- Email: [email protected]
Biochemical and pharmacological similarities and differences among four irreversible enzyme-activated GABA-T inhibitors
GABA Nrurotransmission Brain Rcsecrrch Bulldn, Vol. 5,
Suppl.2, pp.627-631. Printed in the U.S.A
Biochemical and Pharmacological Similarities and Differences Among Four Irreversible Enzyme-Activated GABA-T Inhibitors PAUL J. SCHECHTER Cmtre
AND JEFFREY
GROVE
de ~g~her~h~ ~e~rell ~nt~rn~tio~~~, 16, rue d~Ankur~, 67&34 ~tr~~~~o~r~ Cedex, Frmce
SCHECHTER, P. J. AND J. GROVE. Biochemical andpharmacological similarities and diJferences among four irreversible enzyme-u~tivated GABA-T inhibitors. BRAIN RES. BULL. 5: Suppl. 2,62743i, 1980.-The in viva biochemical and pha~acological effects of four irreversible enzyme-activated GABA-T inhibitors, y-acetylenic GABA (GAG), y-vinyl GABA (GVG), gabaculine (GBL), and isogabaculine (IGBL), were compared in mice following intraperitoneai administration. All four inhibit whole brain GABA-T activity and increase GABA concentrations in a dose-dependent manner with a relative potency of GBL=IGBL>GAG>GVG. Despite having no inhibitory effect on GAD activity in vitro, GBL, IGBL and GVG treatment decreases brain GAD activity in viva. GAG inhibits GADin vitro and in vivo. In all cases, however, the effects of these inhibitors on GABA-T activity are greater than on GAD. All four GABA-‘I’ inhibitors produce sedation, hypothermia, ptosis, piloerection and a characteristic posturing. At high doses all can produce periods of paradoxical excitation and convuisions with a relative incidence of GBL=IGBL>GAG>>GVG. All four protect susceptible mice against audiogenic seizures with a relative potency corresponding to their potency in increasing GABA concentrations. When tested against seizures produced by strychnine, isoniazid and tbiosemicarbazide, the protective effects of GAG and GVG differ from those of GBL and IGBL. Hence, despite similar mechanisms of enzyme inhibition, important biochemical and pharmacological differences exist among these GABA-T inhibitors. Gabaculine Isogabaculine Irreversible enzyme-activated GABA-T inhibitors Mouse brain GABA GABA-T and GAD activities y-Vinyl GABA
THE last 6 or 7 years have witnessed the emergence of a new and rational approach to the design of drugs capable of inactivating enzymes. These drugs have been termed “suicide enzyme inactivators”, “k catalytic inhibitors” or probably “enzyme-activated inhibitors” (for a more appropriately review, see [23]). They share the properties of themselves being relatively unreactive but with a close structural similarity to the natural substrate of the enzyme they are designed to inhibit. This structural similarity allows the molecule to be accepted as substrate by the enzyme and to be converted by the enzyme’s normal mechanism of catalysis to a reactive species which irreversibly inactivates the enzyme. Since, theoretically, only those enzymes able to accept and activate the latent reactive groups of the inhibitor will be affected, these drugs should be highly specific. Two such irreversible enzyme-activated inhibitors of GABA-T were designed and synthesized in our Center several years ago (Fig. I): y-acetylenic GABA (GAG; RMI 71.645) [7] and y-vinyl GABA (GVG; RMI 71.754) [lo]. A third such inhibitor, gabaculine (GBL), was initially isolated from a bacterial culture filtrate and then synthesized chemically in Japan [9]. Isogabaculine (IGBL; RMI 71.932), an isomer of gabaculine, has recently been synthesized in our laboratories [ 131. The proposed mechanisms of action and in vitro biochemical activities of the four GABA-T inhibitors have been de-
Copyright
0 1980 ANKHO
International
y-Acetylenic
GABA
scribed previously [12, 13, 141. In this vivo biochemical and pharmacological the four irreversible enzyme-activated and discuss the possible sign~cance of dissimilarities. In Vivo Biochemical
report we present in comparisons among GABA-T inhibitors their similarities and
Effects in Mice
When administered by parenteral or oral routes, GAG, GVG, GBL and IGBL inhibit brain GABA-T activity and increase brain GABA con~ent~tions. For example (Fig. 2), a single IP dose of 1500 mg/kg GVG produces a rapid decrease in brain GABA-T activity within 1 hour. By 3-4 hours after treatment, GABA-T activity is reduced to about 25% of control values and remains at this low level for at least 24 hours. Even five days after a single dose GABA-T activity is still less than that of untreated mice. Brain GABA concentrations rapidly increase following GVG injection, remain at a stable high level for 24 hours and then descend towards normal over several days. Whereas in vitro GVG has no inhibitory activity on GAD [lo], in vivo we find a slow but prolonged decrease in brain GAD activity reaching about 70% of control values. Following a single injection, GAG produces qualitatively similar alterations of GABA-T activity and brain GABA concentrations as GVG with some quantitative
Inc .-0361-9230/80/080627-05$01.00/O
628
SCHECHTER
/(r lj-ACETYLENIC G&A ( GAG, RMI Tl,645)
go bH
S-VINYL GA8A (WIG, RMI 71,754 1
6ABA
----
GAD ACTIVITY
-
GABA-T ACTIVITY
80
-
60
-
40
-
20
-
‘,L,,
GAG
GABACUCINE (GeLI
ISOGA~CU~INE I IGBL, RMI 71,932
0 IO
1
FIG. 2. Effect of y-vinyl GABA 1500 mgkg IP on whole brain and GAD activities and GABA concentration in mice as a function of time after injection. Each point and vertical bar represents the mean t SE of 6 mice. GABA-T
differences: GAG is approximately 10-15 times more potent than GVG [21] although the elevation in brain GABA is shorter, returning to control concentrations after l-2 days 1223. GBL and IGBL show identical in vivo biochemical effects on the brain GABA-ergic system, as one might expect from their similar chemical structures [20]. The time course of biochemical effects on the GABA system following GBL and IGBL differ from GAG and GVG. Thus, with GBL and IGBL maximum brain GABA concentrations are not reached until 16-24 hours [20] whereas folIowing GAG and GVG peak concentrations of GABA are apparent within 3 to 4 hours after an IP dose.
