The University of Sydney - The University of Sydney - …sydney.edu.au/medicine/pharmacology/adrien-albert/images/... · 2009. 12. 10. · C Rae (1), FA Nasrallah (1) & VJ Balcar

Molecular Pharmacology of Amino Acid

Transmitters: From Cryptic to Contemporary Drug Targets

28-29 November 2009

SYDNEY, AUSTRALIA

HOSTED BY

AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS AND TOXICOLOGISTS

THE ROYAL AUSTRALIAN CHEMICAL INSTITUTE INCORPORATED

CHEMISTRY SERVING AUSTRALIA

GARJfest

MOLECULAR PHARMACOLOGY OF AMINO ACID TRANSMITTERS:

FROM CRYPTIC TO CONTEMPORARY DRUG TARGETS

Sydney, November 2009

Molecular Pharmacology of Amino Acid Transmitters: From Cryptic to Contemporary Drug Target 3

Welcome To GARJFest

Welcome to this event honouring the contributions to Australian and international pharmacology and medicinal chemistry by Professor Graham Johnston AM.

Graham is recognized as a distinguished Australian scientist whose contributions have been instrumental in taking amino acid pharmacology from its infancy to a scenario where it is now a key activity for the international pharmaceutical industry. 2009 has been a big year for Graham who has celebrated his 70th birthday in June.

What stands out from Graham’s enormous list of publications across neurochemistry, medicinal chemistry and pharmacology is how his unique chemical insights have led to idiosyncratic, unexpected advances in neuroscience. Indeed these contributions should probably be viewed within the broadest context of “chemical” neuroscience.

Your registration pack also contains a copy of the special issue of Neurochemical Research honouring his distinguished career. This volume contains articles by former students, colleagues and collaborators, who have worked with Graham scientifically or interacted with him through the activities of learned societies (ASCEPT, RACI, ANS, APSN, ISN).

A few words from various colleagues who could not be with us today:

Norman Bowery - “One could always rely on Graham to make any discussion period come to life. For example, his comments on GABAmodulin are now a part of folklore!”

Arne Schousboe - “It has been a privilege to have known Graham for some 4 decades being able to benefit from his enormous knowledge about neuropharmacology.”

Arthur Duggan - “You may be surprised to learn that I have always been somewhat envious of chemists, even neurochemists, as the ability to isolate or synthesise compounds seemed so fundamental to research on neurotransmitters.”

Abel Lajtha - “I consider you as an important member of the families of pioneers in our field having focused so well on the important area of amino acid transmitters well before we had any idea of the crucially important role they have in neurophysiology and neuropathology, and you did not stay in a narrow subject, you continued with receptors, agonists, antagonist, and their pharmacology with great results – congratulations!”

Sam Enna – “I am personally indebted to Graham in that his research provided me with a lifetime of work and intellectual stimulation. By exploiting his leads, I was able to accumulate the resources necessary to feed, clothe and educate three lovely children and live a comfortable life overall. Many scientists have had a similar parasitic relationship with Graham, sustaining their careers through his energy and insights,”

Special thanks to Jane Hanrahan, Bevyn Jarrott, Vladimir Balcar, Linda Mercer and Madeleine Beart for their support in putting this meeting together. Our sponsors: ASCEPT, RACI, the Adrien Albert Laboratory of Medicinal Chemistry and SpringerLink.

Philip Beart & Mary Collins

4 Molecular Pharmacology of Amino Acid Transmitters: From Cryptic to Contemporary Drug Target

Introductory remarks, Phil Beart

Plenary lecture – Chair: Mary Collins

L1 1.00 GABA Receptors and Neuropharmacology, Wolfgang Froestl

GABA systems and their pharmacology – Chair: Jane Hanrahan

L2 1.45 Now I Know My A,B,C: The Cloning And Characterisation of the GABA Receptor, Peter Schofield

L3 2.05 GABAC Receptors – Are They Simple As “ABC”?, Mary Collins

L4 2.25 The “ABC” Of GABA: Awfully Bloody Complex, Lindy Rae

2.45 – 3.05 Refreshment break

GABA systems and their pharmacology (continued) – Chair: Vladimir Balcar

L5 3.05 Novel GABAc Antagonists Through Bioisosteric Modification Of 4-Aminocyclopent-1-enecarboxylic, Katherine Locock

L6 3.15 Cognitive Enhancement by Selective GABAc Receptor Antagonists, Hye-Lim Kim

L7 3.25 From Amino Acids To Flavonoids, Jane Hanrahan

L8 3.45 Gingko, GABA And Bees, Rujee Duke

L9 4.00 A, B and C: Roles For GABA In Learning and Memory In Chicks, Marie Gibbs

L10 4.20 “Hypo-Glycine-Aemia” to “Relaxin” – A Journey from Obscurity to Uncertainty, Andrew Gundlach

4.40-5.00 Refreshment break

Amino Acids in Way, Shape or Form – Chair: Bevyn Jarrott

L11 5.00 G Protein-Coupled Receptors are Merely an Assembly of Amino Acids, Roger Summers

L12 5.20 CaMKII: An Adaptor Mediating Cross-talk Between Signalling Pathways, John Rostas

L13 5.40 Expression of New EAAT5 Variants in the Mammalian Retina, Aven Lee

L14 5.50 GABA and Graham, Mike Minchin

L15 6.05 Amino Acids: Tales of the Occult and Unexpected, Les Davies

L16 6.20 Trophic Internactions with GARJ, Ian Hendry

6.40 Drinks and refreshments

7.45 Dinner

DAY ONE


Plenary lecture – Chair: Phil Beart

L17 9.00 Glutamate/Aspartate Receptor Jouneys: Personal Tales from Down-under and Back-home, David Lodge

L-Glutamate systems and their pharmacology – Chair: Les Davies

L18 9.45 Glutamate Transport: A Fatal Attraction or Just a Distraction? Vladimir Balcar

L19 10.05 The Interplay Between Ion Coupling and the Pharmacological Properties of Glutamate Transporters, Rob Vandenberg

L20 10.25 Drinking More Than Your Doctor, Peter Dodd

L21 10.45 Constraining L-Glutamate, Robin Allan

11.00-11.30 Refreshment break

Neurochemistry of injury and disease – Chair: Roger Summers

L22 11.30 Glutamate Homeostasis And Ischaemia, David Pow

L23 11.50 The mGlu5 Receptor, Drug-Seeking and Drug-Induced Plasticity, Andrew Lawrence

L24 12.10 The Pharmacologist’s Prayer: “Deliver Us From Brick Dust and Promiscuous Inhibitors”, Bevyn Jarrott

L25 12.25 So Many Molecular Targets: Neuropharmaceuticals, Excitotoxicity and Neuroprotection, Phil Beart

L26 12.40 GABA: Thinking Outside The Brain, Graham Johnston

CONCLUSION

DAY TWO


L1

GABA RECEPTORS AND NEUROPHARMACOLOGY

Wolfgang Froestl, AC Immune EPFL PSE B CH-1015 Lausanne Switzerland

The discovery of GABA as an inhibitory neurotransmitter was a long process, whereas the discovery of the benzodiazepines was pure serendipity. A big step forward was the cloning of GABAA receptors by Schofield et al. in 1987, which opened the way to the understanding of the pharmacology at GABAA receptor subtype combinations. GABAB receptors were first characterized pharmacologically in 1980, whereas the expression cloning was achieved only in 1997. The GABAB receptor antagonist SGS742 showed attention and memory enhancing properties in patients with Mild Cognitive Impairment. This compound also served as starting point for the very recent discovery of selective GABAC receptor antagonists, which have a potential for the treatment of memory and sleep disorders as well as myopia. The many contributions of Prof. G. A. R. Johnston since the hallmark discovery of bicuculline as selective GABAA receptor antagonist in 1970 will be presented.

