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.
Concept
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?
ChallengeThere 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!
ControversyThe 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!
ClonesMolecular 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, β1-β3, γ1-γ3, 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.
ConsensusFrom 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.
ChanceI 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.
CognitionMuch 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.
ColleaguesAbout the author
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