Inhibitor Of Ornithine Aminotransferase Research Articles

Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)cyclopentane-1-carboxylic Acid.

The inhibition of ornithine aminotransferase (OAT), a pyridoxal 5'-phosphate-dependent enzyme, has been implicated as a treatment for hepatocellular carcinoma (HCC), the most common form of liver cancer, for which there is no effective treatment. From a previous evaluation of our aminotransferase inhibitors, (1S,3S)-3-amino-4-(perfluoropropan-2-ylidene)cyclopentane-1-carboxylic acid hydrochloride (1) was found to be a selective and potent inactivator of human OAT (hOAT), which inhibited the growth of HCC in athymic mice implanted with human-derived HCC, even at a dose of 0.1 mg/kg. Currently, investigational new drug (IND)-enabling studies with 1 are underway. The inactivation mechanism of 1, however, has proved to be elusive. Here we propose three possible mechanisms, based on mechanisms of known aminotransferase inactivators: Michael addition, enamine addition, and fluoride ion elimination followed by conjugate addition. On the basis of crystallography and intact protein mass spectrometry, it was determined that 1 inactivates hOAT through fluoride ion elimination to an activated 1,1'-difluoroolefin, followed by conjugate addition and hydrolysis. This result was confirmed with additional studies, including the detection of the cofactor structure by mass spectrometry and through the identification of turnover metabolites. On the basis of this inactivation mechanism and to provide further evidence for the mechanism, analogues of 1 (19, 20) were designed, synthesized, and demonstrated to have the predicted selective inactivation mechanism. These analogues highlight the importance of the trifluoromethyl group and provide a basis for future inactivator design.

Intracellular sources of ornithine for polyamine synthesis in endothelial cells.

Polyamines are essential for proliferation of endothelial cells (EC)and angiogenesis. This study was conducted to identify the metabolic source(s) of ornithine for polyamine synthesis in EC, using N(ω)-hydroxy-nor-L-arginine (Nor-NOHA, an inhibitor of arginase) and gabaculine (an inhibitor of ornithine aminotransferase; OAT). Nor-NOHA inhibited arginase with an IC50 value of 10µM for intact EC. Nor-NOHA (0.5mM) alone inhibited arginase activity in EC by 98%, increased total cellular concentrations of arginine by 14%, and decreased total cellular concentrations of ornithine, putrescine and spermidine by 17, 65 and 74%, respectively. Arginine and glutamine contributed to 73 and 26% of the ornithine produced by EC, respectively. Gabaculine (1mM) alone decreased the total cellular concentrations of arginine, ornithine, putrescine, and spermidine by 14, 96, 32, and 42%, respectively. A combination of both Nor-NOHA and gabaculine completely blocked ornithine production in EC, resulting in no detectable cellular ornithine and almost complete depletion of cellular putrescine and spermidine. Addition of 0.5mM ornithine restored the intracellular concentrations of polyamines in EC treated with Nor-NOHA plus gabaculine, indicating that Nor-NOHA and gabaculine did not inhibit ornithine decarboxylase activity. Our results suggest that the arginase and OAT pathways are the exclusive sources of ornithine in EC when there is little extracellular ornithine and that there is intracellular compartmentalization of arginine and ornithine for endothelial synthesis of polyamines. These novel findings may have important implications for improving placental vascular growth, wound healing, and cancer therapy.

Suppression of Hepatocellular Carcinoma by Inhibition of Overexpressed Ornithine Aminotransferase.

