Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Remarkable and Unexpected Mechanism for (S)-3-Amino-4-(difluoromethylenyl)cyclohex-1-ene-1-carboxylic Acid as a Selective Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via the proline and glutamine metabolic pathways. The selective inhibition of hOAT by compound 10 exhibited potent in vivo antitumor activity. Inspired by the discovery of the aminotransferase inactivator (1S,3S)-3-amino-4-(difluoromethylene)cyclopentane-1-carboxylic acid (5), we rationally designed, synthesized, and evaluated a series of six-membered-ring analogs. Among them, 14 was identified as a new selective hOAT inactivator, which demonstrated a potency 22× greater than that of 10. Three different types of protein mass spectrometry approaches and two crystallographic approaches were employed to identify the structure of hOAT-14 and the formation of a remarkable final adduct (32') in the active site. These spectral studies reveal an enzyme complex heretofore not observed in a PLP-dependent enzyme, which has covalent bonds to two nearby residues. Crystal soaking experiments and molecular dynamics simulations were carried out to identify the structure of the active-site intermediate 27' and elucidate the order of the two covalent bonds that formed, leading to 32'. The initial covalent reaction of the activated warhead occurs with *Thr322 from the second subunit, followed by a subsequent nucleophilic attack by the catalytic residue Lys292. The turnover mechanism of 14 by hOAT was supported by a mass spectrometric analysis of metabolites and fluoride ion release experiments. This novel mechanism for hOAT with 14 will contribute to the further rational design of selective inactivators and an understanding of potential inactivation mechanisms by aminotransferases.

Structural and Kinetic Analyses Reveal the Dual Inhibition Modes of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic Acid (BCF3).

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the leading cause of death among people with cirrhosis. HCC is typically diagnosed in advanced stages when tumors are resistant to both radio- and chemotherapy. Human ornithine aminotransferase (hOAT) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme involved in glutamine and proline metabolism. Because hOAT is overexpressed in HCC cells and a contributing factor for the uncontrolled cellular division that propagates malignant tumors (Ueno et al. J. Hepatol. 2014, 61, 1080-1087), it is a potential drug target for the treatment of HCC. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic acid (BCF3) has been shown in animal models to slow the progression of HCC by acting as a selective and potent mechanism-based inactivator of OAT (Zigmond et al. ACS Med. Chem. Lett. 2015, 6, 840-844). Previous studies have shown that the BCF3-hOAT reaction has a bifurcation in which only 8% of the inhibitor inactivates the enzyme while the remaining 92% ultimately acts as a substrate and undergoes hydrolysis to regenerate the active PLP form of the enzyme. In this manuscript, the rate-limiting step of the inactivation mechanism was determined by stopped-flow spectrophotometry and time-dependent 19F NMR experiments to be the decay of a long-lived external aldimine species. A crystal structure of this transient complex revealed both the structural basis for fractional irreversible inhibition and the principal mode of inhibition of hOAT by BCF3, which is to trap the enzyme in this transient but quasi-stable external aldimine form.

A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of hOAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1R,3S,4S)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (1), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying 8 and 9 as novel selective hOAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated 8 and 9 as covalent inhibitors of hOAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel addition-aromatization inactivation mechanism for 9 contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.

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.

Pharmacologic rescue of hyperammonemia-induced toxicity in zebrafish by inhibition of ornithine aminotransferase.

Hyperammonemia is the common biochemical hallmark of urea cycle disorders, activating neurotoxic pathways. If untreated, affected individuals have a high risk of irreversible brain damage and mortality. Here we show that acute hyperammonemia strongly enhances transamination-dependent formation of osmolytic glutamine and excitatory glutamate, thereby inducing neurotoxicity and death in ammoniotelic zebrafish larvae via synergistically acting overactivation of NMDA receptors and bioenergetic impairment induced by depletion of 2-oxoglutarate. Intriguingly, specific and irreversible inhibition of ornithine aminotransferase (OAT) by 5-fluoromethylornithine rescues zebrafish from lethal concentrations of ammonium acetate and corrects hyperammonemia-induced biochemical alterations. Thus, OAT inhibition is a promising and effective therapeutic approach for preventing neurotoxicity and mortality in acute hyperammonemia.

Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.

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.

Ornithine aminotransferase versus GABA aminotransferase: implications for the design of new anticancer drugs.

Ornithine aminotransferase (OAT) and γ-aminobutyric acid aminotransferase (GABA-AT) are classified under the same evolutionary subgroup and share a large portion of structural, functional, and mechanistic features. Therefore, it is not surprising that many molecules that bind to GABA-AT also bind well to OAT. Unlike GABA-AT, OAT had not been viewed as a potential therapeutic target until recently; consequently, the number of therapeutically viable molecules that target OAT is very limited. In this review the two enzymes are compared with respect to their active-site structures, catalytic and inactivation mechanisms, and selective inhibitors. Insight is offered that could aid in the design and development of new selective inhibitors of OAT for the treatment of cancer.

Protein S-glutathionylation in malaria parasites.

AIMS: Protein S-glutathionylation is a widely distributed post-translational modification of thiol groups with glutathione that can function as a redox-sensitive switch to mediate redox regulation and signal transduction. The malaria parasite Plasmodium falciparum is exposed to intense oxidative stress and possesses the enzymatic system required to regulate protein S-glutathionylation, but despite its potential importance, protein S-glutathionylation has not yet been studied in malaria parasites. In this work we applied a method based on enzymatic deglutathionylation, affinity purification of biotin-maleimide-tagged proteins, and proteomic analyses to characterize the Plasmodium glutathionylome. RESULTS: We identified 493 targets of protein S-glutathionylation in Plasmodium. Functional profiles revealed that the targets are components of central metabolic pathways, such as nitrogen compound metabolism and protein metabolism. Fifteen identified proteins with important functions in metabolic pathways (thioredoxin reductase, thioredoxin, thioredoxin peroxidase 1, glutathione reductase, glutathione S-transferase, plasmoredoxin, mitochondrial dihydrolipoamide dehydrogenase, glutamate dehydrogenase 1, glyoxalase I and II, ornithine δ-aminotransferase, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], pyruvate kinase [PK], and phosphoglycerate mutase) were further analyzed to study their ability to form mixed disulfides with glutathione. We demonstrate that P. falciparum GAPDH, PK, and ornithine δ-aminotransferase are reversibly inhibited by S-glutathionylation. Further, we provide evidence that not only P. falciparum glutaredoxin 1, but also thioredoxin 1 and plasmoredoxin are able to efficiently catalyze protein deglutathionylation. INNOVATION: We used an affinity-purification based proteomic approach to characterize the Plasmodium glutathionylome. CONCLUSION: Our results indicate a wide regulative use of S-glutathionylation in the malaria parasite and contribute to our understanding of redox-regulatory processes in this pathogen.

Adaptative response of nitrogen metabolism in early endotoxemia: role of ornithine aminotransferase.

Arginine (Arg) and glutamine (Gln) utilization is greatly increased during catabolic stress. While the supply of both amino acids has been advocated in this situation, arginine administration is possibly associated with deleterious effects. From a metabolic point of view, these two amino acids are reciprocal precursors via ornithine aminotransferase (OAT). We hypothesized that OAT plays a key role in the interconversion between Arg and Gln. To test this hypothesis, we evaluated the influence of OAT activity in a model of septic shock induced by intraperitoneal injection of lipopolysaccharide (LPS) in wild-type (WT) and transgenic mice overexpressing OAT (OAT) in the liver, kidney and intestine, i.e. the three main organs of OAT expression. Plasma and tissue amino acid concentrations and tissue OAT expression and activity were measured. Five hours after LPS injection, WT and OAT mice showed a similar response to LPS in terms of inflammatory cytokine production and protein catabolism, suggesting that the interconversion between Arg and Gln through this pathway remains limited. Endotoxemia led to a significant decrease in plasma Orn levels and an increase in liver Orn levels. Of note, Orn levels were always lower in OAT mice. While only plasma Arg and Gln remained unaffected by LPS treatment, hepatic Gln was significantly increased without any difference between the two genotypes. In this model of early endotoxemia, arginine and glutamine maintained their metabolic homeostasis. Our results show an inhibition of OAT activity and expression in the liver following LPS treatment. These data highlight the importance of OAT in ornithine metabolism, especially in the liver, and suggest a post-transcriptional regulation of OAT by LPS in the liver.