‘
( mg
I
.
looo3ooo
30501003oD DOSE
FIG. 1. Chemical structure of GABA and of four irreversible enzyme-activated GABA-T inhibitors.
AND GROVF
/
kg
i.p. )
FIG. 3. Dose-response of gabacuhne (GBL), isogabacuhne (IGBL), y-acetylenic GABA (GAG) and y-vinyl GABA (GVG) on brain GABA-T (solid lines) and GAD (dashed lines) activities following IP administration at the time of maximal effect on brain GABA (24 hours post-injection for GBL and IGBL and 4 hours for GAG and GVG).
A direct comparison of the influence of GBL, IGBL, GAG and GVG on whole brain activities of GABA-T and GAD as a function of dose at the time of maximal effects on GABA (4 hours after IP injection for GAG and GVG and 24 hours for GBL and IGBL) is shown in Fig. 3. In terms of GABA-T inhibition, GBL, IGBL and GAG appear approximately equipotent; GVG is considerably less potent. Curiously, GAG is capable of practically abolishing detectable GABA-T activity in brain while with the other inhibitors, a residual amount of enzyme activity is always observed. All four inhibitors produce a decrease in GAD activity to different extents. GAG has been shown to irreversibly inhibit GAD in vitro, although to a lesser extent than GABA-T 112,143. On the other hand, the other GABA-T inhibitors have no in vitro effect on GAD [ 10,131. Comparison of observed increases in brain GABA folfowing various doses of the four inhibitors (Fig 4) yields results not predictable from their effects on the GABA synthetic and catabolic enzymes. The slopes of the dose-response curves for GBL and IGBL are steeper than for GAG and GVG and the maximum GABA elevations possible with GBL and IGBL are approximately twice those attainable with GAG and GVG. Pharmacological
Effects in Mice
General behavior. GAG, GVG, GBL and IGBL all produce a dose-related decrease in spontaneous motor behavior. This is associated with a characteristic posture consisting of a hunched appearance with curved spine, splayed hind paws as well as piloerection, lacrimation, ptosis and
COMPARISON
629
OF GABA-T INHIBITORS .
GABICULINE
^, 2400 -
A
IGOWBACULlNE
\ 2
0
I-ACETYLENIC
0
X-VINYL
-
TABLE AUDIOGENIC
1
SEIZURE PROTECTION
IN MICE*
GABA
GABA
ED;,, (CL,,,)
2000
5 I=
Complete protection
Attenuation of seizure intensity
17 (l&18) 16 (14-19)
I5 (13-17) I4 (12-11)
41 (33-51) 990 (750-1307)
25 (18-33) 540 (367-794)
1600
Gabaculine Isogabaculine y-acetylenic GABA y-vinyl GABA
Values are mg/kg IP 24 hr postinjection hr postinjection for GAG and GVG. *Methods as described [21,22]. lo
30
50
loo
DOSE
300
so0
In00
3ooo
( mg/ kg i.p. 1
FIG. 4. Dose-response of gabaculine, isogabaculine, y-acetylenic GABA and y-vinyl GABA on brain GABA concentrations following IP administration at the time of maximal effect (24 hours postinjection for GBL and IGBL and 4 hours for GAG and GVG).
catatonia [ 191. There is a tendency for the mice to huddle together, perhaps prompted by the hypothermia caused by these agents. Many of these signs have been described following the administration to various species of other agents which augment brain GABA-ergic function, e.g., aminooxyacetic acid [24,26], hydrazinopropionic acid [25], ethanolamine-O-sulfate [2,4] and muscimol [151. Following treatment with moderate to high doses of GBL and IGBL (e.g., 30-50 mg/kg) sedation is interrupted by periods of intense excitation consisting of rapid running, jumping, myoclonus and clonic-type seizures. These are exaggerated by handling and occur in about 50% of the mice. Similar excitatory effects are seen after high doses of GAG (e.g., 100-150 mg/kg) but are less frequent (usually 0 to 20% of mice). GVG rarely produces CNS excitation (< 1% of mice) and myoclonus and convulsions have only been seen at single doses of 3000-3500 mg/kg. Antiseizure effects. Anticonvulsant effects of enhanced GABA-ergic function produced by a variety of means have been repeatedly demonstrated in laboratory animals. Indeed, the prevention of seizures appears to be the obvious pharmacological direction to follow with any new agent suspected of having GABA-facilitating properties. Among the animal models most widely used to test GABA drugs has been the audiogenic seizure mouse. GAG, GVG, GBL and IGBL are highly effective as anti-seizure agents in this model. The ED,,,‘s for audiogenic seizure protection (Table 1) follow the relative potency as GABA-T inhibitors with GBL and IGBL being identical and the most potent and GVG, the least potent. In addition, the time course of seizure protection after GAG and GVG closely parallels the elevations in brain GABA concentrations [21] and a significant linear correlation exists between brain GABA increase at 4 hours after treatment and degree of seizure protection [21]. When tested against other convulsants in mice less concordance among the 4 GABA-T inhibitors is evident. For example, GBL and IGBL do not influence tonic seizures or mortality produced by intravenous strychnine [20] whereas with higher doses,
for GBL and IGBL and 4
GAG and GVG protect 90-100% of mice [ 191. When seizures are induced by isoniazid or thiosemicarbazide, GAG and GVG pre-treatment prevent tonic seizures in 90-100% of mice [19] whereas GBL and IGBL demonstrate less of a protective effect [20]. GAG and/or GVG have also been found to have antiseizure activity in seizures induced by hyperbaric oxygen [27] and by 3-mercaptopropionic acid and pyridoxal phosphate glutamyl-y-hydrazone in mice [ 181, by photic stimulation in photosensitive baboons [ 111, in audiogenic seizures during alcohol withdrawal in rats [3], and in maximal electroshock seizures of rats [5]. GBL and IGBL have been found to be relatively ineffective against 3-mercaptopropionic acid-induced seizures (N. Seiler, personal communication) but, to our knowledge, these GABA-T inhibitors have not yet been tested in these other convulsant models. DISCUSSION
