Ana Farinha Thesis.Pdf

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DEPARTAMENTODEPARTAMENTO DE DE CIÊNCIAS CIÊNCIAS DA DA VIDA VIDA

FACULDADEFACULDADE DE DECIÊNCIAS CIÊNCIAS E TECNOLOGIA E TECNOLOGIA UNIVERSIDADEUNIVERSIDADE DE DECOIMBRA COIMBRA

MappingMapping the the molecular molecular determinants determinants of of positivepositive allosteric allosteric modulators modulators of ofthe the mGlu2mGlu2 receptor receptor

DissertaçãoDissertação apresentada apresentada à Universidade à Universidade de de CoimbraCoimbra para para cumprimento cumprimento dos dosrequisitos requisitos necessáriosnecessários à obtenção à obtenção do graudo grau de Mestrede Mestre em emBiologia Biologia Celular Celular e Molecular,e Molecular, realizada realizada sob soba orientação a orientação científica científica da Doutorada Doutora Hilde Hilde LavreysenLavreysen (Janssen (Janssen Pharmaceutica Pharmaceutica NV) NV)e do e do ProfessorProfessor DoutorDoutor CarlosCarlos DuarteDuarte (Universidade(Universidade de Coimbra)de Coimbra)

AnaAna Isabel Isabel Farinha Farinha

20122012

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 1 28/06/2012 9:58:28 The work described in this thesis resulted from a partnership between the University of Coimbra and Janssen Pharmaceutica NV. All experimental activities were performed at the Janssen Pharmaceutica NV Beerse I, a Johnson and Johnson pharmaceutical research and development facility in Beerse, Belgium.

Beerse, 2012

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Acknowledgements

To my supervisor Hilde Lavreysen, for giving me the opportunity to perform this internship in her group as well as for her constant support and encouragement throughout this year, I am deeply grateful. A great thank you to Luc Peeters, for everything: all the help in the laboratory, all his advice, true caring and friendship. Thank you! A special thanks to the entire group involved in my project, particularly Gary Tresadern for his important help during the writing of this thesis. Thank you Gui D., Marc V. (surrogate fathers indeed!) and Ilse B. for your constant support and honest care and all the staff of Neuroscience department for making all the students part of the team. To University of Coimbra and Janssen Pharmaceutica for this cooperation that enabled me to perform this internship. To Janssen Crew, for all the great times spent during this whole year. And finally, a great great thank you to my parents and my brother, to Miguel and to my friends for the constant and indispensable love and support.

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Table of contents

Abbreviations ...... 7 Resumo ...... 9 Abstract ...... 11 Introduction ...... 13 1.1. G Protein-Coupled Receptors ...... 14 1.1.1. GPCR structure ...... 15 1.1.2. GPCR mode of action – signaling pathways ...... 16 1.1.3. Families of GPCRs ...... 18 1.1.3.1. Family A or Class 1 GPCRs ...... 18 1.1.3.2. Family B or Class 2 GPCRs ...... 19 1.1.3.3. Family C or Class 3 GPCRs ...... 19 1.1.3.4. Families D, E and F or Class 4, 5 and 6 GPCRs ...... 22 1.1.4. GPCRs in the Central Nervous System - Overview ...... 22 1.2. The glutamatergic system in the central nervous system ...... 23 1.2.1. Glutamate receptors ...... 23 1.2.1.1 Ionotropic glutamate receptors ...... 24 1.2.1.2. Metabotropic glutamate receptors ...... 25 1.2.1.3. The mGlu receptor Family ...... 25 Signal Transduction Mechanisms ...... 25 Pharmacology ...... 27 1.2.1.4. Distribution and Functional Roles of mGlu receptors ...... 29 Distribution ...... 29 Functional Roles ...... 31 1.2.3. mGlu2 receptor receptor in central nervous system diseases – mGlu2 receptor as a drug target ...... 32 1.3. Allosteric modulation of GPCRs ...... 34 1.4. Mutational Studies on mGlu receptors ...... 36 Sequence alignment and homology modelling ...... 39 1.5. Goal of the project ...... 41 Materials and Methods ...... 43 2.1. Materials ...... 44 2.2. Positive allosteric modulators tested ...... 44 2.3. Selection of amino acid mutations: sequence alignment and building of an mGlu2 receptor homology model ...... 44

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2.4. Chemical transformation of One Shot® Top10 E. coli cells with point-mutated human mGlu2 receptors and DNA purification ...... 47 2.5. Cell culture ...... 47 2.6. Transient transfection of human mGlu2 receptor cDNA into CHO-K1 cells ...... 48 2.7. Membrane preparation ...... 48 2.8. Western Blot analysis ...... 49 2.9. Radioligand binding assay – [3H]-LY341495 binding ...... 50 Theoretical background...... 50 Procedure ...... 51 2.10. [35S]GTPγS binding assay ...... 52 Theoretical background...... 52 Procedure ...... 53 2.11. Data analysis ...... 53 Results ...... 55 3.1. Expression of WT and mutant mGlu2 receptors in CHO-K1 cells ...... 56 3.2. Glutamate potency for WT and mutated mGlu2 receptors ...... 61 3.3. Effect of mGlu2 mutations on the activity of positive allosteric modulators ...... 63 Discussion ...... 77 4.1. Expression of WT and mutant mGlu2 receptors ...... 78 4.3. Effect of mGlu2 mutations on the activity of positive allosteric modulators ...... 80 Effect of mGlu2 mutations on the different chemical classes ...... 80 mGlu2/3 sequences comparison ...... 84 Overall important amino acids ...... 84 Appendix ...... 89 Appendix 1: Complete names of the compounds mentioned in the text ...... 90 Appendix 2: mGlu2 receptor positive allosteric modulators tested in this study ...... 93 Appendix 3: Complete table screening assay...... 94 Appendix 4: Complete table PAM/mutations results...... 95 References ...... 97

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Abbreviations

[35S]GTPγS - Guanosine 5’-(γ-thio)triphosphate [35S]- 1GZM - rhodopsin receptor

2RH1 - β2-adrenergic receptor 3D – 3-dimensional AMPA - α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid

Bmax – total number of binding sites cAMP - cyclic adenosine monophosphate CHO – Chinese hamster ovary CNS – central nervous system CRD – cystein rich domain DAG - sn-1,2-diacylglycerol DMSO - dimethyl sulphoxide E. coli - Escherichia coli

EC100 – concentration of compound producing 100% of stimulation

EC20 - concentration of compound producing 20% of stimulation

EC50 - concentration of compound producing 50% of stimulation EGFP-N1 - Enhanced Green Fluorescent Protein EL – extracellular loop

Emax – relative maximal stimulation FBS - fetal bovine serum GABA - γ-amino butyric acid GDP – guanosine 5’ diphosphate GPCR – G-protein coupled receptor GRK – G-protein coupled receptor kinases GTP – guanosine 5’ triphosphate hmGluR - human metabotropic glutamate receptor IL – intracellular loop

IP3 - inositol (1,4,5)-trisphosphate JAK – Janus kinase KA - kainate

KD – apparent equilibrium dissociation constant LB - Luria-Bertani

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LTD - long-term depression MAPK/ERK - mitogen-activated protein kinase/extracellular receptor kinase mGluR – metabotropic glutamate receptor MOE - Molecular Operating Environment MTOR - mammalian target of rapamycin NAM - negative allosteric modulator NFDM - Non-Fat Dry Milk NMDA - N-methyl-D-aspartate PAM – positive allosteric modulator PBS - phosphate-buffered saline

PIP2 - phosphatidylinositol (4,5)-bisphosphate PKA –protein kinase A PKC – protein kinase C

PLCβ - phospholipase Cβ RGS – regulators of G-protein signaling rmGluR - rat metabotropic glutamate receptor RT - room temperature TBS-T – Tris-Buffered Saline and Tween 20 TM – transmembrane VFT – venus flytrap domain

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Resumo

O sucesso dos receptores acoplados à proteína G (GPCRs) enquanto alvos terapêuticos para o tratamento de doenças do sistema nervoso central torna estes receptores alvos pertinentes de investigação. Especificamente, a activação de receptores mGlu2 tem mostrado reduzir a transmissão glutamatérgica em áreas do cerebro onde o excesso de sinalização glutamatérgica parece estar implicado na patofisiologia de doenças como a ansiedade e a esquizofrenia. Deste modo, a activação dos receptores mGlu2 está actualmente a ser encarada como uma potencial estratégia para o tratamento destas doenças. Diversos moduladores alostéricos positivos (PAMs), que se ligam a um local do receptor diferente do local de ligação do glutamato, têm revelado modular os receptores mGlu2 de uma forma selectiva. Com o objectivo de identificar aminoácidos potencialmente importantes para a interacção entre o PAM e o receptor mGlu2, foi efectuada a modelação molecular e o docking de PAMs de receptores mGlu2 em paralelo com mutagénese dirigida. Receptores mGlu2 mutantes foram produzidos e o impacto dessas mutações na actividade de diversos PAMs foi avaliado, de forma a confirmar o papel destes aminoácidos na actividade destes compostos. Este estudo identifica aminoácidos importantes para a actividade dos PAMs e sugere um potencial local de ligação destes compostos.

Palavras-chave: GPCR, receptor mGlu2, alosterismo, modulador alostérico positivo, mutagénese.

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Abstract

The proven success of G protein-coupled receptors (GPCRs) as drug targets for the treatment of CNS disorders renders them attractive targets of research. Specifically, activation of the G protein-coupled or metabotropic glutamate (mGlu2) receptor has been shown to reduce glutamatergic transmission in brain regions where excess glutamate signaling may be implicated in the pathophysiology of disorders such as anxiety and schizophrenia. Hence, activation of the mGlu2 receptor is being pursued as a novel therapeutic approach for the treatment of these diseases. Multiple positive allosteric modulators (PAMs), which bind to a site other than that of the endogenous mGlu2 receptor agonist glutamate, have been shown to modulate mGlu2 receptors in a selective way. In order to identify amino acids potentially important for the interaction between PAMs and the mGlu2 receptor, homology modelling and docking of mGlu2 receptor PAMs was performed in parallel with experimental site directed mutagenesis. Mutant mGlu2 receptors were produced and the impact of the selected receptor mutations on the activity of several PAM compounds towards the receptor was evaluated, in order to confirm the role of these amino acids in the activity of the compounds. This study identifies crucial amino acids for the activity of mGlu2 receptor PAMs and suggests a potential binding pocket of PAMs.

Keywords: GPCR, mGlu2 receptor, allosterism, positive allosteric modulator, mutagenesis.

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Chapter 1 Introduction

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1.1. G Protein-Coupled Receptors

Membrane-bound receptors have a leading role in the recognition of intercellular messenger molecules and sensory messages, which makes them fundamental for the communication of the cells with each other and with the environment (Bockaert and Pin, 1999). They have been classified into several families, the most common of which is the G Protein-Coupled Receptors (GPCR) family (Bockaert and Pin, 1999). GPCRs currently constitute one of the principal drug targets in pharmacology (Vauquelin and von Mentzer, 2007; Urwyler, 2011). More than 50% of the marketed therapeutics are based on GPCRs, and a large share of total drug sales and prescriptions (~25%) are directed at these receptors (Lündstrom and Chiu, 2006), rendering them important and pertinent targets of research. GPCRs constitute one of the largest and most diverse protein superfamilies (Urwyler, 2011). Human genome sequencing has revealed that more than 1000 genes encode GPCRs, which represents a substantial part (± 3%) of the human genome (Vauquelin and von Mentzer, 2007). Vassilatis et al., (2003) proposed that 367 human GPCRs have endogenous ligands (‘endoGPCRs’), 143 of which are orphan receptors (i.e. receptors with no known ligand). The remaining receptors are chemosensory (taste, odorant or pheromone receptors), and they respond only to sensory signals of external origin (Vassilatis et al., 2003; Urwyler, 2011). GPCRs are receptors for a variety of extracellular stimuli, such as hormones, neurotransmitters, chemokines, calcium ions, light, odorants (Lündstrom and Chiu, 2006), pheromones, gustative molecules, lipids, peptides, proteins (Urwyler, 2011), small molecules including amino-acid residues and nucleotides (Figure 1). Upon binding of these ligands, they mediate several cellular signal transduction events that are crucial for the cells, tissues, organs and whole organisms to react properly to environmental requirements (Bockaert and Pin, 1999). GPCRs regulate different physiological processes that affect neurological and neurodegenerative functions, cardiovascular mechanisms and metabolic control (Lündstrom and Chiu, 2006).

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Figure 1: Diversity of messages which activate G-protein Coupled Receptors (GPCRs). GPCRs have a central common core made of seven transmembrane helices (TM1-7) connected by three intracellular (i1, i2, i3) and three extracellular (e1, e2, e3) loops. Bockaert and Pin, 1999

1.1.1. GPCR structure

GPCRs consist of a single peptide, whose length can vary from 400 to 1200 amino- acids (Vauquelin and von Mentzer, 2007). They have in common a central core domain composed of seven transmembrane α-helices (TM1 – TM7) connected by three intracellular (IL1 – IL3) and three extracellular (EL1 – EL3) loops (Baldwin, 1993). Each GPCR possesses also an extracellular N-terminal domain and an intracellular C-terminal domain (Lündstrom and Chiu, 2006) (Figure 1). The length and function of the N- and C- terminal domains and of the intracellular loops varies between the different GPCRs and each of these domains confers specific properties to these receptor proteins (Bockaert and Pin, 1999). Two cystein residues, one in EL1 and the other in EL2, are conserved in most GPCRs. These two residues form a disulfide bond which is thought to be important for the packing and stabilization of a restricted number of conformations of the seven TMs (Bockaert and Pin, 1999).