Biochemical characterization of pea ornithine-delta-aminotransferase: substrate specificity and inhibition by di- and polyamines.

Ornithine-delta-aminotransferase (OAT, EC 2.6.1.13) catalyzes the transamination of L-ornithine to L-glutamate-gamma-semialdehyde. The physiological role of OAT in plants is not yet well understood. It is probably related to arginine catabolism resulting in glutamate but the enzyme has also been associated with stress-induced proline biosynthesis. We investigated the enzyme from pea (PsOAT) to assess whether diamines and polyamines may serve as substrates or they show inhibitory properties. First, a cDNA coding for PsOAT was cloned and expressed in Escherichia coli to obtain a recombinant protein with a C-terminal 6xHis tag. Recombinant PsOAT was purified under native conditions by immobilized metal affinity chromatography and its molecular and kinetic properties were characterized. Protein identity was confirmed by peptide mass fingerprinting after proteolytic digestion. The purified PsOAT existed as a monomer of 50 kDa and showed typical spectral properties of enzymes containing pyridoxal-5'-phosphate as a prosthetic group. The cofactor content of PsOAT was estimated to be 0.9 mol per mol of the monomer by a spectrophotometric analysis with phenylhydrazine. L-Ornithine was the best substrate (K(m)=15 mM) but PsOAT also slowly converted N(alpha)-acetyl-L-ornithine. In these reactions, 2-oxoglutarate was the exclusive amino group acceptor (K(m)=2mM). The enzyme had a basic optimal pH of 8.8 and displayed relatively high temperature optimum. Diamines and polyamines were not accepted as substrates. On the other hand, putrescine, spermidine and others represented weak non-competitive inhibitors. A model of the molecular structure of PsOAT was obtained using the crystal structure of human OAT as a template.

Secondary creatine deficiency in ornithine delta-aminotransferase deficiency.

AIMS: Ornithine delta-aminotransferase (OAT) deficiency causes gyrate atrophy (GA) of the retina, as a consequence of high plasma ornithine concentrations. Because creatine synthesis requires the conversion of arginine and glycine into ornithine and guanidinoacetate, high ornithine concentration inhibits this reaction thus causing secondary creatine deficiency. The aim of this study was to evaluate the neuropsychological features and creatine metabolism in patients with GA. METHODS: The study involved 7 GA patients, aged from 11 to 27 years who underwent neuropsychological evaluation and cerebral proton magnetic resonance spectroscopy (MRS). RESULTS: Neurocognitive impairment was found in 5/7 patients, including mental retardation (3/7), school failure (1/7), major visuospatial dyspraxia (1/7), aggressive behavior (3/7) and epilepsy (2/7). Two patients had normal neuropsychological evaluation. Cerebral proton magnetic resonance spectroscopy revealed a profound creatine deficiency in all patients. MRS data were confirmed by decreased levels of creatine and/or guanidinoacetate in plasma and urine in all patients. CONCLUSIONS: In our group of patients with GA, we found a high prevalence of neurological impairment, not reported so far, and possibly related to secondary creatine deficiency and hyperornithinemia. We propose to treat mentally retarded GA patients with high doses of creatine, as it may normalize brain creatine levels and help to reduce ornithine levels.

Polyamines upregulate the mRNA expression of cationic amino acid transporter-1 in human retinal pigment epithelial cells.

We previously showed that ornithine was mainly transported via cationic amino acid transporter (CAT)-1 in human retinal pigment epithelial (RPE) cell line, human telomerase RT (hTERT)-RPE, and that CAT-1 was involved in ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient cell produced by a OAT specific inhibitor, 5-fluoromethylornithine (5-FMO). We showed here that CAT-1 mRNA expression was increased by ornithne in OAT-deficient RPE cells, which was reversed by an inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethylornithine (DFMO). Polyamines, especially spermine, one of the metabolites of ODC, also enhanced the expression of CAT-1 mRNA. ODC mRNA expression was also increased by ornithine and polyamines, and gene silencing of ODC by siRNA decreased ornithine transport activity and its cytotoxicity. In addition, the mRNA of nuclear protein c-myc was also increased in 5-FMO- and ornithine-treated hTERT-RPE cells, and gene silencing of c-myc prevented the induction of CAT-1 and ODC. Increases in expression of CAT-1, ODC, and c-myc, and the inhibition of these stimulated expression by DFMO were also observed in primary porcine RPE cells. These results suggest that spermine plays an important role in stimulation of mRNA expression of CAT-1, which is a crucial role in ornithine cytotoxicity in OAT-deficient hTERT-RPE cells.

