- Email: [email protected]
Control of neurotransmitter release by metabotropic glutamate receptors
S.12. G-protein coupled amino acid receptors: New therapeutic opportunities
S.12. G-protein coupled amino acid receptors: New therapeutic opportunities ~ T h e orphan GPCRs: Targets of the future neuropsychopharmacological drugs O. Civelli, R. Reinscheid, Z. Wang, Y. Saito, S. Lin, S. Clark, H. Nagasaki, T. Takahashi, H.P. Nothacker. University of
California, Iruine, US.A. The principles that underlie most of the drugs used in neuropsychopharmacology were discovered during the 50's to the 70's, long before the actual targets were described molecularly. Indeed mental disorder therapies rely on less than 50 drug targets. The drug discovery programs that led to neuropsychopharmacological drugs were predominantly driven by observations of changes in a particular behavior rather than by a concerted approach. Yet when the targets of the neuropsychopharmacological drugs were finally identified, the vast majority turned out to be G protein-coupled receptors (GPCRs), a fact not surprising since more than 40% of all therapeutically used drugs are directed at GPCRs and since GPCRs are known to be at the center of many CNS functions. Because mental disorders need new and better therapies, the use of GPCRs in drug discovery can be now brought one step further. After sequencing of the human genome, the superfamily of the GPCRs can now be classified into the sensory GPCRs, (500-700) and some 320 other GPCRs that bind all the known neurotransmitters, neuropeptides and peptide hormones. The latter group regulates many functions in the organism and in particular in the CNS. This group also includes all GPCRs that have been sought as drug targets. These last are the ones that include all the GPCRs, which have been targeted in drug discovery. Of the 320 "transmitter" GPCRs, 180 bind known ligands, while the others bind ligands that have not been thus far described. These are the orphan GPCRs. Orphan GPCRs may prove to be a gold mine for neuropsychopharmacological research. First they modulate responses in a manner that is totally novel. Because most of them are expressed in the CNS they may permit to gain a better understanding of either known CNS function from a new stand point or moreover to define novel CNS functions. This could allow us to drastically enrich our understanding of mental disorders. Second, the orphan GPCRs will bind small chemically-friendly molecules which makes them ideally amenable to drug discovery. This should insure that any orphan GPCRs of therapeutic interest will enter drug discovery programs and should lead to the identification of surrogate ligands that will be of great help in basic research. However, orphan GPCR research is handicapped by the lack of the GPCR natural ligands. Consequently, the receptor activity cannot be tested and the ligand biology, which account for half of the system, cannot be studied. To be of value, orphan GPCRs need to be deorphanized, i.e. their natural ligand need to be characterized. We have developed a strategy to identify the natural ligands of orphan GPCRs, the orphan receptor strategy. This strategy relies on the use of expressed orphan GPCRs as targets to isolate their natural ligand from tissue extracts. Since 1995, the date of its first success, the orphan receptor strategy has led to the deorphanizing of nine orphan GPCRs and to the discovery of six novel transmitter families. The novel transmitters (all neuropeptides) are of great interest to neuropsychopharmacology. They have shown
Sl15
to be very diverse in their structures and localization, albeit all expressed in the CNS. The major task resides in defining their principal function(s) as several physiological responses can be associated with the administration of the novel transmitters. The physiological roles of the first two transmitters isolated via the orphan receptor strategy emerge as good examples for the impact this research will have for neuropsychopharmacology. Orphanin FQ/Nociceptin has been shown to be a potent anxiolytic whose activity mimics that of diazepam. Orexins/Hypocretins on the other side are major regulators of narcolepsy. Studies on the principal roles of the other novel transmitters are being carried out. So far we know of some 100 transmitters. These are the cornerstones of all neuropsychopharmacological therapies. The search for the natural ligands of orphan GPCRs promises to lead us to the discovery of some 70 more. When completed this search will immensely enrich our understanding of brain function and consequently of brain disorders and these efforts will undoubtedly lead to novel drugs in a few years. Indeed, every one of the nine deorphanized GPCR systems mentioned above is part of drug discovery programs, some even under intense competition. Small synthetic surrogates have been described for some of the novel neuropeptide system. It is only a matter of time until some of the orphan GPCRs will be targets of marketed drugs. Neuropsychopharmacology will gain immensely of the products from the products of the orphan receptor strategy.