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1.1.2. GPCR mode of action – signaling pathways

GPCRs become functionally active when coupled to guanine nucleotide-binding proteins (G proteins). Typically, the receptor is in its resting or low affinity state in the absence of agonists. This inactive conformation is caused by a number of intramolecular constraining interactions (Vauquelin and von Mentzer, 2007). Upon agonist binding to the receptor, a modification in these constraints occurs, leading to a shift in the receptor conformation. The receptor is now in an active state that leads to the formation of a complex with intracellular G proteins. G proteins constitute a family of closely related membrane-associated polypeptides (Vauquelin and von Mentzer, 2007). They are heterotrimeric proteins that consist of a guanine nucleotide binding Gα subunit (38-52 kDa), a Gβ subunit (35 kDa) and a Gγ (8-10 kDa) subunit. G proteins are anchored to the cytoplasmic side of the plasma membrane (Vauquelin and von Mentzer, 2007). The β and γ subunit are always closely associated, thus creating a βγ complex that is presumed to be interchangeable from one G protein to another (Vauquelin and von Mentzer, 2007). Gα subunit is predominantly hydrophilic but it is anchored to the plasma membrane through its coupling to the βγ complex; this subunit constitutes the receptor-recognizing part of G proteins (Vauquelin and von Mentzer, 2007). As previously mentioned, in the resting state the receptor and the G protein do not interact with each other. In this state, Gα subunit contains tightly bound guanosine-5’- diphosphate (GDP) (Vauquelin and von Mentzer, 2007). Once an agonist binds to the receptor, the conformation of the receptor changes which enables the interaction of the receptor with the Gα subunit of the G protein. GDP is then released and replaced by guanosine-5’-triphosphate (GTP), and the Gα subunit is dissociated from the Gβγ dimer. Gα subunit and Gβγ dimer can then both activate cellular effector molecules (Lündstrom and Chiu, 2006). The G-protein linked effector components are either enzymes or ion channels (Table 1).

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TableTable 1: Principal 1: Principal G protein G protein subunits subunits and and their their primary primary effectors effectors (Adapted (Adapted from from Hermans, Hermans, 2003) 2003) SubunitSubunit FamilyFamily MainMain Subtypes Subtypes PrimaryPrimary effecto effector r α α αS αS GαGαS, GαS, Gαolf olf + Adenylyl+ Adenylyl cyclase cyclase αi/oα i/o GαGαi-1, iGα-1, Gαi-2, iGα-2, Gαi-3 i-3 - Adenylyl- Adenylyl cyclase cyclase + + GαGαoA, oAGα, GαoB oB + K+ Kchannels channels 2+ 2+ GαGαt1, Gαt1, Gαt2 t2 - Ca- Ca channels channels GαGαz z CyclicCyclic GMP GMP + Phosphodiesterase+ Phosphodiesterase αq/11αq/11 GαGαq, Gαq, Gα11, 11Gα, Gα14 14 - Phospholipase- Phospholipase C C GαGα15, 15Gα, Gα16 16 α12α 12 GαGα12, 12Gα, Gα13 13 ? ? β β β1-5β (61-)5 (6) DifferentDifferent assemblies assemblies +/-+/ Adenylyl- Adenylyl cyclase cyclase of β/γof β/γ subunits subunits + Phospholipases+ Phospholipases

PhosphatidylinositolPhosphatidylinositol + 3+-kinase 3-kinase γ γ γ1-11γ1 (12-11 (12) ) + Protein+ Protein kinase kinase C C + Protein+ Protein kinase kinase D D + GPCR+ GPCR kinases kinases Ca2+Ca, 2+K,+ K(and+ (and Na Na+) channels+) channels

TheThe Gα Gα subu subunit nitpossesses possesses an an endogenous endogenous GTP GTPaseas activity,e activity, which which will will cause cause GTP GTP hydrolysishydrolysis into into GDP GDP and and lead lead to to the the termination termination of ofreceptor receptor activity. activity. Rapid Rapid termination termination of of GPCRGPCR function function can can occur occur also also through through other other mechanisms. mechanisms. One One of of them them is isreceptor receptor phosphorylationphosphorylation mediated mediated by byProtein Protein Kinase Kinase A (APKA (PKA) or) Proteinor Protein Kinase Kinase C (CPKC (PKC), which), which will will leadlead to tothe the uncoupling uncoupling of of the the receptor receptor and and the the G protein.G protein. Another Another mechanism mechanism involves involves G G proteinprotein-coupled-coupled receptor receptor kinases kinases (GRKs) (GRKs) which which can can only only phosphorylate phosphorylate the the receptor receptor when when it it is activatedis activated or agonistor agonist-occupied,-occupied, stabilizing stabilizing a conformation a conformation state state necessary necessary for for the the interaction interaction of of the the GPCR GPCR with with arrestins. arrestins. Binding Binding of of arrestin arrestin prevents prevents further further GPCR GPCR and and G Gprotein protein interactionsinteractions or or leads leads to to receptor receptor endocytosi endocytosis. Receptors. Receptor degradation degradation in inlyso lysosomessomes can can also also occur,occur, as aswell well as asregulation regulation of ofgene gene transcription transcription or ortranslation. translation. At Ata posta post-expression-expression level, level, regulatorsregulators of ofG proteinG protein signaling signaling (RGS) (RGS) serve serve as asGTPase GTPase-activating-activating proteins proteins for for G proteins,G proteins, enhancingenhancing the the hydrolysis hydrolysis rate rate of of GTP GTP and and thus thus leading leading to totermination termination of of receptor receptor activity activity (Lündstrom(Lündstrom and and Chiu, Chiu, 2006 2006). ). GPCRsGPCRs are are known known to induceto induce signaling signaling pathways pathways not not only only through through direct direct interaction interaction withwith G proteins,G proteins, but but also also through through interaction interaction with with other other proteins. proteins. These These alternative alternative signaling signaling pathwayspathways may may confer confer a higher a higher specificity specificity to theto the GPCR GPCR signaling signaling and and may may also also allow allow distinct distinct physiologicalphysiological functions functions of theof the different different GPCRs GPCRs (Lündstrom (Lündstrom and and Chiu, Chiu, 2006 2006). Arrestins). Arrestins act actas as adaptersadapters or orscaffolds scaffolds for for signaling signaling proteins proteins involved involved in inthe the ERK/mitogen ERK/mitogen-activated-activated protein protein

17 17

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kinase signaling pathway (Vauquelin and von Mentzer, 2007). GPCRs have also been suggested to interact with the family of the PDZ domain-containing proteins (Kornau et al., 1997). Several GPCRs interact with members of the janus kinase (JAK) family of nonreceptor protein tyrosine kinases, which will recruit and phosphorylate members of the STAT (signal transducers and activators of transcription) family of transcription factors (Ritter and Hall, 2009).

1.1.3. Families of GPCRs

GPCRs are presumed to have evolved from a common ancestor (Vauquelin and von Mentzer, 2007). They have been classified into several families/classes based on sequence and structural similarity (Lündstrom and Chiu, 2006, Vauquelin and von Mentzer, 2007 and Yarnitzky et al., 2010).

1.1.3.1. Family A or Class 1 GPCRs

Family A is the largest group of GPCRs and the subfamily of rhodopsin/β2-adrenegic receptor-like receptors is by far the most studied (Vauquelin and von Mentzer, 2007). Family A consists of light receptors (rhodopsin), adrenaline receptors and olfactory receptors. The homology among the members of this family is overall low (Vauquelin and von Mentzer, 2007) but they all possess an arginine in the highly conserved Asp/Glu-Arg-Tyr (D/ERY) motif positioned at the cytoplasmic side of TM3 (Figure 2) (Lündstrom and Chiu, 2006, Vauquelin and von Mentzer, 2007). The ligands of family A GPCRs bind within the 7TM domain.

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Figure 2: Two-dimensional structure of family A GPCRs and rhodopsin. Adapted from Vauquelin and von Mentzer, 2007.

1.1.3.2. Family B or Class 2 GPCRs

Based on the homology shared by family B receptors (Vauquelin and von Mentzer, 2007), they were divided in three subfamilies: subfamily B1) those recognizing peptide hormones (secretin, glucagon, VIP (Vauquelin and von Mentzer, 2007), calcitonin and corticotrophin-releasing (Lündstrom and Chiu, 2006) hormone receptor family), B2) those containing a GPCR proteolytic site domain (so far, orphan receptors) and B3) those with cystein-rich domains (frizzled and smoothened receptors) (Vauquelin and von Mentzer, 2007). Family B receptors have a propensity to associate with partner/accessory proteins (Vauquelin and von Mentzer, 2007). For the subfamily of secretin/glucagon/VIP receptors, it is shown that the N-terminal domain is required for ligand binding (Unson et al., 1995).

1.1.3.3. Family C or Class 3 GPCRs

Family C receptors include γ-amino butyric acid (GABA) receptors, calcium-sensing receptors, three receptors involved in taste perception and metabotropic glutamate (mGlu) receptors, which represent the focus of this thesis (Vauquelin and von Mentzer, 2007; Lündstrom and Chiu, 2006).

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A B

Figure 3: Structure of family C GPCRs. A) Two-dimensional structure of family C GPCRs and rhodopsin (Adapted from Vauquelin and von Mentzer, 2007); B) Family C GPCRs all have a common core composed of seven-transmembrane helices (the 7TM domain comprised of TM1-7) with a large, bilobal Venus flytrap extracellular N-terminal domain and an intracellular C-terminal domain. (Adapted from Urwyler, 2011).

The typical feature of this GPCR family is the exceptionally large extracellular N- A terminal region (600 amino acids; Vauquelin and von Mentzer, 2007) that is essential for ligand binding and receptor activation (Lündstrom and Chiu, 2006) (Figure 3A). The large extracellular N-terminal domain is also termed the Venus flytrap domain (VFD) (Pin et al., 2003), which is connected to the 7TM domain through a cystein-rich domain (CRD), composed of nine conserved cysteins and present in all family C receptors, except for

GABAB receptors (Vauquelin and von Mentzer, 2007; Niswender and Conn, 2010). Each VFD is composed of two lobes, and the cleft between them constitutes the binding site for the natural messenger (Vauquelin and von Mentzer, 2007, Niswender and Conn, 2010) (Figure 3B). Interestingly, the VFD shares sequence similarity with bacterial periplasmic-binding proteins, which are involved in the transport of small molecules (Vauquelin and von Mentzer, 2007). The TM loops of family C GPCRs show an overall low sequence similarity with the homologous loops of family A. However, the two families share several conserved amino acid residues, as well as a conserved disulfide bond between the top of TM3 and the second extracellular loop, suggesting a common ancestor gene for both and, for family C GPCRs, a fusion of that gene with the gene for a periplasmic-binding protein (Vauquelin and von Mentzer, 2007). Class C GPCRs have been found to form homo- and heterodimers (Vauquelin and von Mentzer, 2007). For mGlu receptors, there is evidence suggesting that they form homodimers stabilized by a disulphide bridge (through VFD or CRD). Accordingly, studies focusing on the structure of mGlu receptors revealed that the binding of the agonist to one or both domains induces large conformational changes (Jingami et al., 2003). There are three main

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configurations of the VFD dimer: open-open (inactive), which is stabilized by antagonists; open-closed and closed-closed, which are induced by the binding of the agonist to one or two protomers, respectively (Figure 4). The configuration of the flytrap domains is important for receptor activation; it has for example been shown that mutations in the residues that prevent closure of the VFD are capable of switching the pharmacology of antagonists to agonists (Bessis et al., 2002). In the case of mGlu receptors, VFDs not only bind glutamate, but also bind magnesium and calcium, which can potentiate or activate the receptor (Francesconi and Duvoisin 2004; Kubo et al., 1998; Kunishima et al., 2000).

Figure 4: Scheme of the mGlu receptor dimer in different activity states. mGlu receptor dimers possess two large extracellular domains called the Venus flytrap domains (VFDs), which bind glutamate and other so-called orthosteric ligands. The cysteine-rich domain links the VFDs to seven transmembrane-spanning domains; the C-terminus is intracellular and is often subject to alternative splicing to generate different C-terminal protein tails. The open-open state is the inactive state and can be stabilized by antagonists. One or both VFDs can bind glutamate, which will lead to active receptor conformations. Niswender and Conn, 2010

Once an agonist binds to the VFD, it induces conformational changes that are propagated to the transmembrane domain (TM) and C-terminal tail, through the CRD (Huang et al., 2011). A binding pocket located in the TM is the binding site for the majority of characterized allosteric modulators (discussed in Section 1.3) of mGlu receptors, thus allowing the modulation of receptor activity by those ligands (Brauner-Osborne et al., 2007). The C- terminal domain is target of alternative splicing, regulation by phosphorylation, and modulatory protein-protein interactions in several GPCRs, which make it an important region for the modulation of G protein coupling (Niswender and Conn, 2010; Bockaert and Pin, 1999).

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1.1.3.4. Families D, E and F or Class 4, 5 and 6 GPCRs

The yeast pheromone receptors – families D (STE2 receptors) and E (STE3 receptors) and four cAMP receptors from Dictyostelium discoideum (family F) form three smaller families of GPCRs (Vauquelin and von Mentzer, 2007; Lündstrom and Chiu, 2006).