Therapeutic anticancer efficacy of a synthetic diazonamide analog in the absence of overt toxicity.

Blocking cell division through the inhibition of mitosis is one of the most successful clinical strategies for the treatment of cancer. Taxanes and vinca alkaloids are in widespread use and have demonstrated substantive therapeutic efficacy. Both classes of compounds bind directly to tubulin, a structural component of the mitotic spindle. The ubiquitous utilization of tubulin in cell division in both cancerous and normal cells, however, tempers the broad spectrum of activity of currently used antimitotics by significant toxicities in normal dividing tissue. Moreover, peripheral nerve cells that rely on microtubules to shuttle cargo along axonal processes are also damaged by tubulin-binding drugs. Thus, neutropenia and peripheral neuropathy are the most frequently cited dose-limiting toxicities of this class of chemotherapeutics. Here we report the preclinical assessment of AB-5, a structural and functional analog of the natural product diazonamide A. AB-5, like taxanes and vinca alkaloids, blocks cell division during mitosis. However, AB-5 works not by binding tubulin but rather through inhibition of a newly discovered role for ornithine-delta-aminotransferase in mitosis. We hereby report that, unlike other antimitotics, AB-5 is extremely well tolerated by mice when administered under conditions where the drug cures xenografted tumors as effectively as taxanes and vinca alkaloids. AB-5-treated mice show no weight loss, no change in overall physical appearance, and no evidence of neutropenia. These observations raise the possibility that AB-5 may have clinical utility for cancer therapy under conditions largely devoid of chemotherapeutic toxicity and suggest that further preclinical evaluation of AB-5 is warranted.

Prevention of ornithine cytotoxicity by nonpolar side chain amino acids in retinal pigment epithelial cells.

PURPOSE: To investigate the effect of amino acids on ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy (GA) of the choroid and retina. METHODS: RPE cells were treated with 0.5 mM 5-fluoromethylornithine (5-FMOrn), a specific and irreversible OAT inhibitor. OAT-deficient RPE cells were incubated with 10 mM ornithine in the presence of 20 mM of 1 of 18 amino acids or 10 mM 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of the amino acid transporter system L. Ornithine cytotoxicity and cytoprotective effects of each amino acid was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay 72 hours after treatment with ornithine in OAT-deficient RPE cells. Ornithine incorporation into RPE cells was evaluated using DL-[14C]ornithine. RESULTS: An MTT colorimetric assay revealed that small and large zwitterionic amino acids, but not acidic or basic amino acids, decreased ornithine cytotoxicity in OAT-deficient RPE cells. Incorporation of DL-[14C]ornithine by RPE cells decreased to 79% of the control level after incubation for 48 hours with 20 mM leucine, the most effective cytoprotective amino acid. Further, BCH prevented ornithine cytotoxicity in a dose-dependent manner. Both light and heavy chains of L-type amino acid transporter (LAT)-1, LAT2, y+LAT1, and 4F2hc were expressed in RPE cells. CONCLUSIONS: The present results demonstrate that L-type amino acid transporter(s) may be involved in protection against ornithine cytotoxicity in human RPE cells. Thus, amino acid transportation in RPE cells may be a good target for a new therapy for GA as well as other kinds of chorioretinal degeneration.

Is hyperprolinemia type I actually a benign trait? Report of a case with severe neurologic involvement and vigabatrin intolerance.