Hepatocellular carcinoma is the second leading cause of cancer death worldwide. DNA microarray analysis identified the ornithine aminotransferase (OAT) gene as a prominent gene overexpressed in hepatocellular carcinoma (HCC) from Psammomys obesus. In vitro studies demonstrated inactivation of OAT by gabaculine (1), a neurotoxic natural product, which suppressed in vitro proliferation of two HCC cell lines. Alpha-fetoprotein (AFP) secretion, a biomarker for HCC, was suppressed by gabaculine in both cell lines, but not significantly. Because of the active site similarity between GABA aminotransferase (GABA-AT) and OAT, a library of 24 GABA-AT inhibitors was screened to identify a more selective inhibitor of OAT. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidene)cyclopentane-1-carboxylic acid (2) was found to be an inactivator of OAT that only weakly inhibits GABA-AT, l-aspartate aminotransferase, and l-alanine aminotransferase. In vitro administration of 2 significantly suppressed AFP secretion in both Hep3B and HepG2 HCC cells; in vivo, 2 significantly suppressed AFP serum levels and tumor growth in HCC-harboring mice, even at 0.1 mg/kg. Overexpression of the OAT gene in HCC and the ability to block the growth of HCC by OAT inhibitors support the role of OAT as a potential therapeutic target to inhibit HCC growth. This is the first demonstration of suppression of HCC by an OAT inactivator.

Plasma citrulline and arginine kinetics in juvenile channel catfish, Ictalurus punctatus, given oral gabaculine

Experiments with young mammals have indicated that renal arginine synthesis is a major endogenous source of this amino acid. The kidney readily synthesizes arginine from intestinally derived citrulline, which is produced from glutamate, the so-called intestinal-renal axis. The present experiment was conducted to ascertain whether or not citrulline and arginine are synthesized in channel catfish via this pathway by administering gabaculine, an effective inhibitor of ornithine aminotransferase, and then monitoring circulating levels of arginine, citrulline, glutamate and ornithine. A conditioning diet was fed to apparent satiation to triplicate groups of channel catfish initially averaging 150 g/fish. After 2 weeks of acclimation, fish were lightly anesthetized with MS-222 and were intubated with a gelatin capsule containing gabaculine (3 mg/kg body weight) in one dose. Then, 24 and 48 h after gabaculine treatment, blood plasma was obtained and analyzed for amino acids via HPLC. Oral gabaculine administration significantly (p < 0.05) decreased plasma concentrations of citrulline (−41.8%) and arginine (−8.9%). It also increased (p < 0.05) plasma glutamine (+17.5%) and ornithine (+256.6%). A reasonable explanation for the increase in plasma glutamine and the decrease in citrulline and arginine is offered by the inhibition of glutamine degradation and a decrease in synthesis of citrulline caused by gabaculine. Results of this experiment suggest that endogenous synthesis of citrulline occurs in channel catfish, most likely at the small intestine, and it may serve as a de novo source of arginine. Therefore, the physiological needs of fish tissues and organs for arginine appear to be met via both dietary arginine and the endogenous synthesis of this amino acid.

Ornithine metabolism along the female mouse nephron: localization of ornithine decarboxylase and ornithine aminotransferase.

The fate of ornithine in the nephron of the female OF-1 Swiss mouse remains unknown. The aim of the present study was to identify the nephron segments containing the key enzymes involved in ornithine metabolism: ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). Viable tubules isolated by microdissection were incubated with [1-14C]ornithine to study the oxidative pathway. Other tubules were permeabilized to measure the ODC activity. Ornithine was decarboxylated in all intact tubules. Gabaculine, a suicide inhibitor of OAT, and rotenone sharply decreased the production of 14CO2 from [1-14C]ornithine. No ODC activity was found in permeabilized tubules isolated from untreated mice. Testosterone increased ODC activity in the proximal tubule substantially and to a minor extent in other nephron segments. In situ hybridization showed ODC messenger ribonucleic acid (mRNA) to be absent in kidneys of untreated females but abundant in the cortex and the outer stripe of the outer medulla of testosterone-treated female mice. The whole proximal tubule contained a great density of silver grains corresponding to ODC mRNA. In conclusion, no basal ODC activity was found in the nephron of female mice. The testosterone-inducible ODC is localized mainly in the proximal tubule, but is also present in distal tubules and collecting ducts. OAT is distributed along the whole nephron, but its activity is higher in proximal tubules than in distal tubules.

Catabolism of arginine and ornithine in perfused rat liver; localisation and regulation.

