GABAA antagonists

seminars in THE NEUROSCIENCES, Vol 3, 1991 : pp 2 0 5 -210

GABAA antagonists Graham A . R . Johnston The GABA antagonist action of the convulsant alkaloid bicuculline was discovered 20 years ago . This action is now used to define GABA A receptors . Other more potent GABA A receptor antagonists with a range of molecular structures have been discovered subsequently . The existence of subclasses of GABA A receptors, proposed on the basis of agonist structureactivity data and molecular modelling, has been supported by recent cDNA studies which indicate a substantial molecular heterogeneity of GABAA receptor subunit proteins . Further studies on GABAA antagonists with a range of GABAA agonists acting on receptors of known molecular composition may provide a pharmacologically useful subclassification of GABA A receptors and lead to the development of new therapeutic agents acting on specific subclasses of GABAA receptors . Key words :

Dicentra and Adlumia . Its convulsant action was reported in 1934 by Welch and Henderson . 5 Several investigators are now known to have examined the action of bicuculline on various synaptic processes in order to explain its convulsant processes ; indeed Zou Gang and his colleagues in Shanghai in 1965 showed that bicuculline could block synaptic inhibition (as I have noted elsewhere, this work was not published until 1976 due to the Chinese Cultural Revolution) . 6 Our discovery ] in 1970 of the GABA antagonist action of bicuculline came from a systematic study of convulsant alkaloids following our work published in 1967 on the glycine antagonist action of strychnine .? Three years of investigation of convulsant alkaloids and related substances finally yielded the GABA antagonist action of bicuculline and the finding that, while many isoquinoline alkaloids produce convulsions on systemic administration to mammals, most are glycine antagonists, with GABA antagonism being restricted to the phthalide isoquinoline alkaloids that have the IS, 9R configuration . 8

bicuculline / GABA A receptor subclasses

THE DISCOVERY, in 1970, of bicuculline as an antagonist of certain inhibitory actions of GABA (-y-aminobutyric acid) on central neurons provided vital pharmacological evidence for the role of GABA as an inhibitory neurotransmitter in the CNS . 1 Mammalian GABA receptors are currently classified into at least two pharmacological classes : GABAA receptors are antagonised by bicuculline and insensitive to activation by baclofen, whereas GABA B receptors are activated by baclofen and insensitive to bicuculline, 2 and see Bowery et al in this issue . 3 The GABA antagonist action of bicuculline provided a relatively convenient pharmacological means to support other evidence for the likely action of GABA as an inhibitory synaptic transmitter released by defined neurones in the CNS . By 1975, GABA was generally considered to be the major inhibitory transmitter in mammalian brain . Bicuculline is a phthalide isoquinoline alkaloid first isolated from the plant Dicentra cucullaria4 and subsequently from a variety of species of Corydalis,

Why is bicuculline an antagonist? We put forward a theory for the GABA antagonist action of bicuculline using Drieding stereomodels to identify equivalent groupings in bicuculline (the N-atom and the 012=C 11 -C T group) that could be isosteric with the amino- and carboxylgroupings in GABA and equivalent groupings other bicuculline-sensitive GABA agonists including muscimol . 1 Ligand binding studies indicated that bicuculline, GABA and the GABA agonist muscimol shared some common binding sites on brain membranes . The interactions between bicuculline, GABA and muscimol and their common binding sites appeared to be competitive, consistent with bicuculline acting as a competitive antagonist of GABA and muscimol action . On the basis of computer-aided molecular modelling studies, we speculated 9 on why bicuculline acts as an antagonist, whereas GABA and muscimol act as agonists, suggesting that receptor interactions with the aromatic rings of bicuculline were important .