1.1.4. GPCRs in the Central Nervous System - Overview

GPCRs are widely expressed in the central nervous system (CNS) (Lündstrom and Chiu, 2006; Lim, 2007). They are expressed in different types of neurons and in astrocytes and glial cells, and mediate a slow modulation of neuronal activity, in contrast to ion channels, which mediate fast synaptic excitatory neurotransmission (Lündstrom and Chiu, 2006, Dingledine et al., 1999). Postsynaptically, GPCRs can induce changes in intracellular cAMP levels, increase phosphoinositol turnover and/or indirectly modulate ion channel activity through the activation of kinases. Presynaptically, they act as autoreceptors to decrease the release of the respective neurotransmitter or as heteroreceptors that modulate the release of neurotransmitters other than their equivalent ligands (Lündstrom and Chiu, 2006). GPCRs play a critical role in developmental processes and synaptic transmission, and they also mediate other important physiological processes such as cognition, thought and emotional state, motor and hormonal control and pain sensation (Lündstrom and Chiu, 2006; Lim, 2007). Given the wide distribution of GPCRs in the CNS, their involvement in CNS disorders is expectable. Abnormalities in the regulation of GPCRs have been related to psychiatric disorders such as schizophrenia and mood disorders (Catapano and Manji, 2007). GPCRs have also been shown to play important roles in key neurotransmitter systems that are disrupted in Alzheimer’s disease and hence have been related to Alzheimer’s disease pathogenesis (Thathiah and De Strooper, 2011). GPCRs have been proven successful as drug targets in the CNS: they constitute the target for some of the most efficacious analgesics, and many drugs for depression, anxiety and schizophrenia act on GPCRs. Additional GPCRs are emerging as potential targets for Parkinson and Alzheimer’s diseases (Lündstrom and Chiu, 2006).

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In the past decade, mGlu receptors have received much attention due to their strong potential as a drug target (Lavreysen and Dautzenberg, 2008). mGlu receptors are of interest for the treatment of several neurological and psychiatric disorders, such as depression, anxiety, schizophrenia, chronic pain, epilepsy, Alzheimer’s disease and Parkinson’s disease (Conn et al., 2009b); Marino and Conn, 2006). mGlu receptors will be discussed in more detail in Section 1.2.2.2.

1.2. The glutamatergic system in the central nervous system

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Approximately 90% of neurons in the brain utilize glutamate as their primary neurotransmitter, and 80-90% of the synapses in the brain are glutamatergic (Breitenberg and Schüz, 1998). Glutamate is the major mediator of sensory information, motor coordination, emotions and cognition (Siegel et al., 2006). The concentration of this neurotransmitter in brain gray matter ranges between 10 and 15 µmol per gram of tissue, whereas in the white matter it varies between 4 and 6 µmol/g (Siegel et al., 2006). Once synthesized in the presynaptic cytoplasm, glutamate accumulates in synaptic vesicles, by specific transporters, termed vesicular glutamate transporters. Glutamate participates in many reactions in the brain: its formation is a step in the metabolism of glucose and amino-acids; it is a precursor for GABA in GABAergic neurons and for glutamine in glial cells; it is a constituent of proteins and peptides. As a result, glutamate is found in all cells of the brain (neuronal and glial cell bodies and their processes), both in the cytosol and the mitochondria (Siegel et al., 2006). Abnormal glutamate neurotransmission has been related to many neurological disorders, including Alzheimer’s disease, Parkinson’s disease, addiction, depression, epilepsy, pain, anxiety, and schizophrenia (Rowe et al., 2008).

1.2.1. Glutamate receptors

Glutamate binds to and activates two main categories of receptors: ionotropic and metabotropic (Figure 5).

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1.1.2.1.12.1.1 Ionotropic Ionotropic glutamate glutamate receptors receptors

IonotropicIonotropic glutamateglutamate receptorsreceptors –– oror ligand ligand-gated-gated ionion channels channels –– mediatemediate the the vast vast majoritymajority of of fast fast synaptic synaptic excitatory excitatory neurotransmission neurotransmission in in the the brain brain (Dingledine (Dingledine et et al. al., ,1999) 1999) andand they they comprise comprise two two functional functional domains: domains: an an extracellular extracellular domain domain which which is is the the neurotransmitterneurotransmitter binding binding site site and and a a membrane membrane-crossi-crossingng domain domain that that forms forms an an ion ion channel channel (Purves(Purves et et al. al., ,2004). 2004). NeurotransmitterNeurotransmitter binding binding induces induces a a conformational conformational change change in in the the receptor, receptor, increasing increasing thethe probability probability of of channel channel opening opening andand thus thus a a cation cation influx influx. . Three Three classes classes of of ionotropic ionotropic glutamateglutamate receptors receptors ha haveve been been identified identified and and named named after after the the agonist agonist that that activates activates them: them: N N-- methylmethyl-D-D-aspartate-aspartate (NMDA)(NMDA) receptors, receptors, α α-amino-amino-3-3-hydroxy-hydroxy-5-5-methyl-methyl-4-4-isoxazole-isoxazole propionic propionic acidacid (AMPA) (AMPA) receptors receptors and and kainate kainate (KA) (KA) receptors. receptors. The Theyy all all havehave a a different different affinity affinity for for glutamateglutamate (Javitt, (Javitt, 2004). 2004). Ligand Ligand-gated-gated ion ion channel channel receptors receptors appear appear to to be be a a multimeric multimeric aggregationaggregation of of different different protein protein subunits. subunits. For For NMDA NMDA receptors, receptors, NMDAR1 NMDAR1 is is the the obligatory obligatory subunitsubunit that that combines combines with with NMDAR2A NMDAR2A-2D-2D subunits subunits to to form form a a gated gated channel. channel. AMPA AMPA receptorsreceptors consist consist of of four four different different subunits, subunits, GluR1 GluR1-4,-4, all all of of which which can can exist exist in in two two different different forms.forms. Kai Kainatenate receptors receptors are are formed formed by by five five kainate kainate subunits, subunits, GluR5 GluR5-7-7 and and KA1 KA1-2,-2, which which can can undergoundergo alternative alternative splicing splicing and and editing editing (Nakanishi, (Nakanishi, 1992). 1992). DueDue to to the the fact fact that that ionotropic ionotropic glutamate glutamate receptors receptors are are expressed expressed by by nearly nearly all all types types of of neuronsneurons and and mediate mediate fast fast excitatoryexcitatory neurotransmission neurotransmission throughout throughout the the brain, brain, direct direct pharmacologicalpharmacological manipul manipulationation of of these these receptors receptors is is un undesireddesired as as their their inhibition inhibition could could produceproduce widespread widespread disruption disruption of of brain brain function, function, which which could could lead lead to to serious serious side side effects effects (Rowe(Rowe et et al. al., ,2008; 2008; S Swansonwanson et et al. al., ,2005). 2005).

FigureFigure 5 5: :Molecular Molecular subtypes subtypes of of glutamate glutamate receptors. receptors. Eac Eachh subtype subtype includes includes three three functionally functionally defin defineded groups groups (classes) (classes) ofof receptor, receptor, which which are are made made up up of of numerous numerous individual individual subunits subunits that that are are encoded encoded by by different different gene genes.s . Adapted Adapted from from SiegelSiegel et et al. al., 2006, 2006 2424

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1.2.1.2. Metabotropic glutamate receptors

mGlu receptors are, as discussed above (see section 1.1.3.3.), members of the family C GPCRs and they are involved in the slower action of glutamate (Lim, 2007). These neuromodulatory receptors enable glutamate to modulate cell excitability and synaptic transmission through second messenger signalling pathways (Niswender and Conn, 2010).

1.2.1.3. The mGlu receptor Family

To date, eight mGlu receptors have been identified and they can be divided in three groups (I, II and III) according to their amino acid sequence homology, pharmacology and the preferred signal transduction mechanisms they couple to when expressed in vitro (See Table 2 for a summary) (Swanson et al., 2005). In addition to these eight mGlu receptors, several isoforms of these subtypes are produced by the alternative splicing of their RNAs, which generates splice variants in the cytoplasmic C-terminal domain (Litschig et al., 1999; Urwyler, 2011).

Signal Transduction Mechanisms

Group I receptors (mGlu1/5 receptor) preferentially activate phospholipase Cβ (PLCβ)

via Gq/G11 proteins. This results in the hydrolysis of phosphatidylinositol (4,5)-bisphosphate

(PIP2) and the consequent generation of inositol (1,4,5)-trisphosphate (IP3) and sn-1,2- diacylglycerol (DAG), which initiate a second messenger cascade: IP3 will mobilize Ca2+ from intracellular calcium stores, and DAG will increase the activity of membrane-bound PKC (Figure 6A). These receptors have been shown to modulate additional signaling

pathways, including other cascades downstream of Gαq, as well as pathways originating from

Gαi/o, GαS and other molecules independent of G proteins (Hermans et al., 2001). Accordingly, depending on the cell type or neuronal population, group I mGlu receptors are capable of inducing the activation of several downstream effectors: phospholipase D, protein kinase pathways such as casein kinase 1, cyclin-dependent protein kinase 5, Jun kinase, components of the mitogen-activated protein kinase/extracellular receptor kinase (MAPK/ERK) pathway, and the mammalian target of rapamycin (MTOR)/p70 S6 kinase

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pathway (Niswender and Conn, 2010). The last two pathways are presumably important for the regulation of synaptic plasticity by group I mGlu receptors (Niswender and Conn, 2010). Additionally, when mGlu1a receptors are expressed in several cellular systems they can enhance the formation of cAMP and the release of arachidonic acid (Aramori and Nakanishi, 1992). Groups II (mGlu2/3) and III (mGlu4/6/7/8) inhibit adenylyl cyclase activity – which will lead to decreases in the cyclic adenosine monophosphate (cAMP) levels – and directly

regulate ion channels and other downstream signaling partners via Gαi/o proteins (Figure 6B). As for group I, group II and III mGlu receptors have been associated with other signaling pathways, including activation of MAPK and phosphatidyl inositol 3-kinase PI3 kinase pathways (Iacovelli et al., 2002), which shows that the regulation of synaptic transmission by these receptors involves different mechanisms (Spooren et al., 2003; Niswender and Conn, 2010; Wood et al., 2011).

Figure 6: General signal transduction mechanisms of mGlu receptors. A) Group I mGlu receptors signal transduction pathway; B) Group II and III mGlu receptors signal transduction pathway (Adapted from Gasparini and Spooren, 2007). .

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Pharmacology

The pharmacology of mGlu receptors has been ascertained by studies in both native preparations (as brain slice and cultured neurons) and non-neuronal cell lines expressing recombinant rat or human mGlu receptors. L-glutamate, (1S, 3R)-ACPD, ABH x D-I (agonists) and LY341495 (antagonist) are common ligands for the three different mGlu receptor groups (Niswender and Conn, 2010; Schoepp et al., 1999), but specific agonists and antagonists for each of the three groups of mGlu receptors have been identified. Table 2 summarizes the pharmacological profiles of group I, II and III mGlu receptors (full name of the compounds can be found in Appendix 1). Besides orthosteric ligands, allosteric ligands – negative (NAM) and positive (PAM) – are also presented. The two concepts (orthosteric and allosteric) will be discussed in section 1.3. The pharmacology of group III mGlu receptors is not as extensively described as for groups I and II, due to the lack of subtype-selective pharmacological tools to study these targets (Schoepp et al., 1999). Some of the compounds, of which MPEP and SIB-1893 are examples, have shown activity at multiple mGlu receptor subtypes, which require caution when interpreting their effect, as it can be arising from different mGlu receptor subtypes.

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Table 2: Pharmacological profile of mGlu receptors. PAM: positive allosteric modulator; NAM: negative allosteric modulator

Pharmacodynamic profile Selectivity Compound References (S )-3,5-DHPG Desai et al., 1995 Group I Quisqualate Parmentier et al. , 1998 Orthosteric agonist Z-CBQA Littman et al, 1999 mGluR5 E-CBQA CHPG Doherty et al., 1997 MCPG and derivatives Niswender and Conn, 2010 Orthosteric antagonist Group I LY367385 Clark et al., 1998 CPCCOEt Litschig et al. , 2000 R214127 Lavreysen et al. , 2003 CTZ Surin et al., 2007 Bay 36-7620 Carroll et al., 2001 mGluR1 JNJ 16259685 Lavreysen et al., 2004 FTIDC Suzuki et al., 2007 NAM YM 298198 Kohara et al., 2005 EM-TBPC Malherbe et al., 2003 a) SIB-1757 Group I Varney et al., 1999 SIB-1893 mGluR5 MPEP Gasparini et al., 1999 MTEP Cosford et al., 2003 Ro 67-7476 Ro 67-4853 Knoflach et al., 2001 Ro 01-6128 VU60 mGluR1 VU54 VU48 Hemstapat et al., 2006 PAM VU34 VU71 DFB Zhang et al. , 2005 CPPHA mGluR5 CDPPB de Paulis et al., 2006 VU29 Chen, 2007 ADX47273 Liu et al., 2008 DCG-IV Hayashi et al., 1993 (2R,4R)-APDC Schoepp et al., 1996 1S ,3S- ACPD Pin et al., 1994 LY354740 Schoepp et al., 1997 LY379268 Monn et al., 1999 S -4MeGlu Bräuner-Osborne et al., 1997a) Orthosteric agonist 2S, 4S -4MG Bräuner-Osborne et al., 1997a) Group II NAAG Wroblewska et al., 1997 Group II L-CBG-I Tsujishima et al., 1998 L-F2CCG-I Saitoh et al., 1998 LY389795 (?) Monn et al., 1999 LCCG-I Hayashi et al., 1992 MGS0028 Nakazato et al, 2000 LY341495 Kingston et al. , 1995 Orthosteric antagonist HYDIA Lundström et al. , 2009 LY487379 Schaffhauser et al., 2003 PAM mGlu2 BINA Galici et al., 2006 Acetophenone series Trabanco et al., 2011 MNI-135 MNI-136 Hemstapat et al. , 2007 NAM Group II MNI-137 RO4988546 Lundström et al., 2011 RO5488608 Group III AP4 Cartmell et al., 1998 S -SOP Laurie et al., 1997 mGluR8 (RS )-PPG Flor et al. , 1998 Orthosteric agonist (+)-ACPT-III Acher et al., 1997 ACPT-I Acher et al., 1997 Group III mGluR6 S -Homo-AMPA Ahmadian et al, 1997 mGluR4 S -AP4 Laurie et al., 1997 Orthosteric antagonist Group III CPPG Kowal et al., 1998 SIB-1893 PAM mGluR4 Mathiesen et al., 2003 MPEP

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1.2.1.4. Distribution and Functional Roles of mGlu receptors

Distribution

mGlu receptors are widely distributed in the CNS, suggesting that these receptors have the ability to play a role in numerous functions throughout the brain (Niswender and Conn, 2010). They are specifically concentrated at discrete synaptic and extrasynaptic sites in neurons and glia in nearly every major brain region and their activation results in diverse actions on neuronal excitability and synaptic transmission through the modulation of ion channels and other regulatory and signaling proteins (Niswender and Conn, 2010) (Figure 7). In general, group I mGlu receptors are localized in the postsynaptic terminal, and, when activated, often lead to cell depolarization and increases in neuronal excitability. mGlu1 receptor immunoreactivity was shown in the caudate putamen, nucleus accumbens and globus pallidus (Martin et al., 1992; Baude et al., 1993; Petralia et al., 1997; Testa et al., 1998; Lavreysen et al., 2004). Moderate expression was found in the substantia nigra pars reticulata. Additionally, mGlu1 receptor binding in rat brain was found in the cerebellum, thalamus, dentate gyrus and medial central gray. Moderate binding was also found in the CA3 of the hippocampus and in the hypothalamus, and, in lower levels, in the basal ganglia and cortex (Lavreysen et al., 2004). mGlu5 receptors are widely expressed in the hippocampus, caudate/putamen, lateral septum, cortex and olfactory bulb (Shigemoto et al., 1993; Romano et al., 1995). Astrocytes from the cortex, thalamus, tegmentum, hippocampus

and striatum have also been found to express mGlu5 (Bradley and Challiss, 2012). In contrast to group I, group II and III mGlu receptors are often localized presynaptically or in preterminal axons where they inhibit neurotransmitter release (Figure 7). This can take place on excitatory, inhibitory and neuromodulatory synapses (Niswender and Conn, 2010).