GBL, IGBL, GAG and GVG are four irreversible enzyme-induced inhibitors of GABA-T. Although they share a common mechanism of enzyme inactivation the biochemical consequences of treatment, in the whole brain of animals, differ. Whereas the degree of GABA-T inhibition is similar among the 4 inhibitors and the influence on GAD activity similar among GBL, IGBL and GVG, the elevations of brain GABA attainable with GBL and IGBL are considerably greater than with either GAG or GVG. These results suggest important dissimilarities in the compartmentalization of the increased GABA between GBL and IGBL on one hand and GAG and GVG on the other. Despite different biochemical effects, the 4 GABA-T inhibitors share various behavioral and pharmacological properties in mice. They all produce a generalized sedation with the assumption of a characteristic posture by the animals. They all decrease body temperature. In addition, they share the ability to protect susceptible mice against audiogenically-induced seizures. Many of these properties seem to be a common denomination of all agents capable of augmenting brain GABA-ergic function whether by inhibition of GABA-T or as direct-acting GABA agonists. On the other hand, GBL, IGBL, GAG and GVG have certain dissimilarities. They do not show the same anticonvulsant activity in other seizure models and differ markedly in their ability to produce excitation, myoclonus and frank convulsions. Thus, such stimulatory behavior is often
630
SCHECHTER
seen following GBL and IGBL treatment in mice and is rarely seen with GVG. The propensity of other, less specific, GABA-T inhibitors to induce CNS excitation and seizures has been related to their concomitant inhibition of GAD activity [28]. Such a mechanism is unlikely to be involved in the excitation seen following treatment with GBL, IGBL, GAG and GVG since we find no correlation between degree of GAD inhibition and relative frequency of excitation. The biochemical correlates of the behavioral and pharmacological similarities and dissimilarities among the 4 GABA-T inhibitors, therefore, are not obvious. It is apparent that a consideration of changes in whole brain GABA metabolism represents only a first approach and probably an oversimplification. Regional alterations in brain GABA metabolism following GABA-T inhibition [6] are unlikely to illuminate this point. Recently, differences in the distribution of the increased GABA between synaptosomal and nonsynaptosomal fractions prepared from cortex of mice treated with the four GABA-T inhibitors have been noted [ 181. Such an approach begins to address the question of what fraction of the GABA increase is found in the pre-synaptic compartment, which presumably is available for release upon
AND GROVE
neuronal stimulation, and which fraction is contained in other cellular loci. Such studies need to be extended and
correlations made between these differences in distribution and the spectrum of pharmacological activities seen. It is also likely that many of the effects observed with GAG, GVG, GBL and IGBL are due to actions other than those related to their effects on GABA metabolism per se. Despite a high degree of biochemical specificity, predictable from their proposed enzyme-activated mechanism. enzymes other than their intended targets may be inhibited. Thus for example, GAG, GBL and IGBL but not GVG also inhibit ornithine aminotransferase 18,131. These agents may also act as direct GABA antagonists [ 16,171. In addition, GBL has been reported to inhibit sodium-dependent uptake of GABA in rat brain slices [ 11. Hence the pharmacological activity of GAG, GVG, GBL and IGBL may involve a complex interplay of several independent actions, each action having a different dose-, time-, and, possibly, site-dependency. It is hoped that further studies of these 4 inhibitors and comparisons with other agents which augment GABA-ergic function, may help to unravel the complexities of GABA’s effects in the CNS.
REFERENCES 1. Allan, R. D., G. A. R. Johnston and B. Twitchin. Effects of gabaculine on the uptake, binding and metabolism of GABA.
12. Metcalf,
Neurosci. Lett. 4: 5i-54, 1977. _ 2. Baxter. M. G.. L. J. Fowler. A. A. Miller and J. M. G. Walker.
13. Metcalf, B. W. and M. J. Jung. Molecular basis for the irreversible inhibition of 4aminobutyric acid:2-oxoghttarate and L-omithine:2-oxoacid amino-transferases by 3-amino-1,5cyclohexadienyl carboxylic acid (isogabaculine). Molec. Phur-
Some behavioral and anticonvulsant actions in mice of ethanolamine-0-sulphate, an inhibitor of 4-aminobutyrate aminotransferase. Br. J. Pharmac. 47: 68lP, 1973. 3. Cooper, B. R., K. Viik, R. M. Ferris and H. L. White. Antagonisn of the enhanced susceptibility to audiogenic seizures during alcohol withdrawal in the rat by y-aminobutyric acid (GABA) and “GABA-mimetic” agents. J. Phnrmac. exp. Ther. 209: 396-403, 1979. 4. Horton, R. W., G. M. Anlezark,
C. B. Sawaya and B. S. Meldrum. Monoamine and GABA metabolism and the anticonvulsant action of di-n-propylacetate and ethanolamine-0-sulphate.
