Publications by authors named "Klemens Kaupmann"

54 Publications

Discovery and Optimization of Novel SUCNR1 Inhibitors: Design of Zwitterionic Derivatives with a Salt Bridge for the Improvement of Oral Exposure.

J Med Chem 2020 09 28;63(17):9856-9875. Epub 2020 Aug 28.

Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland.

G-protein-coupled receptor SUCNR1 (succinate receptor 1 or GPR91) senses the citric cycle intermediate succinate and is implicated in various pathological conditions such as rheumatoid arthritis, liver fibrosis, or obesity. Here, we describe a novel SUCNR1 antagonist scaffold discovered by high-throughput screening. The poor permeation and absorption properties of the most potent compounds, which were zwitterionic in nature, could be improved by the formation of an internal salt bridge, which helped in shielding the two opposite charges and thus also the high polarity of zwitterions with separated charges. The designed compounds containing such a salt bridge reached high oral bioavailability and oral exposure. We believe that this principle could find a broad interest in the medicinal chemistry field as it can be useful not only for the modulation of properties in zwitterionic compounds but also in acidic or basic compounds with poor permeation.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01020DOI Listing
September 2020

Structure-Based and Property-Driven Optimization of -Aryl Imidazoles toward Potent and Selective Oral RORγt Inhibitors.

J Med Chem 2019 12 3;62(23):10816-10832. Epub 2019 Dec 3.

Global Discovery Chemistry , 181 Massachusetts Avenue , 02139 Cambridge , Massachusetts , United States.

Retinoic acid receptor-related orphan receptor gamma-t (RORγt) is considered to be the master transcription factor for the development of Th17 cells that produce proinflammatory cytokines such as IL-17A. Overproportionate Th17 cell abundance is associated with the pathogenesis of many inflammatory conditions including psoriasis. In a high-throughput fluorescence resonance energy transfer (FRET) screen, we identified compound 1 as a hit with promising lipophilic efficiency (LipE). Using structure-based drug design based on a number of X-ray cocrystal structures, we morphed this hit class into potent imidazoles, exemplified by compound . To improve the poor absorption, distribution, metabolism, and excretion (ADME) properties of neutral imidazoles, we extended our ligands with carboxylic acid substituents toward a polar, water-rich area of the protein. This highly lipophilicity-efficient modification ultimately led to the discovery of compound , a potent and selective inhibitor of RORγt with good ADME properties and excellent in vivo pharmacokinetics. This compound showed good efficacy in an in vivo delayed-type hypersensitivity pharmacology model in rats.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01291DOI Listing
December 2019

Structural basis of species-selective antagonist binding to the succinate receptor.

Nature 2019 10 23;574(7779):581-585. Epub 2019 Oct 23.

Chemical Biology & Therapeutics, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation. Because SUCNR1 senses succinate as an immunological danger signal-which has relevance for diseases including ulcerative colitis, liver fibrosis, diabetes and rheumatoid arthritis-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.
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http://dx.doi.org/10.1038/s41586-019-1663-8DOI Listing
October 2019

Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses.

Front Immunol 2019 26;10:577. Epub 2019 Mar 26.

Autoimmunity, Transplantation, and Inflammation Disease Area, Novartis Institutes for BioMedical Research, Basel, Switzerland.

The nuclear hormone receptor retinoic acid receptor-related-orphan-receptor-gamma t (RORγt) is the key transcription factor required for Th17 cell differentiation and for production of IL-17 family cytokines by innate and adaptive immune cells. Dysregulated Th17 immune responses have been associated with the pathogenesis of several inflammatory and autoimmune diseases such as psoriasis, psoriatic arthritis, and ankylosing spondylitis. In this article, we describe the pharmacology of a potent and selective low molecular weight RORγt inhibitor identified after a structure-based hit-to-lead optimization effort. The compound interfered with co-activator binding to the RORγt ligand binding domain and impaired the transcriptional activity of RORγt as evidenced by blocked IL-17A secretion and RORE-mediated transactivation of a luciferase reporter gene. The inhibitor effectively reduced IL-17A production by human naive and memory T-cells and attenuated transcription of pro-inflammatory Th17 signature genes, such as , and . The compound selectively suppressed the Th17/IL-17 pathway and did not interfere with polarization of other T helper cell lineages. Furthermore, the inhibitor was selective for RORγt and did not modify the transcriptional activity of the closely related family members RORα and RORβ. Using human keratinocytes cultured with supernatants from compound treated Th17 cells we showed that pharmacological inhibition of RORγt translated to suppressed IL-17-regulated gene expression in keratinocyte cell cultures. Furthermore, in immersion skin cultures our RORγt inhibitor suppressed IL-17A production by Th17-skewed skin resident cells which correlated with reduced human β defensin 2 expression in the skin. Our data suggests that inhibiting RORγt transcriptional activity by a low molecular weight inhibitor may hold utility for the treatment of Th17/IL-17-mediated skin pathologies.
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http://dx.doi.org/10.3389/fimmu.2019.00577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443933PMC
July 2020

Optimizing a Weakly Binding Fragment into a Potent RORγt Inverse Agonist with Efficacy in an in Vivo Inflammation Model.

J Med Chem 2018 08 24;61(15):6724-6735. Epub 2018 Jul 24.