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Figure 7: Scheme of general mGlu receptor localization at the synapse. Niswender and Conn, 2010.

Accordingly, group II mGlu receptors (mGlu2 and mGlu3) localize primarily presynaptically in the hippocampus, cortex, striatum, thalamus and amygdala (Ohishi et al., 1993a,b). Studies of in situ hybridization (Ohishi et al., 1993a,b), immunohistochemistry (Carlton et al., 2001) and autoradiography (Schaffhauser et al., 1998) demonstrated the expression of mGlu2/3 in the hippocampus, olfactory bulb, neocortical regions, cerebellar Golgi neurons and, with a lower level of expression, in thalamic nuclei and striatum (Schaffhauser et al., 2003). Astrocytes have also been found to express mGlu3 (Bradley and Challiss, 2012). Group III mGlu receptors show an heterogeneous distribution: mGlu4 is highly expressed in cerebellar granule cells (Lavreysen and Dautzenberg, 2008) and is present at lower expression levels in regions like the hippocampus, amygdala, striatum and olfactory bulb (Lavreysen and Dautzenberg, 2008); mGlu6 is found primarily in retinal ON bipolar cells (Nakajima et al., 1993), but there are indications of low level expression in the hippocampus, limbic, cerebellar and cerebral areas (Lavreysen and Dautzenberg, 2008); mGlu7 is broadly distributed in the entire brain, and localizes in the active zones of the synapses (Shigemoto et al., 1997; Kinoshita et al., 1998); finally, mGlu8 is also widely distributed throughout the brain but it is expressed in lower levels than mGlu4 and mGlu7 (Niswender and Conn, 2010); it localizes predominantly presynaptically, but also in some postsynaptic locations and in the periphery (Lavreysen and Dautzenberg, 2008).

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Functional Roles

The physiological roles of different mGlu receptor subtypes are greatly specific to the neuronal population and even subcellular localization (Niswender and Conn, 2010). mGlu receptor genetic deletion in mice has helped revealing the potential roles for each receptor. High expression of mGlu1 in the hippocampus indicates a potential role in learning and memory (Niswender and Conn, 2010). In the same lines, mGlu1 deficiency has been shown to lead to a deficit in long-term depression in the cerebellum (Aiba et al., 1994). Lack of mGlu1 seems to lead to abnormal levels of regression of climbing fibers from cerebellar Purkinje cells, suggesting that mGlu1 plays a critical role in maintaining adequate levels of innervations of cerebellar neurons (Levenes et al., 1997). Additionally, it was recently shown that several spontaneous mutations which cause spontaneous ataxia in mice are related to the ligand-binding domain of mGlu1 (hence, mGlu1 function is suggested to be critical for this ataxic phenotype) (Sachs et al., 2007; Niswender and Conn, 2010). mGlu5 is understood to have a role in learning and memory, addiction, motor regulation and obesity and it has been recently related to the treatment of fragile X syndrome (Niswender and Conn, 2010). Deficits in prepulse inhibition (measure of sensorimotor gating that is impaired in schizophrenia patients) were seen in mGlu1 and mGlu5 knock-out animals (Brody et al., 2003 and 2004). Group II and III mGlu receptors play important roles in the induction of long-term depression (LTD), thus reducing the efficacy of transmission (Bellone et al., 2008; Pinheiro and Mulle, 2008). Activation of mGlu2/3 decreases synaptic transmission and glutamate release in the hippocampus (Macek et al., 1996). mGlu3 seems to have an important role in astrocytes: studies with wild-type, mGlu2 and mGlu3 knock-out mice showed that an agonist of group II mGlu receptors has neuroprotective effects when NMDA is administered to the cells, but that the neuroprotective effect is lost when mGlu3 is absent from astrocytes in the cell culture. In the same studies, mGlu2 activation seemed harmful in terms of excitotoxicity (Corti et al., 2007). mGlu4 knock-out mice show impairments in cerebellar synaptic plasticity and in learning complicated motor tasks (Pekhletski et al., 1996). They also exhibit impaired abilities in spatial memory performance (Gerlai et al., 1998). mGlu4 has furthermore been

shown to modulate GABAA receptor-mediated seizure activity (Snead et al., 2000). mGlu6, which is found primarily in the retinal ON bipolar cells, seems to have a fundamental role in ON responses to light (Masu et al., 1995; Sugihara et al., 1997). mGlu7 has low affinity for glutamate and the deletion of this receptor caused an epileptic phenotype, suggesting that this receptor only becomes active when the levels of glutamate are very high, working as a break

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for overstimulation by glutamate (Niswender and Conn, 2010). Furthermore, knockout animals revealed abnormalities in learning tasks. As such, mGlu7 has been implicated in amygdala-dependent learning (Masugi et al., 1999). The wide presence and range of functions of mGlu receptors throughout the CNS renders them important players in CNS disorders. Based on the distribution and physiological roles of each mGlu receptor subtype, it is possible to relate them to brain circuits or regions implicated in the pathology of CNS-related disorders. Indeed, members of the three groups of mGlu receptors have been related to neurological and psychiatric disorders, such as pain, schizophrenia, anxiety, depression, epilepsy, fragile X syndrome, cognitive disorders, Alzheimer’s and Parkinson’s disease (Gregory et al., 2011). Conversely to ionotropic glutamate receptors, whose pharmacological manipulation would need to be highly sensitive to avoid disruption of brain function, mGlu receptors represent an opportunity for developing drugs that regulate glutamate neurotransmission in an indirect and selective manner (Rowe et al., 2008). Due to the wide and heterogeneous distribution of these receptors in the CNS, they may act as selective targets for the development of new treatment strategies for psychiatric and neurological disorders (Niswender and Conn, 2010).

1.2.3. mGlu2 receptor receptor in central nervous system diseases – mGlu2 receptor as a drug target

The distribution and major functions of group II mGlu receptors in neuronal excitability and synaptic transmission, renders the modulation of these receptors a promising strategy for the treatment of neurological and neuropsychiatric disorders (Rowe et al., 2008). Preclinical and clinical studies strongly suggest that several agonists of this group of receptors have potential as a new strategy for the treatment of anxiety disorders and schizophrenia (Niswender and Conn, 2010). mGlu2 receptor, particularly, was shown to selectively mediate the beneficial effects of group II agonists in rodent models of psychosis (Fell et al., 2008; Woolley et al., 2008). The mGlu2 receptor role and its potential for the treatment of schizophrenia and anxiety disorders will be discussed in more detail here. DCG-IV and (2R, 4R)-APDC were the first selective group II mGlu receptor agonists (Schoepp et al., 1999). More recently, systemically active and highly selective agonists of group II mGlu receptors have been developed, enabling the investigation of in vitro and in

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vivo functions of these receptors. The mGlu2/3 receptor agonists LY379268 and LY354740 cause anxiolytic-like effects and antipsychotic-like activity in animal models used to predict efficacy in the treatment of anxiety disorders and schizophrenia (Conn et al., 2009a; Carter et al., 2004; Linden et al., 2005) and there are even clinical data supporting the use of mGlu2/3 agonists for the treatment of anxiety in humans (Grillon et al., 2003). Additionally, LY- 2140023, the prodrug of the mGlu2/3 agonist LY-404039, has been shown to improve positive and negative symptoms in schizophrenic patients (Patil et al., 2007; Mezler et al., 2010). Unlike currently marketed antipsychotic drugs, there were no major adverse effects reported for the mGlu2/3 agonist in the clinical studies to date. However, chronic administration of group II mGlu receptor agonists induces tolerance in at least one rodent model that has been used to predict antipsychotic efficacy (Conn et al., 2009b). While these compounds are highly selective for group II mGlu receptors, they do not distinguish between mGlu2 and mGlu3, possibly representing a disadvantage since preclinical studies with mGlu2 and mGlu3 knockout mice suggest that mGlu2 is likely to be the responsible for preclinical efficacy (Spooren et al., 2000). However, novel compounds that specifically act on mGlu2 have been identified; they act as so-called positive allosteric modulators (PAMs) of mGlu2. Early examples of mGlu2 receptor PAMs include LY487379 and BINA (Conn et al., 2009a). These compounds have been shown to be highly selective for mGlu2 (Conn et al., 2009a) and they have efficacy in animal models that predict antipsychotic activity that are very similar to those observed with mGlu2/3 agonists (Galici et al., 2005). In recent years there was great progress in the discovery, optimization and clinical development of several GPCR subtypes allosteric modulators (Conn et al., 2009 b). Hence, the concept of allosteric modulation will now be further discussed.

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1.1.3.3. AllostericAllosteric modulationmodulation ofof GPCRsGPCRs

AllostericAllosteric –– fromfrom thethe GreekGreek “αλλο”“αλλο” (other)(other) andand “στερεο”“στερεο” (object,(object, shape)shape) –– modulationmodulation refersrefers toto thethe regulationregulation ofof aa proteinprotein byby thethe bindingbinding ofof aa ligandligand toto aa sitesite thatthat isis topographicallytopographically distinctdistinct fromfrom itsits orthosteric orthosteric –– “ορτηο”“ορτηο” (correct) (correct) –– sitesite (the (the binding binding site site for for the the naturalnatural or or exogenousexogenous/competitive/competitive agonistsagonists onon a a recepto receptor)r) (Urw(Urwyler,yler, 2011; 2011; Conn Conn etet al al.,., 2009b 2009b)).. AllostericAllosteric modulatorsmodulators induceinduce aa changechange inin thethe threethree--dimensionaldimensional receptorreceptor conformation,conformation, thusthus affectingaffecting thethe affinities affinities and/or and/or efficaciesefficacies ofof orthostericorthosteric ligandsligands (Urw(Urwyyler,ler, 2011; 2011; Conn Conn etet al al.,., 2009b2009b)).. AllostericAllosteric ligandsligands areare,, inin generalgeneral,, structurallystructurally diversediverse andand differentdifferent fromfrom orthostericorthosteric ligands,ligands, particularlyparticularly fromfrom thethe endogenousendogenous naturalnatural agonistsagonists (Urwyler,(Urwyler, 2011)2011).. AllostericAllosteric modulatorsmodulators cancan bebe classifclassifiedied accordingaccording toto theirtheir effecteffect onon thethe affinity/affinity/ efficacyefficacy ofof thethe naturalnatural agonistagonist toto thethe receptor:receptor: lligandsigands thatthat enhanceenhance agonistagonist--inducedinduced receptorreceptor activityactivity areare referred referred to to as as positive positive allosteric allosteric modulators modulators (PAMs) (PAMs) (or (or allosteric allosteric activators), activators), whereas whereas thethesese thatthat decrease decrease agonistagonist activitactivityy are are termed termed negative negative allosteric allosteric modulators modulators (N (NAMs)AMs) (or (or allostericallosteric inhibitors);inhibitors); nneutraleutral allostericallosteric ligandsligands bindbind toto thethe allostericallosteric site,site, butbut havehave nono effectseffects onon thethe responsesresponses toto tthehe orthostericorthosteric agonistagonist.. Furthermore,Furthermore, thesethese compoundscompounds havehave oneone oror moremore ofof the the fol followinglowing properties properties (Figure (Figure 8): 8): affinity affinity modulation modulation (the (the receptor receptor conformational conformational changechange affectsaffects thethe receptorreceptor bindingbinding pocket,pocket, inducinginducing modificationsmodifications inin thethe associationassociation and/orand/or dissociationdissociation rate rate of of the the orthosteric orthosteric ligand), ligand), efficacy efficacy modulation modulation (the (the allosteric allosteric mo modulatordulator bindingbinding inducesinduces aa changechange inin thethe intracellularintracellular responses,responses, thusthus modifyingmodifying thethe orthostericorthosteric ligandligand signallingsignalling capacity)capacity) andand agonism/inverseagonism/inverse agonismagonism (the(the allostericallosteric modulatormodulator affectsaffects receptorreceptor signallingsignalling inin eithereither aa positivepositive (agonism)(agonism) oror negativenegative (invers(inversee agonism)agonism) way,way, irrespectiveirrespective ofof thethe presencepresence oror absenceabsence ofof anan orthostericorthosteric ligand)ligand) (Conn(Conn etet al.al.,, 20092009 bb).).

