the trans isomer was more potent than GABA as a bicuculline-sensitive depressant of the firing of cat spinal neurons in vivo , whereas the cis-isomer was less potent than GABA and its effects appeared not to be sensitive to bicuculline methochloride. Trans -2-(aminomethyl) cyclopropanecarboxylic acid was a weak inhibitor of the sodium-dependent uptake of GABA by mini slices of rat cerebral cortex and a substrate for the GABA: 2-oxoglutarate aminotransferase activity in extracts of rat brain mitochondria. The cis isomer did not influence GABA uptake or aminotransferase activity and neither isomer reduced glutamate decar-boxylase activity in rat brain homogenates. Both cyclopropane isomers inhibited the sodium-independent binding of GABA to synaptic membranes from rat brain and their relative potencies together with those found for the stereochemically related unsaturated derivatives, cis -and trans -4-aminocrotonic acid, were broadly consistent with the activity observed for these compounds in vivo on cat spinal neurons. These studies reinforce the evidence that extended rather than folded conformations of GABA are active at most GABA recognition sites within the mammalian central nervous system.     

DIHYDROMUSCIMOL, THIOMUSCIMOL AND RELATED HETEROCYCLIC COMPOUNDS AS GABA ANALOGUES

A series of heterocyclic GABA analogues related to muscimol (5-aminomethyl-3-isoxazolol) were tested as depressants of the firing of GABA sensitive neurones on the cat spinal cord, and as inhibitors of the sodium-independent binding of GABA to rat brain membranes. Furthermore, the compounds were examined as inhibitors of GABA uptake into rat brain slices and as inhibitors of the activities of the GABA-metabolizing enzymes L-glutamate 1-carboxylyase and GABA:2-oxoglutarate aminotransferase. Dihydromuscimol [(RS)-4,5-dihydromuscimol] and thiomuscimol (5-aminomethyl-3-isothiazolol) were approximately equipotent to muscimol as bicuculline-sensitive depressants of neuronal firing and as inhibitors of GABA binding. The structurally related compounds isomuscimol (3-aminomethyl-5-isoxa-zolol) and azamuscimol (5-aminomethyl-3-pyrazolol) were much weaker than muscimol as GABA agonists. The affinity of the compounds for GABA receptor sites in vitro is in agreement with their relative potency as GABA receptor agonists in vivo. The rat brain synaptic membranes used for the GABA receptor binding studies were prepared by two procedures, which were shown to have a pronounced influence on the observed potency of the inhibitors of GABA binding. The compounds were weak or inactive as inhibitors of the uptake of GABA into rat brain slices and of the activity of GABA: 2-oxoglutarate aminotransferase in vitro. Azamuscimol and 2-methylaza-muscimol were moderately potent inhibitors.of the activity of L-glutamate 1-carboxylyase in vitro. This inhibition by azamuscimol was timedependent following pseudo-first-order kinetics, consistent with azamuscimol acting as a catalytic inhibitor. The structure of the heterocyclic rings of these zwitterionic compounds is a factor of critical importance for interaction with GABA receptors. The present structure-activity analysis demonstrates that heterocyclic GABA analogues having a high degree of delocalization of the negative charges have low affinity for the GABA receptors.     

Degradation of Pyrimidines in Saccharomyces Kluyveri: Transamination of β-Alanine

Beta-alanine is an intermediate in the reductive degradation of uracil. Recently we have identified and characterized the Saccharomyces kluyveri PYD4 gene and the corresponding enzyme β -alanine aminotransferase ( ^Sk Pyd4p), highly homologous to eukaryotic γ-aminobutyrate aminotransferase (GABA-AT). S. kluyveri has two aminotransferases, GABA aminotransferase ( ^Sk Uga1p) with 80% and ^Sk Pyd4p with 55% identity to S. cerevisiae GABA-AT. ^Sk Pyd4p is a typical pyridoxal phosphate-dependent aminotransferase, specific for α -ketoglutarate (α KG), β -alanine (BAL) and γ -aminobutyrate (GABA), showing a ping-pong kinetic mechanism involving two half-reactions and substrate inhibition. ^Sk Uga1p accepts only α KG and GABA but not BAL, thus only ^Sk Pydy4p belongs to the uracil degradative pathway.     

Purification and expression of a processing protease on beta-alanine-oxoglutarate aminotransferase from rat liver mitochondria

GABA[arrow beta]AlaAT convertase is an endopeptidase that processes brain-type 4-aminobutyrate aminotransferase (GABA AT; EC 2.6.1.19) to liver-type beta-alanine-oxoglutarate aminotransferase (beta-AlaAT I) in rats. Its molecular mass was 180 kDa as determined by gel filtration. A subunit molecular mass of 97652 Da was measured using MALDI-TOF MS. The N-terminal sequence of the purified GABA[arrow beta]AlaAT convertase was SRVEVSKVLILGSGGLSIGQAGEFDYSGSQAV- and was identical to residues 418-449 of carbamoyl-phosphate synthetase I (CPS I; EC 1.2.1.27) purified from rat liver. The subunit molecular mass and the N-terminal amino acid sequence suggested that GABA[arrow beta]AlaAT convertase was the 418-1305 peptide of CPS I. An expression vector containing the coding region of the 418-1305 peptide of rat CPS I was transfected into NIH3T3 cells and the extract of the cells showed GABA[arrow beta]AlaAT convertase activity.     