Hyperprolinemia type I is a deficiency of proline oxidase (McKusick 23950), leading to hyperprolinemia and iminoglycinuria, usually with renal involvement. Hyperprolinemia type I is considered a benign trait. We reported a case of hyperprolinemia type I with a severe neurologic disorder and without renal involvement. The patient had marked psychomotor delay and right hemiparesis. Epilepsy was characterized by status epilepticus or a cluster of seizures. Laboratory findings revealed elevated levels of proline in the serum, urine, and cerebrospinal fluid without delta1-pyrroline 5-carboxylate dehydrogenase in the plasma or urine. Fluorescence in situ hybridization excluded a chromosome 22q11 deletion. Vigabatrin inhibits ornithine transaminase. Thus, vigabatrin could lead to a depletion of the normal pool of pyrroline 5-carboxylate dehydrogenase and could aggravate the clinical condition of the child. In this study, vigabatrin was discontinued. In the following months, the patient had marked psychomotor improvement, without modification of the epilepsy. We suggest that vigabatrin should be avoided in hyperprolinemia type I.

Heterogeneity in ornithine cytotoxicity of bovine retinal pigment epithelial cells in primary culture.

Gyrate atrophy of the choroid and retina is a chorioretinal degeneration caused by hyperornithinemia and a deficiency of ornithine-delta-aminotransferase (OAT). We recently showed that ornithine exhibits cytotoxicity to human retinal pigment epithelial (RPE) cell lines treated with the OAT inhibitor, 5-fluoromethylornithine (5-FMOrn), and suggested that this system may be an in vitro model of gyrate atrophy. In the present study, in order to apply this system to primary cultured RPE cells, we freshly prepared RPE cells from bovine eyes and studied the effect of ornithine on cell damage. Two phenotypes, epithelioid and fusiform, which coexisted in the primary culture and epithelioid phenotype cells, but not fusiform ones, were severely damaged and partially detached from the substrate by 10 m m ornithine and 0.5 m m 5-FMOrn. Neither ornithine nor 5-FMOrn alone exhibited such cytotoxicity to both phenotypes of RPE cells. Proline significantly prevented the ornithine-induced cytotoxicity. Epithelioid and fusiform phenotypes isolated from the primary culture showed different distribution of actin filaments. A combination of ornithine and 5-FMOrn time-dependently inhibited [(3)H]thymidine incorporation in the epithelioid, but not fusiform, cells. Proline prevented the inhibition of [(3)H]thymidine incorporation by ornithine in 5-FMOrn-treated epithelioid cells. Furthermore, l -azetidine-2-carboxylic acid, a collagen synthesis inhibitor, reduced [(3)H]thymidine incorporation in epithelioid, but not fusiform, cells, which was reversed by proline. These results demonstrate that the epithelioid phenotype of bovine RPE cells becomes susceptible to ornithine following inactivation of OAT. The phenotypic cells and its prevention by proline may provide insight into biochemical triggers that induce gyrate atrophy.

Ornithine aminotransferase, a potential target for the treatment of hyperammonemias.

Ornithine-delta-aminotransferase (OAT) (EC 2.6.1.13) is a pyridoxal-5' phosphate dependent mitochondrial matrix enzyme. It controls the L-ornithine (Orn) level in tissues by catalysing the transfer of the delta-amino group of Orn to 2-oxoglutarate. The products of this reaction are L-glutamate-gamma-semialdehyde and L-glutamate. Among the compounds known to inhibit (or inactivate) OAT, only L-canaline and (SS)-5-(fluoromethyl)ornithine [(SS)-5FMOrn] are selective for OAT. Treatment of laboratory animals with 5FMOrn causes a dramatic accumulation of Orn in most tissues and organs, and the enhanced formation of urea due to saturation of ornithine:carbamoyltransferase with its substrate. The enhancement of urea formation by increased endogenous levels of Orn is comparable with that produced by large doses of Orn and arginine, a treatment known to enhance the detoxification of ammonia. However, protection to lethal doses of ammonium salts by exogenous Orn is rapidly fading. In contrast, inactivation of OAT by a small dose of 5FMOrn renders a long-lasting protective effect against various forms of hyperammonemic states. Among these the reduction of ammonia concentrations in blood and tissues, and the reduction of the pathologic excretion of orotic acid to normal levels in mice with hereditary defects of the urea cycle, were most impressive. In human hereditary OAT deficiency the elevated intraocular concentrations of Orn are considered to be a cause of gyrate atrophy. This is presumably the reason, why OAT has not been considered as a therapeutically useful target. Chronic inactivation of OAT by repeated administration of 5FMOrn, caused elevated intraocular Orn concentrations, but this treatment had no effect on the function and histology of the visual system, or the behaviour of adult mice. The confirmation of this and related observations in higher species will show, whether OAT inactivation has potentials in the treatment of hyperammonemic states.