From the Adrien Albert Laboratory of Medicinal Chemistry, Department of Pharmacology, The University of Sydney, NSW 2006, Australia ©1991 by W B . Saunders Company 1044-5765/91/0303-0004$5 .00/0 205



G .A .R . Johnston

206 Such speculation continues on the basis of more detailed computer-aided analyses (e .g . ref 10) with the latest proposal involving an area of increased electron density on bicuculline, compared to GABA, which serves to block rather than open the chloride channel that is part of the multiunit GABA receptorionophore complex ." This is interesting in view of the observation that bicuculline acts only extracellularly as a GABA antagonist, whereas the chloride channel antagonist picrotoxin can work both intra- and extracellularly ; 12 this is consistent with the idea that bicuculline interacts with GABA recognition sites on the extracellular part of the GABA receptor proteins .

substituted phthalide ring system may also be important as (+ )-hydrastine hydrochloride appears to be significantly more stable than bicuculline salts in aqueous solution with respect to the lactone moiety . (+ )-Hydrastine may offer some advantages over bicuculline in terms of potency, solubility and stability in aqueous solution . Recent chemical studies on modifying the structure of bicuculline have been directed towards making derivatives suitable for the preparation of affinity columns for the purification of bicuculline-binding proteins and for affinity labelling studies . 14 Derivatives with substituents in the 5 position seem to be the most promising since certain substitutions here make little difference to GABAA antagonist activity .

Bicuculline analogues Instability of bicuculline The current structure-activity data on bicuculline as a GABAA antagonist may be summarised as in Figure 1 . Recent work has shown that ( + )-hydrastine, isolated from Corydalis stricta in China and the enantiomer of the commercially available ( - )-hydrastine, is a more potent competitive antagonist at GABA A receptors than bicuculline . 13 (+ )-Hydrastine shows a pA a value of 6 .5 as a GABAA receptor antagonist in the guinea-pig ileum, compared with that of bicuculline, pA a 6.1 . In binding studies on rat brain membranes, it is eight times more potent than bicuculline . The structural difference between the two alkaloids is that (+ )-hydrastine has methoxy substitutions at carbons 3' and 4' whereas bicuculline has a methylenedioxy bridge at these carbons . As well as steric differences between bicuculline and (+ )-hydrastine, differences in electron density in the

The relative instability of bicuculline salts in aqueous solution at physiological pH has been known for many years 15 but needs to be re-emphasised . The lactone ring opens up to yield bicucine which appears to be inactive as a GABA antagonist . The half life of bicuculline is many hours at 0 °C, 45 min at 24 °C and only a few minutes at 37 °C . Activity can be restored over 24 h at pH 2 or lower, under which conditions the lactone ring is reformed . The chemical changes can be monitored by u .v . spectroscopy . The quarternary salts, such as bicuculline methochloride, are somewhat more stable but can present problems . We recently found that some commercial samples of bicuculline methochloride, including tritium labelled material, were much less active than anticipated due to opening of the lactone ring . It is

May be substituted with -CH2-X 1

May be replaced by 2 methoxy groups = (+)-hydrastine

. O

May be replaced by methyl as in bicuculline methochloride

O

Figure 1 . Summary of structure-activity data on bicuculline as a GABA antagonist .

207

GA BA A antagonists

important to check the efficacy of all samples, particularly when trying to use bicuculline or its methochloride to block out bicuculline-sensitive phenomena, e .g . studying GABAB receptors .

Subclasses of GABAA receptors Our early computer-aided molecular modelling studies over a range of GABA agonists led to the proposal in 1979 of `the existence of a multiplicity of GABA receptors' (this predates the current GABAA/B terminology) and that `certain of the conformationally restricted GABA agonists may show selectivity for different classes of bicucullinesensitive receptors' .9 This proposal was supported by our subsequent findings of different agonist profiles for bicuculline- sensitive activity on `synaptic' and 'non-synaptic' GABA receptors in the spinal cord, dorsal and ventral roots of immature rats . 16 GABA binds to two kinetically and pharmacologically distinct classes of bicuculline-sensitive GABAA binding sites on rat brain membranes, with the low affinity binding site being linked to benzodiazepine receptors 17 (and see Richards et al, this issue 18 ) . This is consistent with electrophysiological findings that not all GABAA receptors appear to be linked to benzodiazepine receptors . Furthermore it is well established that there are subclasses of benzodiazepine receptors, not all of which are linked to GABA receptors . Both bicuculline and (+ )-hydrastine are more potent antagonists of the low affinity GABA A binding sites than of the high affinity sites . 13 Thus on the basis of agonist profiles, molecular modelling, and benzodiazepine interactions there was a growing body of evidence for subclasses of GABAA receptors and binding sites . But who suspected the diversity of potential subclasses to be revealed by molecular biology? Early molecular biology studies using benzodiazepine-linked affinity columns to purify GABAbinding proteins suggested the GABAA receptor complex consisted of two subunits, a and ß, but subsequent work using DNA homology cloning revealed further subunits, 'y, 6 and e, and subtypes of subunits, al-a4, 01-03, -yl, y2 and others (for reviews, see ref 19 and article by Tobin, this issue 20) . Reconstitution studies indicate that, although single class subunits can form functional GABA-activated chloride channels ('homomeric channels'), the physiology and pharmacology of these channels are different from those of in vivo channels . Mixtures of