The transcription factor RORγt is an attractive drug-target due to its role in the differentiation of IL-17 producing Th17 cells that play a critical role in the etiopathology of several autoimmune diseases. Identification of starting points for RORγt inverse agonists with good properties has been a challenge. We report the identification of a fragment hit and its conversion into a potent inverse agonist through fragment optimization, growing and merging efforts. Further analysis of the binding mode revealed that inverse agonism was achieved by an unusual mechanism. In contrast to other reported inverse agonists, there is no direct interaction or displacement of helix 12 observed in the crystal structure. Nevertheless, compound 9 proved to be efficacious in a delayed-type hypersensitivity (DTH) inflammation model in rats.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00529DOI Listing
August 2018

PET Imaging of T Cells: Target Identification and Feasibility Assessment.

ChemMedChem 2018 08 2;13(15):1566-1579. Epub 2018 Jul 2.

Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 141 Klybeckstrasse, 4057, Basel, Switzerland.

Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are, however, no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: protein kinase C isozyme θ (PKC θ), lymphocyte-specific protein tyrosine kinase (Lck), zeta-chain-associated protein kinase 70 (ZAP70), and interleukin-2-inducible T-cell kinase (Itk). Ultimately, we focused on Itk and identified a tool molecule with properties suitable for in vivo imaging of T cells: (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxamide (23). Although it does not have the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of T cells in patients.
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http://dx.doi.org/10.1002/cmdc.201800241DOI Listing
August 2018

Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo.

PLoS One 2017 20;12(11):e0188391. Epub 2017 Nov 20.

Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, Basel, Switzerland.

Retinoic acid receptor-related-orphan-receptor-C (RORγt) is the key transcription factor that is driving the differentiation of IL-17 producing T-helper 17 (Th17) cells that are implicated in the pathology of various autoimmune and inflammatory diseases. Based on the importance of RORγt in promoting Th17-driven pathology, there is considerable interest to develop low-molecular-weight compounds with the aim of inhibiting the transcriptional activity of this nuclear hormone receptor. In this article, we describe the in vitro and in vivo pharmacology of a potent and selective small-molecular-weight RORγt inverse agonist. The compound binds to the ligand binding domain (LBD) of RORγt leading to displacement of a co-activator peptide. We show for the first time that a RORγt inverse agonist down-regulates permissive histone H3 acetylation and methylation at the IL17A and IL23R promoter regions, thereby providing insight into the transcriptional inhibition of RORγt-dependent genes. Consistent with this, the compound effectively reduced IL-17A production by polarized human T-cells and γδT-cells and attenuated transcription of RORγt target genes. The inhibitor showed good in vivo efficacy in an antigen-induced arthritis model in rats and reduced the frequencies of IL-17A producing cells in ex vivo recall assays. In summary, we demonstrate that inhibiting RORγt by a low-molecular-weight inhibitor results in efficient and selective blockade of the pro-inflammatory Th17/IL-17A pathway making it an attractive target for Th17-mediated disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188391PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695821PMC
December 2017

Structural States of RORγt: X-ray Elucidation of Molecular Mechanisms and Binding Interactions for Natural and Synthetic Compounds.

ChemMedChem 2017 07 20;12(13):1014-1021. Epub 2017 Jun 20.

Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Campus, 4002, Basel, Switzerland.

The T-cell-specific retinoic acid receptor (RAR)-related orphan receptor-γ (RORγt) is a key transcription factor for the production of pro-inflammatory Th17 cytokines, which are implicated in the pathogenesis of autoimmune diseases. Over the years, several structurally diverse RORγt inverse agonists have been reported, but combining high potency and good physicochemical properties has remained a challenging task. We recently reported a new series of inverse agonists based on an imidazopyridine core with good physicochemical properties and excellent selectivity. Herein we report eight new X-ray crystal structures for different classes of natural and synthetic compounds, including examples selected from the patent literature. Analysis of their respective binding modes revealed insight into the molecular mechanisms that lead to agonism, antagonism, or inverse agonism. We report new molecular mechanisms for RORγt agonism and propose a separation of the inverse agonists into two classes: those that act via steric clash and those that act via other mechanisms (for the latter, co-crystallization with a co-activator peptide and helix 12 in the agonist position is still possible). For the non-steric clash inverse agonists, we propose a new mechanism ("water trapping") which can be combined with other mechanisms (e.g., close contacts with H479). In addition, we compare the interactions made for selected compounds in the "back pocket" near S404 and in the "sulfate pocket" near R364 and R367. Taken together, these new mechanistic insights should prove useful for the design and optimization of further RORγt modulators.
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http://dx.doi.org/10.1002/cmdc.201700278DOI Listing
July 2017

Retinoic-acid-orphan-receptor-C inhibition suppresses Th17 cells and induces thymic aberrations.

JCI Insight 2017 03 9;2(5):e91127. Epub 2017 Mar 9.

Preclinical Safety, Novartis Institutes for BioMedical Research, Basel, Switzerland.

