Inhibitors of brain GABA aminotransferase

Comp. Biochem. PhysioL Vol. 93A, No. 1, pp. 247-254, 1989

0300-9629/89 $3.00 + 0.00 © 1989 Pergamon Press plc

Printed in Great Britain

MINI REVIEW INHIBITORS OF BRAIN GABA AMINOTRANSFERASE G. TUNNICLIFF Laboratory of Neurochemistry, Indiana University School of Medicine, 8600 University Boulevard, Evansville, IN 47712, USA (Received 9 August 1988)

INTRODUCTION

4-Aminobutyrate (GABA) plays a particularly crucial role in brain function and, indeed, is the most widely distributed inhibitory chemical transmitter in the central nervous system (Fonnum and Storm-Mathison, 1978). This amino acid is released from GABAergic nerve terminals upon stimulation and its action is initiated after it binds to its recognition site on the GABA/benzodiazepine receptor complex situated in the membrane of the postsynaptic neuron (Olsen, 1981). Binding of transmitter to the GABA^ site of the receptor complex opens up specific ion channels, allowing the entry of CI- ions into nerve cell. This results in a hypcrpolarization of the postsynaptic target cell and an increase in the threshold for firing (Krnjevic, 1974). The antagonist, bicuculline, acts as a competitive inhibitor at the GABA^ site (Curtis et al., 1971; Zukin et al., 1974). Comparatively recently, a second GABA receptor has been discovered that is insensitive to bicuculline but requires Ca 2+ for ligand binding. This recognition site, which is not linked to CI- channels, is known as the GABAB receptor (Bowery, 1983). The rapid removal of the amino acid from the immediate area of the relevant receptor terminates GABA transmission. This is achieved through the action of a Na+-dependent GABA pump located in the presynaptic membrane and in surrounding glial cell outer membranes (Martin, 1976). The biosynthesis of GABA takes place in the cytoplasm of the nerve terminal, whereas its metabolic degradation occurs in the mitochondria of both the presynaptic neuron and adjacent glial cells. This catabolism is occasioned by the combined catalytic action of two enzymes--GABA amino-transferase (GABA-T; EC 2.6.1.19) and succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24)--bound together in the form of a stable complex in the mitochondrial matrix (Head and Churchich, 1984). This enzyme complex converts GABA to succinate via the intermediate succinic semi-aldehyde. During the reaction, ~-ketoglutarate acts as an amino group acceptor and, as a consequence, is converted to glutamate. Pyridoxal 5'phosphate is required as cofactor for the initial transamination step, and NAD + is a coenzyme for the accompanying oxidation.

Based on the theory of Cleland (1963), experimental data obtained by a number of workers (Schousboe et aL, 1974; Maitre et al., 1975; White and Salto, 1978) suggests that the mechanism of the transamination is of the ping pong, bi-bi type. This means that GABA is the first substrate to bind at the active site and by giving up its amino group to pyridoxal 5'-phosphate, is converted to succinic semialdehyde, which can dissociate from the GABA-T active site and find its way to the adjacent catalytic site of SSADH. The ~,-ketoglutarate then binds to the active site of GABA-T and receives the amino group from the cofactor. This results in the formation of the second product, glutamate. There is virtually no accumulation of succinic semialdehyde since the activity of the dehydrogenase is greater than that of the aminotransferase (Miller and Pitts, 1967). The rate of catabolism of GABA is about twice that of its biosynthesis, thus the rate limiting step in GABA metabolism is the conversion of glutamate to GABA by means of L-glutamate decarboxylase (see review by Tunnicliff and Ngo, 1986).

INHIBITORS

lnhibitors interfering with GABA binding Many compounds are now known that can adversely affect the catalytic activity of the GABAT/SSADH complex. The vast majority of these substances are inhibitors of GABA-T, and most can be classified as structural analogues of GABA that behave as competitive inhibitors of the enzyme. One of the earliest reports of inhibition of brain GABA-T described the effects of hydroxylamine (Baxter and Roberts, 1961). Although this compound does not bare a striking structural resemblance to GABA, it was reported to be a competitive inhibitor when GABA was the varied substrate, and to exhibit an inhibition constant (Kt) of about 0.2 raM. Intraperitoneal injection of hydroxylamine HCI to rats (75 mg/kg) resulted in an elevation in GABA coneentrations and an inhibition of GABA-T activity in homogenates of brain prepared 90 min later. As seems to be the case with many of the competitive inhibitors of GABA-T, hydroxylamine lacks specificity since it is a carbonyl trapping agelat which is capable of inhibiting other pyridoxal Y-phosphate