Arginine catabolism in liver, requires arginase to convert arginine to ornithine and ornithine aminotransferase (OAT) to initiate ornithine catabolism. OAT is restricted to a few cells of the hepatic aanus, the perivenous hepatocytes (1). The question arises as to whether these cells contain an arginase or whether arginase in the periportal cells, where the urea cycle occurs, must cleave arginine and release ornithine downstream to the OAT-containing hepatocytes. To discriminate between these two hypotheses we examined CO, production from U-14C arginine in livers perfused in antegrade and retrograde directions. Rat liver contains no arginine decarboxylase and only very low activity of ornithine decarboxylase (2). Furthermore, l4CO, production from U-C arginine in our perfusions is inhibited by gabaculine, an inhibitor of OAT (3). Therefore, we used CO, production, from U-C arginine as a measure of arginine catabolism through the OAT pathway. Conversion of C arginine to 14C02 would be impossible in retrograde perfusions if the perivenous, OAT-containing cells, lacked an arginase. Figure 1. represents data for the catabolism of U-C arginine to CO, in livers perfused in both antegrade and retrograde directions. The maximum rate of CO, production in the

Effects of water-stress on proline content and ornithine aminotransferase activity of radish cotyledons

The concentration of proline and the level of ornithine aminotransferase (OAT) activity in light-grown cotyledons of radish ( Raphanus sativus cv National) are increased as an inverse function of the relative water content. Gabaculine, which is an irreversible inhibitor of plant OAT, reduced the water-stress induced proline accumulation considerably. It can be assumed that the ornithine pathway contributes via an increase of OAT activity, to proline synthesis, in addition to the glutamate pathway, in water-stressed cotyledons. Drought-induced OAT activity appears to be due to an increase in one of the two OAT isoforms which have been separated by anion-exchange chromatography.

Gabaculine as a tool to investigate the polyamine biosynthesis pathway in habituated callus of Beta vulgaris (L.)

The polyamine content of a habituated callus of Beta vulgaris (L.) is strongly diminished after treatment with gabaculine, a potent inhibitor of ornithine aminotransferase. The inhibitory effect of gabaculine is reversed if ornithine is supplied. This result may indicate that proline catabolism provides ornithine for polyamine synthesis.

Disturbed nitrogen metabolism associated with the hyperhydric status of fully habituated callus of sugarbeet

The content of polyamines and proline was much lower in a normal (N) callus of Beta vulgaris L. than in a fully habituated hyperhydric (H) callus. The H callus also contained more glutamate and had a higher glutamate dehydrogenase activity. The excess of glutamate, in this chlorophyll‐deficient callus, was linked to accumulation of proline and polyamines. Experiments with α‐difluoromethylornithine (DFMO) and α‐difluoromethylarginine (DFMA) showed that both ornithine decarboxylase and arginine decarboxylase participated in the synthesis of polyamines (especially spermidine and putrescine) and removal of ammonia. It is hypothesized that the H callus was subjected to ammonia stress from the start of the culture. Experiments with gabaculine, an inhibitor of ornithine aminotransferase, showed that this enzyme linked proline degradation to polyamine synthesis through the production of ornithine. This disturbed nitrogen metabolism appeared to be characteristic of the fully habituated callus and might explain the low growth of this hyperhydric tissue.

Inhibition of ornithine aminotransferase by 5-fluoromethylornithine: protection against acute thioacetamide intoxication by elevated tissue ornithine levels.

5-Fluoromethylornithine (5FMOrn) is a selective inactivator of ornithine aminotransferase. Its administration causes a dramatic increase of ornithine concentrations in all tissues. Treatment of mice with 20 mg.kg-1 5FMOrn shortly before or after a lethal dose (600 mg.kg-1, intraperitoneally) of thioacetamide (TAA), followed by a second dose 24 hr later, prevented death of 60% of the mice. Pathologic symptoms of TAA intoxication (liver haemorrhage, elevation of amino acids in blood and tissues, diminution of liver spermidine and spermine concentrations, elevation of the activity of liver enzymes in the plasma) were significantly ameliorated by the treatment. The liver protective action of 5FMOrn is related to the elevation of ornithine concentration, as appears from the fact that other, less selective inactivators of ornithine aminotransferase, also produced some protection against acute intoxication with TAA, but not a structurally related compound with no effect on this enzyme.