Eur. J. Pharmac. 41: 387-397, 1977. 5. Iadarola, M. J. and K. Gale. Evaluation
of increases in nervecomnerve-terminal-independent terminal-dependent vs partments of GABA in vivo: Correlation with anticonvulsant effects of GABA-T inhibition. Brain Res. Bull. 5: Suppl. 2, 13-19, 1980. 6. Jung, M. J. In viva biochemistry of GABA transaminase inhibition. In: Enzyme-Activated Irreversible Inhibitors, edited by N. Seiler, M. J. Jung and J. Koch-Weser. Amsterdam: Elsevier/ North Holland Biomedical Press, 1978, pp. 135-148. 7. Jung, M. J. and B. W. Metcalf. Catalytic inhibition of y-aminobutyric acid-u-ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog. Biochem. biophys. Res. Commun. 67: 301-306, 1975. 8. Jung, M. J. and N. Seiler. Enzyme-activated
irreversible inhibitors of L-ornithine:2-oxoacid aminotransferase. J. biol. Chem. 253: 7431-7439, 1978. 9. Kobayashi, K., S. Miyazawa, A. Terahara, H. Mishima and H. Kurihara. Gabaculine: y-Aminobutyrate aminotransferase inhibitor of microbial origin. Tetrahedron Lett. 7: 537-540, 1976. 10. Lippert, B., B. W. Metcalf, M. J. Jung and P. Casara. 4-Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase in mammalian brain. Eur. J. Biochem.
74: 441-445,
1977.
11. Meldrum. B. and R. Horton. Blockade of epileptic responses in the photosensitive baboon, Papio papio, by two irreversible inhibitors of GABA-transaminase. y-acetylenic GABA (I-aminohex-S-ynoic acid) and -y-vinyl GABA (4-amino-hex-5-enoic acid). Psychopharmacology 59: 47-50, 1978.
Pharmac.
muc.
B. W. Inhibitors 28: 1705-1712,
16: 539-545,
of GABA metabolism.
Biochcm.
1979.
1979.
14. Metcalf, B. W., B. Lippert and P. Casara. Enzyme-activated irreversible inhibition of transaminases. In: Enzyme-Activated Irreversible Inhibitors, edited by N. Seiler, M. J. Jung and J. Koch-Weser. Amsterdam: ElsevieriNorth Holland Biomedical Press, 1978, pp. 123-133. 15. Naik, S. R., A. Guidotti and E. Costa. Central GABA receptor agonists: Comparison of muscimol and baclofen. Neuropharmacology 15: 479-484, 1976. 16. Palfreyman, M. G., M. M. Robin, M. Zraika, C. R. Gardner and P. J. Schechter. Dyskinesia induced by intracerebral injection of GABA-T inhibitors: A striatal or cortical phenomenon? Brain Res. Bull. 5: Suppl. 2, 613-619, 1980. 17. Robin, M. M., M. G. Palfreyman and P. J. Schechter. Dyskinetic effects of intrastriatally injected GABA-transaminase inhibitors. Lifp Sci. 25: 1103-1110, 1979. 18. Sarhan, S. and N. Seiler. Metabolic inhibitors and subcellular distribution of GABA. J. Neurosci. Res. 4: 399-421, 1979. 19. Schechter, P. J. and Y. Tranier. The pharmacology of enzymeactivated inhibitors of GABA-transaminase. In: EnzymeActivated Irreversible Inhibitors, edited by N. Seiler, M. J. Jung and J. Koch-Weser. Amsterdam: Elsevier/North Holland Biomedical Press, 1978, pp. 149-162. 20. Schechter, P. J., Y. Tranier and J. Grove. Gabaculine and isogabaculine: In vivo biochemistry and pharmacology in mice. f,@ Sci. 24: 1173-1182, 1979. 21. Schechter, P. J., Y. Tranier, M. J. Jung and P. Bohlen. Audiogenic seizure protection by elevated brain GABA concentration in mice: Effects of y-acetylenic GABA and y-vinyl GABA, two irreversible GABA-T inhibitors. Eur. J. Pharmac. 45: 31%328,
1977.
22. Schechter, P. J., Y. Tranier, M. J. Jung and A. Sjoerdsma. Antiseizure activity of y-acetylenic y-aminobutyric acid: A catalytic irreversible inhibitor of y-aminobutyric acid transaminase. J. Pharmac. exp. Ther. 201: 606-612, 1977. 23. Seiler, N., M. J. Jung and J. Koch-Weser, (eds.) EnzymeActivated Irreversible Inhibitors. Amsterdam: Elsevier/North Holland Biomedical Press, 1978.
COMPARISON
OF GABA-T
INHIBITORS
24. Van Gelder, N. M. The effect of aminooxyacetic acid on the metabolism of y-aminobutyric acid in brain. Biochem. Phurmclc. 15: 533-539, 1966. 25. Van Gelder, N. M. Hydrazinopropionic acid: A new inhibitor of aminobutyrate transaminase and glutamate decarboxylase. .I. Neurochem. 15: 747-757, 1968. 26. Wallach, D. P. Studies on the GABA pathway. I. The inhibition of y-aminobutyric acid-a-ketoglutaric acid transaminase in vitro and in Live by U-7524 (amino-oxyacetic acid). Biochem. Phat’mJcJc.. 5: 323-331, 1961.
631
27. Wood, J. D., J. S. Durham and S. J. Peesker. Effect of dt-npropylacetate and y-acetylenic GABA on hyperbaric oxygeninduced seizures and GABA metabolism. Neurochem. Res. 2: 707-715, 1977. 28. Wood, J. D. and S. J. Peesker. Development of an expression which relates the excitable state of the brain to the level of GAD activity and GABA content, with particular reference to the action of hydrazine and its derivatives. J. Neurochem. 23: 703112. 1974.