247

248

G. TUNN1CLIFF

dependent enzymes by forming oximes with the cofactor (Roberts and Simonsen, 1963). Another of the earlier known inhibitors was amino-oxyacetic acid (AOAA) (Wallach, 1960, 1961), a drug that structurally resembles G A B A much more closely than does hydroxylamine. The kinetic study of the inhibition was carried out on GABA-T extracted from E. coli rather than on the enzyme from mammalian brain. The kinetics were consistent with the idea that the drug competes with G A B A binding to the active site of GABA-T and with a very high potency (K~ = 7.5/~M). More recently, the inhibition kinetics have been investigated employing the enzyme from rat brain. It was confirmed that A O A A competed with G A B A binding in a remarkably potent manner and, in fact, the enzyme was about 10 times as sensitive to the drug as was the bacterial GABA-T (Tunnicliff et al., 1977b; Ngo and Tunnicliff, 1978). Following peripheral administration (500mg/kg), A O A A was shown to cross the blood-brain barrier and after about 4 hr to dramatically increase brain levels of G A B A (Van Gelder, 1966). There was a concomitant inhibition of GABA-T which appeared to be responsible for the elevated amino acid concentration. The effects of this drug are not confined to GABA-T, however. Other studies have revealed that L-glutamate decarboxylase is also inhibited which might account for the seizures often associated with A O A A administration (Wood, 1975). The lack of specificity of this compound can be attributed to its being a carbonyl-trapping agent and therefore able to inhibit any pyridoxal 5'-phosphate dependent reaction. Another structural analogue of G A B A that can inhibit GABA-T activity is L-cycloserine which was reported to compete with G A B A binding with moderate potency (Scotto et al., 1963). Intraperitoneal injection of the drug into mice (300 mg/kg) caused a pronounced elevation of brain G A B A concentration, 2 hr later, together with an inhibition of GABA-T, the time course of which tended to follow that of the changes in GABA. Cycloserine was not very specific for GABA-T since L-glutamate decarboxylase also underwent some inhibition, albeit much less than the transaminase. Van Gelder (1968) reported that hydrazinopropionic acid, another structural analogue of GABA, appears to bind to the active site of GABA-T and in so doing acts as a competitive inhibitor. The K~ was calculated as 0.23pM. The administration of 20 mg/kg of hydrazinopropionic acid to mice gave rise to the complete inhibition of GABA-T, although the author failed to state at what time after administration this effect occurred. Brain G A B A levels were markedly elevated 6 hr after the injection of the drug. L-Glutamate decarboxylase was also inhibited; however, this enzyme was far less sensitive than GABA-T, for it underwent a maximum inhibition of only 20%, 4 hr after drug administration. The stable adduct 4-aminobutyrate-pyridoxal Y-phosphate has been synthesized and tested on rat brain GABA-T activity (Tunnicliff et al., 1977a). It was found to be a powerful inhibitor of the enzyme (Ki = 1.4/~M) and probably acts by competing for the binding of G A B A to the active site. D,L-Homocysteine, by contrast, is a comparatively

weak inhibitor of mouse brain GABA-T (Tunnicliff and Ngo, 1977). This amino acid exhibits a dual mode of action. It competes with G A B A for the active site (Ki = 6 mM), but also forms a complex with pyridoxal Y-phosphate, thus interfering with the catalytic process by a second mechanism. In common with several GABA-T inhibitors, homocysteine lacks specificity since it also inhibits the activity of L-glutamate decarboxylase. Buu and Van Gelder (1974) observed that the analogues, 4-amino-3-chlorobutyric acid and 4-amino-3-phenylbutyric acid, were competitive inhibitors of mouse brain G A B A aminotransferase. For the synaptosomal enzyme the K~ values were calculated as 0.18 and 6.7raM, respectively. A related compound, 3-chlorophenyiGABA, is the antispastic drug, baclofen, which has a high affinity for GABAB receptors (Bowery, 1983) but is a relatively weak inhibitor of GABA-T (Curtis et al., 1974). 2,4-Diaminobutyric acid and 4-N-hydroxydiaminobutyric acid also have an affinity for the active site of GABA-T (Iversen and Kelly, 1975; Tunnicliff et al., 1977b; Beart and Johnston, 1973). Both drugs, however, are weak inhibitors. A number of straight chain and branched chain fatty acids have been tested as inhibitors of GABA-T (Fowler et al., 1975; Maitre et al., 1978). Those fatty acids that had an effect did so as competitive inhibitors with respect to GABA. One of these was valproic acid, now a clinically important antiepileptic agent, particularly useful in the treatment of absence and myoclonic seizures. This drug appears to be an extremely weak inhibitor, however (K~ = 4 2 m M ) ; because of this there is some doubt that dipropylacetate's antiepileptic action in humans is related to its ability to inhibit GABA-T in vivo despite the fact that in mice administration leads to increased brain concentrations of G A B A and to an inhibition of GABA-T (Simler et al., 1973; Anlezark et al., 1976). Propionic acid, n-butyric acid and n-valeric acid were also inhibitors and were more potent than valproic acid. Imidazoleacetic acid, a structural analogue and strong agonist at the GABAA receptor, has also been reported to be an inhibitor of GABA-T in vitro. When G A B A was the varied substrate, however, a noncompetitive-type inhibition was observed (Clifford et al., 1973). The administration of imidazoleacetic acid to mice induced a hypnotic state (Roberts and Simonsen, 1963) and whole brain levels of GABA have been found to increase (Clifford et al., 1973). The in vivo effects of this drug on GABA-T have yet to be evaluated. Trans-4-Aminocrotonic acid and 2-methylaminocrotonic acid are apparent inhibitors of the enzyme by competing with GABA. However, both of these compounds are able to act as amino group donors and thus are substrates for the transamination reaction. In fact, they appear to be even better substrates than G A B A itself (Allan and Johnston, 1983). Silverman and Levy (1981a) have shown that 4-amino-3-chlorobutyric acid, the competitive inhibitor studied by Buu and Van Gelder (1974), can act as a substrate for GABA-T, being about oneseventh as efficient as GABA. The compound has a relatively low Km value (about 0.05mM) when

