Inhibition of Ornithine Aminotransferase - American Chemical Society

Chapter 15

Inhibition of Ornithine Aminotransferase: A New Target for Therapeutic Intervention 1

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Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 9, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0639.ch015

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J. B. Dueep , K. Jund , B. Lesur , S. Sarhan , M. Schleimer , P. R. Zimmermann , and N. Seiler 2

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Strasbourg Center, Marion Merrell Dow Research Institute, 16 rue d'Ankara, 67080 Strasbourg Cedex, France Groupe de Recherche en Thérapeutique Anticancéreuse, Unité de Recherche Associé au Centre National de la Recherche Scientifique 1529, Institut de Recherche Contre le Cancer, Faculté de Médecine, Université de Rennes, 2 avenue du Pr. Léon Bernard, F-35043 Rennes Cedex, France 2

5-Fluoromethylornithine (5-FMorn) is an irreversible inhibitor of ornithine aminotransferase (OAT). Among the four enantiomers, only one enantiomer had inhibitory activity. (S,S)-5FMorn 2a was synthesized and found to be the active enantiomer. OAT inhibition was shown to enhance other ornithine metabolic pathways, such as the urea cycle and polyamine formation. Thus, 2a improved ammonia detoxification through enhancement of urea formation. Therefore it can be expected that 2a will be of therapeutic value in diseases characterized by elevated concentrations of ammonia in blood and cerebrospinal fluid. Increased polyamine formation may support tissue and nerve regeneration after trauma.

Ornithine (2,5-diaminopentanoic acid; Orn) and the most abundant inhibitory neurotransmitter amino acid, 4-aminobutyric acid (GABA), have certain features in common. Their major catabolic pathways are initiated by the transfer of the ω-amino groups to 2-oxoglutarate. These reactions are catalyzed by similar pyridoxal phosphate-dependent enzymes. Both amino acids are present in virtually all tissues of the vertebrate organism, though at different concentrations: in accordance with its neurotransmitter function GABA concentrations are highest in brain (1), whereas highest Orn concentrations are found in liver (2,3), in agreement with its role in urea formation. Due to its well established physiological role all aspects of GABA metabolism have been extensively studied (4), and several inhibitors of 4-aminobutyrate:2oxoglutarate aminotransferase (GABA-T) were synthesized. One of the inactivators of GABA-T became an antiepileptic drug (5). In contrast, our knowledge of the physiological and pharmacological aspects of Orn is incomplete. Only with the availability of the racemic mixture of 5-fluoromethylornithine (MDL 72912, (R/S)-

0097-6156/96/0639-0196S15.00/0 © 1996 American Chemical Society

Ojima et al.; Biomedical Frontiers of Fluorine Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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5FM0rn) (6), it became possible to study consequences of the selective inactivation of ornithine:2-oxoglutarate aminotransferase (OAT) in vivo. It became apparent (7) that only one out of the four enantiomers, namely (S,S)-6-fluoro-2,5-diarninohexanoic acid, was an inactivator of OAT. Therefore, the synthesis of this compound became of major interest.


2. Biochemical Implications 2.1. Ornithine Metabolism. In liver ornithine is at the crossing of three major metabolic pathways (Fig. 1): a) . Orn reacts with carbamoyl phosphate to form citrulline. This reaction, catalyzed by ornithinexarbamoyltransferase (OCT), is the first step of the urea cycle. The final product of the reaction sequence is arginine, which is hydrolyzed by arginase to form Orn and urea. The role of the urea cycle is the elimination of ammonia from the organism, the most important endogenous CNS toxicant. Citrulline formation occurs nearly exclusively in the liver. In most other organs OCT activity is low or absent. However, most tissues contain arginase. In muscle Orn is formed by transfer of the amidino group of arginine to glycine (Fig. 1). b) . Orn is decarboxylated by ornithine decarboxylase (ODC) to form putrescine (1,4butanediamine). This reaction is the initial, highly regulated step of polyamine formation (8,9). The transformation of putrescine to spermidine, and the formation of spermine from spermidine is catalyzed by specific synthases. The polyamines are constitutents of all cells. They are required to ensure basic functions, such as growth, proliferation and differentiation. In agreement with their ubiquitous occurrence the decarboxylation of Orn is a general reaction, it plays, however, quantitatively a minor role, compared to the other reactions of Orn. c) Orn reacts with 2-oxoglutarate to form glutamic acid and glutamic acid semialdehyde. OAT, the enzyme which catalyzes this reaction, is a mitochondrial matrix enzyme. It is presumably present in most vertebrate tissues. Glutamic acid semialdehyde is mainly transformed to glutamic acid by glutamic acid semialdehyde dehydrogenase, but it is also a precursor of proline, which may be formed via Δ pyrroline 5-carboxylic acid (Fig. 1). There are not only metabolic, but also regulatory interrelationships between Orn and GABA (10), even though most of the glutamate used in the brain to form GABA is formed by hydrolysis of glutamine, not by the pathway shown in Fig. 1. It is evident from the above statements that the transamination of Orn and its role as precursor of putrescine are general, whereas its function within the urea cycle is nearly exclusive for liver. The relative rates of the three reactions of Orn change with physiologic and also under pathologic conditions. To mention only one example: In the case of malignant liver tumors, the activities of both OCT and OAT are reduced, whereas ODC activity is enhanced (11). Increased activities of ODC and enhanced formation of putrescine are usually signalling the increase of cell proliferation rates of both normal (embryonal development) and tumor cells (12). 1