Suppl.2, pp.627-631. Printed in the U.S.A
Biochemical and Pharmacological Similarities and Differences Among Four Irreversible Enzyme-Activated GABA-T Inhibitors PAUL J. SCHECHTER Cmtre
AND JEFFREY
GROVE
de ~g~her~h~ ~e~rell ~nt~rn~tio~~~, 16, rue d~Ankur~, 67&34 ~tr~~~~o~r~ Cedex, Frmce
SCHECHTER, P. J. AND J. GROVE. Biochemical andpharmacological similarities and diJferences among four irreversible enzyme-u~tivated GABA-T inhibitors. BRAIN RES. BULL. 5: Suppl. 2,62743i, 1980.-The in viva biochemical and pha~acological effects of four irreversible enzyme-activated GABA-T inhibitors, y-acetylenic GABA (GAG), y-vinyl GABA (GVG), gabaculine (GBL), and isogabaculine (IGBL), were compared in mice following intraperitoneai administration. All four inhibit whole brain GABA-T activity and increase GABA concentrations in a dose-dependent manner with a relative potency of GBL=IGBL>GAG>GVG. Despite having no inhibitory effect on GAD activity in vitro, GBL, IGBL and GVG treatment decreases brain GAD activity in viva. GAG inhibits GADin vitro and in vivo. In all cases, however, the effects of these inhibitors on GABA-T activity are greater than on GAD. All four GABA-‘I’ inhibitors produce sedation, hypothermia, ptosis, piloerection and a characteristic posturing. At high doses all can produce periods of paradoxical excitation and convuisions with a relative incidence of GBL=IGBL>GAG>>GVG. All four protect susceptible mice against audiogenic seizures with a relative potency corresponding to their potency in increasing GABA concentrations. When tested against seizures produced by strychnine, isoniazid and tbiosemicarbazide, the protective effects of GAG and GVG differ from those of GBL and IGBL. Hence, despite similar mechanisms of enzyme inhibition, important biochemical and pharmacological differences exist among these GABA-T inhibitors. Gabaculine Isogabaculine Irreversible enzyme-activated GABA-T inhibitors Mouse brain GABA GABA-T and GAD activities y-Vinyl GABA
THE last 6 or 7 years have witnessed the emergence of a new and rational approach to the design of drugs capable of inactivating enzymes. These drugs have been termed “suicide enzyme inactivators”, “k catalytic inhibitors” or probably “enzyme-activated inhibitors” (for a more appropriately review, see [23]). They share the properties of themselves being relatively unreactive but with a close structural similarity to the natural substrate of the enzyme they are designed to inhibit. This structural similarity allows the molecule to be accepted as substrate by the enzyme and to be converted by the enzyme’s normal mechanism of catalysis to a reactive species which irreversibly inactivates the enzyme. Since, theoretically, only those enzymes able to accept and activate the latent reactive groups of the inhibitor will be affected, these drugs should be highly specific. Two such irreversible enzyme-activated inhibitors of GABA-T were designed and synthesized in our Center several years ago (Fig. I): y-acetylenic GABA (GAG; RMI 71.645) [7] and y-vinyl GABA (GVG; RMI 71.754) [lo]. A third such inhibitor, gabaculine (GBL), was initially isolated from a bacterial culture filtrate and then synthesized chemically in Japan [9]. Isogabaculine (IGBL; RMI 71.932), an isomer of gabaculine, has recently been synthesized in our laboratories [ 131. The proposed mechanisms of action and in vitro biochemical activities of the four GABA-T inhibitors have been de-
Copyright
0 1980 ANKHO
International
y-Acetylenic
GABA
scribed previously [12, 13, 141. In this vivo biochemical and pharmacological the four irreversible enzyme-activated and discuss the possible sign~cance of dissimilarities. In Vivo Biochemical
report we present in comparisons among GABA-T inhibitors their similarities and
Effects in Mice
When administered by parenteral or oral routes, GAG, GVG, GBL and IGBL inhibit brain GABA-T activity and increase brain GABA con~ent~tions. For example (Fig. 2), a single IP dose of 1500 mg/kg GVG produces a rapid decrease in brain GABA-T activity within 1 hour. By 3-4 hours after treatment, GABA-T activity is reduced to about 25% of control values and remains at this low level for at least 24 hours. Even five days after a single dose GABA-T activity is still less than that of untreated mice. Brain GABA concentrations rapidly increase following GVG injection, remain at a stable high level for 24 hours and then descend towards normal over several days. Whereas in vitro GVG has no inhibitory activity on GAD [lo], in vivo we find a slow but prolonged decrease in brain GAD activity reaching about 70% of control values. Following a single injection, GAG produces qualitatively similar alterations of GABA-T activity and brain GABA concentrations as GVG with some quantitative
Inc .-0361-9230/80/080627-05$01.00/O
628
SCHECHTER
/(r lj-ACETYLENIC G&A ( GAG, RMI Tl,645)
go bH
S-VINYL GA8A (WIG, RMI 71,754 1
6ABA
----
GAD ACTIVITY
-
GABA-T ACTIVITY
80
-
60
-
40
-
20
-
‘,L,,
GAG
GABACUCINE (GeLI
ISOGA~CU~INE I IGBL, RMI 71,932
0 IO
1
FIG. 2. Effect of y-vinyl GABA 1500 mgkg IP on whole brain and GAD activities and GABA concentration in mice as a function of time after injection. Each point and vertical bar represents the mean t SE of 6 mice. GABA-T
differences: GAG is approximately 10-15 times more potent than GVG [21] although the elevation in brain GABA is shorter, returning to control concentrations after l-2 days 1223. GBL and IGBL show identical in vivo biochemical effects on the brain GABA-ergic system, as one might expect from their similar chemical structures [20]. The time course of biochemical effects on the GABA system following GBL and IGBL differ from GAG and GVG. Thus, with GBL and IGBL maximum brain GABA concentrations are not reached until 16-24 hours [20] whereas folIowing GAG and GVG peak concentrations of GABA are apparent within 3 to 4 hours after an IP dose.