L2

NOW I KNOW MY A, B, C: THE CLONING AND CHARACTERISATION OF THE GABA RECEPTOR

PR Schofield, Prince of Wales Medical Research Institute, NSW 2031.

The cloning of the genes encoding the subunits of the brain GABAA receptor in 1987 defined the primary structure of this important neurotransmitter receptor. In addition to defining the primary structure, this work revealed the existence of the ligand-gated ion channel receptor superfamily, now referred to as the Cys-loop receptor superfamily. The molecular identification of the extent of receptor subunit and subtype diversity in both the GABA and glycine receptor systems included the definition and characterisation of multiple receptor subunit subtypes such as the α1-α6 and β1-β3 subunits; the identification and analysis of novel receptor subunits such as γ1-3, δ, ε and ρ especially the γ subunits which encode the benzodiazepine response of the GABAA receptor; and the functional and anatomical analysis of receptor subtype diversity. These advances in the understanding of receptor subtype diversity in both the GABAA and the related glycine receptor provided the basis for defining the molecular mechanisms of ligand binding and signal transduction at these receptors. The identification of heritable mutations in the glycine receptor in hyperekplexia (startle disease) patients and animal models provided yet another opportunity to advance our understanding of the molecular basis of signal transduction in the ligand-gated ion channel receptors.


L3

GABAC RECEPTORS – ARE THEY AS SIMPLE AS “ABC”?

M Chebib Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006

GABAC receptors are members of the ligand-gated ion channels (LGIC) superfamily that include GABAA, nicotinic acetylcholine, glycine and serotonin type 3 receptors. GABAC receptors form homomeric pentamers that conduct chloride ions to regulate excitatory and inhibitory processes. Such receptors were first described by Professors Graham Johnston, Phil Beart and David Curtis, in 1975, when they observed that the effects of GABA on cat spinal cord neurons were not completely inhibited by bicuculline but were activated by cis-aminocrotonic acid (CACA). It was almost 10 years later when Professor Graham Johnston and Dr Coleen Drew showed that such receptors were not activated by baclofen and therefore not GABAB receptors. Thus the “GABAC receptor” terminology was proposed. Subsequently, it was shown that ρ-subunits made up the GABAC receptor. These subunits are largely expressed in the retina and modestly expressed throughout the CNS. Their pharmacology is distinct from both GABAA and GABAB receptors. Research over the last 15 years within our team led to GABAC-selective antagonists, in particular, phosphinic acid analogues of GABA. Such ligands were found to be enhancers of learning and memory in rodent models. Could such a simple receptor produce such powerful effects in vivo?

Johnston GA, Curtis DR, Beart PM, Game CJ, McCulloch RM, Twitchin B. (1975) J Neurochem. 24, 157-60 Drew CA, Johnston GA, Weatherby RP. (1984) Neurosci Lett. 52, 317-21

L4

THE “ABC” OF GABA: AWFULLY BLOODY COMPLEX.

C Rae (1), FA Nasrallah (1) & VJ Balcar (2). (1) Prince of Wales Medical Research Institute, and Brain Sciences, UNSW 2031 (2) Bosch Institute & School of Med. Sciences, Sydney Medical School, The University of Sydney, NSW 2006.

Although the relationship between excitatory activity and metabolism is reasonably well understood (and held largely to be linear), the relationship between inhibitory (i.e. GABAergic) activity and metabolism is largely unexplored. The advent of whole body MR scanners capable of measuring GABA levels non-invasively in the brain has meant that GABA concentrations are measured regularly around the globe and conclusions drawn about their meaning with little empirical basis for these conclusions. Several years ago we set out to determine the relationship between the activity of components in the GABAergic system and metabolism using our cortical brain slice model of metabolism. In this system, metabolism of [3-13C]pyruvate is examined in the absence (control) and presence of selected drugs active in the GABAergic system and the data subjected to multivariate statistical analysis (e.g. Nasrallah et al 2007, 2009, in press, Rae et al 2009). More than 350 data sets later we have generated a GABAergic “footprint” which clearly shows distinct metabolic activities for more than 6 separate GABAergic entities which are mostly representative of responses to distinct concentrations of GABA. Excitingly, this footprint allows us to distinguish the activities of “dirty” drugs, such as GHB and is allowing us to begin to make predictions about GABAergic metabolic behaviour.

Nasrallah et al (2007) JCBFM 27, 1510-1520 Nasrallah et al (2009) J Neurochem 108, 57-71. Rae et al (2009) J Neurochem 109 (s1) 109-116. Nasrallah et al (in press) Metabolomics [epub 28th Aug 2009]


L5

NOVEL GABAC ANTAGONISTS THROUGH BIOISOSTERIC MODIFICATION OF 4-AMINOCYCLOPENT-1-ENECARBOXYLIC ACID.

Katherine ES Locock, Graham AR Johnston, Robin D Allan. Department of Pharmacology, University of Sydney, NSW 2006.

The simple amino acid GABA is the predominant inhibitory neurotransmitter of the mammalian CNS. Its effects are mediated via action at three membrane-bound receptor subtypes – the ionotropic GABAA and GABAC, and via the metabotropic GABAB. Recent evidence suggests that GABAC antagonists may facilitate learning and memory in rats (Chebib et al., 2009). Therefore, the development of new agents that target this system may have applications in the treatment of cognitive disorders such as Alzheimer’s disease. Gains in potency and selectivity can be achieved by bioisosteric replacement of known GABA agents. This study chose the non-selective GABAA agonist and GABAC antagonist, 4-ACPCA (4-aminocyclopent-1-enecarboxylic acid) as a parent for such manipulations with the aim of deriving GABAC selective agents. A novel series of agents was derived via replacement of the carboxylic acid (COOH) group of 4-ACPCA with functionalised amide (CONH2, CONH(CH2)2CH3, CONH(CH2)4CH3, CONHCH2Ar and COArCH3) and hydroxamate (CONHOH and CONHOCH2Ar) groups. The potency/selectivity of these compounds was ascertained using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes expressing wild type human recombinant GABAA (α1β2γ2L) and GABAC (ρ1) receptors. The substitution rationale utilised in this study resulted in gains in selectivity at the GABAC site as all compounds tested were found to be selective GABAC antagonists. Significant gains in potency at this site were also observed in the case of the primary amide (CONH2, IC50=9.5±1.4 µM, n=4) and hydroxamic acid (CONHOH, IC50=13±4.3 µM, n=4) derivatives compared to the non-selective parent 4-ACPCA (COOH, IC50=30±6.8 µM, n=4). Hence this study has led to the development of novel potent GABAC selective antagonists through the structural modification of known GABA agents.