GABA-T inhibitors compared to the natural substrate. Interestingly though, the enzyme catalyzes an exclusive elimination of the chloride group and results in the formation of succinic semialdehyde. This analogue also undergoes a slight transamination but this represents only about 0.2% of the elimination reaction. The related compound, 4-amino-3-fluorobutyric acid, behaved similarly to the chloro analogue. Subsequent work originating from Silverman's laboratory has revealed that 4-amino-2-(substituted methyl)butenoic acids are competitive inhibitors with a potencies far greater than other known substituted GABA derivatives (Silverman et al., 1986). The hydroxy-, chloro- and fluoro-derivatives exhibited Ki values in the micfomolar range. 4-Aminotetrolic acid and trans-2-(aminomethyl)cyclopropane carboxylic acid are conformationally restricted analogues of GABA and both have been demonstrated to be inhibitors of brain GABA-T (Beart et al., 1972; Allan et al., 1980; Tunnicliff et al., 1988). The first of these compounds, however, did not display great potency (Ki = 0.58 mM). Moreover, the second compound can act as a substrate and is about half as efficient as GABA as an amino group donor for the transamination reaction. The drug 1-(N-decyl)-3-pyrazolidinone (BW 357U) has been synthesized at the Welcome Research Laboratories and found to be a potent inhibitor of GABA-T (White et al., 1983). There is evidence, though, that the ring moiety of this compound undergoes hydrolysis in aqueous solution to yield a structural analogue of hydrazinopropionic acid and that it is the latter substance that is the actual inhibitor. The presence of 1 mM GABA completely abolished the inhibitory effect of 0.4 #M BW 357U. Oral administration of this compound (30 mg/kg) to rats resulted in a large increase in GABA levels in the brain and in a corresponding decrease in GABA-T activity; L-glutamate decarboxylase activity was also slightly inhibited. lnhibitors interfering with o~-ketoglutarate binding

Very few compounds are known that interfere with ~-ketoglutarate binding to the catalytic site of the enzyme, at least with reasonable potency. One of the few inhibitors discovered is l-propyl-3,5-dicyano-4-phenyl-6-hydroxy-2-pyridone that has been reported to irreversibly inhibit GABA-T in vitro. The rate of inactivation is reduced in the presence of ~-ketoglutarate, whereas GABA has no such effect (Ciesielski et al., 1979). The mechanism of this inactivation is unknown although it does seem to depend on an initial binding at the cc-ketoglutarate recognition site on the enzyme. Inhibitors interfering with cofactor binding

One of the most potent inhibitors of GABA-T is AOAA-pyridoxal 5'-phosphate, a stable adduct formed when the cofactor is allowed to react with amino-oxyacetic acid (Churchich, 1982). This compound competes with pyridoxal 5'-phosphate binding and has an exceptionally high affinity for the enzyme (Ki = 0.01/~M). Another pyridoxal 5'-phosphate analogue, 4-vinylpyridoxal Y-phosphate, is also a powerful inhibitor (Ki--0.2/~M).

249

Using GABA-T extracted from pig kidney, Vasil'ev and Nikolaeva (1972) demonstrated that a series of cofactor analogues competitively inhibited the enzyme with respect to pyridoxal Y-phosphate. For example, 5-hydroxypropylpyridoxal 5'-phosphate, the strongest of the inhibitors, yielded an inhibition constant of I0 pM. Medina 0963) studied the action of a series of hydrazines on GABA metabolism and reported that the administration of hydrazine, dimethylhydrazine and monomethylhydrazine each inhibited brain GABA-T activity. These drugs act by forming complexes with pyridoxal Y-phosphate and consequently are not specific inhibitors of the enzyme. Similarly, Massieu et al. 0964) demonstrated that the carbonyltrapping agent, L-glutamic acid-T-hydrazide, inhibited brain GABA-T in vivo and increased GABA levels after administration to adult mice; L-glutamate decarboxylase activity was also decreased, although to a lesser extent. Irreversible inhibitors Suicide inhibitors. A suicide, mechanism-based (or k~,J inhibitor is an unreactive substrate analogue that binds to the active site and is converted by the enzyme to a highly reactive species which typically forms a covalent bond with an essential amino acid residue, thus inactivating the enzyme. Ethanolamine-O-sulphate (Fowler and John, 1972) binds to the catalytic site of GABA-T and undergoes an enzyme-induced sulphate group elimination. This generated a//,y-unsaturated imine, capable of alkylating nucleophilic residues. This compound does not significantly penetrate the blood-brain barrier but in vivo studies have been carried out by intracerebroventricular administration to audiogenic seizure susceptible mice. Twenty-four hr later, brain GABA concentrations were increased 4--10 fold, and GABA-T activity was inhibited over 50%. Moreover, these animals were completely protected against seizures (Anlezark et ai., 1976). A number of halogen-substituted analogues of GABA have been found to act as mechanism-based irreversible inhibitors of GABA-T. Silverman and Levy (1980, 1981b) reported that 4-amino-5fluoropentanoic acid inactivated the pig brain enzyme if the latter was in the pyridoxal Y-phosphate form. Apparently, this compound crossed the blood-brain barrier, since an intraperitoneal injection to mice (100mg/kg) produced sedation and raised brain GABA concentrations dramatically. Subsequent work has demonstrated that this compound does, indeed, inactivate GABA-T in vivo (Silverman et al., 1983). A close analogue, 4-amino-5-fluoropent-2enoic acid, also behaves as a mechanism-based inactivator (Silverman et al., 1986; Silverman and George, 1988a). Further work from the same laboratory has led to the discovery that 4-amino-2-fluorobut-2-enoic acid inactivates pig brain GABA-T by normal catalytic isomerization followed by active site nucleophilic attack (Silverman and George, 1988b). Bey et al. (1981) studied nine different fluoroderivatives of GABA and related structures and observed that five of them inactivated GABA-T isolated from pig brain in a time-dependent manner.