the various subunits and subtypes of subunits are needed to approach the physiology and pharmacology observed in vivo of `normal heterooligomeric' GABA receptors (see also Tobin, this issue 20 ) . The potential for GABAA receptor heterogeneity based on different subunit/subtype protein combinations is thus substantial! Furthermore, it should be noted that these are the GABAA receptor proteins purified on the basis of their association with benzodiazepine binding capacity and that not all GABAA receptors are necessarily linked to benzodiazepine receptors .

New GABA antagonists A variety of substances have now been shown to act as competitive GABA antagonists . The structures of some of these antagonists are shown in Figure 2, in relative order of potency . The most potent GABAA antagonist described is probably cortisone which acts at concentrations as low as 1 pM ; 21 however, cortisone is a non-competitive antagonist and thus may not act directly with the active site of GABAA receptors, though it, cortisol and other steroids may be involved as endogenous modulators of GABAA receptor properties, 22 and see Simmonds, this issue . 23 The amidine steroid analogue RU5135 is the most potent competitive GABA antagonist yet described, being some 500 times more potent than bicuculline in inhibiting GABA enhancement of diazepam binding . 24 RU5135 is a very potent inhibitor of muscimol (IC50 11 nM) and bicuculline (IC50 0 .8 nM) binding ;25 however, its action is not restricted to GABA antagonism as it has a more effective role as a glycine antagonist in the cat spinal cord in vivo . 26 In vitro in the cuneate nucleus, RU5135 is a competitive antagonist of muscimol (pA2 8 .31), 27 and in the ileum it is a competitive GABA antagonist (pA2 8 .0), 28 while in the optic nerve it antagonises glycine action (pA2 7 .67) . 27 Pitrazepin was first reported to be at least ten times more potent than bicuculline as a selective GABA A antagonist . 29 On cerebellar slices pitrazepin is a competitive antagonist of muscimol action (pA2 5 .97) only marginally more potent than bicuculline methiodide (pA2 5 .92), 30 and on hippocampal slices it is some three times more potent than bicuculline methobromide as an antagonist of isoguvacine action (pA2 6 .70 for pitrazepin and 6 .18 for bicuculline methobromide) .31 Pitrazepin (IC 50 24 nM) is five times more potent than bicuculline (IC50 15 nM) in

G. A . R . Johnston

208

NH

RU5135

PITRAZEPIN

10

H 3 CO

SR95531

(+)-HYDRASTINE

BICUCULLINE

SECURININE

Figure 2 . Structures of some competitive antagonists of GABAA receptors in approximate rank order of potency . displacing [ 3H ] -bicuculline bound to rat brain membranes . 31 A series of pyridazinyl-GABA derivatives have been described as GABAA antagonists . Of these SR95531 acts as a selective GABAA antagonist in vivo in the spinal cord 32 and the cuneate nucleus, being approximately equipotent with bicuculline methochloride, 33 binding studies using [ 3H]-GABA and GABA-stimulated [ 3H ] -diazepam binding to rat brain membranes indicate that SR95531 is a competitive inhibitor of high affinity GABA binding sites and a non-competitive inhibitor of low affinity sites. 34 This indicates a difference between SR95531 and bicuculline in their relative potencies for high and low affinity binding sites, with SR95531 being more potent at the high affinity sites and bicuculline being more potent at the low affinity sites . The