‘
( mg
I
.
looo3ooo
30501003oD DOSE
FIG. 1. Chemical structure of GABA and of four irreversible enzyme-activated GABA-T inhibitors.
AND GROVF
/
kg
i.p. )
FIG. 3. Dose-response of gabacuhne (GBL), isogabacuhne (IGBL), y-acetylenic GABA (GAG) and y-vinyl GABA (GVG) on brain GABA-T (solid lines) and GAD (dashed lines) activities following IP administration at the time of maximal effect on brain GABA (24 hours post-injection for GBL and IGBL and 4 hours for GAG and GVG).
A direct comparison of the influence of GBL, IGBL, GAG and GVG on whole brain activities of GABA-T and GAD as a function of dose at the time of maximal effects on GABA (4 hours after IP injection for GAG and GVG and 24 hours for GBL and IGBL) is shown in Fig. 3. In terms of GABA-T inhibition, GBL, IGBL and GAG appear approximately equipotent; GVG is considerably less potent. Curiously, GAG is capable of practically abolishing detectable GABA-T activity in brain while with the other inhibitors, a residual amount of enzyme activity is always observed. All four inhibitors produce a decrease in GAD activity to different extents. GAG has been shown to irreversibly inhibit GAD in vitro, although to a lesser extent than GABA-T 112,143. On the other hand, the other GABA-T inhibitors have no in vitro effect on GAD [ 10,131. Comparison of observed increases in brain GABA folfowing various doses of the four inhibitors (Fig 4) yields results not predictable from their effects on the GABA synthetic and catabolic enzymes. The slopes of the dose-response curves for GBL and IGBL are steeper than for GAG and GVG and the maximum GABA elevations possible with GBL and IGBL are approximately twice those attainable with GAG and GVG. Pharmacological
Effects in Mice
General behavior. GAG, GVG, GBL and IGBL all produce a dose-related decrease in spontaneous motor behavior. This is associated with a characteristic posture consisting of a hunched appearance with curved spine, splayed hind paws as well as piloerection, lacrimation, ptosis and
COMPARISON
629
OF GABA-T INHIBITORS .
GABICULINE
^, 2400 -
A
IGOWBACULlNE
\ 2
0
I-ACETYLENIC
0
X-VINYL
-
TABLE AUDIOGENIC
1
SEIZURE PROTECTION
IN MICE*
GABA
GABA
ED;,, (CL,,,)
2000
5 I=
Complete protection
Attenuation of seizure intensity
17 (l&18) 16 (14-19)
I5 (13-17) I4 (12-11)
41 (33-51) 990 (750-1307)
25 (18-33) 540 (367-794)
1600
Gabaculine Isogabaculine y-acetylenic GABA y-vinyl GABA
Values are mg/kg IP 24 hr postinjection hr postinjection for GAG and GVG. *Methods as described [21,22]. lo
30
50
loo
DOSE
300
so0
In00
3ooo
( mg/ kg i.p. 1
FIG. 4. Dose-response of gabaculine, isogabaculine, y-acetylenic GABA and y-vinyl GABA on brain GABA concentrations following IP administration at the time of maximal effect (24 hours postinjection for GBL and IGBL and 4 hours for GAG and GVG).
catatonia [ 191. There is a tendency for the mice to huddle together, perhaps prompted by the hypothermia caused by these agents. Many of these signs have been described following the administration to various species of other agents which augment brain GABA-ergic function, e.g., aminooxyacetic acid [24,26], hydrazinopropionic acid [25], ethanolamine-O-sulfate [2,4] and muscimol [151. Following treatment with moderate to high doses of GBL and IGBL (e.g., 30-50 mg/kg) sedation is interrupted by periods of intense excitation consisting of rapid running, jumping, myoclonus and clonic-type seizures. These are exaggerated by handling and occur in about 50% of the mice. Similar excitatory effects are seen after high doses of GAG (e.g., 100-150 mg/kg) but are less frequent (usually 0 to 20% of mice). GVG rarely produces CNS excitation (< 1% of mice) and myoclonus and convulsions have only been seen at single doses of 3000-3500 mg/kg. Antiseizure effects. Anticonvulsant effects of enhanced GABA-ergic function produced by a variety of means have been repeatedly demonstrated in laboratory animals. Indeed, the prevention of seizures appears to be the obvious pharmacological direction to follow with any new agent suspected of having GABA-facilitating properties. Among the animal models most widely used to test GABA drugs has been the audiogenic seizure mouse. GAG, GVG, GBL and IGBL are highly effective as anti-seizure agents in this model. The ED,,,‘s for audiogenic seizure protection (Table 1) follow the relative potency as GABA-T inhibitors with GBL and IGBL being identical and the most potent and GVG, the least potent. In addition, the time course of seizure protection after GAG and GVG closely parallels the elevations in brain GABA concentrations [21] and a significant linear correlation exists between brain GABA increase at 4 hours after treatment and degree of seizure protection [21]. When tested against other convulsants in mice less concordance among the 4 GABA-T inhibitors is evident. For example, GBL and IGBL do not influence tonic seizures or mortality produced by intravenous strychnine [20] whereas with higher doses,
for GBL and IGBL and 4
GAG and GVG protect 90-100% of mice [ 191. When seizures are induced by isoniazid or thiosemicarbazide, GAG and GVG pre-treatment prevent tonic seizures in 90-100% of mice [19] whereas GBL and IGBL demonstrate less of a protective effect [20]. GAG and/or GVG have also been found to have antiseizure activity in seizures induced by hyperbaric oxygen [27] and by 3-mercaptopropionic acid and pyridoxal phosphate glutamyl-y-hydrazone in mice [ 181, by photic stimulation in photosensitive baboons [ 111, in audiogenic seizures during alcohol withdrawal in rats [3], and in maximal electroshock seizures of rats [5]. GBL and IGBL have been found to be relatively ineffective against 3-mercaptopropionic acid-induced seizures (N. Seiler, personal communication) but, to our knowledge, these GABA-T inhibitors have not yet been tested in these other convulsant models. DISCUSSION