250

G. TUNNICLIFF

The most potent of these inhibitors were 3-amino-4fluorobutyric and 3-amino-4,4-difluorobutyric acids (Ki = 1.7 and 2 mM, respectively). Administration of either drug to mice produced inhibition of GABA-T and a rapid elevation of brain GABA. In vivo, the most potent inhibitor was 3-amino-4-fluorobutyric acid (ICs0 = 7 mg/kg). Indeed, this is one of the most effective inhibitors of GABA-T known. L-Glutamate decarboxylase activity was also modestly inhibited. y-Acetylenic G A B A inactivates partially purified pig brain GABA-T in a time-dependent manner (Metcalf et aL, 1979). The presence of G A B A dramatically slows the rate of inactivation. If ~-ketoglutarate is added at this stage, the rate of inactivation again increases. It was concluded that the enzyme has to be in the pyridoxal form in order for the inhibitor to bind at the active site. y-Acetylenic G A B A is not specific for GABA-T since it can also inactivate L-glutamate decarboxylase, albeit not nearly as effectively, y-Vinyl GABA, on the other hand, is a similar structural analogue that is much more specific as an inhibitor (Lippert et al., 1977). It is virtually without effect on L-glutamate decarboxylase. When y-acetylenic G A B A was administered to rats it caused a substantial inhibition of brain GABA-T and a dose-dependent increase in G A B A levels (Jung et al., 1977). y-Vinyl G A B A also inhibits GABA-T and elevates brain G A B A after peripheral administration to mice (Fig. 1). This latter drug has been demonstrated to offer protection against many types of experimentally induced seizures (Schechter et al., 1979; Meldrum and Horton, 1978; Iadarola and Gale, 1981). As a result of this anticonvulsant action, y-vinyl G A B A has undergone a number of clinical trials in patients suffering from intractable seizures. On the whole the results have been encouraging. Both severity and frequency of epileptic episodes were reduced in many of the patients (see review by Hammond and Wilder, 1985). The related compound, y-allenyl GABA, inactivates isolated GABA-T in a similar manner to the latter two drugs, only it is more potent. In vivo also, this drug is 2-3 times more effective at elevating brain G A B A levels and inhibiting GABA-T (Jung et al., 1984; Casara et al., 1984; Castelhano and Kranz, 1984).

-ii\o

The naturally occurring GABA analogue, gabaculline, has been isolated from Streptomyces toyocaenis and shown to be a potent irreversible inhibitor of mouse brain GABA-T (Rando and Bangerter, 1976). As with the previous kcat inhibitors, inactivation by gabaculline requires its catalytic turnover at the active site. The inhibitor undergoes a transamination and finally m-carboxyphenylpyridoxamine 5'-phosphate is formed after a spontaneous aromatization (Rando, 1977). The effects of gabaculline are extremely potent, with a K~ of 0.6/~M for binding to the enzyme. Gabaculline penetrates the blood-brain barrier; thus, after an intraperitoneal injection, GABA-T is inactivated and a concomitant increase in brain G A B A levels are observed (Rando and Bangerter, 1977). Mice, so treated, usually exhibit seizures followed by prolonged somnolence. Active site modifiers

Many chemicals are known that can react with amino acids and form stable complexes. The most useful of these compounds are those that are reasonably specific in their choice of amino acid. The chief difference between these agents and suicide inhibitors is that active site modifiers are reactive when they bind at the active site and do not require the catalytic activity of the enzyme to produce a reactive product. These agents are useful at pinpointing essential residues at the active site of enzymes. For instance, an enzyme can be incubated with an amino acid-specific reagent and will undergo an inactivation if that amino acid is crucial for catalytic activity. The effects of several of these reagents have been studied on GABA-T activity. Phenylglyoxal is fairly specific for arginine residues and when the enzyme was exposed to this reagent for varying lengths of time, a time-dependent inactivation was observed (Tunnicliff, 1980). Although neither G A B A nor -ketoglutarate was able to affect the rate of inactivation, pyridoxal 5'-phosphate was. Consequently, the evidence supported the idea that an arginine residue was present at or near the cofactor binding site of GABA-T. Schousboe et al. (1974) reported that p-chloromercurybenzoate and 5,5'-dithiobis-2-nitrobenzoate can inhibit GABA-T activity. Both compounds are sulfhydryl-group reactive and thus are indicative of 1400

.-.1oo ~ o

c~

75

•

GAD

e

~

•

1000

• "~

co >

50

o

~

~'~ --

•

GABA

-i

5°°

°-

• 10O

0

i

i

i

0

24

48

time after

i

a

i

i

72

96

120

144

injection (hr)

Fig, 1. Effects of an intraperitoneal dose of y-vinyl GABA (1.5 g/kg) on mouse brain GABA metabolism. Each value represents the mean of live animals. Redrawn from Metcalf et al. (1979).

GABA-T inhibitors the involvement of cysteine residues in GABA-T activity. Neither substrate, though, afforded protection against loss of activity. Corroborative results were obtained by Moses and Churchich (1980) who concluded that exposure of the enzyme to 5,5'-dithiobis-2-nitrobenzoate or N-iodoacetylaminoethyl 5-naphthylamine-l-sulfonate resulted in the loss of one sulfhydryl group per dimer of enzyme. These authors speculated that the reaction of an essential sulfhydryl group led to a conformational change in the enzyme, reducing its catalytic activity. Later work emanating from the same laboratory revealed that 2 SH groups per dimer led to an inactivation of the enzyme (Choi and Churchich, 1985) in the presence of N-(1-pyrene)maleimide. Confirmation of this result was obtained in the same laboratory by Kim and Churchich (1987a) who used iodosobenzoate to induce an inactivation by reacting with cysteine residues. As with N-(l-pyrene)maleimide, the reaction of 2 SH residues per dimer was shown to be responsible for the loss of catalytic activity. The presence of ct-ketoglutarate, but not GABA, protected the enzyme from inhibition. The binding of ~t-ketoglutarate to the active site may involve a lysine residue. It has been demonstrated that o-phthalaldehyde modifies six lysine residues per dimer but this effect can be prevented by the presence of substrate (Kim and Churchich, 1981). This conclusion was supported by later data which showed that adenosine triphosphopyridoxai and a dialdehyde derivative of l,Nr-etheno-ATP reacted with lysine residues at the active site and produced a time-dependent inactivation (Kim and Churchich, 1987b). Both effects could be reduced by the presence of ~-ketoglutarate. Other inhibitors