Retinoic-acid-orphan-receptor-C (RORC) is a master regulator of Th17 cells, which are pathogenic in several autoimmune diseases. Genetic deficiency in mice, while preventing autoimmunity, causes early lethality due to metastatic thymic T cell lymphomas. We sought to determine whether pharmacological RORC inhibition could be an effective and safe therapy for autoimmune diseases by evaluating its effects on Th17 cell functions and intrathymic T cell development. RORC inhibitors effectively inhibited Th17 differentiation and IL-17A production, and delayed-type hypersensitivity reactions. In vitro, RORC inhibitors induced apoptosis, as well as and mRNA downregulation, in mouse and nonhuman primate thymocytes, respectively. Chronic, 13-week RORC inhibitor treatment in rats caused progressive thymic alterations in all analyzed rats similar to those in -deficient mice prior to T cell lymphoma development. One rat developed thymic cortical hyperplasia with preneoplastic features, including increased mitosis and reduced IKAROS expression, albeit without skewed T cell clonality. In summary, pharmacological inhibition of RORC not only blocks Th17 cell development and related cytokine production, but also recapitulates thymic aberrations seen in -deficient mice. While RORC inhibition may offer an effective therapeutic principle for Th17-mediated diseases, T cell lymphoma with chronic therapy remains an apparent risk.
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http://dx.doi.org/10.1172/jci.insight.91127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333964PMC
March 2017

Blunted 5-HT receptor-mediated responses and antidepressant-like behavior in mice lacking the GABA but not GABA subunit isoforms.

Psychopharmacology (Berl) 2017 05 9;234(9-10):1511-1523. Epub 2017 Jan 9.

APC Microbiome Institute, University College Cork, Cork, Ireland.

Rationale: There is accumulating evidence for a role of GABA receptors in depression. GABA receptors are heterodimers of GABA and GABA receptor subunits. The predominant GABA subunit isoforms are GABA and GABA. GABA isoforms in mice differentially influence cognition, conditioned fear, and susceptibility to stress, yet their influence in tests of antidepressant-like activity has not been fully investigated.

Objectives: Given the interactions between GABA receptors and the serotonergic system and the involvement of 5-HT receptors (5-HTR) in antidepressant action, we sought to evaluate 5-HTR function in GABA and GABA mice.

Methods: GABA and GABA mice were assessed in the forced swim test (FST), and body temperature and hypothalamic-pituitary-adrenal (HPA) responses to the 5-HTR agonist 8-OH-DPAT were determined. Brain 5-HTR expression was assessed by [H]-MPPF and [H]-8-OH-DPAT autoradiography and 5-HTR G-protein coupling by [S]GTP-γ-S autoradiography.

Results: As previously described, GABA mice showed an antidepressant-like profile in the FST. GABA mice also demonstrated profoundly blunted hypothermic and motoric responses to 8-OH-DPAT. Furthermore, 8-OH-DPAT-induced corticosterone and adrenocorticotropic hormone (ACTH) release were both attenuated in GABA mice. Interestingly, [H]-MPPF and [H]-8-OH-DPAT binding was largely unaffected by genotype. [S]GTP-γ-S autoradiography suggested that altered 5-HTR G-protein coupling only partially contributes to the functional presynaptic 5-HTR desensitization, and not at all to the blunted postsynaptic 5-HTR-mediated responses, seen in GABA mice.

Conclusion: These data demonstrate distinct functional links between 5-HTRs and the GABA subunit isoform and suggest that the GABA isoform may be implicated in the antidepressant-like effects of GABA receptor antagonists and in neurobiological mechanisms underlying depression.
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http://dx.doi.org/10.1007/s00213-016-4521-5DOI Listing
May 2017

Synthesis and Biological Evaluation of New Triazolo- and Imidazolopyridine RORγt Inverse Agonists.

ChemMedChem 2016 12 30;11(24):2640-2648. Epub 2016 Nov 30.

Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Campus, 4002, Basel, Switzerland.

Retinoic-acid-related orphan receptor γt (RORγt) is a key transcription factor implicated in the production of pro-inflammatory Th17 cytokines, which drive a number of autoimmune diseases. Despite diverse chemical series having been reported, combining high potency with a good physicochemical profile has been a very challenging task in the RORγt inhibitor field. Based on available chemical structures and incorporating in-house knowledge, a new series of triazolo- and imidazopyridine RORγt inverse agonists was designed. In addition, replacement of the terminal cyclopentylamide metabolic soft spot by five-membered heterocycles was investigated. From our efforts, we identified an optimal 6,7,8-substituted imidazo[1,2-a]pyridine core system and a 5-tert-butyl-1,2,4-oxadiazole as cyclopentylamide replacement leading to compounds 10 ((S)-N-(8-((4-(cyclopentanecarbonyl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide) and 33 ((S)-N-(8-((4-(5-(tert-butyl)-1,2,4-oxadiazol-3-yl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide). Both derivatives showed good pharmacological potencies in biochemical and cell-based assays combined with excellent physicochemical properties, including low to medium plasma protein binding across species. Finally, 10 and 33 were shown to be active in a rodent pharmacokinetic/pharmacodynamic (PK/PD) model after oral gavage at 15 mg kg , lowering IL-17 cytokine production in ex vivo antigen recall assays.
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http://dx.doi.org/10.1002/cmdc.201600500DOI Listing
December 2016

Differential roles of GABAB1 subunit isoforms on locomotor responses to acute and repeated administration of cocaine.

Behav Brain Res 2016 Feb 27;298(Pt B):12-6. Epub 2015 Oct 27.

Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland; APC Microbiome Institute, University College Cork, Cork, Ireland. Electronic address:

GABAB receptors are crucial modulators of the behavioural effects of drug abuse, and agonists and positive allosteric modulators show promise as pharmacological strategies for anti-addiction therapeutics. GABAB receptors are functional heterodimers of GABAB1 and GABAB2 subunits. The predominant neuronal GABAB1 subunit isoforms are GABAB1a and GABAB1b. Selective ablation of these isoforms in mice revealed differential behavioural responses in fear, cognition and stress sensitivity. However, the influence of the two GABAB1 isoforms on responses to drugs of abuse is unclear. Therefore we examined the responses of GABAB1 subunit isoform null mice to cocaine in acute locomotor activity and conditioned place preference (CPP) paradigms. During habituation for the acute locomotor activity assay, GABAB1b(-/-) mice showed higher levels of locomotor activity relative to wild-type (WT) and GABAB1a(-/-) mice, in accordance with previous studies. Acute cocaine (10 mg/kg) increased locomotor activity in habituated mice of all three genotypes, with GABAB1a(-/-) mice showing sustained hyperlocomotor responses 30 min after cocaine relative to WT and GABAB1b(-/-) mice. No genotypes demonstrated a cocaine-induced place preference, however, GABAB1a(-/-) mice demonstrated enhanced locomotor sensitisation to chronic cocaine in the CPP paradigm in comparison to WT mice, whereas GABAB1b(-/-) mice failed to develop locomotor sensitisation, despite higher levels of basal locomotor activity. These findings indicate that GABAB1a and GABAB1b isoforms differentially regulate behavioural responses to cocaine, with deletion of GABAB1a enhancing cocaine-induced locomotor activity and deletion of GABAB1b protecting from cocaine-induced sensitisation.
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http://dx.doi.org/10.1016/j.bbr.2015.10.039DOI Listing
February 2016

Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate.

FASEB J 2015 Jul 8;29(7):2980-92. Epub 2015 Apr 8.

*Novartis Institutes for Biomedical Research, Basel, Switzerland; and Department of Neurosciences, Research Group for Neurobiology and Gene Therapy, Katholieke Universiteit (KU) Leuven, Leuven, Belgium

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant forms of Parkinson's disease. LRRK2 is a modular, multidomain protein containing 2 enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling. Although enormous efforts have been made, the exact pathophysiologic mechanisms of LRRK2 are still not completely known. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. This approach allows the identification of substrates of 1 particular kinase in a complex cellular environment. Several of the identified peptides are involved in the regulation of microtubule (MT) dynamics, including microtubule-associating protein (MAP)/microtubule affinity-regulating kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate MT-binding proteins such as Tau, MAP2, and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic-labeling experiments in living cells confirmed MARK1 as an LRRK2 substrate. Moreover, we also showed that LRRK2 and MARK1 are interacting in eukaryotic cells. Our findings contribute to the identification of physiologic LRRK2 substrates and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morphology.
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http://dx.doi.org/10.1096/fj.14-262329DOI Listing
July 2015

Blocking metabotropic glutamate receptor subtype 7 (mGlu7) via the Venus flytrap domain (VFTD) inhibits amygdala plasticity, stress, and anxiety-related behavior.

J Biol Chem 2014 Apr 4;289(16):10975-87. Epub 2014 Mar 4.

From the Novartis Institutes for BioMedical Research, Novartis AG, CH-4057 Basel, Switzerland.

The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.
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http://dx.doi.org/10.1074/jbc.M113.542654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036238PMC
April 2014

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein.

J Biol Chem 2012 Jan 28;287(2):1406-14. Epub 2011 Nov 28.

Neuroscience Pathway, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.
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http://dx.doi.org/10.1074/jbc.M111.294801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256905PMC
January 2012

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice.

Hum Mol Genet 2011 Nov 9;20(21):4209-23. Epub 2011 Aug 9.

Department of Neuroscience, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.
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http://dx.doi.org/10.1093/hmg/ddr348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3188995PMC
November 2011

Exploring subtype selectivity and metabolic stability of a novel series of ligands for the benzodiazepine binding site of the GABAA receptor.

Bioorg Med Chem Lett 2011 Mar 28;21(5):1523-6. Epub 2010 Dec 28.

Novartis Institutes for BioMedical Research, Neuroscience Research, Basel, Switzerland.

A novel series of agonists at the benzodiazepine binding site of the GABA(A) receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABA(A) receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure-activity relationship and efforts to increase in vitro stabilities are discussed.
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http://dx.doi.org/10.1016/j.bmcl.2010.12.107DOI Listing
March 2011

Both GABA(B) receptor activation and blockade exacerbated anhedonic aspects of nicotine withdrawal in rats.

Eur J Pharmacol 2011 Mar 22;655(1-3):52-8. Epub 2011 Jan 22.

Department of Psychiatry, School of Medicine, University of California San Diego, La Jolla, CA 92093-0603, USA.