Chebib, M, Hinton, T, Schmid, KL, Brinkworth, D, Qian, H, Matos, S, Kim, H-L, Abdel-Halim, H, Kumar, RJ, Johnston, GAR, Hanrahan, JR (2009). J Pharmacol Exper Ther, 328: 448-457.

L6

COGNITIVE ENHANCEMENT BY SELECTIVE GABAC RECEPTOR ANTAGONISTS

Hye-Lim Kim (1), Rohan J Kumar (1), Jane R Hanrahan (1), Sebastian P Fernandez (2), Graham AR Johnston (2) & Mary Chebib (1). (1) Faculty of Pharmacy (2) Department of Pharmacology, Faculty of Medicine The University of Sydney, NSW 2006.

Conformationally restricted phosphinic acid analogues of the nootropic GABAB/C receptor antagonist SGS742 (formally CGP36742) are potent and selective GABAC receptor antagonists. Examination of these analogues may help elucidate a role for hippocampal GABAC receptors in memory processing. The (R) and (S) isomers of 4-(aminocyclopent-1-enyl)-butylphosphinic acid (ACPBPA) were examined for learning and memory enhancing properties in 8-9 week old male Swiss mice using the novel object recognition test (NORT). During training (10 min) the amount of time spent with two identical objects was recorded. 24-hours later during the retention trial (10 min), the amount of time spent exploring one familiar and one novel object was recorded and a recognition index (RI) calculated. Compounds were injected ip 20 min prior to training to examine the effects on learning. Compounds were injected ip immediately post training to examine the effects on memory. Results showed that (R)- and (S)-4-ACPBPA can improve learning in a dose-dependent manner with a significant enhancement of the retention RI for both compounds at doses of 10 and 100 mg/kg (n=10, P<0.05 and P<0.001 respectively), compared to saline. 100 mg/kg of both compounds when administered post training show a significant enhancement of the retention RI (n=10, P<0.01), compared to saline. 100 mg/kg of the compounds were also examined to determine if they could reverse the amnesia induced by 0.3 mg/kg scopolamine, or by 4 mg/kg baclofen. (R)-4-ACPBPA reversed the chemically induced amnesia of scopolamine and baclofen (n=10, P<0.001 and P<0.01 respectively). (S)-4-ACPBPA was not as effective reversing the amnesia by scopolamine (n=10, P<0.01), but was as effective as (R)-4-ACPBPA when administered with baclofen (n=10, P<0.01). The nootropic properties of (R)- and (S)-4-ACPBPA demonstrated in mice indicate an involvement of the GABAC receptors in memory processing.


L7

FROM AMINO ACIDS TO FLAVONOIDS

Jane R. Hanrahan Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006

The action of flavonoids at GABAA receptors was first identified by isoflavans isolated from bovine urine displacing diazepam binding to rat brain membranes (Luk et al, 1983). Since that time many flavonoids have been found to displace radiolabelled benzodiazepines with a relatively high affinity (Dekermendjian et al 1999). The behavioural effects of flavonoids in rodents include anxiolysis, myorelaxation, sedation, anticonvulsant activity, amnesic effects and analgesia (Marder et al, 2002). Subsequently, functional electrophysiological studies have suggested the involvement of mechanisms other than via the high affinity flumazenil-sensitive benzodiazepine receptor and that the action of flavonoids at GABAA receptors is quite complex. GARJ and his collaborators have identified 6-substituted flavones as positive modulators at α1β2γ2L and α2β2γ2L receptors; the dietary flavones apigenin, genistein, (-)-epigallocatechin gallate and amentoflavone as negative modulators at α1β2γ2L receptors; and the enhancement of diazepam-induced positive modulation of the GABA response at α1β2γ2L GABAA receptors by apigenin, and (-)-epigallocatechin gallate (Johnston, 2005). Recent studies on simplified synthetic analogues of (-)-epigallocatechin gallate identified a flavan-3-acetate which is selective in terms of efficacy for α2β2γ2L over α1,3,5β2γ2L receptors with anxiolytic activity, but devoid of sedative or myorelaxant effects and a related compound which is an antagonist of modulatory activity at α1β2 receptors (Fernandez et al, 2008). These flavonoids provide useful tools to gain a better understanding of the modulation of GABAA receptors, which is an important mechanism of many classes of therapeutic drugs.

Dekermendjian K, Kahnberg P, Witt MR, Sterner O, Nielsen M. & Liljefors T (1999) J Med Chem, 42, 4343. Fernandez SP, Mewett KN, Hanrahan JR, Chebib M, Johnston GAR (2008) Neuropharmacol, 55 900. Johnston GAR (2005) Curr Pharm Design. 11, 1867. Luk KC, Stern L, Weigele M, O'Brien RA & Spirst N (1983) J Nat Prod, 46, 852. Marder M, Paladini AC. (2002) Curr Top Med Chem. 8, 853.

L8

GINKGO, GABA AND BEES

RK Duke (1), CC Ng (1), A Kaufmann (2), CC Duke (2), VH Tran (2), SW Zhang (3), H Zhu (3), M Pahl (4), ZH Huang (5), T Hinton (1) & GAR Johnston (1), (1) Dept of Pharmacology, Faculty of Medicine, (2) Faculty of Pharmacy, University of Sydney, NSW 2006, (3) Research School of Biological Sciences, Australian National University, ACT 2600, Australia; (4) BEEgroup, Biocentre, University of Würzburg, Würzburg Germany; (5) Department of Entomology, Michigan State University, East Lansing, Michigan 48824, USA

The ginkgo terpene lactones, bilobalide and ginkgolides, are the active constituents of the Ginkgo biloba leaf extract used worldwide for the symptomatic treatment for dementia. Bilobalide and ginkgolides are noncompetitive antagonists of GABAA receptors and are structurally similar to the chloride channel blocker picrotoxinin (Huang et al., 2003; 2004). Similar to picrotoxinin and many GABA receptor noncompetitive antagonists, bilobalide and ginkgolides have insecticidal activity. However, unlike picrotoxinin and other GABA antagonists, they do not cause convulsions. The action of bilobalide and ginkgolides undermines one of the basic tenets of brain function that diminished GABAA receptor–mediated inhibition results in convulsions. To investigate whether this unusual property is due to the difference in their binding sites, we evaluated the effect of bilobalide and ginkgolides on GABAA receptors carrying mutations within the channel using two-electrode voltage clamp electrophysiology and to compare to that of picrotoxinin which is known to bind to the pore of GABAA receptors.