FigurFiguree 88:: ModesModes ofof actionaction ofof allostericallosteric modulators.modulators. a)a) AllostericAllosteric ligandsligands cancan affectaffect orthostericorthosteric ligandligand affinityaffinity (red)(red) and/orand/or efficacyefficacy (blue)(blue),, andand eveneven directlydirectly perturbperturb signallingsignalling inin theirtheir ownown rightright (green).(green). b)b) EffectEffect ofof threethree differentdifferent allostericallosteric potentiatorspotentiators onon thethe bindingbinding (left)(left) oror functionfunction (right)(right) ofof anan orthoorthostericsteric agonistagonist:: thethe firstfirst (red)(red) enhancesenhances orthostericorthosteric agonistagonist affinityaffinity only;only; thethe secondsecond (blue)(blue) enhances enhances orthosteric orthosteric agonist agonist efficacy efficacy only; only; the the thirdthird (green) (green) modestly modestly enhances enhances both both affinity affinity and and efficacy,efficacy, andand alsoalso showsshows allostericallosteric agonism.agonism. ConnConn etet al.al.,, 2009b 2009b

3434

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As mentioned before, GPCRs, including mGlu receptors, have been implicated in several human disorders, so they are the target of many therapeutic agents that are currently in use. However, synthetic ligands exist for only a fraction of the known GPCRs and the production of highly selective compounds suitable as drug leads has proven to be difficult (Wood et al., 2011). Several important issues contribute to the difficulty of discovering small-molecule selective agonists or antagonists that act on the orthosteric site of some GPCRs: orthosteric binding sites are highly conserved for a particular endogenous ligand in members of a single GPCR subfamily, which will limit the achievement of subtype selectivity; moreover, ligands at orthosteric sites for some GPCRs have other physicochemical and pharmacokinetic properties that are incompatible with scaffolds that are useful for small-molecule drug discovery (Conn et al., 2009b); also, in case of mGlu receptors, ligands that bind the orthosteric binding site are frequently glutamate analogues, and for that reason they typically lack the bioavailability and/or CNS penetration desired in a probe or drug candidate (Wood et al., 2011). Finally, a main issue with classical GPCR agonists is that after repeated dosing they often cause receptor desensitization (Rowe et al., 2008). Allosteric modulators can, however, overcome some of these obstacles. Whereas orthosteric agonists stimulate a receptor independently of its physiological state, allosteric ligands that have no agonist activity only exhibit their effects in receptors activated by endogenous agonists, being quiescent in the absence of endogenous orthosteric ligand. Therefore, these modulators act much more in concert with the temporal and spatial organization of endogenous physiological signalling (Conn et al., 2009b), having a lower side effect potential and a lower propensity to induce receptor desensitization (Urwyler, 2011). As opposed to orthosteric binding sites, allosteric binding sites show a higher sequence divergence and hence allosteric modulators can display greater selectivity than orthosteric ligands (Urwyler, 2011; Christopoulos, 2002). Due to the size and number of attachment sites of the recognition domain of the endogenous ligands, it is difficult to design low- molecular-weight synthetic orthosteric ligands that exhibit good bioavailability and/or CNS penetration (Urwyler, 2011). There are, however, some negative aspects of allosteric modulators that are worth to be mentioned: sequence divergence of allosteric binding sites can result in significant differences between species, which will constitute a problem whenever rodent receptors/models are used for drug screening or for in vivo trials of an allosteric compound characterized in vitro at human receptors; the activity-dependence of PAMs might be seen as a disadvantage in neurodegenerative diseases in which the loss of neurons results in

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decreased availability of the endogenous agonist; the “flat”, non-tractable structure-activity relationships that often characterize GPCRs allosteric modulators would be a barrier to the improvement of lead molecules; finally, the complexity of the biology of a disease, which is often not completely understood, makes it still impossible to find an optimal profile of an allosteric drug (Urwyler, 2011). The ubiquity of GPCRs in the CNS renders it important to create a mechanism of smoother and selective activation of these receptors and allosteric modulators can provide that mechanism. This is considered an emerging area of drug discovery. Whilst GPCR ligands likely represent ~50% of all marketed drugs, only two of these, Cinacalcet and Maraviroc, are marketed allosteric modulators. This is due to the different approaches needed to identify allosteric modulators compared to orthosteric ligands and the relative infancy of the field. Although allosteric modulators generally display much better features in specificity when compared to orthosteric ligands, there are, as mentioned before, a few examples of allosteric modulators interacting with multiple receptor subtypes: MPEP and SIB-1893 are both mGlu5 NAMs and mGlu4 PAMs; DFB and CPPHA, mGlu5 PAMs, also display weak NAM activity on mGlu4; and PHCCC has combined mGlu4 PAM and mGlu1 NAM activity. This duality does suggest similarities in allosteric binding sites across different receptors (Gregory et al., 2011). For that reason, the characterization of the binding sites of these compounds is a fundamental step towards the identification of drugs with increased potency and selectivity. Moreover, as more allosteric modulators will likely reach the clinic, it will be crucial to understand more about their binding site and mechanism of action. Several mutagenesis studies, which have been performed to that aim, have been reported in the literature, and will now be further discussed.

1.4. Mutational Studies on mGlu receptors

13 years ago, Litschig et al. (1999) reported the first compound acting at and inhibiting an mGlu receptor without affecting the binding of the endogenous agonist. Based on the sequence alignment of hmGlu1b and hmGlu5a, Litschig et al. identified which amino acids could be playing a role in the selectivity of CPCCOEt for hmGlu1b over hmGluR5a. Accordingly, they systematically exchanged segments and non-conserved single amino acids

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between these two subtypes to precisely identify the amino acids mediating the effect of this compound (Listchig et al., 1999). Since then, a number of mutagenesis studies on mGlu receptors have been published (Table 3) with the purpose of investigating the molecular determinants of these receptors. These studies have been performed through the use of chimeric receptors, which share segments from different receptor groups/subtypes, point mutations on these receptors, and molecular modelling through the alignment of the receptor to the TM membranes of the bovine rhodopsin receptor and in silico docking of compounds. Mutational studies generate important information on the study of mGlu receptors and can be important to reveal how extracellular signals are transmitted into the cells (Listchig et al., 1999). Not only have these studies allowed the mapping of the regions of mGlu receptors that are critical for modulation of agonist activity (Schaffhauser et al., 2003) they also bring insight to the pharmacological profile, site of action and the potential binding mode of novel mGlu receptors ligands, allosteric modulators included. Furthermore, the need to identify novel selective ligands that are specific for each mGlu receptor subtype to characterize the physiological role of individual receptors (Litschig et al., 1999) and to improve selectivity and potency – potentially useful for pharmacological modulation of mGlu receptors – makes these studies extremely relevant (Pagano et al., 2000; Malherbe et al., 2003). In order to predict which amino acids can be playing a role in the interaction ligand/receptor, it is essential not only to know the amino acid sequence of the receptor, but also its structure, which is inferred using a template from a known GPCR structure. This structure can then be used for the docking of allosteric modulators, allowing the establishment of a putative model of ligand/receptor interaction. For this purpose, sequence alignment and homology modelling have to be performed.

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rmGluR2/3 b.s Summary3: o Tabe hmGluR2 hmGluR5 hmGluR1 rmGluR1 rmGluR1 rmGluR5 rmGluR1 rmGluR5 rmGluR1 rmGluR1 rmGlu2 Target - binding site binding

2,4 MNI ; hmGluR (human metabotropic glutamate receptor; rmGluR (rat metabotropic glutamate recep metabotropic rmGluR (rat glutamate receptor; metabotropic (human hmGluR ; - dicarboxypyrroles (NAMs) dicarboxypyrroles BAY36 - 135 f

the amino acids identified as important important molecular determinants of mGlu receptors through mGlureceptors of moleculardeterminants important identifiedas important acids amino the RO5488608 RO4988546 , , Ro 67 Ro 67 Ro 01 Ro EM LY487379 CPCCOEt MNI (NAMs) (NAMs) (NAMs) (PAMs) Ligand (NAM) (NAM) (NAM) (NAM) (PAM) MPEP MPEP - CTZ 7620 (NAM) 7620 - A4 -06094 -Ana Farinha - thesis binnenwerk.indd 38 TBPC

- - - - 7476 4853 6128 136

, ,

MNI Tabe 3: Summary of the amino acids identified as important important molecular determinants of mGlu receptors through mutagenesis studies performed on mGlu receptors. Tabe 3: Summary of the amino acids identified as important important molecular determinants of mGlu receptors through mutagenesis studies performed on mGlu receptors. -

b.s-binding site137 ; hmGluR (human metabotropic glutamate receptor; rmGluR (rat metabotropic glutamate receptor. b.s-binding site; hmGluR (human metabotropic glutamate receptor; rmGluR (rat metabotropic glutamate receptor.

Target Ligand EC2 TM3 TM4 TM5 TM6 TM7 References Remarks Target Ligand EC2 TM3 TM4 TM5 TM6 TM7 References Remarks Asn747 His723

hmGluR1 CPCCOEt EC2 Thr815 hmGluR1 CPCCOEt Thr815 Litschig et al., 1998 (NAM) Ala818 Litschig et al., 1998

(NAM) Ala818

hmGluR5 MPEP Pro655 Ala810 MPEP and CPCCOEt bind to hmGluR5 MPEP Pro655 Ala810 Pagano et al., 2000 MPEP and CPCCOEt bind to Cys671 Arg636 Arg635 Phe643 Tyr658 Pro654 Pro655 Ser668 Ser658 (NAM) TM3 Pagano et al., 2000 overlapping binding pockets (NAM) Ser658 overlapping binding pockets

Ser658 rmGluR1 BAY36-7620 (NAM) TM4 – TM7 Carrol et al., 2001

rmGluR1 BAY36-7620 (NAM) TM4 – TM7 Carrol et al., 2001

rmGluR1 rmGluR1 Ro 01-6128 Ser668 Val757 Gly689 Ro 01-6128 Ser668Ser688 Val757

TM4 Ro 67-4853 Cys671

Ro 67 -4853 Cys671 KnoflachKnoflach et al et., al2001., 2001

rmGluR5 Ro7TM 67-7476 Pro654

rmGluR5 Ro 67-7476 Pro654 (PAMs) Ser657 Asn735

Leu732 (PAMs) Leu743 Ser657

Val757 Val757 Pro654 Ser657 TM5

hmGluR2 LY487379 Ser688 Asn735 TM4 TM4

hmGluR2 LY487379 Ser688 Asn735 SchaffhauserSchaffhauser et al. et, al.2003, 2003

(PAM) Gly689

(PAM) Gly689 38 –

TM7 rmGluR1rmGluR1 EM-TBPC Asn747 Val757 Trp798 Thr815 Phe780 Phe787 Phe80 Trp773 EM-TBPCTyr791 Trp784 Thr780 Tyr805 Asn747Trp798 Val757 Trp798 Thr815

TM6 Malherbe et al., 2003a

(NAM) Phe801 Malherbe et al., 2003a

(NAM) Phe801

Tyr805 Tyr805 Malherbe et al., 2003b Ala81 Thr815 rmGluR5rmGluR5Val798 Ala818 Thr815 MPEP Ala809 Thr815 Pro654Pro654 Leu743Leu743 Ala810 Thr780Thr780 Ala809Ala809 Malherbe et al., 2003b

TM7 tor.

(NAM)(NAM) Tyr658Tyr658 Trp784Trp784

Phe787Phe787

Tyr791Tyr791

mutagenesis studie mutagenesis Schaffhauser Schaffhauser Lundström Lundström Malherbe Malherbe Hemstapat Hemstapat Malherbe rmGluR1 Knoflach 7TM Micheli et al., 2003 7TM Litschig Micheli et al., 2003 rmGluR1 2,4-dicarboxypyrrolesMicheli (NAMs) 2,4-dicarboxypyrroles (NAMs) Pagano Carrol Carrol Surin rmGluR1 Surin et al., 2007 Same site as CPCCOEt rmGluR1 CTZ References Thr815 Surin et al., 2007 Same site as CPCCOEt CTZ Thr815 et al et et al et

(NAMs) al et Ala818 et al et et al et et al et (NAMs) al. et Ala818

al et

al et et al et et al. et

rmGluR2/3 2 ., Hemstapat et al., 2007 Asn735: not important for the binding MNI-135, MNI-136, MNI-137 2001 ., ., 2000 .,

rmGluR2/3 2003 ., Hemstapat et al., 2007 Asn735: not important for the binding ., 1998 ., MNI-135, MNI-136, MNI-1372003b ., ., 2001 ., , 2003a , ., 2007 ., ., 20 ., 007

of these NAMs , (NAMs) mGlureceptors. on sperformed (NAMs) of these NAMs 2003

rmGlu2 Lundström et al., 2011 His723 on EC2: RO4988546 His723 Arg635 Leu732 Trp773 Val798

Lundström et al., 2011 His723 on EC2: rmGlu2 RO4988546 His723 Arg635 Leu732 Trp773 Val798

RO5488608 Arg636 Phe780 selectivityselectivity of mGluR2 of mGluR2 over overmGluR3 mGluR3 RO5488608 Arg636 Phe780 Asn735: Asn735:

selectivity PAMs/NAMs: Overlapping b.s. (NAMs)(NAMs) Phe643Phe643 PAMs/NAMs: Overlapping b.s. PAMs/NAMs: Overlapping b Overlapping PAMs/NAMs: MPEP and CPCCOEt bind to to CPCCOEt bind and MPEP overlapping binding pockets binding overlapping 3838 Same site as CPCCOEt Samesite 28/06/2012 9:58:36 not His723 on EC2: on His723

of of mGluR2 over mGluR3 over mGluR2 of important for the binding the for important these NAMs these Remarks

.s.