谷氨酸棒杆菌S9114摇瓶发酵产γ-氨基丁酸的研究

为实现谷氨酸棒杆菌工业化生产γ-氨基丁酸(GABA),对L-谷氨酸工业生产菌S9114进行代谢途径改造。通过构建一株工程菌株S9114/p JYW-4-gad B1-gad B2,将来源于短乳杆菌Lb85菌株的谷氨酸脱羧酶编码基因gad B1和gad B2进行共表达,实现发酵72 h后发酵液中GABA含量达到32.8 g/L,GABA糖酸转化率达到47.3%。通过敲除该菌株的谷丙转氨酶编码基因ala T,使工程菌株S9114Δala T/p JYW-4-gad B1-gad B2发酵液中L-丙氨酸浓度降低5.5%,进一步降低了发酵副产物的含量。研究结果为利用谷氨酸棒杆菌实现工业化生产γ-氨基丁酸提供了有价值的参考。     

INHIBITION OF GABA UPTAKE IN RAT BRAIN SLICES BY NIPECOTIC ACID, VARIOUS ISOXAZOLES AND RELATED COMPOUNDS

—A variety of isoxazoles structurally related to muscimol (3-hydroxy-5-aminomethylisoxazole) were tested as inhibitors of the uptake of GABA and some other amino acids in rat brain slices, and of the activity of the GABA-metabolizing enzymes l -glutamate 1-carboxylyase and GABA:2-oxo-glutarate aminotransferase. A bicyclic derivative, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol, proved to be a more potent inhibitor of GABA uptake than muscimol. Structure-activity studies on this derivative, which appeared to be a competitive inhibitor of GABA uptake, led to the findings that nipecotic acid (piperidine-3-carboxylic acid) is a powerful non-competitive inhibitor of GABA uptake, and that perhydro-1,2-oxazine-6-carboxylic acid is a relatively weak competitive inhibitor of GABA uptake.     

Gamma-aminobutyric acid production through GABA shunt by synthetic scaffolds introduction in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nylon 4 is a biodegradable polymer which can be produced from the monomer of pyrrolidone. Gammaaminobutyric acid (GABA) is a precursor of pyrrolidone used for the production of bioplastics. In this study, Escherichia coli were engineered to produce gammaaminobutyric acid from glucose via an alternative novel pathway by the introduction of synthetic scaffolds. The GABA pathway constructed contained succinate dehydrogenase, succinate-semialdehyde dehydrogenase and GABA aminotransferase to redirect the Krebs cycle flux to GABA production. By introduction of a synthetic scaffold, production of 0.64 g/L GABA was achieved at 30°C and pH 6.5. Final GABA concentration was increased by 11.3% via the inactivation of competing pathways, and higher initial glucose concentration led to the enhanced final GABA concentration of 1.01 g/L.     

1-Piperideine as an In Vivo Precursor of the γ-Aminobutyric Acid Homologue 5-Aminopentanoic Acid

Intraperitoneal injection of the cyclic imine 1-piperideine in mice resulted in measurable quantities of 5-aminopentanoic acid in brain. 5-Aminopentanoic acid is a methylene homologue of gamma-aminobutyric acid (GABA) that is a weak GABA agonist. 5-Aminopentanoic acid formed in the periphery was ruled out as the source of brain 5-aminopentanoic acid based on the absence of detection in brain following injection of 100 mg/kg of 5-aminopentanoic acid. Deuterium-labeled 1-piperideine was prepared by exchange in deuterated phosphate buffer. Injection of [3.3-2H2]1-piperideine yielded [2.2-2H2]5-aminopentanoic acid in brain. The results are consistent with uptake of 1-piperideine into brain and oxidation of the precursor to 5-aminopentanoic acid. Inhibition of GABA catabolism by pretreatment with aminooxyacetic acid increased brain concentrations of 5-aminopentanoic acid formed from 1-piperideine, suggesting that 5-aminopentanoic acid is an in vivo substrate of 4-aminobutyrate:2-oxoglutarate aminotransferase.     

Amino Acids Content in Germinating Seeds and Seedlings from Castanea sativa L

During germination the chestnut (Castanea sativa L.) var ecotype 33 accumulates a large amount of asparagine in the cotyledons. This compound also accumulates in the growing axis:shoots and roots. In the cotyledons, γ-aminobutyrate (GABA) represents a major amino compound during germination and early seedling growth. In young seedlings, 35 days old, arginine predominates over the other soluble amino acids, particularly in roots. Five enzymic activities involved in arginine and GABA have been measured in the storage organ of the seed: arginase and ornithine carbamyltransferase decrease during germination indicating the slowing down of the urea cycle. In contrast, ornithine aminotransferase increases. Glutamate decarboxylase is particularly active about 21 days after imbibition and GABA aminotransferase activity decreases during germination. These two activities are in good agreement with the likely transport of GABA from cotyledons to growing axis. Asparagine, arginine, and GABA are the three amino compounds obviously involved in the mobilization of nitrogen reserves in the germinating chestnut seeds Castanea sativa.     

Presence of Radiolabelled Metabolites in Release Studies Using [3H]γ-Aminobutyric Acid

Abstract: Most studies on γ-aminobutyric acid (GABA) release from nervous tissue have been conducted using radiolabelled GABA in the presence of aminooxyacetic acid (AOAA) to inhibit GABA: 2-oxoglutarate aminotransferase (GABA-T) to prevent conversion of labelled GABA to labeled catabolites. Here we present data showing that even in the presence of 10 μM-AOAA the spontaneous release of tritium from rat cortical synaptosomes prelabelled with 2,3-[³H]GABA is mainly in the form of tritiated water but that the increase in tritium release in the presence of unlabelled GABA or high potassium-ion concentrations is in the form of authentic [³H]GABA. Interpretation of results should take these facts into account.