"GABABEE Receptors": Tutin, a structural analogue of picrotoxinin, is the poisonous component of New Zealand toxic honey. This honey is highly toxic to mammals but not to honeybees. We have found that picrotoxinin and bilobalide have low toxicity to honeybees indicating that they are resistant to this class of compounds. The ginkgo terpene lactones are candidates for replacing the currently used in-hive miticide tau-fluvalinate to which the major pest of honeybees, Varroa destructor, have developed resistance. We are currently investigating the suitability of ginkgo terpene lactones for use as in-hive miticides by evaluating its sublethal effects on bees (longevity and behavioural aspects such as memory and foraging activity).

Huang SH, Duke RK, Chebib M, Sasaki K, Wada K, Johnston GAR (2003, 2004), Eur J Pharmacol, 464, 1-8; ibid 494, 131-138.


L9

A, B AND C: ROLES FOR GABA IN LEARNING AND MEMORY IN CHICKS. Marie Gibbs, Department of Anatomy and Developmental Biology, Monash University, Clayton, Vic, 3800

There is not a great deal of information regarding the role of GABA in memory processing. This is probably because it can both inhibit and enhance memory depending on the dose (Figure, Gibbs and Johnston 2005). In the sensory mesopallial integration area of the chick brain, bilateral injections of 1 pmol/hemisphere in a volume of 5 µl inhibits memory processing, whereas injections of 100 pmol/hemisphere enhance memory processing. We have established that the effect of the higher dose can be prevented by a suboptimal dose of the GABA-A antagonist bicuculline, whereas the inhibitory action of the lower dose can be prevented by a suboptimal dose of the GABA-C antagonist TMPA. A follow up study on these antagonists revealed memory inhibtion with higher doses of bicuculline and memory enhancement with higher doses of TMPA. A similar pattern of results was seen when these GABAergic antagonists were injected into the hippocampus. GABA-B receptors are also involved in memory processing, with a different pattern of involvement in the mesopallial (cortical) integration area and the hippocampus (Gibbs and Bowser 2009). In addition the result depended on the dose of the drug – a low dose of the GABA-B agonist baclofen enhanced memory whereas a high dose inhibited memory, possibly because of excessive activation. The GABA-B antagonist phaclofen inhibited memory, supporting the role for the GABA-B receptor activation in memory processing. A block to the progress of research into the role of GABA in memory processing is clearly the opposing effects that can be found with different doses of both GABA-B receptor agonists and GABA itself. An important issue now is to identify the cell types (neurons, astrocytes and/or interneurons) and which receptor subtypes of GABA receptors are associated with the response to GABA in memory processing.

Gibbs and Johnston (2005) Neurosci, 131, 567-576

L10

HYPO-GLYCINE-AEMIA’ TO ‘RELAXIN’ – A JOURNEY FROM OBSURITY TO UNCERTAINTY

AL Gundlach, Florey Neuroscience Inst and Dept of Anatomy & Cell Biology, Univ of Melbourne, Victoria 3010

A perennial curiosity in scientific research is what leads each researcher to their topics of study and whether chosen areas and/or methodologies endure for an entire career or whether some ‘adjustments’ are required along the way. There are signs of late that ‘uni-modal’ basic research is being swept aside by ‘big’ research – disease-orientated, multi-faceted research with coverage of genetics, mechanisms, organ and animal systems, and translational studies. In fact, this is the mission statement of my current Institute, but it has not always been like this. In 1986, after an overseas postdoc working on ‘obscure’ sigma opiate receptors (Largent et al 1986), I embarked on another offbeat project with scientists at University of Sydney Dept of Pharmacology and the NSW Department of Agriculture to determine the underlying cause of an inherited myoclonus in young Poll Hereford cattle. This involved use of the then topical radioligand binding and transmitter uptake studies to reveal a dramatic reduction in spinal cord glycine receptors in these calves (Gundlach et al 1988). Subsequent studies revealed a similar condition in Peruvian Pasos, and analogies to human syndromes. In the 20 years since leaving Sydney and amino acids behind, I have studied other non-mainstream topics, including ‘imidazoline’ receptors, the neurogliobiology of galanin, an ‘unsexy’ but somehow enduring pleiotropic peptide (Shen et al 2003); and recently the neurobiological mysteries of relaxins – a newly evolved peptide family with established actions in peripheral organs, which have not been studied in brain. Ironically, and perhaps in a further reflection of how a research path can turn, this has led me back to studying a complex GABA/peptidergic neuron pathway seated in the aptly named ‘nucleus incertus’ (or uncertain nucleus). Hopefully ‘GARJ’ would approve of my return to an amino acid focus and maybe this time, despite initial uncertainty, we might ‘save an important neural network from obscurity’ (Ma et al 2009).

Largent et al (1986) J Pharmacol Exp Ther 238, 739-748 Gundlach AL et al (1988) Science 241, 1807-1810 Shen PJ et al (2003) Eur J Neurosci 18, 1362-1376 Ma S et al (2009) Learn Mem, in press


L11

G PROTEIN-COUPLED RECEPTORS ARE MERELY AN ASSEMBLY OF AMINO-ACIDS

RJ Summers, ET van der Westhuizen, ML Halls and M Kocan. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Dept of Pharmacology, Monash University, Vic 3052.

Relaxins are peptides closely related in structure to insulin. However, unlike insulin they are the cognate ligands for 4 G protein-coupled receptors, the Relaxin Family Peptide Receptors (RXFPs; Bathgate et al 2006). Relaxin acting at RXFP1 shows promise in clinical trials for the treatment of cardiac failure and the RXFP3 has significant potential as a target for anti-anxiety and anti-obesity drugs. Interactions between relaxin and RXFP1 involve 3 sites - the receptor ectodomain, the transmembrane exoloops and the N-terminal LDLa module. cAMP signalling in response to receptor activation involves 3 G proteins, Gαs GαoB and Gαi3(Halls et al 2006). Signalling via Gai3 involves the C-terminal 10 amino acids of the receptor and Arg752 in particular and interaction with membrane lipid rafts (Halls et al 2009).

The cognate ligand for RXFP3 is relaxin-3, a neuropeptide expressed in the nucleus incertus. Profiling of RXFP3 showed high affinity of human relaxin-3 (H3-relaxin) with little effect of human relaxin-2 (H2-relaxin), porcine relaxin or insulin-like peptide 3 (INSL3) (Liu et al 2003). Activation of NF-κB-linked reporter genes by relaxin peptides revealed a similar profile. In contrast, not only was robust activator protein-1 (AP-1) reporter gene activation seen in cells treated with H3-relaxin, but also with H2- and porcine relaxin. Studies with inhibitors (PD98059 - MEK; RWJ67657 - p38MAPK and SP600125 - JNK) suggest that AP-1 activation to H3-relaxin involves JNK and to a lesser extent ERK and p38MAPK, whereas that to H2-relaxin involves p38MAPK but not JNK. The results suggest that several relaxin family peptides can interact with RXFP3 but that the signalling pathway activated is strongly biased by the ligand. Selective activation of these pathways may link RXFP3 activation and effects on stress and food intake that can be exploited therapeutically. These examples illustrate interactions between a few amino acids that can have a pivotal role in determining responses in biological systems.