Sequence alignment and homology modelling

Homology modelling is used to predict the structure of a protein from its sequence (Krieger et al., 2003). It is based on two major observations: the structure of a protein is uniquely determined by its amino acid sequence (Epstain et al., 1963), which means that, theoretically, the structure of a protein can be obtained once its sequence is known (Krieger et al.., 2003); in terms of evolution, the structure is more stable than the associated sequence, so that similar sequences still adopt the same structure (Chothia and Lesk, 1986; Sander and Schneider, 1991). Homology modelling can be described as a seven step procedure: 1) template recognition and initial alignment, 2) alignment correction, 3) backbone generation, 4) loop modelling, 5) side-chain modelling, 6) model optimization and 7) model validation. High-resolution 3-dimensional (3D) structures of proteins provide important information on the form and function of those proteins at a molecular level. Not only does this information elucidate the aspects of protein structure which underlie physiological processes, it is also helpful to examine the interactions of proteins to their ligands and to small molecule drugs (Congreve and Marchall, 2010). Palczewski et al. determined for the first time the 3D crystal structure of a mammalian GPCR, the visual pigment rhodopsin (Figure 9) (Palczewski et al., 2000). The 3D crystal structure of bovine rhodopsin built by Palczweski et al. provided a structural template for other GPCRs, enabling the modeling of other members of this receptor family. The structure of bovine rhodopsin contains many features common to most GPCRs (Palczweski et al., 2000). Furthermore, the molecular size of rhodopsin is intermediate among the members of the GPCR family, and the length of its 7TM helices and extracellular loops are comparable to most of the GPCRs (Palczweski et al., 2000). Homology models based on rhodopsin structure have been used for the study of allosteric interactions at members of class C GPCRs. Even though the sequence similarity between class C GPCRs and rhodopsin-like class A GPCRs is low, the allosteric binding site of class C receptors share structural features with the orthosteric binding site of the class A family (Lundström et al., 2011).

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Figure 9: Three-dimensional representation of bovine rhodopsin (Palczewski et al., 2000)

Until recently, bovine rhodopsin was the best structure of GPCR receptors and the investigation on drug-receptor interaction was limited to models based on homology with this structure or from site-directed mutagenesis experiments (Congreve and Marshall, 2010). Recently, however, the structures of other GPCRs have been identified (namely β1 (Warne et al., 2008) and β2 (Cherezov et al., 2007) adrenergic receptors and adenosine A2a receptor (Jaakola et al., 2008)) enabling a wider analysis of structural differences between the different receptors.

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1.5. Goal of the project

Janssen Pharmaceutica has an mGlu2 receptor PAM in clinical development for the treatment of schizophrenia. In addition, as part of an internal mGlu2 receptor PAM program, other potentially important compounds from different chemical series were identified. In order to understand the mechanisms through which these compounds might produce their effects, it is important to clarify where they bind to the receptor molecule, which receptor region is involved and which amino acids are critical for the binding and/or activity. In order to identify amino acids potentially important for the interaction between PAMs and the mGlu2 receptor, homology modelling and docking of mGlu2 receptor PAMs was performed in parallel with experimental site directed mutagenesis. Accordingly, mutant mGlu2 receptors were produced. The goal of this study is to evaluate the impact of the receptor mutations on the activity of several PAM compounds in order to confirm the role of the selected amino acids in the binding and/or activity of PAMs. This work is an important contribution to the characterization of the allosteric binding site of mGlu2 receptors.

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A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 42 28/06/2012 9:58:37

Chapter 2 Materials and Methods

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2.1. Materials

The fourteen PAMs tested in this study (Appendix 2 for name and chemical series) were synthesized at Janssen Pharmaceutica and dissolved in 100% of dimethyl sulphoxide (DMSO). L-glutamate was purchased from Aldrich® Chemistry. The radioligand [3H]- LY341495 was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA) and Guanosine 5’-(γ-thio)triphosphate [35S]- was purchased from Perkin Elmer® (Boston, MA, USA). The monoclonal anti-metabotropic glutamate receptor 2 [mG2Na-s] antibody Ab15672 was purchased from Abcam (Cambridge, UK).

2.2. Positive allosteric modulators tested

11 compounds used in this study are part of an internal mGlu2 receptor PAM program, and were identified by lead optimization of hits originating from high-throughput screening through a calcium mobilization assay performed on the mGlu2 receptor. The compounds belong to the following chemical classes: 1,4-pyridones, 1,5-pyridones, triazolopyridines, isoquinolones and imidazopyridines. 3 reference PAMs were also tested: BINA (Galici et al., 2006), the acetophenone LY2605740 (Fell et al., 2011) and LY487379 (Schaffhauser et al., 2003). Appendix 2 summarizes the chemical series and name of each compound used.

2.3. Selection of amino acid mutations: sequence alignment and building of an mGlu2 receptor homology model

The identification and selection of mGlu2 receptor amino acids potentially important for PAMs binding, and therefore important targets of mutagenesis studies, was based on different approaches. The work described in this section was performed by a co-worker, Gary Tresadern (Molecular Informatics). Due to the significant difference in the conformation of the EL2, and given the fact that this domain has an important role in ligand binding, the mGlu2 receptor structure was aligned to both rhodopsin (1GZM) and β2-adrenergic receptor (2RH1) structures, and certain conserved motifs between the different receptors were used as constraints to guide the

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alignmentalignmentalignment. The.. The The alignment alignment alignment was was was performed performed performed using usingusing Molecular MolecularMolecular Op Operating Operatingerating Environment Environment Environment (MOE) (MOE) (MOE) softwaresoftwaresoftware with withwith the the theProtein ProteinProtein Align AlignAlign tool tooltool (Chemical (Chemical(Chemical Computing ComputingComputing Group GroupGroup, Canada),, Canada)Canada). .. In I orderInn order order to to establishto establish establish a possible a a possible possible binding binding binding mode mode mode of ofmGlu2 of mGlu2mGlu2 receptor receptor receptor PAMs, PAMs,PAMs, an an anmGlu2 mGlu2mGlu2 receptorreceptorreceptor reference referencereference PAM PAMPAM was waswas manually manuallymanually docked dockeddocked into intointo the the themGlu2 mGlu2mGlu2 receptor receptorreceptor models modelsmodels buil builbuilt fromtt fromfrom the the the bovinebovinebovine rhodopsin rhodopsin rhodopsin and and and β2 - β2 β2adrenergic--adrenergicadrenergic receptor receptor receptor templates, templates,templates, using using using MOE. MOE. MOE. Figure Figure Figure 10 10 10shows showsshows the the the bestbestbest ranked rankedranked docking dockingdocking pose, pose,pose, which whichwhich was waswas obtained obtainedobtained from fromfrom the the themGlu2 mGlu2mGlu2 receptor receptorreceptor model modelmodel built builtbuilt from fromfrom the the the β2-β2β2adrenergic--adrenergicadrenergic receptor receptor receptor template. template. template. The The The potentially potentially potentially important important important amino amino amino acid acid acids weress were were then then then selected selected selected basedbasedbased on on1)on their1)1) theirtheir proxim proximproximity ityityto thetoto the theligand ligandligand (Figure (Figure(Figure 10 )1010 and)) andand 2) literature2)2) literatureliterature referring referringreferring amino aminoamino acids acidsacids at atat thethe thesame samesame posi posipositiontiontion in otherinin otherother mGlu mGlumGlu receptor receptorreceptors (Figuress (Figure(Figure 11 A).1111A).A). Hence, Hence,Hence, Arg636, Arg636,Arg636, Leu639, Leu639,Leu639, Phe643, Phe643,Phe643, Asp725,Asp725,Asp725, Leu732, Leu732,Leu732, Trp773, Trp773,Trp773, Phe776 Phe776Phe776 and andand Phe780 Phe780Phe780 were werewere sele seleselectedctedcted as ascandidatesas candidatescandidates for for forsite sitesite-directed--directeddirected mutagenesismutagenesismutagenesis studies. studies.studies. GivenGivenGiven the the thefact factfact that thatthat most mostmost of theofof the thePAMs PAMsPAMs are are aremGlu2 mGlu2mGlu2 receptor receptorreceptor-selective--selectiveselective, the,, the thesequences sequencessequences of ofof thethe themGlu2 mGlu2mGlu2 and andand mGlu3 mGlu3mGlu3 receptor receptorreceptor were werewere compared comparedcompared in orderinin orderorder to identifytoto identifyidentify which whichwhich amino aminoamino acids acidsacids differ differdiffer betweenbetweenbetween the the thetwo twotwo sequence sequencesequences and,ss and,and, therefore, therefore,therefore, are are arepotentially potentiallypotentially important importantimportant for for forthe the theselective selectiveselective binding bindingbinding of ofmGlu2of mGlu2mGlu2 receptor receptorreceptor PAM PAMPAM compounds compounds compounds (Figure (Figure (Figure 11 B). 11 11B). B). The The The main main main differences differences differences were were were seen seen seen in inthe in the the extracellularextracellularextracellular portion portionportion of TM3,ofof TM3,TM3, TM4, TM4,TM4, EL2 EL2EL2 and andand TM5. TM5.TM5. Overall,Overall,Overall, based based based on on on these these these different different different approaches approachesapproaches (molecula (molecula (molecular modelingrr modeling modeling and and and docking dockingdocking, ,, sequencesequencesequence alignment alignment alignment and andand also also also literature literature literature), 40),), 40 40 amino amino amino acids acids acids were were were identified identified identified as as as potentially potentially potentially importantimportantimportant for for forthe the thePAM PAMPAM-mGlu2--mGlu2mGlu2 receptor receptorreceptor interaction interactioninteraction (Table (Table(Table 4). 4).4).Mutant MutantMutant mGlu2 mGlu2mGlu2 receptor receptorreceptor cDNA cDNAcDNA constructsconstructsconstructs were were were prepared prepared prepared by by byGeneArt GeneArtGeneArt® (Life®® (Life(Life Technologies) Technologies) Technologies) and a a ndnd for for for this this this project project project 21 2121mGlu2 mGlu2mGlu2 receptorreceptorreceptor mutants mutantsmutants (highlighted (highlighted(highlighted in blueinin blueblue in Tableinin TableTable 4) were4)4) werewere further furtherfurther tested. tested.tested.

A AA

B BB FigureFigureFigure 10: 10: 10: Ribbon Ribbon Ribbon diagramdiagramdiagram of ofthe of the the binding binding binding modemodemode of of of a areference a reference reference PAMPAMPAM compound compound compound in inthe in the the mGlu2mGlu2mGlu2 model model model structure structure structure builtbuiltbuilt from from from β2 - adrenergicββ22--adrenergicadrenergic templatetemplatetemplate FigureFigureFigure 11: 11:11:Align AlignAlignmentmentment of mGluR ofof mGluRmGluR 7TMs. 7TMs.7TMs. A) ResiduesA)A) ResiduesResidues highlighted highlightedhighlighted in red inin redredboxes boxesboxes have havehave beenbeenbeen identified identifiedidentified as importantasas importantimportant from from from literature literatureliterature mutagenesis mutagenesis mutagenesis studies; studies;studies; B) B)ComparisonB) ComparisonComparison betweenbetweenbetween hmGluR2 hmGluR2hmGluR2 and and andhmGluR hmGluRhmGluR3, with3,3, withwith highlighted highlightedhighlighted differences differencesdifferences 45 4545

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 45 28/06/2012 9:58:37 Table 4: mGluR2 mutagenesis constructs. The wild type (WT) and the substituting amino acids are indicated, as well as their position and the receptor region where they are located (TM – transmembrane;

EL – extracellular loop). The first 21 mutations, highlighted in light blue, were tested on this study.