GBL, IGBL, GAG and GVG are four irreversible enzyme-induced inhibitors of GABA-T. Although they share a common mechanism of enzyme inactivation the biochemical consequences of treatment, in the whole brain of animals, differ. Whereas the degree of GABA-T inhibition is similar among the 4 inhibitors and the influence on GAD activity similar among GBL, IGBL and GVG, the elevations of brain GABA attainable with GBL and IGBL are considerably greater than with either GAG or GVG. These results suggest important dissimilarities in the compartmentalization of the increased GABA between GBL and IGBL on one hand and GAG and GVG on the other. Despite different biochemical effects, the 4 GABA-T inhibitors share various behavioral and pharmacological properties in mice. They all produce a generalized sedation with the assumption of a characteristic posture by the animals. They all decrease body temperature. In addition, they share the ability to protect susceptible mice against audiogenically-induced seizures. Many of these properties seem to be a common denomination of all agents capable of augmenting brain GABA-ergic function whether by inhibition of GABA-T or as direct-acting GABA agonists. On the other hand, GBL, IGBL, GAG and GVG have certain dissimilarities. They do not show the same anticonvulsant activity in other seizure models and differ markedly in their ability to produce excitation, myoclonus and frank convulsions. Thus, such stimulatory behavior is often
630
SCHECHTER
seen following GBL and IGBL treatment in mice and is rarely seen with GVG. The propensity of other, less specific, GABA-T inhibitors to induce CNS excitation and seizures has been related to their concomitant inhibition of GAD activity [28]. Such a mechanism is unlikely to be involved in the excitation seen following treatment with GBL, IGBL, GAG and GVG since we find no correlation between degree of GAD inhibition and relative frequency of excitation. The biochemical correlates of the behavioral and pharmacological similarities and dissimilarities among the 4 GABA-T inhibitors, therefore, are not obvious. It is apparent that a consideration of changes in whole brain GABA metabolism represents only a first approach and probably an oversimplification. Regional alterations in brain GABA metabolism following GABA-T inhibition [6] are unlikely to illuminate this point. Recently, differences in the distribution of the increased GABA between synaptosomal and nonsynaptosomal fractions prepared from cortex of mice treated with the four GABA-T inhibitors have been noted [ 181. Such an approach begins to address the question of what fraction of the GABA increase is found in the pre-synaptic compartment, which presumably is available for release upon
AND GROVE
neuronal stimulation, and which fraction is contained in other cellular loci. Such studies need to be extended and
correlations made between these differences in distribution and the spectrum of pharmacological activities seen. It is also likely that many of the effects observed with GAG, GVG, GBL and IGBL are due to actions other than those related to their effects on GABA metabolism per se. Despite a high degree of biochemical specificity, predictable from their proposed enzyme-activated mechanism. enzymes other than their intended targets may be inhibited. Thus for example, GAG, GBL and IGBL but not GVG also inhibit ornithine aminotransferase 18,131. These agents may also act as direct GABA antagonists [ 16,171. In addition, GBL has been reported to inhibit sodium-dependent uptake of GABA in rat brain slices [ 11. Hence the pharmacological activity of GAG, GVG, GBL and IGBL may involve a complex interplay of several independent actions, each action having a different dose-, time-, and, possibly, site-dependency. It is hoped that further studies of these 4 inhibitors and comparisons with other agents which augment GABA-ergic function, may help to unravel the complexities of GABA’s effects in the CNS.
REFERENCES 1. Allan, R. D., G. A. R. Johnston and B. Twitchin. Effects of gabaculine on the uptake, binding and metabolism of GABA.
12. Metcalf,
Neurosci. Lett. 4: 5i-54, 1977. _ 2. Baxter. M. G.. L. J. Fowler. A. A. Miller and J. M. G. Walker.
13. Metcalf, B. W. and M. J. Jung. Molecular basis for the irreversible inhibition of 4aminobutyric acid:2-oxoghttarate and L-omithine:2-oxoacid amino-transferases by 3-amino-1,5cyclohexadienyl carboxylic acid (isogabaculine). Molec. Phur-
Some behavioral and anticonvulsant actions in mice of ethanolamine-0-sulphate, an inhibitor of 4-aminobutyrate aminotransferase. Br. J. Pharmac. 47: 68lP, 1973. 3. Cooper, B. R., K. Viik, R. M. Ferris and H. L. White. Antagonisn of the enhanced susceptibility to audiogenic seizures during alcohol withdrawal in the rat by y-aminobutyric acid (GABA) and “GABA-mimetic” agents. J. Phnrmac. exp. Ther. 209: 396-403, 1979. 4. Horton, R. W., G. M. Anlezark,
C. B. Sawaya and B. S. Meldrum. Monoamine and GABA metabolism and the anticonvulsant action of di-n-propylacetate and ethanolamine-0-sulphate.