Recent experiments conducted in this laboratory (unpublished observations) seem to demonstrate that ATP behaves as a competitive inhibitor of partially purified mouse brain GABA-T when G A B A is the varied substrate (Fig. 2). The inhibition constant was calculated as 2.9 mM. These experiments were prompted by the report of Carr et al. (1986) which showed that pig brain GABA-T could be phosphorylated by ATP under appropriate conditions. We, on the other hand, could find no evidence of phosphorylation of the mouse brain enzyme and, indeed, ATP's inhibitory effects were reversible. These results open up the intriguing possibility that within the nervous

251

4 .5

~ . x , ~

• control 0 2ram ATP • 4ram ATP

_

V 2

2

4

6

v/s Fig. 2. Eadie--Hofstee plot of the effects of ATP on partially purified mouse brain GABA-T. Each point is the mean of three determinations. Velocity of reaction (V) = #tool/ rain/rag protein. GABA concentration ($) given in millimolarity, a-Ketogiutarate and pyridoxal phosphate concentrations were I mM and 50 #M, respectively.

system, GABA-T may be under regulatory control by ATP. The short-acting barbiturate thiopental is used clinically as a general anesthetic. It has been demonstrated that this drug can adversely affect the metabolic breakdown of G A B A / n vitro (Cheng and Brunner, 1979). Thiopental behaves as a noncompetitive inhibitor with respect to both G A B A and ~t-ketoglutarate (Ki = 1 mM). In an experiment in which the /n vivo effects of several monoamine oxidase inhibitors on G A B A metabolism were investigated, Popov and Matthies (1969) observed that phenelzine, phenylpropylhydrazine and phenylvalerylhydrazine decreased GABA-T activity 4 hr after administration, and at the same time produced an elevation of G A B A levels. The most likely reason that these drugs were able to inhibit GABA-T is that they are carbonyl-trapping compounds and therefore interfered with cofactor availability for the transamination reaction. SIGNIFICANCE AND SUMMARY

The knowledge gained in studying inhibitors of GABA-T can help us understand the nature of the enzyme, particularly the active site, and the significance of this enzyme in G A B A metabolism. Information on the conformation of the active site can be gained from the structure of those substratc analogues that have the greatest affinity for the

Table 1. S u m m a r y o f effects o f some competitive inhibitors o f G A B A - T in vitro Inhibitor AOAA-PLP VinylPLP B W 357U AOAA Hydrazinopropionic acid GABA-PLP 5-HydroxypropylPLP 2'-lsopropylPLP 4-Amino-2-hydroxymethylbutenoic acid 4-Amino-2-chloromethylbutcnoic acid 4-Amino-2-fluoromethylbutcnoic acid 4-Amino-3-chlorobutyric acid Hydroxylamine

Substrate whose binding is c o m p r o m i z c d Cofactor Cofactor GABA GABA GABA GABA Cofactor Cofactor GABA GABA GABA GABA GABA

Ki (M) 1x 2 x 2 x 6 x 2.3 x 1.4 x 1x 7 x 5 x 2.4 x 1.9 x 1.8 x 2 x

10 -8 10 -7 10 - s 10 - s 10 -7 10 -6 10 -5 10 - s 10 -6 10 -5 10 -5 10 -4 10 -4

252

G. TUNNICLIFF Table 2. Summaryof effect of certain irreversibleinhibitors on GABA-T in vitro Site of interaction Inhibitor on enzyme Ki (M) Ethanolamine-O-sulfate GABA 4.4 × 10 - 4 Gabacuiline GABA 2.9 X 10 - 6 ~,-AcetylenicGABA GABA 8 x 10-5 y-Vinyl GABA GABA n.r. 4-Amino-5-fluoropentanoicacid GABA 4 × 10-4 3-Amino-4-fluorobutyricacid GABA 1.7 x 10-3 3-Amino-4,4-difluorobutyricacid GABA 2 x 10-3 I-Propyldicyanophenylhydroxypyridone • -KG n.r. n.r. = Not ~'ported.

enzyme. A O A A and hydrazinopropionic acid are two of the most potent inhibitors and they have flexible structures, indicating that G A B A does not bind to the catalytic site in a rigid conformation. Indeed, no conformationally restricted analogue is a strong inhibitor of GABA-T. The use of compounds that react specifically with a particular amino acid can yield considerable information on the nature of essential residues at G A B A - T ' s active site. The knowledge gained could assist in the synthesis and development of more powerful inhibitors. In addition, there is little doubt of the importance of manipulating G A B A function in clinical disorders. For example, several anticonvulsant agents could well be effective by inhibiting G A B A - T activity. Valproic acid is such a drug which has wide use in the treatment of certain types o f epilepsy. In addition, the anti-epileptic action of certain experimental drugs has been linked to their ability to inhibit brain G A B A - T activity---ethanolamine-O-sulphate and 7-vinyl G A B A , for instance. Knowledge of the optim u m structural requirements of these antiepileptic agents should lead to the development of more effective inhibitors. In addition to the anti-epileptic action of certain G A B A - T inhibitors, m o u n t i n g evidence points to the potential use of such inhibitors in the reduction of arterial pressure and thus to their potential benefit in the treatment of hypertension (Loscher, 1982). The inhibition of G A B A - T by A T P may not have obvious pharmacological implications, but it does prompt the question as to whether this effect has any tie with the regulation of the enzyme in the intact, functioning central nervous system. Both A T P and G A B A - T are components of the mitochondria. The concentrations of A T P calculated to be present in the vicintiy of the enzyme are in the millimolar range (Mcllwain and Bachelard, 1971), which is apparently quite adequate to have a pronounced effect on enzyme activity and thus to influence the metabolic breakdown of GABA.