References [1] Civelli, O., Nothacker, H.P., Saito, Y., Wang, Z., Lin, S. and Reinscheid R.K. (2001) Novel neurotransmitters as natural ligands of orphan G protein-coupled receptors. Trends in Neuroscience 24:230-237.
•
Control of neurotransmitter release by metabotropic glutamate receptors
D. Schoepp. Eli Lilly and Company, Indianapolis, Indiana, US.A. Glutamate is the major neurotransmitter substance which controls the excitability of the mammalian central nervous system. Importantly, a number of disease states have now been linked to altered glutamate neurotransmitter function. Thus, modulation of glutamate-induced excitation offers therapeutic promise to treat a wide range of neurological and psychiatric disorders. Glutamate receptors include ionotropic (ligand-gated ion channels) receptors (iGlu receptors) that directly mediated glutamate excitations and metabotropic (G-protein coupled receptors or mGlu receptors) that function to modulate glutamate by neuronal and glia mechanisms. In particular, one function of mGlu receptors is to regulate the pre-synaptic release of neurotransmitter glutamate, and in this manner control post-synaptic excitations by glutamate. In general, activation of group I mGlu receptors (mGlul and/or mGlu5) will facilitate the release of synaptic glutamate, while activation of either group II (eg. mGlu2) or group III (eg. mGlu4, mGlu7, mGlu8) leads to a reduction in glutamate release. The role of specific mGlu receptor subtypes in CNS functions involving glutamate release depends on factors which include 1) regional distribution of the mGlu receptor 2) positioning of the mGlu receptor within a neuronal circuit 3) specific synaptic or extra-synaptic localizations for receptors 4) endogenous tone at the receptor under physiological versus pathological conditions (or local concentration of glutamate versus affinity of the receptor for glutamate) 5) direct or indirect mGlu receptor mediated modulation of the release of other
S.12. G-protein coupled amino acid receptors: New therapeutic opportunities
Sl16
neurotransmitter substances (eg. neuropeptides and monoamines) and 6) heterosynaptic regulation of GABA release within neighboring inhibitory synapses. Pharmacological agents which target mGlu receptor subtypes that modulate glutamate release have now been discovered and characterized in animal models, and are progressing in target validation clinical studies. Recent progress on the molecular pharmacology of mGlu receptors along with the use of transgenic animals lacking mGlu receptor subtypes are now further revealing therapeutic directions for mGlu receptor active agents which modulate glutamate release.
References [1] Schoepp D.D., Jane D.E., Monn J.A. (1999) Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38: 1431-1476. [2] Cartmell J. and Schoepp D.D. (2000) Regulation ofneurotransmitter release by metabotropic glutamate receptors. Journal of Neurochemistry 75: 889-907. [3] Schoepp D.D. (2001) Unveiling the fi.mctions of presynaptic metabotropic glutamate receptors in the central nervous system. Journal of Pharmacology and Experimental Therapeutics 299: 12-20.