Nicotine dependence is maintained by the aversive, depression-like effects of nicotine withdrawal and the rewarding effects of acute nicotine. GABA(B) receptor antagonists exhibit antidepressant-like effects in rodents, whereas GABA(B) receptor agonists attenuate the rewarding effects of nicotine. Recent studies with GABA(B) receptor positive modulators showed that these compounds represent potentially improved medications for the treatment of nicotine dependence because of fewer side-effects than GABA(B) receptor agonists. Thus, GABA(B) receptor agonists and antagonists, and GABA(B) receptor positive modulators may have efficacy as smoking cessation aids by targeting different aspects of nicotine dependence and withdrawal. The present study assessed the effects of the GABA(B) receptor agonist CGP44532, the GABA(B) receptor antagonist CGP56433A, and the GABA(B) receptor positive modulator BHF177 on the anhedonic aspects of nicotine withdrawal. Rats were prepared with stimulating electrodes in the posterior lateral hypothalamus. After establishing stable intracranial self-stimulation (ICSS) thresholds, rats were prepared with subcutaneous osmotic minipumps delivering either nicotine or saline for 7 or 14days. ICSS thresholds were assessed 6h post-pump removal. Thirty hours after pump removal, CGP44532, CGP56433A, and BHF177 were administered 30min prior to ICSS testing. Both GABA(B) receptor activation (CGP44532 and BHF177) and blockade (CGP56433A) elevated ICSS thresholds in all groups, resulting in exacerbated effects of nicotine withdrawal in the nicotine-treated groups. These similar effects of GABA(B) receptor activation and blockade on the anhedonic depression-like aspects of nicotine withdrawal were surprising and perhaps reflect differential efficacy of these compounds at presynaptic hetero- and autoreceptors, as well as postsynaptic, GABA(B) receptors.
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http://dx.doi.org/10.1016/j.ejphar.2011.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060559PMC
March 2011

Reduction of alcohol's reinforcing and motivational properties by the positive allosteric modulator of the GABA(B) receptor, BHF177, in alcohol-preferring rats.

Alcohol Clin Exp Res 2009 Oct 1;33(10):1749-56. Epub 2009 Jul 1.

CNR Institute of Neuroscience, Cagliari, Italy.

Background: The positive allosteric modulators of the GABA(B) receptor, CGP7930 and GS39783, have been found to reduce alcohol self-administration in alcohol-preferring rats. The present study was designed to assess the effect of the newly synthesized positive allosteric modulator of the GABA(B) receptor, BHF177, on alcohol's reinforcing and motivational properties in selectively bred Sardinian alcohol-preferring (sP) rats.

Methods: sP rats were initially trained to respond on a lever [on a fixed ratio 4 (FR4) schedule of reinforcement] to orally self-administer alcohol (15%, v/v) or sucrose (1 to 3%, w/v) in daily 30-minute sessions. Once responding reached stable levels, rats were allocated to 2 different experiments: in the first experiment, rats were exposed to sessions with the FR4 schedule of reinforcement; in the second experiment, rats were exposed to sessions with a conventional progressive ratio (PR) schedule of reinforcement. In both experiments, the effect of BHF177 (0, 12.5, 25, and 50 mg/kg; i.g.) on responding for alcohol and sucrose (FR experiment: 1%, w/v; PR experiment: 3%, w/v) was determined.

Results: In the FR experiment, pretreatment with 25 and 50 mg/kg BHF177 produced a 30 and 45% reduction, respectively, in responding for alcohol. In the PR experiment, pretreatment with 50 mg/kg BHF177 resulted in a 35% reduction in breakpoint for alcohol (defined as the lowest response requirement not achieved by each rat and used as index of the motivational strength of alcohol). In both experiments, the effect of BHF177 on alcohol self-administration was specific, since responding for sucrose was unaltered by BHF177 pretreatment.

Conclusions: The present results extend to BHF177 the capacity of the 2 previously tested positive allosteric modulators of the GABA(B) receptor, CGP7930 and GS39783, to specifically suppress alcohol's reinforcing and motivational properties in alcohol-preferring rats.
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http://dx.doi.org/10.1111/j.1530-0277.2009.01012.xDOI Listing
October 2009

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function.

J Biol Chem 2008 Nov 2;283(45):31005-11. Epub 2008 Sep 2.

Department of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, CH-4056 Basel, Switzerland.

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.
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http://dx.doi.org/10.1074/jbc.M804464200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576543PMC
November 2008

Roles of GABAB receptor subtypes in presynaptic auto- and heteroreceptor function regulating GABA and glutamate release.

J Neural Transm (Vienna) 2008 Oct 30;115(10):1401-11. Epub 2008 Jul 30.

Novartis Institutes for BioMedical Research, Neuroscience, Basel, Switzerland.

Gamma-Aminobutyric acid B (GABA B) receptors are heterodimers composed of two subunits GABA B(1) and GABA B(2), the former existing in two isoforms GABA B(1a) and GABA B(1b). The contributions of individual receptor subunits and isoforms to GABA B auto- and heteroreceptor functions were investigated, using release experiments in cortical slice preparations from corresponding knockout mice. Presynaptic GABA B autoreceptors are located on GABAergic terminals and inhibit GABA release, whereas presynaptic GABA B heteroreceptors control the release of other neurotransmitters (e.g. glutamate). Neither baclofen nor the selective antagonist CGP55845 at maximally active concentrations affected [3H]GABA release in slices from GABA B(1)-/- mice. The amount of [3H]GABA released per pulse was unaffected by the stimulation frequency in slices from GABA B(1)-/- and GABA B(2)-/- demonstrating a loss of GABA B autoreceptor function in these knockout animals. The GABA B receptor agonist baclofen was ineffective in modulating glutamate release in cortical slices from GABA B(2)-/- mice, showing that heteroreceptor function was abolished as well. Next we investigated knockout mice for the two predominant GABA B(1) isoforms expressed in brain, GABA B(1a) and GABA B(1b). In cortical, hippocampal and striatal slices from both GABA B(1a)-/- and GABA B(1b)-/- mice, the frequency dependence of [3H]GABA released per pulse was maintained, suggesting that both isoforms participate or can substitute for each other in GABA B autoreceptor function. By contrast, the efficacy of baclofen to inhibit glutamate release was substantially reduced in GABA B(1a)-/-, but essentially unaltered in GABA B(1b)-/- mice. Our data suggest that functional GABA B heteroreceptors regulating glutamate release are predominantly, but not exclusively composed of GABA B(1a) and GABA B(2) subunits.
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http://dx.doi.org/10.1007/s00702-008-0095-7DOI Listing
October 2008

Positive modulation of GABA(B) receptors decreased nicotine self-administration and counteracted nicotine-induced enhancement of brain reward function in rats.