REFERENCES

Allan R. D., Johnston G. A. R. and Twitchin B. (1980) Synthesis of analogues of GABA. V. trans and c/s Isomers of some 4-amino-3-halogenobut-2-enoic acids. Aus. J. chem. 33, 1115-1122. Allan R. D. and Johnston G. A. R. (t983) Synthetic analogs for the study of GABA as a transmitter. Med. Res. Rev. 3, 91-118. Anlezark G., Horton R. W., Meldrurn B, S. and Sawaya M. C. (1976) Anticonvulsant action of ethanolamine-Osulphate and di-n-propylacetate and the metabolism of

7-aminobutyric acid (GABA) in mice with audiogenic seizures. Biochem. Pharmacol. 25, 413--417. Baxter C. F. and Roberts E. (1961) Elevation of 7-aminobutyric acid in brain: selective inhibition of ~/-aminobutyric-,v-ketoglutaric acid transaminase. J. biol. Chem. 236, 3287-3294. Beart P. M. and Johnston G. A. R. (1973) Transamination of analogues of ~,-aminobutyric acid by extracts of rat brain mitochondria. Brain ICes. 49, 459-462. Beart P. M., Uhr M. L. and Johnston G. A. R. (1972) Inhibition of GABA transaminas¢ activity by 4-aminotetrolic acid. J. Neurochem. 19, 1849-1854. Bey P., Jung M. J., Gerhart F., Schirlin D., Van Dorsselaer V, and Casara P. (1981) co-Fluoromethyl analogues of co-amino acids as irreversible inhibitors of 4-aminobutyrate: 2-oxogiutarate arninotransferase. J. Neurochem. 37, 1341-1344. Bowery N. G. (1983) Classification of GABA receptors. In The GABA Receptors (Edited by Enna S. J.), pp. 177-214. Humana Press, Clifton, New Jersey. Buu N. T. and Van Gclder N. M. (1974) Biological actions in vivo and in vitro of two ~-aminobutyric acid (GABA) analogues: ~-Chloro GABA and/~-phenyl GABA. Br. J. Pharmacol. 52, 401-406. Carr R. K., Schlichter D., Spielholtz C. and Wicks W. D. (1986) In vitro phosphorylation of 4-aminobutyrate aminotransferase by cAMP dependent protein kinase. J. cyclic nucl. Prot. Phosph. Res. 11, 11-23. Casara P., Jund K. and Bey P. (1984) General synthetic access to ~-allenyl amines and a-allenyl-,~-aminoacidsas potential enzyme activated irreversible inhibitors of PLP dependent enzymes. Tet. Lett. 25, 1891-1894. Castelhano A. L. and Krantz A. (1984) Allenic amino acids. 1. Synthesis of 7-allenic GABA by a novel aza-Cope rearrangement. J. Am. Chem. Soc. 106, 1877-1879. Cheng S-Z. and Brunner E. A. (1979) Thiopental inhibition of 7-aminobutyrate transaminase in rat brain synaptosomes. Biochem. Pharmacol. 28, 105-109. Choi S-Y. and Churehich J. E. (1985) 4-Aminobutyrate aminotransferase reaction of sulfhydryl residues conneeted with catalytic activity. J. biol. Chem. 260, 993-997. Churchich J. E. (1982) 4-Aminobutyrate aminotransferase: different susceptibility to inhibitors, microenvironment of the cofactor binding site and distance of the catalytic sites. Fur. J. Biochem. 126, 507-511. Ciesielski L., Simler S., Gensburger C., Mandel P., Taillandier G., Benoit-Guyod J. L., Boucherle A., CohenAddad C. and Lajzerowicz J. (1979) GABA transaminase inhibitors. Adv. exp. Med. Biol. 123, 21-41. Cleland W. W. (1963) The kinetics of enzyme-catalyzed reactions with two or more substrates or products. Biochim. biophys. Acta 67, 104-137. Clifford J. M., Tabcner P. V., TunnieliffG., Rick J. T. and Kerkut G. A. (1973) Biochemical and pharmacological actions of imidazoleacetic acid. Biochem. Pharmacol. 22, 535-542. Curtis D. R., Duggan A. W., Felix D. and Johnston G. A. R. (1971) Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord. Brain Res. 32, 69-96.