1,3,4-thiadiazole analogue of SR95531, with a sulphur atom replacing the C=C in the pyridazine ring, is also active as a GABAA antagonist but is five times less potent than bicuculline . 33 The convulsant alkaloid securinine from Securinega suffructicosa is a selective GABAA antagonist in the cat spinal cord in vivo, although binding studies on rat brain membranes suggest that it is some seven times less potent than bicuculline . 36 Studies on the ability of these various classes of GABAA antagonists to reverse the inhibitory effects of GABA on the binding of [ 35S ] -butylbicyclophosphorothionate to rat brain membranes suggest that the rank order of potency is RU5135 > pitrazepin > SR95531 > bicuculline > securinine, which is similar to that found in studies on antagonism of GABAinduced inhibition of neuronal activity . 37 Other

GA BA A antagonists

studies 13 indicate that (+ )-hydrastine is approximately equipotent with SR95531 .

Conclusions After 20 years, the GABA A antagonist activity of bicuculline and related compounds continues to be extensively studied . The recent dramatic advances in the molecular biology of GABA A receptor protein diversity have provided a new impetus in the study of the likely multiplicity of GABAA receptors that has yet to be knowingly exploited in pharmacological terms . We need selective agonists and antagonists for the probable receptor subclasses . The structures of the GABA A receptor antagonists shown in Figure 2 may aid in the design of further antagonists that may show some selectivity for possible subclasses of GABA A receptor sites .

Acknowledgements The author is grateful to Dr Robin Allan and Dr Ken Mewett for helpful discussions and to the National Health and Medical Research Council of Australia for financial support .

References 1 . Curtis DR, Duggan AE, Felix D, Johnston GAR (1970) GABA, bicuculline and central inhibition . Nature 226 : 1222-1224 2 . Hill DR, Bowery NG (1981) 3 H-Baclofen and 3 H-GABA bind to bicuculline insensitive GABAB sites in rat brain . Nature 290 :149-152 3 . Bowery NG, Maguire JJ, Pratt GD (1991) Aspects of the molecular pharmacology of GABAB receptors . Semin Neurosci 3 :241-249 4 . Manske RHF (1932) The alkaloids of fumaraceous plants . II . Dicentra cucullaria (L .) . Can J Res 8 :265-272 5 . Welch AD, Henderson VE (1934) A comparative study of hydrastine, bicuculline and adlumine . J Pharmacol Exp Therap 51 :482-491 6 . Johnston GAR (1985) First Sino-Australian workshop in pharmacology . Trends Pharmacol Sci 4 :159 7 . Curtis DR, Hösli L, Johnston GAR, Johnston IH (1967) Glycine and spinal inhibition . Brain Res 5 :112-114 8 . Curtis DR, Johnston GAR (1974) Convulsant alkaloids, in Neuropoisons, Their Pathophysiological Actions, vol 2Poisons of Plant Origin (Simpson LL, Curtis DR, eds), pp 207-248 . Plenum Press, New York 9 . Andrews PR, Johnston GAR (1979) GABA agonists and antagonists . Biochem Pharmacol 28 :2697-2702 10 . Pooler GW, Steward EG (1988) Structural factors governing agonist and antagonist activity in the GABA A system . Biochem Pharmacol 37 :943-945