J Pharmacol Exp Ther 2008 Jul 29;326(1):306-14. Epub 2008 Apr 29.

Department of Psychiatry, School of Medicine, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0603, USA.

Acute administration of gamma-aminobutyric acid (GABA)-B receptor agonists decreases nicotine, cocaine, ethanol, and heroin self-administration and also decreases food-maintained responding and suppresses locomotor activity at high doses. GABA(B) receptor-positive modulators may represent potentially improved therapeutic compounds because of their fewer side effects than receptor agonists. The present study investigated the effects of administration of the GABA(B) receptor-positive modulators 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and N-[(1R,2R,4S)-bicyclo[2.2.1]hept-2-yl]-2-methyl-5-[4-(trifluoromethyl)phenyl]-4-pyrimidinamine (BHF177) and coadministration of the GABA(B) receptor-positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) with the GABA(B) receptor agonist (3-amino-2[S]-hydroxypropyl)-methylphosphinic acid (CGP44532) on nicotine- and food-maintained responding under fixed ratio (FR) 5 and progressive ratio schedules of reinforcement. Furthermore, the effects of BHF177 and CGP44532 on nicotine-induced enhancement of brain reward function were evaluated. The results indicated that administration of CGP7930 decreased nicotine self-administration under an FR5 schedule. Administration of either GS39783 or CGP44532 selectively decreased nicotine self-administration, whereas coadministration of these compounds had additive effects. BHF177 administration selectively decreased nicotine- but not food-maintained responding under FR5 and progressive ratio schedules. The nicotine-induced enhancement of brain reward function was blocked by BHF177 or CGP44532, although the highest doses of both compounds, particularly CGP44532, decreased brain reward function when administered alone, suggesting an additive, rather than interactive, effect. Overall, the present results indicate that GABA(B) receptor-positive modulators, similarly to GABA(B) receptor agonists, attenuated the reinforcing and reward-enhancing effects of nicotine, perhaps with higher selectivity than GABA(B) receptor agonists. Thus, GABA(B) receptor-positive modulators may be useful antismoking medications.
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http://dx.doi.org/10.1124/jpet.108.139204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2574924PMC
July 2008

Syntheses and optimization of new GS39783 analogues as positive allosteric modulators of GABA B receptors.

Bioorg Med Chem Lett 2007 Nov 8;17(22):6206-11. Epub 2007 Sep 8.

Novartis Institutes for Biomedical Research, Neuroscience, Novartis Pharma A.G., Basel, Switzerland.

The optimization of GS39783 into potent, selective, and safe positive allosteric modulators of GABA(B) receptors is presented.
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http://dx.doi.org/10.1016/j.bmcl.2007.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2278029PMC
November 2007

Metabotropic glutamate 2/3 receptors in the ventral tegmental area and the nucleus accumbens shell are involved in behaviors relating to nicotine dependence.

J Neurosci 2007 Aug;27(34):9077-85

Department of Psychiatry, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.

The motivation to maintain nicotine self-administration and dependence may involve alterations in glutamatergic neurotransmission. Metabotropic glutamate (mGlu) 2/3 receptors regulate glutamate and dopamine release in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) shell, two brain areas critically involved in reward and motivational processes. We found that acute systemic, as well as intra-VTA or intra-NAc, administration of the mGlu2/3 receptor agonist LY379268 [(-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate] decreased nicotine, but not food, self-administration in rats. In addition, nicotine self-administration downregulated mGlu2/3 receptor function in corticolimbic rat brain sites including the VTA and the NAc, demonstrated by decreased coupling of mGlu2/3 receptors to G-proteins in the [35S]GTPgammaS binding assay. Furthermore, repeated treatment with LY379268 reduced nicotine self-administration at the beginning of a 14 d treatment period; however, the number of nicotine infusions earned gradually returned to baseline levels, indicating tolerance to the effects of repeated LY379268 treatment. Finally, LY379268 administration decreased both cue-induced reinstatement of nicotine- and food-seeking behavior. Together, these findings indicate an important role for mGlu2/3 receptors in the posterior VTA and the NAc shell in the mediation of the rewarding effects of nicotine and potentially in cue-induced nicotine-seeking behavior.
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http://dx.doi.org/10.1523/JNEUROSCI.1766-07.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6672208PMC
August 2007

Specific roles of GABA(B(1)) receptor isoforms in cognition.

Behav Brain Res 2007 Jul 5;181(1):158-62. Epub 2007 Apr 5.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.

