10 minutes reading time (1933 words)

    The Leighton Address 1996 Cerebral Chemistry

    G. Johnston
    University of Sydney

    Chemistry is making many contributions to our increased understanding of brain function. Simple chemicals serve as neurotransmitters mediating the transfer of information between nerve cells by activating chemically complex receptors. All nerve cells in the brain have receptors for the inhibitory neurotransmitter, GABA (4-aminobutanoic acid). Until recently, only two major subtypes of GABA receptors were known - GABAA and GABAB receptors. Now there is a third subtype - GABAC receptors. The journey of GABAC receptors, from the initial concept resulting from the synthesis of some key GABA analogues in 1975, through the formal proposal for this subtype of receptors in 1984 and the widespread recognition of their existence only recently, is illustrative of the way scientific concepts sometimes develop, particularly those that need input from a diverse range of disciplines. Chemistry, molecular biology, pharmacology and physiology made important contributions to the. GABAC receptor saga. GABAC receptors represent an important new target for drug design and development. 


    Due to the relatively free rotation around the single bonds that make up its chemical backbone, GABA is a flexible molecule. It can thus exist in a variety of low energy conformations. We began to synthesise analogues of GABA in which the flexibility of the chemical backbone of GABA was restrained by incorporation of unsaturated bonds and/or ring structures. Thus began a systematic study of what became known as "conformationally restricted GABA analogues".

    The underlying concept was that different shapes of GABA might interact with different recognition sites on large molecules that recognise GABA in the course of its function as an inhibitory neurotransmitter. These large molecules include membrane bound receptors, intracellular enzymes and transmembrane transport carriers.

    The key conformationally restricted GABA analogues were the cis- and trans- isomers of 4-aminocrotonic acid, dubbed CACA and TACA. CACA and TACA represented GABA in folded and extended conformations respectively. In 1975 we showed that, while both CACA and TACA inhibited neuronal activity, only the activity of TACA could be antagonised by the convulsant alkaloid, bicuculline(1).

    We noted then that the bicuculline-insensitive action of CACA was similar, but not identical, to that of another GABA analogue, baclofen, the β-(p-chlorophenyl)- derivative of GABA that was used therapeutically to treat spinal spasticity.

    In 1981, David Hill and Norman Bowery proposed two subtypes of GABA receptors, GABAA receptors that could be blocked by bicuculline, and GABAB receptors that were insensitive to bicuculline(2). TACA activated GABAA receptors, while baclofen activated GABAB receptors. Did CACA activate GABAB receptors?

    In 1984, we prepared the active isomer of baclofen, (-)-baclofen, labelled to high specific activity with tritium, and studied its binding to rat brain membranes(3). While GABA and a range of GABA analogues competed with (-)baclofen for binding sites on these membranes, CACA was inactive. This showed that CACA did not interact with GABAB receptors under these conditions. We proposed that existence of a class of bicuculline-insensitive binding sites (GABAC?) for GABA that is insensitive to (-)-baclofen'.

    There was considerable debate about our GABAA receptor proposal at the London IUPHAR Congress in 1984 and at a satellite meeting in Cambridge on GABA Receptors organised by Norman Bowery. While there was, at that time, insufficient evidence for the existence of GABAC receptors, we said that it would take perhaps 10 years to sort out whether or not they really existed. This challenge certainly served as a stimulus for further studies. The appraisal of these studies was always critical!

    The term GABAC appeared very infrequently in peer-reviewed publications until 1993, though much work relevant to GABAC receptors was published. Thus, even in 1992, we were not allowed by a referee to use the term in a paper reporting the bicuculline/baclofen-insensitive binding of GABA in rat cerebellum. We were forced to use the term NANB GABA binding to indicate its non-GABAA and non-GABAB nature(4). I know that many authors had similar problems.

    The controversy was resolved when two papers appeared back to back in Nature late in 1993 using the term GABAC receptors in connection with studies of the bicuculline-insensitive action of GABA in the retina(5,6). I wonder who refereed these papers? Interestingly, one of the senior authors wrote to me asking why I used the term NANB to describe what were clearly GABAC sites!

    Biologists in the USA provided a molecular basis for retinal GABA. receptors by cloning what they termed ρ-receptors from human retinal DNA libraries. It soon became clear that ρ-receptors were in fact GABACreceptors.

    Molecular biology was making great advances in our understanding of GABAA receptors, showing that these were extremely complex receptors made up of 5 protein subunits that could be derived from a range of gene products, named α1- α6, β13, γ13, and δ, named on the basis of sequence homology. This heterogeneity gives rise to a wide diversity of possible heteromeric GABAA receptors. This diversity can be studied by expressing mRNA coding for the various proteins in Xenopus oocytes. The responses of the new receptors in oocytes can then be studied by intracellular recording techniques.

    Initially, it was considered that the ρ-receptors were part of the same family as GABAA receptors, since they show some sequence homology with the GABAA receptor proteins, but recombinant studies showed that the ρ-receptor protein did not form heteromeric receptors with any of the GABAA receptor proteins, instead they formed homomeric receptors with properties very similar to those observed for GABACreceptors in intact retina.

    GABAC receptors are now widely accepted in the neuroscience literature with important articles in Trends in Neurosciences(7) and Trends in Pharmacological Sciences.(8) The first GABAC conference can't be far off! It did take about 10 years for this consensus to be reached. How long will this consensus last? Receptor classification is a highly contentious endeavour. Already, a case is being made for GABAD receptors.