Bathgate RA et al (2006) Pharmacol Rev 58(1): 7-31. Halls ML et al (2006) Mol Pharmacol 70(1): 214-226. Halls ML et al (2009) Mol Pharmacol 75(2): 415-428. Liu C et al (2003) J Biol Chem 278: 50754-50764. L12

CaMKII: AN ADAPTOR MEDIATING CROSS-TALK BETWEEN SIGNALLING PATHWAYS

John AP Rostas & Kathryn A Skelding Faculty of Health and Hunter Medical Research Institute, University of Newcastle, NSW 2308.

Calcium-calmodulin stimulated protein kinase II (CaMKII) is a key regulatory enzyme in many cells. The biological properties of CaMKII are regulated by multi-site autophosphorylation and targeting to cellular microdomains through binding interactions with specific proteins, including receptors, ion channels, enzymes, cytoskeletal and signalling complex scaffold proteins. The role of autophosphorylation at Thr286 has been well characterised and shown to regulate CaMKII function by altering CaMKII activity and CaMKII targeting. We have identified a new autophosphorylation site at Thr253, which regulates CaMKII function exclusively through targeting. We have shown that phosphorylation at either Thr253 or Thr286 can independently enhance or inhibit the binding of CaMKII to specific proteins. The binding interactions with CaMKII are also sensitive to changes in phosphorylation of the binding protein which can be altered by the action of kinases or phosphatases independent of CaMKII. Furthermore, the phosphorylation induced altered binding activity can not only alter the affinity of binding to the same protein partner but can also induce CaMKII to swap protein partners. In this way, by modifying the phosphorylation state of CaMKII and its binding protein partners, the action of multiple intracellular signalling pathways can direct CaMKII to become associated with different signalling complexes in the cell and hence alter the functional response of the cell to a subsequent stimulus. In this way CaMKII can act as an active adaptor mediating cross-talk between intracellular signalling pathways.


L13

EXPRESSION OF NEW EAAT5 VARIANTS IN THE MAMMALIAN RETINA.

A Lee, NL. Barnett & DV. Pow. UQ Centre for Clinical Research, University of Queensland, Royal Brisbane and Womens Hospital, Herston, Qld 4029.

EAAT5 is the predominant glutamate transporter used by photoreceptors in the retina to recover glutamate that is released by their synaptic terminals in response to light stimuli. EAAT5 is unusual in having a large chloride conductance that may therefore cause feedback regulation of release of glutamate by contributing to hyperpolarisation of the synaptic terminals. Accordingly any changes in EAAT5 expression or properties might have a major effect on the functional properties of photoreceptors. Examination of Western blots of rat retina lysate using antibodies to the amino- and carboxyl termini of EAAT5 revealed, contrary to our expectations, several bands at differing molecular weights. These data suggested that variant forms of EAAT5 might exist in the mammalian retina. PCR analysis was performed using primers flanking the coding region of EAAT5. Multiple bands were identified. Bands were excised, inserted into plasmids, expanded in E.Coli and clones sequenced. We identified 6 forms of EAAT5, including the originally described full-length wild-type form. We identified 5 splice variant forms, which skipped, either completely or partially various exons, including exon 3, exon 7 exon 8, exon 9 and exon 10. The exon 8 and exon 10-skipping forms generated frame shifts that should lead to truncated proteins. Similarly we have generated splice-specific primers and have currently verified that exon 3, 7 and 9 skipping forms are detectable. Current studies include raising antibodies to the slice variant forms and expression of cloned proteins in mammalian cell lines. Initial immunocytochemical data appear to support the view that protein coded by the exon-9 skipping form of EAAT5 may be expressed in the retina. Exon-skipping forms of EAATs have been implicated in human disease, so ongoing studies include analysis of transport properties and determination of expression profiles in the normal human retina, and in retinas with disease including macular degeneration.

L14

GABA AND GRAHAM

Mike Minchin, Astellas Pharma Europe, UK

My interest in GABA started with my Ph.D. studies in Les Iversen’s lab in the early seventies and continued well into the industrial phase of my career. In those early years I was interested in GABA release, first from glia and then from neurons. I was also keen to try and differentiate the two, by using radiolabelled precursors that preferentially labelled GABA in either glia or neurons. The first papers I published on the compartmentation theme were done in collaboration with Phil Beart, who was doing a postdoc in Les’ lab. I then moved to the John Curtin School, where I met Graham for the first time. Everybody made me and my new wife very welcome, including Graham and Helen, and their hospitality and kindness have continued down the years. I persevered with the idea of using compartmented precursors for release experiments, but now using the much more difficult tissue, spinal cord, which was the tissue of interest in the lab at the time. I also made use of the action of protoveratrine to depolarise neurons selectively.

I regret to say that in the two years that I was in Canberra, I never managed to publish a paper with Graham, but he was always very supportive of my neurochemical efforts.

On my return to the UK I worked first with Tony Angel on the effects of anaesthetics on amino acid release and then with Richard Green and David Nutt on the possible role of GABA in depression. The latter studies involved electroconvulsive shock (ECS) in rodents, using parameters which were similar to those used in severely depressed patients. This led to studies which showed that a single ECS had a profound effect on GABA release from brain slices. I translated these findings into a drug discovery project when I moved into industry at Wyeth UK. In this project we analysed the presynaptic GABA receptor which regulated GABA release and found what we thought was a novel type of receptor, which was more sensitive to antagonism by bicuculline than was the GABAA receptor. We found N-substituted GABA derivatives which were selective agonists at the autoreceptor, but the project was terminated when the medicinal chemistry reached an impasse. So, like most industrial compounds, our GABA autoreceptor agonists remained on the shelf. Meanwhile, Graham’s interest in GABA has not waned – far from it. After his groundbreaking identification of bicuculline with David Curtis in 1971, he has continued to dissect GABA receptors, and in the mid-nineties identified a new one, the GABAC receptor.

Since the Wyeth days I have not worked on GABA again, but those early years in Cambridge, Canberra and then Oxford were some of the most intellectually stimulating times of my life and Graham played a large part in them.


L15

AMINO ACIDS: TALES OF THE OCCULT AND UNEXPECTED.

LP Davies, Australian Pesticides & Veterinary Medicines Authority, Canberra, ACT 2603

In these days of Global Financial Crises and weasel words, of optimising synergies, outcomes-based deliverables and mutually-agreed action plans, it is comforting to think back to those simpler times in the early 1970s when the amino acid laboratories of David Curtis and Graham Johnston in Canberra were a focal point for researchers coming from many parts of the world. Probably the most adventurous of them was David Lodge who drove from England in an old ‘Land Rover’, accompanied by his family. Curtis’s section of the Physiology Department (which later in my time at the John Curtin School of Medical Research [1972-1975] broke away to form the Pharmacology Department) was populated by electrophysiologists, chemists and biochemists; those of us in Graham Johnston’s neurochemistry laboratory felt somewhat sorry for the electrophysiologists who spent long afternoons in the warm, darkened and cramped space of David Curtis’s shielded room (the ‘Tardis’) or, later in that period, in Arthur Duggan’s; many was the time that someone almost dozed off and overbalanced on their stool while watching the CRO as glutamate, GABA or glycine was being ‘squirted’ onto a spinal cord neuron of an anaesthetised cat.