The GABA(B) receptor is a heterodimer of GABA(B(1)) and GABA(B(2)) subunits. There are two isoforms of the GABA(B(1)) subunit: GABA(B(1a)) and GABA(B(1b)). Recent studies with mutant mice suggest a differential role for the two GABA(B(1)) isoforms in behavioural processes. As pharmacological and genetic studies have implicated GABA(B) receptors in cognition we investigated the behaviour of GABA(B(1a))(-/-) and GABA(B(1b))(-/-) mice in different types of cognitive paradigms. GABA(B(1a))(-/-) and GABA(B(1b))(-/-) mice were both impaired relative to wildtype controls in a continuous spontaneous alternation behaviour test of working spatial memory. In contrast to the reported phenotype of GABA(B(1))(-/-) mice, however, neither GABA(B(1a))(-/-) nor GABA(B(1b))(-/-) mice were deficient in a passive avoidance task. On the other hand, GABA(B(1a))(-/-) mice were impaired in familiar and novel object recognition. We conclude that GABA(B(1)) isoforms contribute differentially to GABA(B) receptor-mediated cognitive processes.
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http://dx.doi.org/10.1016/j.bbr.2007.03.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2980901PMC
July 2007

GABAB receptor-positive modulation-induced blockade of the rewarding properties of nicotine is associated with a reduction in nucleus accumbens DeltaFosB accumulation.

J Pharmacol Exp Ther 2007 Apr 10;321(1):172-7. Epub 2007 Jan 10.

Neuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

There is an increasing demand for a novel non-nicotinic, nondopaminergic therapeutic approach to nicotine addiction. GABAergic mechanisms have been implicated in drug dependence. Recently, a novel GABAB receptor allosteric-positive modulator, GS39783, was characterized. There are no investigations to date on the effects of GABAB receptor-positive modulators in animal models of nicotine reinforcement. Conditioned place preference (CPP) paradigms are based on the principle that animals, like humans, would learn to seek environmental stimuli that have been previously associated with rewarding events. Here we show that nicotine (0.06 mg/kg s.c.) induced a robust CPP response. Furthermore, GS39783 (30-100 mg/kg p.o.) during the conditioning phase blocked the rewarding effects of nicotine in the CPP paradigm in rats. However, GS39783 did not significantly alter the CPP effects of nicotine when given only immediately before the CPP test. A growing body of evidence suggests that repeated administration of drugs of abuse induced long-term molecular changes in brain plasticity, most notably an accumulation of DeltaFosB, in the striatal complex that contribute to the manifestation of dependence. There was a significant accumulation of DeltaFosB in the nucleus accumbens, but not in the dorsal striatum, of rats treated daily for 5 days with nicotine (0.06 mg/kg i.p.). GS39783 completely (30-100 mg/kg p.o.) counteracted these nicotine-induced molecular adaptations when given before the CPP acquisition phase but not when administered immediately before the test phase. Taken together, the behavioral and molecular changes induced by nicotine occur in concert and are concomitantly amenable to reversal by GABAB receptor-positive modulators.
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http://dx.doi.org/10.1124/jpet.106.116228DOI Listing
April 2007

Behavioral evaluation of mice deficient in GABA(B(1)) receptor isoforms in tests of unconditioned anxiety.

Psychopharmacology (Berl) 2007 Mar 15;190(4):541-53. Epub 2006 Dec 15.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4002 Basel, Switzerland.

Rationale: Emerging data support a role for GABA(B) receptors in anxiety. GABA(B) receptors are comprised of a heterodimeric complex of GABA(B1) and GABA(B2) receptor subunits. The predominant neuronal GABA(B1) receptor isoforms are GABA(B(1a)) and GABA(B(1b)). Recent findings indicate specific roles for these isoforms in conditioned fear responses, although their influence on behavior in tests of unconditioned anxiety is unknown.

Objective: The aim of this study was to examine the role of the GABA(B(1)) isoforms in unconditioned anxiety.

Materials And Methods: Mice deficient in the GABA(B(1a)) or GABA(B(1b)) receptor isoforms were examined in a battery of anxiety tests.

Results: In most tests, genotype did not significantly affect anxious behavior, including the elevated plus maze, marble burying, and stress-induced hypothermia tests. Corticosterone and adrenocorticotropic hormone levels were similarly unaffected by genotype. Female, but not male, GABA(-/-)B(1a) and GABA(-/-)B(1b) mice showed increased anxiety relative to wild-type controls in the elevated zero maze. In the staircase test, male GABA(-/-)B(1b) mice defecated more than male GABA(-/-)B(1a) mice, although no other test parameter was influenced by genotype. In the light-dark box, female GABA(-/-)B(1a) mice spent less time in the light compartment compared to the GABA(-/-)B(1b) females, whereas male GABA(-/-)B(1b) mice made fewer light-dark transitions than GABA(-/-)B(1a) males.

Conclusions: Specific roles for either GABA(B(1)) isoform in unconditioned anxiety were not explicit. This differs from their contribution in conditioned anxiety and from the anxious phenotype of GABA(B1) and GABA(B2) subunit knockout mice. The findings suggest that the GABA(B(1)) isoforms have specific relevance for anxiety with a cognitive component, rather than for innate anxiety per se.
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http://dx.doi.org/10.1007/s00213-006-0631-9DOI Listing
March 2007

GABAB1 receptor subunit isoforms exert a differential influence on baseline but not GABAB receptor agonist-induced changes in mice.