Eur. J. Pharmac. 41: 387-397, 1977. 5. Iadarola, M. J. and K. Gale. Evaluation
of increases in nervecomnerve-terminal-independent terminal-dependent vs partments of GABA in vivo: Correlation with anticonvulsant effects of GABA-T inhibition. Brain Res. Bull. 5: Suppl. 2, 13-19, 1980. 6. Jung, M. J. In viva biochemistry of GABA transaminase inhibition. In: Enzyme-Activated Irreversible Inhibitors, edited by N. Seiler, M. J. Jung and J. Koch-Weser. Amsterdam: Elsevier/ North Holland Biomedical Press, 1978, pp. 135-148. 7. Jung, M. J. and B. W. Metcalf. Catalytic inhibition of y-aminobutyric acid-u-ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog. Biochem. biophys. Res. Commun. 67: 301-306, 1975. 8. Jung, M. J. and N. Seiler. Enzyme-activated
irreversible inhibitors of L-ornithine:2-oxoacid aminotransferase. J. biol. Chem. 253: 7431-7439, 1978. 9. Kobayashi, K., S. Miyazawa, A. Terahara, H. Mishima and H. Kurihara. Gabaculine: y-Aminobutyrate aminotransferase inhibitor of microbial origin. Tetrahedron Lett. 7: 537-540, 1976. 10. Lippert, B., B. W. Metcalf, M. J. Jung and P. Casara. 4-Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase in mammalian brain. Eur. J. Biochem.
74: 441-445,
1977.
11. Meldrum. B. and R. Horton. Blockade of epileptic responses in the photosensitive baboon, Papio papio, by two irreversible inhibitors of GABA-transaminase. y-acetylenic GABA (I-aminohex-S-ynoic acid) and -y-vinyl GABA (4-amino-hex-5-enoic acid). Psychopharmacology 59: 47-50, 1978.
Pharmac.
muc.
B. W. Inhibitors 28: 1705-1712,
16: 539-545,
of GABA metabolism.
Biochcm.
1979.
1979.
14. Metcalf, B. W., B. Lippert and P. Casara. Enzyme-activated irreversible inhibition of transaminases. In: Enzyme-Activated Irreversible Inhibitors, edited by N. Seiler, M. J. Jung and J. Koch-Weser. Amsterdam: ElsevieriNorth Holland Biomedical Press, 1978, pp. 123-133. 15. Naik, S. R., A. Guidotti and E. Costa. Central GABA receptor agonists: Comparison of muscimol and baclofen. Neuropharmacology 15: 479-484, 1976. 16. Palfreyman, M. G., M. M. Robin, M. Zraika, C. R. Gardner and P. J. Schechter. Dyskinesia induced by intracerebral injection of GABA-T inhibitors: A striatal or cortical phenomenon? Brain Res. Bull. 5: Suppl. 2, 613-619, 1980. 17. Robin, M. M., M. G. Palfreyman and P. J. Schechter. Dyskinetic effects of intrastriatally injected GABA-transaminase inhibitors. Lifp Sci. 25: 1103-1110, 1979. 18. Sarhan, S. and N. Seiler. Metabolic inhibitors and subcellular distribution of GABA. J. Neurosci. Res. 4: 399-421, 1979. 19. Schechter, P. J. and Y. Tranier. The pharmacology of enzymeactivated inhibitors of GABA-transaminase. In: EnzymeActivated Irreversible Inhibitors, edited by N. Seiler, M. J. Jung and J. Koch-Weser. Amsterdam: Elsevier/North Holland Biomedical Press, 1978, pp. 149-162. 20. Schechter, P. J., Y. Tranier and J. Grove. Gabaculine and isogabaculine: In vivo biochemistry and pharmacology in mice. f,@ Sci. 24: 1173-1182, 1979. 21. Schechter, P. J., Y. Tranier, M. J. Jung and P. Bohlen. Audiogenic seizure protection by elevated brain GABA concentration in mice: Effects of y-acetylenic GABA and y-vinyl GABA, two irreversible GABA-T inhibitors. Eur. J. Pharmac. 45: 31%328,
1977.
22. Schechter, P. J., Y. Tranier, M. J. Jung and A. Sjoerdsma. Antiseizure activity of y-acetylenic y-aminobutyric acid: A catalytic irreversible inhibitor of y-aminobutyric acid transaminase. J. Pharmac. exp. Ther. 201: 606-612, 1977. 23. Seiler, N., M. J. Jung and J. Koch-Weser, (eds.) EnzymeActivated Irreversible Inhibitors. Amsterdam: Elsevier/North Holland Biomedical Press, 1978.
COMPARISON
OF GABA-T
INHIBITORS
24. Van Gelder, N. M. The effect of aminooxyacetic acid on the metabolism of y-aminobutyric acid in brain. Biochem. Phurmclc. 15: 533-539, 1966. 25. Van Gelder, N. M. Hydrazinopropionic acid: A new inhibitor of aminobutyrate transaminase and glutamate decarboxylase. .I. Neurochem. 15: 747-757, 1968. 26. Wallach, D. P. Studies on the GABA pathway. I. The inhibition of y-aminobutyric acid-a-ketoglutaric acid transaminase in vitro and in Live by U-7524 (amino-oxyacetic acid). Biochem. Phat’mJcJc.. 5: 323-331, 1961.
631
27. Wood, J. D., J. S. Durham and S. J. Peesker. Effect of dt-npropylacetate and y-acetylenic GABA on hyperbaric oxygeninduced seizures and GABA metabolism. Neurochem. Res. 2: 707-715, 1977. 28. Wood, J. D. and S. J. Peesker. Development of an expression which relates the excitable state of the brain to the level of GAD activity and GABA content, with particular reference to the action of hydrazine and its derivatives. J. Neurochem. 23: 703112. 1974.