Graham’s neurochemistry laboratory ran on the smell of an oily rag. We nicknamed the old shaking water bath (used for experiments on amino acid uptake into brain slices) the ‘African Queen’ because it sounded like an ancient steam-driven paddle steamer. Our spectrophotometer was a ‘Shimadzu’ of an age, shape and size not too different from the Japanese midget submarine which raided Sydney Harbour in WWII. The laboratory didn’t have a spare inch of room, with Graham, his first three PhD students (Phil Beart, Vladimir Balcar, and me) and a laboratory technician (Bruce Twitchin). A narrow walkway into Graham’s office was also the location for my very small desk. Marie-Louise Uhr (working on glycine and serine metabolism) had the laboratory immediately opposite. Morning and afternoon tea at ‘The John’ were daily rituals when everyone adjourned to the tearoom and was served tea from huge aluminium teapots at a servery staffed by kindly ladies who seemed part of the fixtures. Relaxing in easy chairs, it was enthralling to listen to Arthur Chapman tell stories of those heady days when he worked as laboratory technician for Sir John Eccles. I often thought that it was a great pity that he never wrote his memoirs – but perhaps some of the stories told were better left unrecorded! No doubt many stories about GARJ will be just as enthralling.

L17

GLUTAMATE/ASPARTATE RECEPTOR JOURNEYS: PERSONAL TALES FROM DOWN-UNDER AND BACK-HOME.

David Lodge, MRC Centre for Synaptic Plasticity, Department of Anatomy, Medical School, University of Bristol, BS8 1TD, United Kingdom.

In this review, the beginnings of glutamate pharmacology (Curtis and Johnston, 1974) will be traced from the early doubts about ‘non-specific’ excitatory effects, through glutamate- and aspartate-preferring receptors, to NMDA, quisqualate/AMPA and kainate receptor subtypes (Johnston et al., 1974; McLennan and Lodge, 1979), and finally to the cloning of genes for these receptor subunits. The development of selective agonists and antagonists, both orthosteric and allosteric (Lodge and Johnston, 1985), was crucial to subtype classification, and coupled with neurochemical advances in synthesis, release and uptake of glutamate (Balcar and Johnston, 1972; Duggan and Johnston, 1974) established its transmitter role. Symbiosis between biologists and chemists was the key to this success, which allowed the fundamental importance of glutamate receptors to synaptic activity in health and disease to be realised. The subsequent ability to clone, express and manipulate glutamate receptor subunits has led to further advances in our understanding of these receptors but also in nomenclature issues (Lodge, 2009).

From small beginnings in Canberra, Bristol, Vancouver, Copenhagen, Tokyo, etc., the glutamate field is reaching fruition with compounds now in the clinic for a variety of neurological and psychiatric diseases.

Balcar VJ and Johnston GAR (1972) J Neurochem 19, 2556-2566 Curtis DR and Johnston GAR (1974) Ergebn Physiol 69, 97-188 Duggan AW and Johnston GAR (1974) J Neurochem 17, 1205-1208 Johnston GAR et al (1974) Nature 248, 804-805 Lodge D (2009) Neuropharmacology 56, 6-21 Lodge D and Johnston GAR (1985) Neurosci Lett 56, 371-375 McLennan H and Lodge D (1979) Brain Res 169, 83-90


L18

GLUTAMATE TRANSPORT: A FATAL ATTRACTION OR JUST A DISTRACTION?

VJ Balcar (1) and C Rae (2), (1) Bosch Institute & School of Med. Sciences, Sydney Medical School, The University of Sydney F 13, NSW 2006. (2) Prince of Wales Medical Research Institute and The University of New South Wales, NSW 2031

Synapses in the CNS incorporate substrate-specific, efficient transport of neurotransmitters. Inhibiting such transport should increase the efficacy of a selected group of synapses. This has been exploited in designing some very successful drugs. What about glutamatergic synapses? Excess synaptic (extracellular) L-glutamate (L-Glu) can damage neurons and, therefore, inhibitors of L-Glu transport (GluT) would be neurotoxic rather than beneficial. Graham Johnston’s work on the structural specificity of GluT (Balcar and Johnston 1972, Davies and Johnston 1976) has greatly facilitated the development of specific GluT substrates/inhibitors. However, inhibition of GluT does not always produce rapid effects in vivo. Administration of GluT inhibitors for days or weeks may often be needed to demonstrate the “expected” neurodegeneration. Fluctuations in glutamatergic activity for a short time may not be enough to harm otherwise healthy neurons but a long-term inhibition of GluT, while perhaps disrupting brain function in a less obvious manner, could lead to a “slow” neuronal death. Could metabolic changes be involved? Indeed, inhibition of GluT in brain tissue in vitro significantly modifies metabolism (Moussa et al. 2007). The changes vary from one GluT inhibitor to another, thus reflecting differences in their mode of action or selectivity for individual transporters. Similarly, results of studies on activity-related regulation of GluT in cultured astrocytes depend on which GluT substrate/inhibitor is used. The underlying mechanisms are complex and may be related to energy production and activity of Na+,K+-ATPase. Since its initial characterization in Graham Johnston’s laboratory, GluT has attracted many scientists around the world mainly because of its potential importance and apparent key position in synaptic physiology. Those very traits imply that GluT is tightly controlled so that it is not easily perturbed in vivo. Studying GluT in the CNS sometimes starts as a minor distraction - just a quick clarification of a few almost obvious points - but may end up as a very long-term challenge that keeps coming back again and again.

Balcar VJ and Johnston GARJ (1972) J Neurochem, 19, 2657-2666 Davies LP and Johnston GARJ (1976) J Neurochem, 26, 1007-1014 Moussa CEH et al. (2007) J Neurosci Res, 85, 342-350

L19

THE INTERPLAY BETWEEN ION COUPLING AND THE PHARMACOLOGICAL PROPERTIES OF GLUTAMATE TRANSPORTERS

Renae Ryan, Nicholas Kortt and Robert Vandenberg, Department of Pharmacology, School of Medical Sciences, Bosch Institute, University of Sydney, NSW, 2006

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and activates a wide range of receptors to mediate a complex array of functions. Extracellular glutamate levels are tightly controlled by a family of transporters known as Excitatory Amino Acid Transporter (EAATs) which serve to maintain a dynamic signalling system between neurons. The crystal structure of a prokaryotic homologue of the EAATs has recently been determined, which has aided our ability to understand the structural basis of the function of these proteins. However, the prokaryotic homologue, GltPh, shows some differences in substrate affinity and specificity and in ion coupling mechanisms when compared to the EAATs. We have exploited subtle differences in amino acid sequences of the EAATs and GltPh to better understand the structural basis for differences in function between these two transporters. An arginine residue is in close 3 dimensional proximity to the substrate binding site of the both the EAATs and GltPh, but is found on different structural elements; transmembrane domain 8 in the EAATs and hairpin loop 1 in GltPh. We have switched the location of the arginine residue in EAAT1 and also in GltPh and investigated the change in functional properties. The EAAT1 mutant has a 150 fold higher affinity for substrates and does not require K+ counter-transport, which are similar to GltPh. The GltPh mutant shows lower affinity for substrates, which is more EAAT1-like, but K+ coupling has not been introduced. These results demonstrate that the location of an arginine residue influences both substrate affinity and K+ coupling of EAAT1 and provides a potential explanation for the interplay between substrate binding and K+ coupling.