J Pharmacol Exp Ther 2006 Dec 21;319(3):1317-26. Epub 2006 Sep 21.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

GABA(B) receptor agonists produce hypothermia and motor incoordination. Two GABA(B(1)) receptor subunit isoforms exist, but because of lack of specific molecular or pharmacological tools, the relevance of these isoforms in controlling basal body temperature, locomotor activity, or in vivo responses to GABA(B) receptor agonists has been unknown. Here, we used mice deficient in the GABA(B(1a)) and GABA(B(1b)) subunit isoforms to examine the influence of these isoforms on both baseline motor behavior and body temperature and on the motor-incoordinating and hypothermic responses to the GABA(B) receptor agonists l-baclofen and gamma-hydroxybutyrate (GHB). GABA(B(1b))(-/-) mice were hyperactive in a novel environment and showed slower habituation than either GABA(B(1a))(-/-) or wild-type mice. GABA(B(1b))(-/-) mice were hyperactive throughout the circadian dark phase. Hypothermia in response to l-baclofen (6 and 12 mg/kg) or GHB (1 g/kg), baclofen-induced ataxia as determined on the fixed-speed Rotarod, and GHB-induced hypolocomotion were significantly, but for the most part similarly, attenuated in both GABA(B(1a))(-/-) and GABA(B(1b))(-/-) mice. We conclude that l-baclofen and GHB are nonselective for either GABA(B(1)) receptor isoform in terms of in vivo responses. However, GABA(B(1)) receptor isoforms have distinct and different roles in mediating locomotor behavioral responses to a novel environment. Therefore, GABA(B(1a)) and GABA(B(1b)) isoforms are functionally relevant molecular variants of the GABA(B(1)) receptor subunit, which are differentially involved in specific neurophysiological processes and behaviors.
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http://dx.doi.org/10.1124/jpet.106.111971DOI Listing
December 2006

Point mutations in the transmembrane region of GABAB2 facilitate activation by the positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) in the absence of the GABAB1 subunit.

Mol Pharmacol 2006 Dec 11;70(6):2027-36. Epub 2006 Sep 11.

Neuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

GABA(B) receptors are heterodimers of two subunits, GABA(B1) (GB1) and GABA(B2) (GB2). Agonists such as GABA and baclofen bind to the GB1 subunit only, whereas GB2 is essential for G protein activation. Positive allosteric modulators enhance the potency and efficacy of agonists at GABA(B) receptors and are of particular interest because they lack the sedative and muscle relaxant properties of agonists. In this study, we aimed to characterize the interaction of the positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) with the GABA(B) receptor heterodimer. Using functional guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, we observed positive modulation by GS39783 in different vertebrate species but not in Drosophila melanogaster. However, coexpression of D. melanogaster GB1 with rat GB2 yielded functional receptors positively modulated by GS39783. Together with data from rat/D. melanogaster GB2 subunit chimeras, this pointed to a critical role of the GB2 transmembrane region for positive modulation. We further characterized GS39783 function using point mutations. GS39783 positively modulated GABA responses but also showed considerable agonistic activity at heterodimers containing a mutant rat GB2 subunit with three amino acid substitutions in transmembrane domain VI. It was surprising that in contrast to wild-type rat GB2, this mutant subunit was also activated by GS39783 when expressed without GB1. The mutations of both G706T and A708P are necessary and sufficient for activation and identify a key region for the effect of GS39783 in the GB2 transmembrane region. Our data show that mutations of specific amino acids in GB2 can induce agonism in addition to positive modulation and facilitate GB2 activation in the absence of GB1.
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http://dx.doi.org/10.1124/mol.106.028183DOI Listing
December 2006

GABA(B(1)) receptor isoforms differentially mediate the acquisition and extinction of aversive taste memories.

J Neurosci 2006 Aug;26(34):8800-3

Novartis Institutes for BioMedical Research, Novartis Pharma, CH-4002 Basel, Switzerland.

Conditioned taste aversion (CTA) is a form of aversive memory in which an association is made between a consumed substance and a subsequent malaise. CTA is a critical mechanism for the successful survival, and hence evolution, of most animal species. The role of excitatory neurotransmitters in the neurochemical mechanisms of CTA is well recognized; however, less is known about the involvement of inhibitory receptor systems. In particular, the potential functions of metabotropic GABA(B) receptors in CTA have not yet been fully explored. GABA(B) receptors are metabotropic GABA receptors that are comprised of two subunits, GABA(B(1)) and GABA(B(2)), which form heterodimers. The Gabbr1 gene is transcribed into two predominant isoforms, GABA(B(1a)) and GABA(B(1b)), which differ in sequence primarily by the inclusion of a pair of sushi domains (also known as short consensus repeats) in the GABA(B(1a)) N terminus. The behavioral function of mammalian GABA(B(1)) receptor isoforms is currently unknown. Here, using a point mutation strategy in mice, we demonstrate that these two GABA(B(1)) receptor isoforms are differentially involved in critical components of CTA. In contrast to GABA(B(1b))-/- and wild-type mice, GABA(B(1a))-/- mice failed to acquire CTA. In contrast, GABA(B(1b))-/- mice robustly acquired CTA but failed to show any extinction of this aversion. The data demonstrate that GABA(B) receptors are involved in both the acquisition and extinction of CTA; however, receptors containing the GABA(B(1a)) or the GABA(B(1b)) isoform differentially contribute to the mechanisms used to learn and remember the salience of aversive stimuli.
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http://dx.doi.org/10.1523/JNEUROSCI.2076-06.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6674388PMC
August 2006