•
Anatomy, physiology and pharmacology of GABAB receptors
N.G. Bowery. Department of Pharmacology, Medical School
University of Birmingham, B15 2TT, United Kingdom G-protein coupled receptors can be expressed in cell membranes in different ways. They may simply reside as a single protein unit after initial transcription in the endoplasmic reticulum or in some instances the functional receptor may be expressed as a combination of two identical protein molecules linked to form a homodimer. The structure of the metabotropic GABAB receptor introduces even further complexity to the situation as it exists as a unique heterodimer. Detection of this structural dimer was initiated from studies with the cloned receptor subtmit, GABABI. A second receptor subunit was shown to be linked to GABABl through coiled-coil domains at the C-terminal in a stoichiometric 1:1 ratio. This appears to be an absolute requirement for functional expression of the GABAB receptor. This second subunit was designated GABAB2 and has many of the structural features of GABAm including a large molecular weight (110 KDa), 7 transmembrane domains, a long extracellular chain at the N-terminus with 54% similarity (35% homology) with GABABI. At least six isoforms ( l a - l f ) of subunit GABAB1 and three forms of subunit GABAI32 have been reported but whether these all make functional forms of the receptor is unclear. The ligand binding domain of the heterodimer appears to reside in the N-terminal segment of GABAB1 with no evidence for any ligand binding to GABAB2. Current evidence would suggest that even though GABAB2 has a large N-terminus, comparable in length to that in GABAm with considerable homology between them, GABAB2 is more important for effector coupling and signalling of the receptor. On this basis one could predict that G-protein coupling is only mediated through the GABAB2 subunit and this seems to be the current view. The interaction of the C-terminal coiled-coil domains of the subunits appears to mask the action of the retention motif present in the C-terminal of GABAB1 and this is believed to provide a 'trafficking checkpoint' to ensure organized assembly of the functional receptor. The coiled-coil structures seem not to be essential for the heterodimerization to occur. Other proteins that can associate with GABAB], such as mGlu4, will facilitate
expression. However, unlike GABAB2, they are unable to provide effector coupling within the cell membrane. GABAB receptor distribution in CNS: Distribution studies of mRNA for two of the splice variants of the GABAm subunit, GABAB(la) and GABAB(lb) using in situ hybridisation techniques has revealed that they appear to be differentially distributed. Initial studies in rat and human cerebellum and spinal cord indicate that GABAB0a) is associated with presynaptic receptors whereas GABAB(lb) may be responsible for postsynaptic receptor formation in the cerebellum. However, the contrary arrangement has been observed in, for example, the thalamo-cortical circuitry where GABABIa subunits appear to be postsynaptic on cell bodies. As a consequence it seems unlikely that functional role and cellular location can be assigned in a general manner to specific GABAB receptor subunit splice variants. The regional distributions of GABABI and GABAB2 protein subunits are in broad alignment with that of the native receptor although in some brain areas, such as the caudate putamen, GABAB1 and the native receptor are present whereas GABAB2 appears to be absent. A wide distribution of the receptor subunits has also been detected throughout the periphery. However, again the GABAB2 subnnit was not always present, for example, in the uterus and spleen where GABAm subnnits could be detected. This would surely support the existence of an additional, as yet undiscovered, subunit. GABAB Receptor Effector mechanisms: The effector mechanisms to which neuronal GABAB receptors are coupled are adenylate cyclase and Ca 2+ and K + channels. Whilst it can be demonstrated that the majority of these effects are mediated via G-proteins, in particular Gi2c~, not all effects of GABAB receptor activation appear to be coupled. Pertussis toxin insensitive effects of baclofen have been noted, for example, in rat spinal cord and magnocellular neurones of the paraventricular and supraoptic nuclei. GABAB receptor activation decreases neuronal membrane conductance for Ca 2+ but increases conductance to K + ions. The decrease in Ca 2+ conductance appears to be primarily associated with presynaptic sites suppressing 'P/Q' and 'N' type channels whereas modulation of K + conductances appears to be primarily linked with postsynaptic GABAB sites and possibly more than one type of K + channel. Numerous effects have been attributed to the action of GABAB receptor agonists. Some of these inelude: centrally-mediated muscle relaxation, an antitussive action, bronchiolar relaxation, urinary bladder inhibition, gastric motility increase, epileptogenesis, suppression of self-administered cocaine, nicotine and opiates, antinociception, yawning, hypotension, brown fat thermogenesis, cognitive impairment, reduction in release of hormones such as corticotrophin releasing hormone, prolactin releasing hormone, luteinizing hormone and melanocyte stimulating hormone and reduced gastric acid secretion. The centrally-mediated muscle relaxant action of baclofen is the most well recognised of its effects and the mechanism underlying this appears to derive from its ability to reduce the release of excitatory neurotransmitter on to motoneurones in the ventral horn of the spinal cord. Although pain relief has been noted with baclofen in trigeminal neuralgia in man, its usefulness as an analgesic has always been questioned. Nevertheless, more recent clinical observations have indicated that baclofen can reduce pain due to stroke or spinal cord injury and musculoskeletal pain when administered by intrathecal infusion. Recent observations with baclofen indicate that it may also be a very effective treatment for cocaine, nicotine and opiate addiction by reducing the craving for the drug. In rats, baclofen, administered at doses of 1-5 mg/kg, suppressed the self-administration of cocaine without affecting