L20

DRINKING MORE THAN YOUR DOCTOR.

PR Dodd, CP Ng & JP Ridge, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072.

Real-time RT-PCR was used to assay N-methyl-D-aspartate (NMDA) receptor NR1, NR2A and NR2B subunit mRNA expression, normalized to GAPDH, in human cortex tissue obtained at autopsy under informed written consent from chronic alcoholics with and without comorbid cirrhosis of the liver and matched controls. Subunit mRNA expression was influenced by the subject’s genotype. The TaqIA polymorphism selectively modulated NMDA receptor mean transcript expression in cirrhotic-alcoholic superior frontal cortex, in diametrically opposite ways in male and female subjects. Genetic make-up may differentially influence vulnerability to brain damage by altering the excitation:inhibition balance, particularly in alcoholics with comorbid cirrhosis of the liver. The TaqIA polymorphism, long associated with the DRD2 genotype, resides within the coding region of a contiguous gene that was thought not to be expressed in the brain, the poorly characterized Ankyrin-Repeat-containing Kinase 1 (ANKK1) gene. Using PCR, ANKK1 mRNA transcript was detected in inferior temporal, occipital, superior frontal and primary motor cortex of control human brain. ANKK1 expression may mediate the influence of the TaqIA polymorphism on phenotype. Supported by NHMRC

L21

CONSTRAINING L-GLUTAMATE.

RD Allan, Department of Pharmacology, University of Sydney, NSW 2006.

Conformationally restricted analogues of neurotransmitters have allowed an understanding of basic requirements for receptor binding, and have led to the development of many sub-type selective agents. However, a feature that is not widely appreciated is that, when conformationally restricted analogues show appreciable potency and selectivity, the molecular constraints are such that a limited amount of conformational flexibility is still retained. This point will be emphasised in describing the development of analogues in which the orientations available to the binding groups of glutamate (1) is restricted by a ring system. Investigation of the simple cyclobutane analogues by assessing excitatory activity on the rat cortical wedge preparation led to the discovery of trans-1-aminocyclobutane-1,3-dicarboxylic acid (2) as a potent agonist, acting selectively at the NMDA receptor subtype. In contrast, the other cyclobutane stereoisomer is much less active. The 3-dimensional arrangement of binding groups can also be restricted as a result of bioisosteric replacement of one of the binding groups. In particular, replacement of the chain carboxylic acid group with a particular 3-hydroxypyridazines N-oxide as in compound 3 ((RS)-2-amino-3-(3-hydroxypyridiazin-4-yl 1-oxide)propionic acid) shows that this group is an outstanding carboxylate mimic, giving rise to a potent and selective agonist, but with selectivity for the AMPA rather than the NMDA subtype. The fact that additional conformational restriction on these pyridazine analogues leads to a loss of activity is consistent with the idea that a limited amount of molecular flexibility, with one binding group moving in a specific direction relative to the other binding groups, is important for agonist activity.

Oc SF PM IT

1 2 3


L22

GLUTAMATE HOMEOSTASIS AND ISCHAEMIA.

DV. Pow(1) A. Lee (1) P.Poronnik (2) & S. Sullivan (1). (1) UQ Centre for Clinical Research, University of Queensland, Royal Brisbane and Womens Hospital, Herston, Qld 4029, (2) School of Medical Sciences, RMIT, Melbourne Vic 3083.

Ischaemia is a complex biochemical insult, but the damage that ensues includes a major contribution from the effects of hypoxia. Glutamate dys-homeostasis is evident in response to hypoxia-ischaemia, both short-term, during the insult when there is energetic compromise, and longer term when oxygen and energy substrates are again available. Glutamate homeostasis may readily be delineated into four functional processes, including the release of glutamate from neurons, the uptake of glutamate by transporters (EAATs), the metabolic interconversions of glutamate and glutamine and the trafficking of glutamine. We have examined the expression of glutamate transporters and the expression of the glutamate catabolising enzyme glutamine synthetase in the pig brain after hypoxic insults. Our data indicate that there is a good spatial concordance between the patterns of glutamate transporter loss from astrocytes and the patterns of evoked damage in the brain. In addition we demonstrate that immunolabeling for glutamine synthetase (which is essential for the catabolism of glutamate after recovery into astrocytes), reveals a similar pattern of spatially-related loss, but the loses of glutamine synthetase precede any anatomical evidence for damage. Our initial data indicate that there is an intimate molecular complex formed by EAAT1 and Glutamine synthetase, suggesting transport and catabolism are tightly linked. Similarly we demonstrate that the sodium/potassium ATPase, which generates the sodium gradient needed for glutamate transport, appears to be part of this complex. Our data further suggest that EAATs are spatially constrained in the plasmamembranes of astrocytes by interactions with cytoskeletal proteins. Collectively these data suggest that dys-regulation of glutamate uptake and catabolism may have a common origin in the stability of complexes of the type we describe, and thus such complexes may represent pharmacological targets.

L23

THE mGLU5 RECEPTOR, DRUG-SEEKING & DRUG-INDUCED PLASTICITY. A.J. Lawrence, Florey Neuroscience Institutes & Centre for Neuroscience, University of Melbourne, Vic 3010

The mGlu5 receptor is a target for regulation of alcohol and drug abuse, drug-induced plasticity and contextual associations. For example, treatment of rodents with a selective mGlu5 antagonist, MTEP, can reduce both the self-administration of alcohol and cocaine and also relapse drug-seeking. These data suggest that mGlu5 receptors are implicated in appetitive and consummatory properties of drugs of abuse. In accordance, mice lacking mGlu5 receptors show reduced self-administration of ethanol that is paralleled by an increased sensitivity to the hypnotic effects of ethanol. mGlu5 lacking mice can still metabolise ethanol and also find ethanol positively reinforcing. In relation to cocaine, while mice lacking mGlu5 receptors develop a place preference to cocaine, the ability of cocaine to cause synaptic adaptation at excitatory inputs onto dopaminergic neurons of the ventral tegmental area is absent. This lack of cocaine-induced synaptic plasticity is mirrored by a differential profile of hyperactivity to acute cocaine. Interestingly however, mGlu5 deficient mice still sensitize to cocaine, providing evidence for dissociation between cocaine-induced plasticity onto tegmental dopaminergic neurons and psychomotor sensitization. Parallel studies in wild-type mice demonstrate that MTEP pre-treatment can attenuate acute cocaine hyperactivity but has no effect on the development of sensitization to cocaine. These data collectively suggest that the mGlu5 receptor is implicated in drug-seeking and also specific aspects of drug-induced plasticity.