2.2. Ornithine: 2-Oxoacid Aminotransferase (OAT). The analysis of the gene structure suggests one expressed gene and several pseudogenes (13,14), but there are observations in favor of the existence of more than one form of OAT. The enzyme has been purified e.g. by Peraino et al. (15) from several sources ( mol. wt. 43 - 45 kDa). Its physical properties have been extensively studied. For example, a preliminary x-ray diffraction study of human recombinant OAT has been reported (16), the amino acid sequence, including that of the active site of the enzyme is known (17,18), and the quaternary structure of the enzyme from pig kidney has been studied by high-resolution electron microscopy (19). The activity of the liver OAT is affected by multiple factors among which hormones and diet are most important.


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Putrescine dAdoMet

Spermidine dAdoMet

Spermine Glu

GABA

Homocamosine

2rOxogu ltarate

IGABA-Tj

Proline

Glutamate

τ SSAFig. 1. Majorreactionsinvolved in ornithine metabolism arginase; arginine:glycine amidinotransferase; ornithine:2-oxoacid aminotransferase (OAT), ornithinexarbamoyltransferase (OCT); ornithine decarboxylase (ODC). Abbreviations: Arg arginine, Cit citrulline, Gly glycine, Glu glutamic acid, GluSA glutamic acid semialdehyde, GAD glutamic acid decarboxylase, GABA 4-aminobutyric acid, GABA-T 4-aminobutyric acid:2-oxoacid aminotransferase, dAdoMet decarboxylated S-adenosylmethionine. Enzymes:


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2.3. Inhibitors of OAT. It has been mentioned that OAT is a pyridoxal phosphatedependent enzyme (15). The key step in the transamination sequence is the abstraction of the pro-S hydrogen atom at C5 (20). These properties suggested the search for inhibitors of OAT. 2.3.1. Pyridoxal Phosphate Scavengers. Non-specific inhibition of OAT by carbonyl reagents was predictable. For example 0.2 mM amino-oxyacetic acid inhibits almost completely OAT from rat liver mitochondria (21). This compound is known to inhibit also GABA-T, and many other transaminases and glutamate decarboxylase (22,23). /-Canaline [(5)-2-amino-amino-oxybutyric acid], a close structural analog of Orn, is a rather potent and selective inhibitor of OAT (24). Scavenging of pyridoxal phosphate as the mechanism of inactivation has been established (25,26). Inactivation is not restricted to the natural S enantiomer, however, the R enantiomer reacts considerably slower with the enzyme (7). Although rather selective in vitro, /-canaline is not well suited for in vivo studies. It appears to be rapidly metabolized. Consequently high doses are required, and the effects are relatively short lasting, as has been shown for the racemate (7,10). 2.3.2. Enzyme-activated Irreversible Inhibitors. Enzyme-activated irreversible inhibitors (mechanism based inactivators) are relatively unreactive molecules which require a considerable structural similarity to the natural substrate, in order to allow competition for binding within the active site of the enzyme, and activation by the catalytic reaction (27,28). The close relationship between GABA-T and OAT is documented by the fact that most enzyme-activated irreversible inhibitors of GABA-T [(S/ft)-4-arnino-5-hexynoic acid; (S)-4-amino-,5,6-heptadienoic acid; 5amino-l,3-cyclohexadienyl carboxylic acid (gabaculin)] are potent inactivators of OAT, both in vitro and in vivo (29-31). Mono or difluorinated methyl analogs of 3-aminopropionic acid, GABA and homoGABA were also found to be irreversible inhibitors of both GABA-T and/or OAT. (Table 1). Based on this knowledge, (/fc/S)-5FMOrn was conceived as a potential inactivator of OAT. Indeed, (/Ê/S)-5FMOrn exhibits concentration- and time-dependent inactivation of OAT in vitro. The inactivation rate is reduced in the presence of Orn, indicating competition for binding at the active site of the enzyme. No significant inhibitory effect is exerted on OCT, but (&$)-5-FMOrn is decarboxylated by ODC at a rate approximately 100-times slower than Orn. The compound did not inactivate GABA-T, and even long-term administration of the compound did not show any evidence for the accumulation of GABA in brain (3). In mice it caused a dose- and time-dependent decrease of OAT activity, virtually in all tissues. A dose of 25 mg/kg given intraperitoneally produced maximal (85 - 95%) inactivation between 1 and 24 h. The extent of OAT inhibition depended on the tissue; a fraction of OAT activity present in most tissues was refractory to inactivation by (/£/S)-5FMOrn, but was inhibited competitively (33). During the time of maximal inhibition of OAT, Orn concentrations increased dramatically in all organs, including the brain. However, even chronic administration of the compound to mice at daily intraperitoneal doses of 10 - 20 mg/kg, or by oral administration (with the drinking fluid) at an average daily dose of 40 mg/kg did not produce any toxicology, or gross behavioral changes (3). Specific attention was paid to the possibility of the development of gyrate atrophy, since it was believed (34) that elevated Orn concentration in the eye is a major pathogenetic factor in gyrate atrophy of the choroid and retina. 3. Consequences and Therapeutic Rationale of OAT Inhibition (i£/S)-5FMOrn is the first compound that allows one to produce long lasting elevations of tissue Orn concentrations without producing toxic effects. Therefore, potential therapeutic applications of OAT inhibition were envisaged. Since Orn accumulates rapidly in virtually all organs of the vertebrate organism one has to Ojima et al.; Biomedical Frontiers of Fluorine Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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assume that transamination is indeed the major general catabolic pathway of Orn, and that Orn cannot be eliminated at an appropriate rate by urinary excretion. A consequence of the elevation of Orn concentrations during long-term blockade of OAT is its channeling into the remaining pathways (Fig. 1). The enhanced formation of putrescine, and of citrulline could be expected, unless the enzymes responsible for the key reactions were saturated under physiological conditions. 3.1. Enhancement of Polyamine Metabolism. Administration of (#/S>5FMOrn elevates only moderately putrescine concentrations in tissues, and has no significant effect on the concentrations of spermidine and spermine. However, as was established for mouse brain, the turnover of spermidine is increased by 100% (35). The increase of polyamine concentrations is avoided due to the close regulation of their intracellular concentration (8,9), but as the experiments demonstrated, an enhanced flux of the putrescine moiety along the polyamine metabolic pathway was obvious. Major efforts of the last 15 years were devoted to establish methods for the inhibition of polyamine formation, in order to prevent neoplastic growth (36). Potential targets for the opposite, namely the enhancement of polyamine biosynthesis, are therapeutically required growth processes, specifically the regeneration of injured tissues. It is known that administration of the 2-oxoglutarate salt of Orn has, among others, beneficial effects in surgical and other trauma (37,38), and in burn injury (39). Most probably the enhancement of polyamine formation contributes to the favorable effect of this treatment (40,41). After injury of their axons, neurons appear to shift their metabolic activity into a reparative mode, aimed at survival and regeneration. Alternatively they undergo degeneration and die. Treatment with polyamines is known to enhance functional regeneration of peripheral nerves after axotomy (42-45) and favors the survival of neurons after axonal injury or ischemia (46,47). These few examples may be sufficient to indicate potential applications for the inhibition of OAT in an area of great therapeutic importance. Up to now very little work has been done concerning effects of (/2/5)-5FMOrn on models of tissue regeneration. This may change with the general availability of the active enantiomer of 5FMOrn. 3.2. Detoxification of Ammonia by Enhancement of Urea Formation. Inactivation of OAT by (/Ê/S)-5FMOrn is not only evident from the enhanced citrulline and urea formation and the suppression of the ammonia-induced pathologic excretion of orotic acid (48,49), but also from the impressive protection of treated animals from lethal intoxication with ammonium salts. Pretreatment with 0.042 mmol/kg of the drug before administration of 13 mmol/kg ammonium acetate is sufficient to protect 90% of the animals from death for more than 10 h (50). By comparison, 20 mmol/kg of arginine or Orn are needed to achieve a short lasting (

Inhibition of Ornithine Aminotransferase - American Chemical Society

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