Two continuous coupled assays for ornithine-δ-aminotransferase

Juncosa, Jose I.; Lee, Hyunbeom; Silverman, Richard B.

doi:10.1016/j.ab.2013.05.025

Cited by 11 publications

(12 citation statements)

References 12 publications

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“…These assays were performed using a modification of the procedure by Juncosa, Lee, and Silverman. 39 OAT (0.25 μg) was incubated with various concentrations of 5 (0.5 μM, 2 μM, 5 μM, 10 μM, 20 μM) in 100 mM potassium pyrophosphate buffer, pH 8.0, containing 1 mM α-ketoglutarate in a total volume of 20 μL at room temperature. At time intervals, 80 μL of assay solution, preincubated at 37 °C for 10 min, containing PYCR1 (0.5 μg), 12.5 mM α-ketoglutarate, 1 mM NADH, 0.03 mM PLP, and 25 mM L- ornithine in 100 mM potassium pyrophosphate buffer, pH 8.0, were added to the incubation mixture and assayed for OAT activity at 37 °C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction

Juncosa

Takaya

et al. 2018

J. Am. Chem. Soc.

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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.

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Section: Methodsmentioning

confidence: 99%

Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction

Juncosa

Takaya

et al. 2018

J. Am. Chem. Soc.

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“…OAT activity was assayed by a modification of the method developed by Juncosa, Lee and Silverman [23] . Wells in a 96-well microtiter plate were loaded with 160 μl of a mixture containing 100 mM potassium pyrophosphate (pH 8.0), 10 mM α-ketoglutarate, 0.4 mM NADH, 0.025 mM pyridoxal 5′-phosphate, and 80 mM L-ornithine (all from Sigma-Aldrich, St. Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

The retarded hair growth ( rhg ) mutation in mice is an allele of ornithine aminotransferase ( Oat )

Bisaillon

Radden

Szabo

et al. 2014

Molecular Genetics and Metabolism Reports

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Because of the similar phenotypes they generate and their proximate reported locations on Chromosome 7, we tested the recessive retarded hair growth (rhg) and frizzy (fr) mouse mutations for allelism, but found instead that these defects complement. To discover the molecular basis of rhg, we analyzed a large intraspecific backcross panel that segregated for rhg and restricted this locus to a 0.9 Mb region that includes fewer than ten genes, only five of which have been reported to be expressed in skin. Complementation testing between rhg and a recessive null allele of fibroblast growth factor receptor 2 eliminated Fgfr2 as the possible basis of the retarded hair growth phenotype, but DNA sequencing of another of these candidates, ornithine aminotransferase (Oat), revealed a G to C transversion specifically associated with the rhg allele that would result in a glycine to alanine substitution at residue 353 of the gene product. To test whether this missense mutation might cause the mutant phenotype, we crossed rhg/rhg mice with mice that carried a recessive, perinatal-lethal, null mutation in Oat (designated OatΔ herein). Hybrid offspring that inherited both rhg and OatΔ displayed markedly delayed postnatal growth and hair development, indicating that these two mutations are allelic, and suggesting strongly that the G to C mutation in Oat is responsible for the retarded hair growth phenotype. Comparisons among +/+, rhg/+, rhg/rhg and rhg/OatΔ mice showed plasma ornithine levels and ornithine aminotransferase activities (in liver lysates) consistent with this assignment. Because histology of 7- and 12-month-old rhg/rhg and rhg/OatΔ retinas revealed chorioretinal degeneration similar to that described previously for OatΔ/OatΔ mice, we suggest that the rhg mutant may offer an ideal model for gyrate atrophy of the choroid and retina (GACR) in humans, which is also caused by the substitution of glycine 353 in some families.

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“…Ornithine-δ-aminotransferase (OAT) is a nuclear encoded, pyridoxal-5'-phosphate (PLP)-dependent enzyme, found in the mitochondrial matrix of most human tissues [ 26 , 27 ]. It involves in the synthesis of glutamate from ornithine, and further synthesizes proline and glutamine, as well as regulating the levels of ornithine in the cells [ 28 – 30 ].…”

Section: Discussionmentioning

confidence: 99%

Preliminary Proteomic Analysis of A549 Cells Infected with Avian Influenza Virus H7N9 and Influenza A Virus H1N1

Ding

et al. 2016

PLoS ONE

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A newly emerged H7N9 influenza virus poses high risk to human beings. However, the pathogenic mechanism of the virus remains unclear. The temporal response of primary human alveolar adenocarcinoma epithelial cells (A549) infected with H7N9 influenza virus and H1N1 influenza A virus (H1N1, pdm09) were evaluated using the proteomics approaches (2D-DIGE combined with MALDI-TOF-MS/MS) at 24, 48 and 72 hours post of the infection (hpi). There were 11, 12 and 33 proteins with significant different expressions (P<0.05) at 24, 48 and 72hpi, especially F-actin-capping protein subunit alpha-1 (CAPZA1), Ornithine aminotransferase (OAT), Poly(rC)-binding protein 1 (PCBP1), Eukaryotic translation initiation factor 5A-1 (EIF5A) and Platelet-activating factor acetylhydrolaseⅠb subunit beta (PAFAH1B2) were validated by western-blot analysis. The functional analysis revealed that the differential proteins in A549 cells involved in regulating cytopathic effect. Among them, the down-regulation of CAPZA1, OAT, PCBP1, EIF5A are related to the death of cells infected by H7N9 influenza virus. This is the first time show that the down-regulation of PAFAH1B2 is related to the later clinical symptoms of patients infected by H7N9 influenza virus. These findings may improve our understanding of pathogenic mechanism of H7N9 influenza virus in proteomics.

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Two continuous coupled assays for ornithine-δ-aminotransferase

Cited by 11 publications

References 12 publications

Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction

Design and Mechanism of (S)-3-Amino-4-(difluoromethylenyl)cyclopent-1-ene-1-carboxylic Acid, a Highly Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Addiction

The retarded hair growth ( rhg ) mutation in mice is an allele of ornithine aminotransferase ( Oat )

Preliminary Proteomic Analysis of A549 Cells Infected with Avian Influenza Virus H7N9 and Influenza A Virus H1N1

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