S.12. G-protein coupled amino acid receptors: New therapeutic opportunities ~ T h e orphan GPCRs: Targets of the future neuropsychopharmacological drugs O. Civelli, R. Reinscheid, Z. Wang, Y. Saito, S. Lin, S. Clark, H. Nagasaki, T. Takahashi, H.P. Nothacker. University of
California, Iruine, US.A. The principles that underlie most of the drugs used in neuropsychopharmacology were discovered during the 50's to the 70's, long before the actual targets were described molecularly. Indeed mental disorder therapies rely on less than 50 drug targets. The drug discovery programs that led to neuropsychopharmacological drugs were predominantly driven by observations of changes in a particular behavior rather than by a concerted approach. Yet when the targets of the neuropsychopharmacological drugs were finally identified, the vast majority turned out to be G protein-coupled receptors (GPCRs), a fact not surprising since more than 40% of all therapeutically used drugs are directed at GPCRs and since GPCRs are known to be at the center of many CNS functions. Because mental disorders need new and better therapies, the use of GPCRs in drug discovery can be now brought one step further. After sequencing of the human genome, the superfamily of the GPCRs can now be classified into the sensory GPCRs, (500-700) and some 320 other GPCRs that bind all the known neurotransmitters, neuropeptides and peptide hormones. The latter group regulates many functions in the organism and in particular in the CNS. This group also includes all GPCRs that have been sought as drug targets. These last are the ones that include all the GPCRs, which have been targeted in drug discovery. Of the 320 "transmitter" GPCRs, 180 bind known ligands, while the others bind ligands that have not been thus far described. These are the orphan GPCRs. Orphan GPCRs may prove to be a gold mine for neuropsychopharmacological research. First they modulate responses in a manner that is totally novel. Because most of them are expressed in the CNS they may permit to gain a better understanding of either known CNS function from a new stand point or moreover to define novel CNS functions. This could allow us to drastically enrich our understanding of mental disorders. Second, the orphan GPCRs will bind small chemically-friendly molecules which makes them ideally amenable to drug discovery. This should insure that any orphan GPCRs of therapeutic interest will enter drug discovery programs and should lead to the identification of surrogate ligands that will be of great help in basic research. However, orphan GPCR research is handicapped by the lack of the GPCR natural ligands. Consequently, the receptor activity cannot be tested and the ligand biology, which account for half of the system, cannot be studied. To be of value, orphan GPCRs need to be deorphanized, i.e. their natural ligand need to be characterized. We have developed a strategy to identify the natural ligands of orphan GPCRs, the orphan receptor strategy. This strategy relies on the use of expressed orphan GPCRs as targets to isolate their natural ligand from tissue extracts. Since 1995, the date of its first success, the orphan receptor strategy has led to the deorphanizing of nine orphan GPCRs and to the discovery of six novel transmitter families. The novel transmitters (all neuropeptides) are of great interest to neuropsychopharmacology. They have shown
Sl15
to be very diverse in their structures and localization, albeit all expressed in the CNS. The major task resides in defining their principal function(s) as several physiological responses can be associated with the administration of the novel transmitters. The physiological roles of the first two transmitters isolated via the orphan receptor strategy emerge as good examples for the impact this research will have for neuropsychopharmacology. Orphanin FQ/Nociceptin has been shown to be a potent anxiolytic whose activity mimics that of diazepam. Orexins/Hypocretins on the other side are major regulators of narcolepsy. Studies on the principal roles of the other novel transmitters are being carried out. So far we know of some 100 transmitters. These are the cornerstones of all neuropsychopharmacological therapies. The search for the natural ligands of orphan GPCRs promises to lead us to the discovery of some 70 more. When completed this search will immensely enrich our understanding of brain function and consequently of brain disorders and these efforts will undoubtedly lead to novel drugs in a few years. Indeed, every one of the nine deorphanized GPCR systems mentioned above is part of drug discovery programs, some even under intense competition. Small synthetic surrogates have been described for some of the novel neuropeptide system. It is only a matter of time until some of the orphan GPCRs will be targets of marketed drugs. Neuropsychopharmacology will gain immensely of the products from the products of the orphan receptor strategy.