    From a combination of medicinal chemistry, molecular biology, pharmacology and physiology, it is now known that GABAC receptors are relatively simple GABA-gated chloride ion channels(7,8). In contrast, GABAA receptors are very complex, need high concentrations of GABA to activate them, desensitise more quickly and their channels open for a shorter time. GABAA receptors may have evolved from GABACreceptors.

    While GABAA receptors have a rich medicinal chemistry, being influenced by a variety of therapeutically important drugs, including benzodiazepines, barbiturates and neurosteroids, the medicinal chemistry of GABAC receptors is only just beginning. A chance meeting at the Collegium Internationale Neuropsychopharmacologicum Congress in Melbourne in June 1996 may have important consequences for GABACmedicinal chemistry.

    I had been invited to the Congress to talk about neurosteroid modulation of GABAA receptors. By chance, I sat next to a Swiss colleague from Ciba-Geigy, Wolfgang Froestl, at one of the lectures that was of marginal interest to either of us. He asked me how the GABAC work was going. I told him it was going very well and said we were after the phosphonic acid analogues of CACA and TACA. To my surprise, he told me that he had made them as part of a major study on GABAB receptors(9) and he offered to send them to me. Within a month, Mary Chebib and Robert Vandenberg had tested these compounds, and some others that he had sent, on GABAC receptors expressed in oocytes. The results were not what we had hoped for, but quite interesting nonetheless. He sent us some further compounds and some of these were very interesting indeed.

    The Ciba-Geigy team had developed a range of GABAB receptor antagonists that they were investigating for their effects on cognition. There is much interest cognitive enhancers to treat cognitive deficits in disorders such as Alzheimer's disease and schizophrenia. One of their key compounds turned out to be a GABAC receptor antagonist. This compound has very interesting properties that could not be satisfactorily explained on the basis of its action as a GABAB receptor antagonist. Could its additional GABAC receptor antagonist properties be important?Commercial-in-confidenceAs must happen with 'commercially sensitive' scientific findings, a curtain of confidentiality now falls over many aspects of GABAC medicinal chemistry. GABAC receptors now represent an important new therapeutic target.

    Much of science is concerned with discovering 'nature's secrets'. GABAC receptors were there waiting to be discovered. But science is also creative. The emerging medicinal chemistry of GABAC receptors will have a major creative component. The race is on to design, discover and develop new chemical entities acting on GABAC receptors. It is a race we intend to win! We have an excellent commercial partner in the Melbourne high technology company Circadian Technologies Ltd, and experience in memory drug development from participating in an R&D syndicate comprising AMRAD, CSIRO and the Macquarie Bank.

    What started out as pure research in 1975, took more than 20 years to show its commercial potential. This is not at all unusual, given the multidisciplinary nature of the work, much of which was 'ahead of its time'. What was important was that the pure research was nurtured in an international peer-reviewed competitive environment. The long term view of such work presents a great challenge to formulating science policy as governments, of whatever persuasion, are usually short sighted.

    It is a pleasure to acknowledge my many colleagues that have contributed to the studies on GABAC receptors, including Robin Allan, Phil Beart, Peter Burden, Mary Chebib, David Curtis, Colleen Drew, Rujee Duke, Ken Mewett, Hue Tran, Bruce Twitchin, Robert Vandenberg and Robert Weatherby, most of whom trained as chemists. The award of the RACI's 1996 Leighton Memorial Medal, for services to Australian chemistry in the broadest sense, does great honour to me and my colleagues. I have greatly enjoyed my interaction with the RACI ever since I joined as a student member. I look forward to continuing to serve the institute and Australian chemistry - currently I am a member of the RACI international Relations committee and the RACI representative on the organising committee of Pacifichem 2000, providing me with many opportunities to further the cause of Australian chemistry in the Asian Pacific region.

    1. Johnston, G.A.R., et al., J. Neurochem. 1975, 24, 157-160.
    2. Hill, D.R., and Bowery, N.G., Nature, 1981, 29O, 149-152.
    3. Drew, C.A., Johnston, G.A.R., and Weatherby, R.P., Neurosci. Lett., 1984,52, 317-321.
    4. Drew, C.A', and Johnston, G.A.R., J. Neurochem.,1992, 58, 1087-1092.
    5. Feigenspan, H., Wassle, H., and Bormann, J., Nature, 1993, 361, 159-162.
    6. Qian H., and Dowling, J.E., Nature, 1993, 361, 162-164.
    7. Bormann, J., and Feigenspan, A., Trends Neurosci.,l995, 18, 515-519.
    8. Johnston, G.A.R., Trends Pharmacol. Sci.,1996, 17, 319-323.
    9. Froestl, W., et al., J. Med. Chem., 1995, 38, 3313-3331.

    About the author

    Graham Johnston has been Professor of Pharmacology at the University of Sydney since 1980. This article is based on his 1996 Leighton Memorial Medal lectures at the University of Sydney and Monash University. The Leighton Medal is his fourth scientific medal - he received the Sydney University Medal in Organic Chemistry in 1960, the RACI's H.G. Smith Memorial Medal in 1989 and the inaugural Rand Medal of the Australasian Society of Clinical and Experimental pharmacologists and Toxicologists in 1993. He also won a medal at the European Winter Brain Research Conference in France in 1988 - for the most spectacular fall in the ski race! He has served the RACI as National President, NSW Branch President and Chair of the Medicinal and Agricultural Chemistry Division. He has been President of the Federation of Australian Scientific and Technological Societies (FASTS) and is a member of various Scientific Advisory Boards, including those of the Australian Jockey Club, Biota Holdings and the Neuroscience Institute for Schizophrenia and Allied Disorders, and is a member of the Commonwealth Administrative Appeals Tribunal. His research is funded by the NH&MRC, ARC and Circadian Technologies Ltd.
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