Mutant Receptor WT AA Position Mutation WT codon Name codon region Ser 688 Leu TCG TTG S688L

TM4 Literature Gly 689 Val GGC GTC G689V

Asn 735 Asp AAT GAT N735D TM5

Ser 688 Leu TCG TTG S688L

TM4 (PAM Gly 689 Val GGC GTC G689V

Ser 688 Leu TCG TTG S688L 1 ) Gly 689 Val GGC GTC G689V TM4/5

Asn 735 Asp AAT GAT N735D

Arg 636 Ala CGT GCT R636A

Leu 639 Ala TTG GCG L639A TM3

Phe 643 Ala TTC GCC F643A D Asp 725 Ala GAT GCT D A ocking 725 TM5 Leu 732 Ala CTG GCG L732A

Trp 773 Ala TGG GCG W773A

Phe 776 Ala TTC GCC F776A TM6

Phe 780 Ala TTC GCC F780A

Ser 644 Ala TCT GCT S644A TM3 comparison mGluR2/3 Val 700 Leu GTC CTC V700L TM4

His 723 Val CAC GTC H723V EL2

Ser 644 Ala TCT GCT S644A

Val 700 Leu GTC CTC V700L TM3/4/EL2

His 723 Val CAC GTC H723V

Arg 635 Ala AGA GCG R635A TM3 Literature (NAMs Met 728 Ala ATG GCG M728A

Ser 731 Ala AGC GCC S731A TM5 2 ) Val 736 Ala GTG GCG V736A

Val 798 Ala GTG GCG V798A TM7

Ala 681 Phe GCC TTC A681F Ile 693 Met ATT ATG I M 693 TM4 Val 695 Ser GTG TCG V695S

Ala 696 Val GCC GTC A696V

Gly 706 Arg GGA CGA G706R

Glu 708 Tyr GAG TAC E708Y mGluR2 vs mGluR3 Ala 710 Leu GCC CTC A L 710 EL2 Pro 711 Ala CCC GCC P711A

Val 716 Thr GTG ACG V716T

Thr 718 Ile ACC ATC T718I

Ala 726 Ser GCC TCC A726S

Gly 730 Ile GGC ATC G I 730 TM5 Ala 733 Thr GCC ACC A733T

Ala 740 Ile GCC ATC A740I

Cys 616 Ser TGC TCC C616S Ile 622 Phe ATC TTC I F 622 TM2 Thr 641 Ser ACC TCC T641S

Ala 642 Ser GCC TCC A642S 1) Baez et al, 2002; Schaffhauser et al., 2003; Hemstapat et al., 2007; Rowe et al., 2008. 2) Lündstrom et al., 2011 46

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2.4. Chemical transformation of One Shot® Top10 E. coli cells with point-mutated human mGlu2 receptors and DNA purification

1 µl of cDNA was added to 50 µl of One ShotTM competent cells (Life Technology) and the mixture was incubated 30 min on ice. After that, cells were heat shocked (30 sec at 42ºC) and rapidly returned on ice for 2 min. 250 µl of S.O.C. medium (InvitrogenTM; formulation per liter: 2% tryptone, 0.5% yeast extract, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulphate, 20 mM glucose) was added and the cells were incubated for 1 h at 37ºC in a rotary shaking incubator (InnovaTM 4230, New Brunswick Scientific, Edison, NJ, USA), at 225 rpm. From this, 20 µl was spread in a plate and incubated for 18 h at 37ºC. In order to prepare a starter culture, one colony was picked from the transformation plate to inoculate 5 ml Luria-Bertani (LB) medium containing 100 µg/ml ampicilin. The culture was incubated for approximately 6 h at 37 ºC and 300 rpm, in a rotary shaking incubator (InnovaTM 4230, New Brunswick Scientific, Edison, NJ, USA). For large scale preparation, 500 ml LB medium supplemented with 100 µg/ml ampicilin were inoculated with 2.5 ml of the starter culture and incubated overnight at 37ºC and 300 rpm (Model G25 Incubator Shaker, New Brunswick Scientific, Edison, NJ, USA). The cells were then harvested by centrifugation (Multifuge 3 S-R, Heraeus, Buckinghamshire, England). DNA purification from the recombinant E. coli cells was performed using the Endofree® Plasmid Maxi/Mega Purification kit (Qiagen GmbH, Hilden, Germany), according to manufacturer’s instructions. After purification, the DNA was analyzed through sequencing.

2.5. Cell culture

CHO-K1 cells (ATCC: CCL-61) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; HyClone®, Thermo Scientific, Cramlington, UK) and 2% (v/v) Solution A. Solution A consists of Penicillin G (Serva, Bioconnect, Huissen, The Netherlands) 5.10e6 IU/L, Streptomycin sulphate (Serva) 5 g/L, Pyruvic acid (Sigma) 5.5 g/L and L-Glutamine (Sigma,

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St. Louis, MO, USA) 14.6 g/L. Cells were kept at 37ºC in a humidified atmosphere with 5%

CO2.

2.6. Transient transfection of human mGlu2 receptor cDNA into CHO-K1 cells

CHO-K1 cells were seeded in 145 cm2 Petri dishes (NuncTM, Roskilde, Denmark) at a seeding density of 20000 cells/cm2 in DMEM supplemented with 10% heat-inactivated FBS and 2% Solution A. 24h later, when a confluence of 50-70% was reached, human mGlu2 receptor (hmGluR2) cDNA, WT and mutated (Table 4, mutation 1 – 21) was transiently transfected into CHO-K1 cells using Lipofectamine® (InvitrogenTM, Life TechnologiesTM), and the recombinant cell lines were incubated overnight in the same medium, at 37ºC and in

an atmosphere of 5% CO2. One plate was used for transfection with the plasmid encoding the Enhanced Green Fluorescent Protein (EGFP-N1) to be used as a control of the transfection efficiency. 20-24 h after the transfection, the medium was replaced by fresh medium, and 20- 24 h after the medium replacement butyrate (final concentration of 5 mM) was added to each plate. The transfection efficiency was qualitatively evaluated at this point through the visualization of the cells transfected with EGFP-N1, using a fluorescence microscope (Axiovert 135, Zeiss, N.V., S.A.). 20-24h after the addition of butyrate, the plates with recombinant cells were washed twice with ice-cold phosphate-buffered saline (PBS) and stored at -80 ºC, or used readily for membrane preparation.

2.7. Membrane preparation

Confluent plates with transfected CHO-K1 cells expressing the wild-type or mutant hmGlu2 receptors were harvested with a cell scraper and resuspended in ice-cold 50 mM Tris-HCl buffer, pH 7.4. The cell suspension was, from this moment on, always kept on ice. The cell suspension was centrifuged for 10 min at 16000 rpm in a Sorvall 5C PLUS SS34 centrifuge at 4ºC. The resulting cell pellet was resuspended and homogenized in ice-cold 5 mM Tris-HCl, pH 7.4, using an Ultra Turrax homogenizer (IKA TE5) at 24000 rpm. Additional 5 mM Tris-HCl was added to the homogenate and it was centrifuged again for 20 min at 18000 rpm in a Sorvall RC 5B/RC 28S centrifuge at 4ºC. The final pellet was resuspended and homogenized in 50 mM Tris-HCl using the Ultra Turrax homogenizer, and

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the membrane suspension was aliquoted in cryovials and frozen at -80ºC. The protein concentration was measured using the Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories, Munich, Germany) using bovine serum albumin (Sigma, St. Louis, MO, USA) as standard.

2.8. Western Blot analysis

hmGlu2 receptor membranes were thawed and homogenized using the Ultra Turrax homogenizer. 200 μl of membrane suspension was transferred to a tube and 400 μl of RIPA buffer (150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0, Sigma) complemented with phosphatase and protease inhibitors (Roche) was added. The lysate was incubated 30 min on ice, and centrifuged at 14000 rpm (20 min, 4ºC). The supernatant fraction was recovered to a fresh tube and the protein concentration was determined by BCATM Protein Assay (Sigma-Aldrich). 3 μg of protein was loaded on NuPAGE® Novex® Tris-Acetate 4-12% Bis-Tris Gel (Life Technologies). The electrophoresis ran initially at 100 V, after which the voltage was increased to 170-200 V. The proteins on the gel were blotted for 8 min on a nitrocellulose membrane through a dry blotting system (iBlot, Life Technologies). Membranes were blocked for 1h at RT with Non-Fat Dry Milk (NFDM) (Santa Cruz, Technology) (5% w/v) diluted in Tris buffer (10mM Tris pH8.0, 150mM NaCl and 0.05% v/v Tween 20) (Amersham Biosciences, GE Healthcare, Little Chalfond Buckinghamshire, UK) (TBS-T), after which they were washed three times in TBS-T. The membranes were then incubated with the primary antibody (anti-metabotropic glutamate receptor 2 antibody; final concentration 0.75 μg/ml) diluted in 5% NFDM in TBS-T, overnight at 4ºC, with gentle agitation. Primary antibodies were detected through the HRP-linked secondary antibody (1:10000 in TBS-T, Amersham Biosciences) via SuperSignal® West Dura Extended Duration Substrate (Pierce, Thermoscientific). Signals were captured and quantified by chemiluminescence (G-box Syngene, Syngene).

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2.9. Radioligand binding assay – [3H]-LY341495 binding

Theoretical background

The binding of a ligand to its receptor is the first and indispensable step in the chain of reactions that will lead to a pharmacological effect (de Jong et al., 2005). Therefore, the process of ligand binding can be measured to investigate receptor molecules and the binding properties of a given compound acting on the receptor (Vauquelin and von Mentzer, 2007). Radioligand binding assays are based on the ability of an analyte (unlabelled ligand) to displace a radioactively labelled ligand (radioligand) that binds to the receptor of interest. The radioactively labelled ligand must be appropriate to be used in a radioligand binding assay, and therefore it must obey several criteria: it should exhibit high affinity and selectivity to the receptor that is being studied; it should display a high specific activity towards the receptor; and it should be radiochemically pure, stable and resistant to enzymatic degradation (de Jong et al., 2005; Vauquelin and von Mentzer, 2007). Tritium (3H) and iodine (125I) are the most commonly used isotopes for ligand radio labelling: tritium exhibits a longer half-life than iodine (12.3 years over 60 days, respectively), which enables longer periods of storage, but since it has a lower specific radioactivity than iodine (29 Ci/mmol over 2125 Ci/mmol, respectively) it is only suitable when the tissue under study contains sufficient amounts of the receptor of interest (Vauquelin and von Mentzer, 2007). Radioligand binding studies involve three important concepts: specific binding (the binding to the receptor of interest), non-specific binding (the observed binding in the presence of an appropriate excess of unlabeled competitor ligand – corresponds to binding to filters, absorption to the tissue, dissolution in membrane lipids) and total binding (the combination of specific and non-specific binding). Specific binding is determined by subtracting the non- specific binding from the total binding. Receptor-bound radioligand is separated from free through filtration, centrifugation or suction, followed by the measurement of the remaining radioactivity. Radioligand binding studies can be performed through different experimental approaches. Saturation binding experiments are performed using a constant amount of target receptor and increasing concentrations of radioligand. From these experiments the amount of

the receptor of interest can be determined. Furthermore, the KD of a radioligand, i.e. the concentration of radioligand needed to bind 50% of the receptors and hence a measure of the affinity of the radioligand for the receptor, can also be determined. Competition binding

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experiments use constant amounts of protein and radioligand, but increasing concentrations of an unlabeled ligand to be tested. These experiments provide information on the affinity of that ligand to a receptor. Finally, kinetic experiments enable the determination of the rate constants for radioligand association and dissociation.

Procedure

In this study, both saturation and competition binding experiments were performed. hmGluR2-CHO-K1 membranes were thawed and homogenized using the Ultra Turrax homogenizer and the protein concentration was measured using the Bradford Method (Bio- Rad Protein Assay, Bio-Rad Laboratories, Munich, Germany). Membranes were diluted in

ice-cold binding buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2 and 2 mM CaCl2. The reaction mixture contained 10 µg of membrane protein and 3 nM of [3H]-LY341495 in a total volume of 500 µl. In order to measure the non-specific binding, 1 mM glutamate was used. The reaction mixture was incubated for 60 min at room temperature (RT). The incubation was stopped with a filtration step using Unifilter-96 GF/B filter plates in a 96-well PerkinElmer filtermate harvester, and the plates were dried overnight at RT. The remaining radioactivity was measured in a Microplate scintillation and luminescence counter (Packard).

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2.10. [35S]GTPγS binding assay

Theoretical background

This functional assay measures the level of G protein activation that follows agonist occupation of a GPCR. The activation of a GPCR involves the exchange of GDP for GTP at the Gα-subunit of G-protein heterotrimers. This event is followed by the dissociation of the G-protein α-subunit from the βγ complex, which will then both elicit downstream signal transduction events. The Gα subunit possesses an endogenous GTPase activity, which will cause the GTP hydrolysis into GDP, an event enabling the reassembly of Gα and Gβγ subunits and the termination of the receptor activity, providing a mechanism to restrain the duration of the signal (Figure 2.1a). 35S-labeled guanosine 5′-O-(γ-thio)triphosphate ([35S]GTPγS) is an analogue of GTP that is also able to bind to the α-subunit, but is resistant to its GTPase activity; because this non-hydrolysable form of GTP remains bound for a sufficient period of time, it allows counting of the amount of [35S] label incorporated (Lazareno, 1997; Kowal et al., 1998) (Figure 2.1 b).

Figure 12: a) Once an agonist binds to a receptor, GDP is replaced by GTP on the Gα subunit of the G protein; Gα subunit and Gβγ complex both activate cellular effectors; the GTPase activity of α subunit hydrolyses GTP to GDP, the three subunits reassemble and the receptor is turned off. b) When [35S]GTPγS is present, its exchange for GDP also occurs, but the GTPase activity of the Gα subunit is unable to hydrolyse [35S]GTPγS, which accumulates. Harrison and Traynor, 2003

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This functional assay measures an early receptor-mediated functional response – important to avoid amplification that can occur when analyzing parameters further downstream of the receptor – and it enables the determination of basic pharmacological parameters of compounds acting at GPCRs, such as potency, efficacy and antagonist affinity (Harrison and Traynor, 2003).

Procedure

hmGluR2-CHO-K1 membranes were thawed on ice and homogenized using the Ultra Turrax homogenizer and the protein concentration was measured using the Bradford method Bio-Rad Protein Assay kit. The membranes were diluted in assay buffer (pH 7.4 solution of

10 mM HEPES acid and 10 mM HEPES, containing 100 mM NaCl, 3 mM MgCl2, 10 µM GDP and 14.3 µg/ml saponin). The assay mixture (final total volume of 200 µl) contained 18 µl of assay buffer, 2 µl of test compound (at 10-fold final concentration), 20 µl of glutamate and 10 µg of membrane protein, after the addition of which a pre-incubation step of 30 min at 30 ºC was performed. Finally, 0.1 nM [35S] GTPγS was added and the assay mixture was incubated for another 30 min at 30 ºC. The reaction was stopped through filtration using Unifilter-96 GF/B filter plates (PerkinElmer Life Sciences, Boston, MA) in a 96-well PerkinElmer filtermate harvester, in order to remove the unbound [35S] GTPγS. The filters were washed 3 times with ice-cold 10

mM NaH2PO4/ 10 mM Na2HPO4 buffer, pH 7.4, and dried overnight at room temperature. 40 μl of MicroscintTMO (Perkin Elmer, MA, USA) was added to each well, covered with a plate sealer, and 30 min later the remaining radioactivity was counted in a Microplate scintillation and luminescence counter (Packard). When testing compounds for their PAM effect, the

GTPγS assay was performed using 4 μM glutamate (corresponding to the EC20 of glutamate) as agonist.