References [1] Civelli, O., Nothacker, H.P., Saito, Y., Wang, Z., Lin, S. and Reinscheid R.K. (2001) Novel neurotransmitters as natural ligands of orphan G protein-coupled receptors. Trends in Neuroscience 24:230-237.
•
Control of neurotransmitter release by metabotropic glutamate receptors
D. Schoepp. Eli Lilly and Company, Indianapolis, Indiana, US.A. Glutamate is the major neurotransmitter substance which controls the excitability of the mammalian central nervous system. Importantly, a number of disease states have now been linked to altered glutamate neurotransmitter function. Thus, modulation of glutamate-induced excitation offers therapeutic promise to treat a wide range of neurological and psychiatric disorders. Glutamate receptors include ionotropic (ligand-gated ion channels) receptors (iGlu receptors) that directly mediated glutamate excitations and metabotropic (G-protein coupled receptors or mGlu receptors) that function to modulate glutamate by neuronal and glia mechanisms. In particular, one function of mGlu receptors is to regulate the pre-synaptic release of neurotransmitter glutamate, and in this manner control post-synaptic excitations by glutamate. In general, activation of group I mGlu receptors (mGlul and/or mGlu5) will facilitate the release of synaptic glutamate, while activation of either group II (eg. mGlu2) or group III (eg. mGlu4, mGlu7, mGlu8) leads to a reduction in glutamate release. The role of specific mGlu receptor subtypes in CNS functions involving glutamate release depends on factors which include 1) regional distribution of the mGlu receptor 2) positioning of the mGlu receptor within a neuronal circuit 3) specific synaptic or extra-synaptic localizations for receptors 4) endogenous tone at the receptor under physiological versus pathological conditions (or local concentration of glutamate versus affinity of the receptor for glutamate) 5) direct or indirect mGlu receptor mediated modulation of the release of other
S.12. G-protein coupled amino acid receptors: New therapeutic opportunities
Sl16
neurotransmitter substances (eg. neuropeptides and monoamines) and 6) heterosynaptic regulation of GABA release within neighboring inhibitory synapses. Pharmacological agents which target mGlu receptor subtypes that modulate glutamate release have now been discovered and characterized in animal models, and are progressing in target validation clinical studies. Recent progress on the molecular pharmacology of mGlu receptors along with the use of transgenic animals lacking mGlu receptor subtypes are now further revealing therapeutic directions for mGlu receptor active agents which modulate glutamate release.
References [1] Schoepp D.D., Jane D.E., Monn J.A. (1999) Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38: 1431-1476. [2] Cartmell J. and Schoepp D.D. (2000) Regulation ofneurotransmitter release by metabotropic glutamate receptors. Journal of Neurochemistry 75: 889-907. [3] Schoepp D.D. (2001) Unveiling the fi.mctions of presynaptic metabotropic glutamate receptors in the central nervous system. Journal of Pharmacology and Experimental Therapeutics 299: 12-20.