Crystal structure of human ornithine aminotransferase complexed with the highly specific and potent inhibitor 5-fluoromethylornithine.

Ornithine aminotransferase (l-ornithine:2-oxoacid delta-aminotransferase; EC 2.6.1.13), a pyridoxal-5'-phosphate-dependent mitochondrial enzyme controls the l-ornithine level in tissues by catalyzing the transfer of the delta-amino group of l-ornithine to 2-oxoglutarate, producing l-glutamate- gamma-semialdehyde and l-glutamate. (2S, 5S)-5-Fluoromethylornithine is the only inhibitor exclusively specific for ornithine aminotransferase known to date. Both in vitro and in vivo, it blocks the enzyme by a suicide reaction leading to a covalent adduct with the cofactor. The crystal structure of the enzyme-inhibitor complex was solved at a resolution of 1.95 A. No significant conformational changes compared with the native enzyme structure were observed. The structure reveals the atomic details of the cofactor-inhibitor adduct and its interactions with the active site of the enzyme. The main residues responsible for specific binding of the inhibitor are Arg180, which forms a strong salt bridge with the alpha-carboxylate and Tyr55, which is involved in a short hydrogen bond with the alpha-amino group. The experimental observation that in the racemic mixture, (2S, 5S)-5-fluoromethylornithine is exclusively responsible for the enzyme inhibition can be explained on the basis of the active site topology. Model building studies strongly suggest that the natural substrate l-ornithine, in its external aldimine adduct with the enzyme, makes use of the same recognition site as the inhibitor. It is proposed that the neutralization of the active site Arg413 by a salt bridge with Glu235 also plays an important role in productive binding of both 5-fluoromethylornithine and l-ornithine. Arg180 and Arg413 are believed to be instrumental in recognition of l-glutamate, by binding its gamma and alpha-carboxylate groups, respectively. This requires a different side-chain conformation of Glu235. Lys292 is the only obvious candidate for catalyzing the rate-limiting proton transfer steps in the transamination reaction.

Prevention of ornithine cytotoxicity by proline in human retinal pigment epithelial cells.

PURPOSE: To investigate the relationship between ornithine-delta-aminotransferase (OAT) deficiency and ornithine accumulation and the specific degeneration of retinal pigment epithelial (RPE) cells in gyrate atrophy. METHODS: Human RPE cells, human hepatoma cells, and human fibroblast cells were treated with 5-fluoromethylornithine (5-FMOrn), a specific irreversible inhibitor of OAT. Ornithine cytotoxicity was determined by using a [3H]thymidine incorporation assay and immunohistochemical staining for cytokeratin. The effects of various metabolites of ornithine and arginine, such as creatine, creatine phosphate, I-delta 1-pyrroline-5-carboxylic acid (L-P5C), and proline, which may be deficient in gyrate atrophy on RPE cell damage by ornithine, were determined by the same procedures. RESULTS: When the human RPE cells, HepG2 hepatoma cells, and WI-38 fibroblast cells were treated with 0.5 mM 5-FMOrn for 30 minutes, which inactivated OAT, ornithine exhibited severe time- and dose-dependent inhibition of DNA synthesis in the human RPE cells but not in the HepG2 hepatoma cells or WI-38 fibroblast cells. The inhibition of DNA synthesis was accompanied by drastic changes in morphologic appearance, disorganization of the cytoskeleton, and cell death. Ornithine or 5-FMOrn alone did not exhibit such cytotoxicity to the RPE cells. Proline prevented the cytotoxicity of ornithine. CONCLUSIONS: These findings suggest that an elevated level of ornithine combined with an increased sensitivity to ornithine as a result of OAT deficiency may be crucial to the specific RPE degeneration in gyrate atrophy. They suggest also that abnormalities of proline metabolism may be involved in the progress of gyrate atrophy.