GABA-T inhibitors Curtis D. R., Game C. J. A., Johnston G. A. R. and MeCulloch R. M. (1974) Central effects of #-(p-chlorophenyl)-~,-aminobutyric acid. Brain Res. 70, 493-499. Fonnum F. and Storm-Mathisen J. (1978) Localization of GABAergic neurons in the CNS. Hndbk Psychopharmacol. 9, 357-401. Fowler L. J. and John R. A. (1972) Active-site-directed irreversible inhibition of rat brain 4-aminobutyrate aminotransferase by ethanolamine O-sulphate in vitro and in vivo. Biochem. J. 130, 569-573. Fowler L. J., Beckford J. and John R. A. (1975) An analysis of the kinetics of the inhibition of rabbit brain-aminobutyrate aminotransferase by sodium n-dipropylacetate and some other simple carboxylic acids. Biochem. Pharmacol. 24, 1267-1270. Hammond E. J. and Wilder B. J. (1985) Gamma-Vinyl GABA. Gen. Pharmacol. 16, 441-447. Hearl W. G. and Churchich J. E. (1984) Interactions between 4-aminobutyrate aminotransferase and succinic semialdehyde dehydrogenase, two mitochondrial enzymes. J. biol. Chem. 259, 11459-11463. Iadarola M. J. and Gale K. (1981) Cellular compartments of GABA in brain and their relationship to anticonvulsant activity. Molec. Cell. Biochem. 39, 305-330. Iversen L. L. and Kelly J. S. (1975) Uptake and metabolism of )'-aminobutyric acid by neurones and glial cells. Biochem. Pharmacol. 24, 933-938. Jung M. J., Lippert B., Metcalf B. W., Sehechter P. J., Bohlen P. and Sjoerdsma A. (1977) The effect of 4-aminohex-5-ynoic acid ()'-acetylenic GABA, )'-ethynyl GABA), a catalytic inhibitor of GABA-transaminase, on brain GABA metabolism in vivo. J. Neurochem. 28, 717-723. Jung M. J., Heydt J-G. and Casara P. (1984) ~,-Allenyl GABA, a new inhibitor of 4-amino butyrate amino transferase. A comparison with other inhibitors of this enzyme. Biochem. Pharmacol. 33, 3717-3720. Kim D. S. and Churchich J. E. (1981) 4-Aminobutyrate aminotransferase, the reaction of lysine residues connected with enzymatic activity. Biochem. Biophys. Res. Commun. 99, 1333-1340. Kim D. S. and Churchich J. E. (1987a) The reversible oxidation of vicinal SH groups in 4-aminobutyrate aminotransferase. J. biol. Chem. 262, 14250-14254. Kim D. S. and Churchich J. E. (1987b) Active site modification of 4-aminobutyrate aminotransferase with ATP analogs. Biochim. Biophys. Acta 916, 265-270. Krnjevic K. (1974) Chemical nature of synaptic transmission in vertebrates. Physiol. Rev. 54, 418-540. Lippert B., Metcalf B. W., Jung M. J. and Casara P. (1977) 4-Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric acid aminotransferase in mammalian brain. Eur. J. Biochem. 74, 441--445. Loscher W. (1982) Cardiovascular effects of GABA, GABA-aminotransferase inhibitors and valproic acid following systemic administration in rats, cats and dogs: Pharmacological approach to localize the site of action. Arch. Int. Pharmacodyn. 257, 32-58. Maitre M., Ciesielski L., Cash C. and Mandel P. (1975) Purification and studies on some properties of the 4-aminobutyrate: 2-oxoglutarate transaminase from rat brain. Eur. J. Biochem. 52, 157-169. Maitre M., Ciesielski L., Cash C. and Mandel P. (1978) Comparison of the structural characteristics of the 4-aminobutyrate: 2-oxoglutarate transaminases from rat and human brain, and of their affinities for certain inhibitors. Biochim. biophys Acta 522, 385-399. Martin D. L. (1976) Carrier-mediated transport and removal of GABA from synaptic regions. In GABA in Nervous System Function (Edited by Roberts E., Chase T. N. and Tower D. B.), pp. 347-386. Raven Press, New York.

253

Massieu G. H., Tapia R. I., Pasantes H. and Ortega B. G. (1964) Convulsant effect of L-glutamic acid-y-hydrazide by simultaneous treatment with pyridoxal phosphate. Biochem. Pharmacol. 13, 118-120. McIlwain H. and Bachelard H. S. (1971) Biochemistry and the Central Nervous System, 4th Edn. Churchill Livingstone, Edinburgh. Medina M. A. (1963) The in vivo effects of hydrazines and vitamin B6 on the metabolism of gamma-aminobutyric acid. J. Pharmacol. exp. Ther. 140, 133-137. Meldrum B. S. and Horton R. (1978) Blockade of epileptic responses in the photosensitive baboon Papio papio, by two irreversible inhibitors of GABA-transaminase, ~,-acetylenic GABA (4-amino-hex-5-ynoic acid) and )'-vinyl GABA (4-amino-hex-5-enoic acid). Psychopharmacology 59, 47-50. Metcalf B. W., Jung M. J., Casara P., Bthlen P. and Schechter P. J. (1979))'-Acetylenic GABA and ),-vinyl GABA--Two enzyme activated irreversible inhibitors of GABA aminotransferase. In GABA-Neurotransmitters (Edited by Krogsgaard-Larsen P., Scheel-Kriiger J. and Kofod H.), pp. 236-246. Munksgaard, Copenhagen. Miller A. L. and Pitts F. N. (1967) Brain succinate semialdehyde dehydrogenase--II. Activities in twenty-four regions of human brain. J. Neurochem. 14, 579-584. Moses U. and Churchich J. E. (1980) Reactivity of one thiol group in the dimeric protein, 4-aminobutyrate aminotransferase. Biochim. biophys. Acta 613, 392-400. Ngo T. T. and Tunnicliff G. (1978) Further evidence for the existence of isozyrnes of brain )'-aminobutyrate aminotransferase. Comp. Biochem. Physiol. 59C, 101-104. Olsen R. W. (1981) GABA-benzodiazepine-barbiturate receptor interactions. J. Neurochem. 37, 1-13. Perry T. L. and Hansen S. (1973) Sustained drug-induced elevation of brain GABA in the rat. J. Neurochem. 21, ! 167-1175. Popov M. and Matthies H. (1969) Some effects of monoamine oxidase inhibitors on the metabolism of )'-aminobutyric acid in the brain. J. Neurochem. 16, 899-907. Rando R. R. (1974) The chemistry and enzymology of kcat inhibitors. Science 185, 320-324. Rando R. R. (1977) Mechanism of the irreversible inhibition of v-aminobutyric acid ~-ketoglutaric acid acid transaminase by the neurotoxin gabaculline. Biochemistry 16, 4604-4610. Rando R. R. and Bangerter F. W. (1976) The irreversible inhibition of mouse brain GABA transaminase by gabaculline. J. Am. Chem. Soc. 98, 6762-6764. Roberts E. and Simonsen D. G. (1963) Some properties of L-glutamic decarboxylase in mouse brain. Biochem. Pharmacol. 12, 113-134. Schechter P. J., Tranier Y. and Grove Y. (1979) Attempts to correlate alterations in brain GABA metabolism by GABA-T inhibitors with their anticonvulsant effects. Adv. exp. Med. Biol. 123, 43-57. Schousboe A., Wu J.-Y. and Roberts E. (1974) Subunit structure and kinetic properties of 4-aminobutyrate-2ketogiutarate transaminase purified from mouse brain. J. Neurochem. 23, 1189-1195. Scotto P., Monaco P., Scardi V. and Bonavita V. (1963) Neurochemical studies with L-cycloserine, a central depressant agent. J. Neurochem. 10, 831-839. Silverman R. B., Durkee S. C. and Invergo B. J. (1986) 4-Amino-2-(substituted methyl)-2-butenoic acids: substrates and potent inhibitors of )'-aminobutyric acid aminotransferase. J. Med. Chem. 29, 764-770. Silverman R. B. and George C. (1988a) Mechanism of inactivation of )'-aminobutyric acid aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid. Biochem. biophys. Res. Commun. 150, 942-946. Silverman R. B. and George C. (1988b) Inactivation of )'-aminobutyric acid aminotransferase by (Z)-4-amino-2fluorobut-2-enoic acid. Biochemistry 27, 3285-3289.