•
Anatomy, physiology and pharmacology of GABAB receptors
N.G. Bowery. Department of Pharmacology, Medical School
University of Birmingham, B15 2TT, United Kingdom G-protein coupled receptors can be expressed in cell membranes in different ways. They may simply reside as a single protein unit after initial transcription in the endoplasmic reticulum or in some instances the functional receptor may be expressed as a combination of two identical protein molecules linked to form a homodimer. The structure of the metabotropic GABAB receptor introduces even further complexity to the situation as it exists as a unique heterodimer. Detection of this structural dimer was initiated from studies with the cloned receptor subtmit, GABABI. A second receptor subunit was shown to be linked to GABABl through coiled-coil domains at the C-terminal in a stoichiometric 1:1 ratio. This appears to be an absolute requirement for functional expression of the GABAB receptor. This second subunit was designated GABAB2 and has many of the structural features of GABAm including a large molecular weight (110 KDa), 7 transmembrane domains, a long extracellular chain at the N-terminus with 54% similarity (35% homology) with GABABI. At least six isoforms ( l a - l f ) of subunit GABAB1 and three forms of subunit GABAI32 have been reported but whether these all make functional forms of the receptor is unclear. The ligand binding domain of the heterodimer appears to reside in the N-terminal segment of GABAB1 with no evidence for any ligand binding to GABAB2. Current evidence would suggest that even though GABAB2 has a large N-terminus, comparable in length to that in GABAm with considerable homology between them, GABAB2 is more important for effector coupling and signalling of the receptor. On this basis one could predict that G-protein coupling is only mediated through the GABAB2 subunit and this seems to be the current view. The interaction of the C-terminal coiled-coil domains of the subunits appears to mask the action of the retention motif present in the C-terminal of GABAB1 and this is believed to provide a 'trafficking checkpoint' to ensure organized assembly of the functional receptor. The coiled-coil structures seem not to be essential for the heterodimerization to occur. Other proteins that can associate with GABAB], such as mGlu4, will facilitate
expression. However, unlike GABAB2, they are unable to provide effector coupling within the cell membrane. GABAB receptor distribution in CNS: Distribution studies of mRNA for two of the splice variants of the GABAm subunit, GABAB(la) and GABAB(lb) using in situ hybridisation techniques has revealed that they appear to be differentially distributed. Initial studies in rat and human cerebellum and spinal cord indicate that GABAB0a) is associated with presynaptic receptors whereas GABAB(lb) may be responsible for postsynaptic receptor formation in the cerebellum. However, the contrary arrangement has been observed in, for example, the thalamo-cortical circuitry where GABABIa subunits appear to be postsynaptic on cell bodies. As a consequence it seems unlikely that functional role and cellular location can be assigned in a general manner to specific GABAB receptor subunit splice variants. The regional distributions of GABABI and GABAB2 protein subunits are in broad alignment with that of the native receptor although in some brain areas, such as the caudate putamen, GABAB1 and the native receptor are present whereas GABAB2 appears to be absent. A wide distribution of the receptor subunits has also been detected throughout the periphery. However, again the GABAB2 subnnit was not always present, for example, in the uterus and spleen where GABAm subnnits could be detected. This would surely support the existence of an additional, as yet undiscovered, subunit. GABAB Receptor Effector mechanisms: The effector mechanisms to which neuronal GABAB receptors are coupled are adenylate cyclase and Ca 2+ and K + channels. Whilst it can be demonstrated that the majority of these effects are mediated via G-proteins, in particular Gi2c~, not all effects of GABAB receptor activation appear to be coupled. Pertussis toxin insensitive effects of baclofen have been noted, for example, in rat spinal cord and magnocellular neurones of the paraventricular and supraoptic nuclei. GABAB receptor activation decreases neuronal membrane conductance for Ca 2+ but increases conductance to K + ions. The decrease in Ca 2+ conductance appears to be primarily associated with presynaptic sites suppressing 'P/Q' and 'N' type channels whereas modulation of K + conductances appears to be primarily linked with postsynaptic GABAB sites and possibly more than one type of K + channel. Numerous effects have been attributed to the action of GABAB receptor agonists. Some of these inelude: centrally-mediated muscle relaxation, an antitussive action, bronchiolar relaxation, urinary bladder inhibition, gastric motility increase, epileptogenesis, suppression of self-administered cocaine, nicotine and opiates, antinociception, yawning, hypotension, brown fat thermogenesis, cognitive impairment, reduction in release of hormones such as corticotrophin releasing hormone, prolactin releasing hormone, luteinizing hormone and melanocyte stimulating hormone and reduced gastric acid secretion. The centrally-mediated muscle relaxant action of baclofen is the most well recognised of its effects and the mechanism underlying this appears to derive from its ability to reduce the release of excitatory neurotransmitter on to motoneurones in the ventral horn of the spinal cord. Although pain relief has been noted with baclofen in trigeminal neuralgia in man, its usefulness as an analgesic has always been questioned. Nevertheless, more recent clinical observations have indicated that baclofen can reduce pain due to stroke or spinal cord injury and musculoskeletal pain when administered by intrathecal infusion. Recent observations with baclofen indicate that it may also be a very effective treatment for cocaine, nicotine and opiate addiction by reducing the craving for the drug. In rats, baclofen, administered at doses of 1-5 mg/kg, suppressed the self-administration of cocaine without affecting