L24

THE PHARMACOLOGIST’S PRAYER: “DELIVER US FROM BRICK DUST AND PROMISCUOUS INHIBITORS”.

B Jarrott, Florey Neuroscience Institutes, Univ of Melbourne, Vic 3010

The application of combinatorial chemistry/parallel phase synthesis has resulted in a multitude of novel compounds for pharmacologists to assess for potential drug actions and this has created a bottleneck in the conventional drug discovery process. Even the development of high throughput screens using either radioreceptor assays or intracellular signalling in intact cells has not markedly speeded up the discovery of innovative new drugs because high throughput screens often give false positives. This is now believed to be due to ‘promiscuous’ compounds that act noncompetitively at enzymes or receptors and also show little relationship between structure and function (Seidler et al 2003). A contributing factor is that combinatorial chemistry tends to produce multi-ring, hydrophobic compounds with log Ps > 4 that are highly insoluble in water (‘brick dust’). While these compounds can dissolve in solvents such as dimethyl sulfoxide that can be added to radioreceptor assays, they often precipitate out when re-examined in more relevant pharmacological screens such as in vitro organ-bath preparations and in vivo animal models of diseases. Even if these compounds do not appear to precipitate out upon dilution of the dimethyl sulfoxide vehicle, they have been found to form aggregates which are particles of 30-400 nm in diameter and these aggregates are the active inhibitory species often on a range of unrelated enzymes or membrane-bound receptors – hence the term ‘promiscuous inhibitors’ (Seidler et al 2003). These compounds are unlikely to make good leads for drug discovery projects but fortunately, can now be identified early by a dynamic laser light scattering technique. In our research into cytoprotection against ischemia based on synthetic flavonol compounds and butylated hydroxy toluene analogues, we have encountered this problem with these lipophilic compounds. However, we have found that formation of prodrugs by esterification of a hydroxyl group either with adipic acid or phosphonic acid gives ionisable compounds that are sufficiently water soluble for parenteral injection (Jarrott et al 2009) and also the water soluble esters do not form aggregates. This also should allow investigations into their mechanism of action without the complication of aggregates causing promiscuous interactions with receptors and enzymes.

Seidler J et al (2003) J Med Chem, 46, 4477-4486 Jarrott B et al (2009) USA Patent 2009/0130051, 1-31

L25

SO MANY MOLECULAR TARGETS: NEUROPHARMACEUTICALS, EXCITOTOXICITY AND NEUROPROTECTION.

PM Beart, Molecular Neuropharmacology, Florey Neuroscience Institutes and Department of Pharmacology, University of Melbourne, Vic 3010

Academic researchers have played major roles in drug development, including the design and syntheses of diverse neuro-pharmaceuticals. Typical of medicinal chemistry, my introduction to the syntheses of water soluble, small molecules proved both rewarding and frustrating. 4-Aminotetrolic acid was an initial success (Beart et al 1971), but probably represented the last personal efforts by Graham Johnston in the synthetic arena. Ironically at this time parallel work in Canberra explored the in vivo neurotoxicity of excitant amino acids (Johnston 1973) providing some initial observations relevant to excitotoxicity, which subsequently became a topic of long-term commitment. Structure activity relationships remained an area of interest and analyses of phencyclidine/sigma drugs (Manallack et al 1988) led to the development of novel bifunctional molecules that were neuroprotective in stroke injury (Callaway et al 1999). Our patents with the pharmaceutical industry have fallen by the wayside due its lack of commitment to neuroprotection. Mechanistic investigations of excitotoxicity and neuronal injury have produced insights into programmed cell death. The importance of a healthy diet rich in naturally occurring, cytoprotective molecules remains a great consolation (Mercer et al 2005). Recent efforts focus on glial genes as targets for preserving neuronal function.

Beart PM et al (1971) Nat New Biol, 234, 80-81 Callaway JK et al (1999) Stroke, 30, 2704-2712 Johnston GAR (1973) Biochem Pharmacol, 22, 137-140 Manallack DT et al (1988) Mol Pharmacol, 34, 863-879 Mercer LD et al (2005) Biochem Pharmacol, 69, 339-345


L26

GABA: THINKING OUTSIDE THE BRAIN.

GAR Johnston, Adrien Albert Laboratory of Medicinal Chemistry, Department of Pharmacology, The University of Sydney, NSW, 2006, Australia

During the course of helping to write a major review on the synthesis of GABA analogs (Hanrahan & Johnston 2009) I was reminded of the widespread occurrence of GABA systems and of GABA motifs. GABA has been described as a conserved and ubiquitous signaling system (Bouché et al., 2003) and occurs extensively in plants, fungi, bacteria, insects, invertebrates, and vertebrates.

Several biologically active GABA analogues occur naturally. These include (S)-4-amino-3-hydroxybutanoic acid (GABOB), also found in the cyclic depsipeptide hapalosin, and L-carnitine (used in the treatment of myopathies) found in mammalian tissues. Statine [(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid] is regarded as the key pharmacophore in the rennin inhibitor pepstatin isolated from bacteria. Statine and its analogs are useful as building blocks for peptidomimetics. Homoisoserine (2-hydroxy-4-aminobutyric acid) acts as inhibitor of GABA uptake, exhibits antitumor activity, is found in many antibiotics, and is also used to construct peptidomimetics. The GABA motif is found in drugs such as baclofen, diclofenac, haloperidol, gabapentin, pregabalin and zanamivir.

In mammals, GABA is found in many organs outside of the CNS where it serves various functions. GABA is involved in cell proliferation and migration, and may play a role in cancer. Recent evidence implicates GABA receptors in mucus overproduction in asthma acting on airway epithelial cells. GABA regulates insulin secretion from pancreatic β cells in concert with changes in glucose concentration and may be involved with type 1 diabetes. Functional GABA receptors have also been described in T cells and macrophages. Thus in addition to brain disorders, asthma, cancer, diabetes, and the immune system may also be important targets for GABA analogs.

Bouché N, Lacombe B & Fromm H (2003) Trends Cell Biology, 13, 607-610 Hanrahan JR & Johnston GAR (2009) Amino Acids, Peptides and Proteins in Organic Chemistry, Volume 1, ed. Hughes AB, pp. 573-689, Weinheim, Wiley-VCH

Documents

The University of Sydney - The University of Sydney - …sydney.edu.au/medicine/pharmacology/adrien-albert/images/... · 2009. 12. 10. · C Rae (1), FA Nasrallah (1) & VJ Balcar