254

G. TUNNICLIFI;

Silverman R. B. and Levy M. A. (1980) Irreversible inactivation of pig brain 1,-aminobutyric acid-~t-ketoglutarate transaminase by 4-amino-5-halopentanoic acids. Biochem. biophys. Res. Comraun. 95, 250--255. Silverman R. B. and Levy M. A. (1981a) Substituted 4-aminobutanoic acids: Substrates for y-aminobutyric acid a-ketoglutaric acid aminotransferase. J. biol. Chem. 256, 11565-11568. Silverman R. B. and Levy M. A. (1981b) Mechanism of inactivation of ~,-aminobutyric acid-~t-ketoglutaric acid aminotransferase by 4-amino-5-halopentanoic acids. Biochemistry 20, 1197-1203. Silverman R. B., Levy M. A., Muztar A. J. and Hirseh J. D. (1983) In vivo inactivation of ~-aminobutyric acid-~tketogtutarate transaminase by 4-amino-5-fluoropentanoic acid. Biochem. biophys. Res. Commun 102, 520--523. Simler S., Ciesielski L., Maitre M., Randrianarisoa H. and Mandel P. (1973) Effect of sodium n-dipropylacetate on audiogenic seizures and brain ~,-aminobutyric acid level. Biochem. Pharmacol. 22, 1701-1708. Tunnicliff G. (1980) Essential arginine residues at the pyridoxal phosphate binding site of brain y-aminobutyrate aminotransferase. Biochem. biophys. Res. Commun. 97, 160-165. Tunnicliff G. and Ngo T. T. (1977) The mode of action of homocysteine on mouse brain glutamic decarboxylase and ~-aminobutyrate aminotransferase. Can. J. Biochem. 55, 1013-1018. Tunnicliff G. and Ngo T. T. (1986) Regulation of 7-aminobutyric acid synthesis in the vertebrate nervous system. Neurochem. Int. 8, 287-297. Tunnieliff G., Ngo T. T. and Barbeau A. (1977a) N-(5'Phosphopyridoxyl)-4-aminobutyric acid: a stable bisubstrate adduct inhibitor of rat brain 4-aminobutyric acid aminotransferase. Experientia 33, 20-21. Tunnicliff G., Rogier C. J. and Youngs T. L. (1988) Inhibition of neuronal membrane GABAB receptor binding by GABA structural analogues. Int. J. Biochem. 20, 179-182.

Tunnieliff G., Ngo T. T., Rojo-Ortega J. M. and Barbeau A. (1977b) The inhibition by substrate analogues of ~,-aminobutyrate aminotransferase from mitochondria of different subcellular fractions of rat brain. Can. J. Biochem. 55, 479-484. Van Gelder N. M. (1966) The effect of aminooxyacetic acid on the metabolism of y-aminobutyric acid in brain. Biochem. Pharmacol. 15, 533-539. Van Gelder N. M. (1968) Hydrazinopropionic acid: A new inhibitor of aminobutyrate transaminase and glutamate decarboxylase. J. Neurochem. 15, 747-757. Yu Vasil'ev V. and Nikolaeva Z. K. (1973) A study of the effects of some analogs of pyridoxal 5'-phosphate and apo-~,-aminobutyrate ct-ketoglutarate aminotransferase. Biokhimiya 38, 1006-1014. Wallach D. P. (1960) The inhibition of gamma-aminobutyric acid-alpha-ketoglutaric acid transaminase/n vitro and in vivo by amino-oxyacetic acid. Biochem. Pharmacol. 5, 166-167. Wallach D. P. (1961) Studies on the GABA pathway--I. The inhibition of ~,,-aminobutyric acid-~t-ketoglutaric acid transaminase/n vitro and/n vivo by U-7524 (aminooxyacetie acid). Biochem. PharmacoL 5, 323-331. White H. L., Cooper B. R. and Howard J. L. (1983) Regulation of GABA-T: inhibition of GABA-T by BW 357U. In Glutamine, Glutamate, and GABA in the Central Nervous System (Edited by Hertz L., Kvamme E. MeGeer E. G. and Schousboe A.), pp. 145-159. Alan R. Liss, New York. White H. L. and Sato T. L. (1978) GABA-transaminase of human brain and peripheral tissues---kinetic and molecular properties. J. Neurochem. 31, 41-47. Wood J. D. (1975) The role of ~-aminobutyric acid in the mechanism of seizures. Progr. NeurobioL 5, 77-95. Zukin S. R., Young A. B. and Snyder S. H. (1974) Gammaaminobutyric acid binding to receptor sites in the rat central nervous system. Proc. natn. Acad. Sci. USA 71, 4802-4807.