The Leighton Address 1996 Cerebral Chemistry

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 GABA_B receptors under these conditions. We proposed that existence of a class of bicuculline-insensitive binding sites (GABA_C?) for GABA that is insensitive to (-)-baclofen'.

There was considerable debate about our GABA_A 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 GABA_C 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!

Controversy

The term GABA_C appeared very infrequently in peer-reviewed publications until 1993, though much work relevant to GABA_C 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-GABA_A and non-GABA_B 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 GABA_C 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 GABA_C sites!

Clones

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 GABA_Creceptors.

Molecular biology was making great advances in our understanding of GABA_A 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, β₁-β₃, γ₁-γ₃, and δ, named on the basis of sequence homology. This heterogeneity gives rise to a wide diversity of possible heteromeric GABA_A 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 GABA_A receptors, since they show some sequence homology with the GABA_A receptor proteins, but recombinant studies showed that the ρ-receptor protein did not form heteromeric receptors with any of the GABA_A receptor proteins, instead they formed homomeric receptors with properties very similar to those observed for GABA_Creceptors in intact retina.

Consensus

GABA_C 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 GABA_C 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 GABA_D receptors.

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

Chance

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

I had been invited to the Congress to talk about neurosteroid modulation of GABA_A 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 GABA_C 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 GABA_B 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 GABA_C 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.

Cognition

The Ciba-Geigy team had developed a range of GABA_B 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 GABA_C receptor antagonist. This compound has very interesting properties that could not be satisfactorily explained on the basis of its action as a GABA_B receptor antagonist. Could its additional GABA_C 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 GABA_C medicinal chemistry. GABA_C receptors now represent an important new therapeutic target.

Much of science is concerned with discovering 'nature's secrets'. GABA_C receptors were there waiting to be discovered. But science is also creative. The emerging medicinal chemistry of GABA_C receptors will have a major creative component. The race is on to design, discover and develop new chemical entities acting on GABA_C 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.

Colleagues

It is a pleasure to acknowledge my many colleagues that have contributed to the studies on GABA_C 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.

References

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.