209 11 . Aprison MH, Lipkowitz KB (1989) On the GABA A receptor : a molecular modelling approach . J Neurosci Res 23 :129-135 12 .- Akaike N, Hattori K, Oomura Y, Carpenter DO (1985) Bicuculline and picrotoxin block y-aminobutyric acid-gated Cl - conductance by different mechanisms . Experientia 41 :70-71 13 . Huang J-H, Johnston GAR (1990) (+ )-Hydrastine, a potent competitive antagonist at mammalian GABAA receptors . Br j Pharmacol 99 :727-730 14 . Allan RD, Apostopoulos C (1990) Synthesis of substituted (+ )-bicuculline derivatives through chloromethylation . Aust J Chem 43 :1259-1268 15 . Olsen RW, Ban M, Miller T, Johnston GAR (1975) Chemical instability of the GABA antagonist bicuculline under physiological conditions . Brain Res 98 :383-387 16 . Allan RD, Evans RH, Johnston GAR (1980) y-Aminobutyric acid agonists : an in vitro comparison between depression of spinal synaptic activity and depolarization of spinal root fibres in the rat . Br J Pharmacol 70 :609-615 17 . Johnston GAR (1986) Multiplicity of GABA receptors, in BenzodiazepinelGABA Receptors and Chloride Channels : Structural and Functional Properties (Olsen R, VenterJC, eds), pp 57-71 . Alan R Liss, New York 18 . Richards G, Schoch P, Haefely W (1991) Benzodiazepine receptors : new vistas . Semin Neurosci 3 :191-203 19 . Schofield PR (1989) The GABAA receptor: molecular biology reveals a complex picture . Trends Pharmacol Sci 10 :476-478 20 . Tobin AJ (1991) Molecular biological approaches to the synthesis and action of GABA . Semin Neurosci 3 :183-190 21 . Ong J, Kerr DIB, Capper H, Johnston GAR (1990) Cortisone : a potent GABAA antagonist in the guinea-pig isolated ileum . J Pharm Pharmac 42 :662-664 22 . Johnston GAR, Kerr DIB, Ong J (1987) Stress, steroids and GABA receptors, in Pharmacology (Rand MJ, Raper C, eds), pp 121-124 . Elsevier, Amsterdam 23 . Simmonds MA (1991) Modulation of the GABA A receptor by steroids . Semin Neurosci 3 :231-239 24 . Hunt P, Clements Jewery S (1981) A steroid derivative, R 5135, antagonises the GABA/benzodiazepine receptor interaction . Neuropharmacology 20 :357-361 25 . Olsen RW (1984) y-Aminobutyric acid receptor binding antagonism by the amidine steroid RU5135 . Eur J Pharmacol 103 :333-337 26 . Curtis DR, Malik R (1985) Glycine antagonism by RU 5135 . Eur J Pharmacol 110 :383-384 27 . Simmonds MA, Turner JP (1985) Antagonism of inhibitory amino acids by the steroid derivative RU5135 . Br J Pharmacol 84 :631-635 28 . Ong J, Kerr DIB (1989) Modulation of spontaneous motility by GABAA receptor antagonism in the guinea pig isolated ileum . Neurosci Lett 101 :203-208 29 . Gähwiller BH, Maurer R, Wüthrich HJ (1984) Pitrazepin, a novel GABAA antagonist . Neurosci Lett 45 :311-316 30 . Bagust J, Gardner CR, Hussain S, Walker RJ (1987) Quantitative analysis of gamma-aminobutyric acid (GABA) agonists and antagonists on cell activity in rat cerebellar slices . Br J Pharmacol 91 :446P 31 . Kemp JA, Marshall GR, Wong EHF, Woodruff GN (1985) Pharmacological studies on pitrazepin, a GABAA receptor antagonist . Br j Pharmacol 85 :237P 32 . Gynther BD, Curtis DR (1986) Pyridazinyl-GABA derivatives as GABA and glycine antagonists in the spinal cord of the cat . Neurosci Lett 68 :211-215 33 . Michaud JC, Mienwille JM, Chambon JP, Biziere K (1986) Interactions between three pyridazinyl-GABA derivatives and central GABA and glycine receptors in the rat, an in vivo microiontophoretic study. Neuropharmacology 25 :1197-1203

210 34 . Heaulme M, Chambon J-P, Leyris R, Molimard J-C, Wermuth CG, Biziere K (1986) Biochemical characterisation of the interaction of three pyrdazinyl-GABA derivatives with the GABAA receptor site . Brain Res 384 :224-231 35 . Allan RD, Apostopoulos C, Richardson JA (1990) 2-Imino-1,3,4-thiadiazole derivatives of GABA as GABA A antagonists . Aust J Chem 43 :1767-1772

G . A . R . Johnston 36 . Beutler JA, Karbon EW, Brubaker AN, Malik R, Curtis DR, Enna SJ (1985) Securinine alkaloids : a new class of GABA receptor antagonist . Brain Res 330 :135-140 37 . Squires RF, Saederup E (1987) GABAA receptor blockers reverse the inhibitory effect of GABA on brain-specific [ 35 S]TBPS binding . Brain Res 414 :357-364