2.11. Data analysis

Data analysis was performed using GraphPad Prism version 4.02 for Windows (GraphPad Software, San Diego, USA). Concentration-response curves were fitted using non-linear regression analysis fitting the equation: Y=Bottom + (Top-

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Bottom)/(1+10^((LogEC50-X)*HillSlope)). Saturation binding experiments were analyzed using a non-linear regression analysis.

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Chapter 3 Results

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3.3.1.1 Expression. Expression of of WT WT and and mutant mutant mGlu mGlu2 2receptors receptors in in CHO CHO-K1-K1 cells cells

OnceOnce purified, purified, DNA DNA from from WT WT and and mutant mutant hmGlu2 hmGlu2 receptors receptors was was analyzed analyzed through through sequencing,sequencing, which which confirmed confirmed the the correct correct sequence sequence for for all all cas cases.es. TheThe efficiency efficiency of of transient transient transfections transfections into into CHO CHO-K1-K1 was was qualitatively qualitatively evaluated evaluated throughthrough EGFP EGFP-N1-N1 expression expression and and consequent consequent fluorescence fluorescence each each time time a anew new transfection transfection was was performed,performed, which which always always revealed revealed an an efficiency efficiency of of about about 30 30-35%-35% (Figure (Figure 13). 13).

FigureFigure 13: 13: Visualization Visualization of of transient transient transfection transfection efficiency efficiency in in CHO CHO-K1-K1 cells, cells, through through expression expression of of EGFP EGFP-N1;-N1; thisthis procedure procedure was was repeated repeated each each time time a a transfection transfection was was perform performed;ed; the the displayed displayed images images are are representative representative

examplesexamples of of this this evaluation, evaluation, and and correspond correspond to to the the transfection transfection of of A: A: WT WT hmGlu2, hmGlu2, B: B: Mutation Mutation G689V G689V and and C: C: MutationMutation R636A R636A

InIn order order to to assess assess the the expression expression of of WT WT and and mutated mutated receptors receptors in in membranes membranes of of CHO CHO transfectedtransfected cells, cells, Western Western blots blots were were performed performed using using an an antibody antibody that that recognizes recognizes a a47 47 amino amino acidacid sequence sequence of of the the C C-terminal-terminal tail tail of of mGlu2. mGlu2. Non Non-transfected-transfected CHO CHO-K1-K1 (referre (referred dto to as as CHO CHO WT),WT), CHO CHO-K1-K1 stably stably transfected transfected with with WT WT hmGlu2 hmGlu2 (referred (referred to to as as CHO CHO mGlu2 mGlu2 Stable) Stable) and and CHOCHO-K1-K1 transiently transiently transfected transfected with with WT WT hmGlu2 hmGlu2 (referred (referred to to as as CHO CHO mGlu2 mGlu2 Trans.) Trans.) were were usedused as as controls. controls. Except Except for for the the non non-transfected-transfected cells, cells, for for each each sample, sample, tw two omain main bands bands were were detected:detected: a aimmunoreactive immunoreactive band band running running at at ~ ~100 100 kDa kDa corresponding corresponding to to the the monomeric monomeric form form ofof mGlu2 mGlu2 and and a aband band running running at at ~200 ~200 kda kda corresponding corresponding to to the the homodimer. homodimer. The The immuno immunoblotblot analysisanalysis (Figure (Figure 14 14) )revealed revealed the the differences differences in in expression expression le levelsvels for for the the different different samples, samples, butbut overall overall confirms confirms the the expression expression of of each each mGlu2 mGlu2 receptor receptor (WT (WT and and mutant) mutant) in in CHO CHO-K1-K1 cells.cells. Mutant Mutant W773A W773A (#11) (#11) displays displays a arather rather low low expression expression level level of of the the mGlu2 mGlu2 homodimer. homodimer.

5656

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#1 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 #21 220

100

Figure 14: Western blot analysis on mGlu2 WT and mutant receptors to illustrate the expression of the receptors in CHO-K1 cells after stable (for WT hmGlu2) or transient transfection (for WT and mutant hmGlu2). Mutants are indicated with the corresponding mutation number (See Table 6, Methods section). As expected, the monomer (~100 kDa) and dimer (~200 kDa) are observed and this staining pattern is the same for WT and mutant mGlu2. Actin (antibody diluted 1:2000 in TBS-T) is presented as a loading control. Molecular weight markers are indicated in kDa. A representative immunoblot of 2 independent experiments is shown.

To further confirm the expression of mGlu2 in CHO-K1 cells, and to observe whether any of the mutations affected the orthosteric binding site or the receptor conformation needed for binding of orthosteric molecules like glutamate, radioligand binding studies were performed using the orthosteric mGlu2/3 receptor antagonist [3H]-LY341495. All mutated receptors showed similar specific [3H]-LY341495 binding (in the range of 90%), confirming that the orthosteric site had remained intact in the mutated receptor forms. Furthermore, glutamate was able to displace [3H]-LY341495 in a concentration- dependent manner, both from the receptor stably expressed in CHO-K1 cells and from transiently transfected WT or mutant receptors (Figure 15), confirming that mutant receptors were still able to bind glutamate.

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3500 A Stable WT 130 B Trans. WT F643A (#8) 3000 110 S688L (#1) H723A (#16) 2500 90 cpm) L732A (#10)

W773A (#11) H]-LY341495 2000 3 70

1500 50

1000 30 H]-LY341495 binding ( binding H]-LY341495 3 [

500 [ binding, %specific 10

0 -10 -9 -8 -7 -6 -5 -4 -3 -2 -9 -8 -7 -6 -5 -4 -3 -2 log [glutamate], M log [glutamate], M

3 Figure 15: Displacement of [ H]-LY341495 binding from hmGlu2-WT (stably and transiently transfected) and hmGlu2 mutants by glutamate. The affinity of glutamate is not altered by any of the mutations (see Table 5 for all the results). A) Total binding results are presented as counts per minute (cpm); data presented as mean±S.D. of one experiment performed in triplicate. B) Results are presented as percentage of specific binding, i.e. total binding (binding in the absence of glutamate; corresponds to 100%) minus non-specific binding (binding in the presence of 1 mM glutamate)); data is presented as mean±S.D. of one experiment performed in triplicate.

Figure 15A does show that, for different mGlu2 constructs, the absolute values of total binding vary, which is likely due to differences in receptor expression levels. Namely mutation W773A (#11) shows a prominent decrease in total binding, consistent with the

results obtained in Western blot analysis. pIC50 values for glutamate binding inhibition were

similar for all receptors (pIC50 of 5 corresponding to an IC50 of 10 μM) (Table 5) (n=1 or n=2).

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TableTable 5: 5:Summary Summary of ofpIC50 pIC50 values values for for displacement displacement of of[3 H][3H]-LY341495-LY341495 binding binding from from hmGlu2 hmGlu2-WT-WT (stably (stably andand transiently transiently transfected) transfected) and and hmGlu2 hmGlu2 mutants mutants by by glutamate. glutamate. Mutations Mutations are are sorted sorted by by receptor receptor region. region. For For WT,WT, data data is ispresented presented as asmean±SD mean±SD of ofat atleast least 3 concentration3 concentration-response-response measurements measurements performed performed in intriplicate; triplicate; forfor mutants mutants S688L S688L (#1), (#1), F643A F643A (#8), (#8), L732A L732A (#10), (#10), W773A W773A (#11) (#11) and and H723V H723V (# 16) (#16) data data is is presented presented as as mean±SDmean±SD of ofat atleast least 2 concentration2 concentration-response-response measurements measurements performed performed in intriplicate; triplicate; for for the the restant restant mutations, mutations, thethe results results of ofone one concentration concentration-response-response measurement measurement performed performed in intriplicate triplicate are are presented. presented.

ReceptorReceptor Region Region MutMutationation pIC50pIC50 glutamate glutamate ------StableStable WT WT 4.94.9 ± ±0.10 0.10 ------TransientTransient WT WT 4.94.9 ± ±0.07 0.07 R635AR635A (#18) (#18) 4.94.9

R636AR636A (#6) (#6) 4.84.8 L639AL639A (#7) (#7) 4.94.9 TM3 TM3 F643AF643A (#8) (#8) 4.84.8 ± ±0.06 0.06 S644AS644A (#14) (#14) 4.74.7 S688LS688L (#1) (#1) 4.74.7 ± ±0.06 0.06 G689VG689V (#2) (#2) 4.74.7

S688L/G689VS688L/G689V (#4) (#4) 4.74.7

TM4 TM4 S688L/G689V/N735DS688L/G689V/N735D (#5) (#5) 4.74.7 V700LV700L (#15) (#15) 4.94.9 S644A/V700L/H723VS644A/V700L/H723V (#17) (#17) 4.84.8 H723VH723V (#16) (#16) 4.64.6 D725AD725A (#9) (#9) 5.05.0

M728AM728A (#19) (#19) 4.94.9 S731AS731A (#20) (#20) 5.05.0 L732AL732A (#10) (#10) 4.94.9 ± ±0.06 0.06 EL2/TM 5 EL2/TM 5 EL2/TM N7N735D35D (#3) (#3) 4.84.8 V736AV736A (#21) (#21) 4.94.9

W773AW773A (#11) (#11) 4.94.9 ± ±0.12 0.12 F776AF776A (#12) (#12) 4.84.8 TM 6 TM 6 TM F780AF780A (#13) (#13) 4.94.9

It Iist isof of note note that that the the differences differences in in expression expression level level were were also also evaluated evaluated with with a amodest modest setset of of saturation saturation experiments experiments using using [3 [H]3H]-LY341495;-LY341495; as as can can be be seen seen from from Table Table 6 6and and Figure Figure 16,16, for for the the tested tested mutations mutations (S688L (S688L (#1), (#1), G689V G689V (#2), (#2), R636A R636A (#6) (#6) and and L639A L639A (#7)), (#7)), these these studiesstudies furthermore furthermore confirmed confirmed that that expression expression levels levels in in the the transiently transiently transfected transfected cell cell membranesmembranes could could vary vary and and that that expression expression levels levels were were low lowerer than than in in the the stably stably transfected transfected 3 3 cells.cells. Nevertheless, Nevertheless, the the K KD Dof of [ H][ H]-LY341495-LY341495 was was unchanged, unchanged, indicating indicating that that also also orthosteric orthosteric antagonistsantagonists bind bind with with similar similar affinity affinity to to the the orthosteric orthosteric binding binding site. site.

5959

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0.150 0.150

0.125 SB 0.125

0.100 0.100 SB/F (nM) SB/F Bound (nM) Bound 0.075 0.075 BL

0.050 0.050

0.025 0.025

0.000 0.000 0 1 2 3 4 5 6 7 8 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 Free (nM) SB (nM)

0.200 CHO WT mGlu2 Trans. 0.20 CHO WT mGlu2 Trans. SB/F

0.175 TB

0.150 0.15

0.125 SB

0.100 0.10 SB/F (nM) SB/F

Bound (nM) Bound 0.075 BL

0.050 0.05

0.025

0.000 0.00 0 1 2 3 4 5 6 7 8 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 Free (nM) SB (nM)

0.200 0.20 CHO mGlu2 S688L CHO mGlu2 S688L SB/F

0.175 TB

0.150 0.15

0.125 SB

0.100 0.10 SB/F (nM) SB/F

Bound (nM) Bound 0.075 BL

0.050 0.05

0.025

0.000 0.00 0 1 2 3 4 5 6 7 8 9 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 Free (nM) SB (nM)

0.20 CHO mGlu2 G689V 0.20 CHO mGlu2 G689V SB/F TB

0.15 0.15

0.10 0.10 SB/F (nM) SB/F Bound (nM) Bound BL

0.05 0.05

0.00 0.00 0 1 2 3 4 5 6 7 8 9 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 Free (nM) SB (nM)

0.20 SB/F 0.200 CHO mGlu2 R636A CHO mGlu2 R636A

0.175 TB

0.150 0.15

0.125 SB

0.100 0.10 SB/F (nM) SB/F

Bound (nM) Bound 0.075 BL

0.050 0.05

0.025

0.000 0.00 0 1 2 3 4 5 6 7 8 9 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 Free (nM) SB (nM)

0.200 CHO mGlu2 L639A 0.20 CHO mGlu2 L639A SB/F

0.175 TB

0.150 0.15

0.125 SB

0.100 0.10 SB/F (nM) SB/F

Bound (nM) Bound 0.075 BL

0.050 0.05

0.025

0.000 0.00 0 1 2 3 4 5 6 7 8 9 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 Free (nM) SB (nM)

Figure 16: Saturation binding curves and correspondent Scatchard plots of [3H]-LY341495 binding to WT (stably and transiently transfected) and mutant mGlu2 receptors (S688L, G689V, R636A and L639SA) expressed in CHO-K1 membranes. Data are presented as nanomolar specifically bound. Data points were determined in triplicate (n=1). 60

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TableTableTable 6: 6: 6: Equilibrium Equilibrium Equilibrium binding binding binding constants constants constants of of of [ [3 [3H]3H]H]--LY341495-LY341495LY341495 binding binding binding to to to CHO CHO CHO membranes membranes membranes expressingexpressingexpressing WTWT WT andand and mutantmutant mutant mGlu2mGlu2 mGlu2 receptorsreceptors receptors (n=1).(n=1). (n=1).

KKK (nM)(nM) (nM) BmaxBmaxBmax (fmol/mg(fmol/mg (fmol/mg ofof of protein)protein) protein) DDD mGlu2mGlu2mGlu2 SS Stabletabletable WTWT WT 0.870.870.87 648064806480 mGlu2mGlu2mGlu2 Trans.Trans. Trans. WTWT WT 0.720.720.72 690069006900

S688LS688LS688L (#1)(#1) (#1) 0.670.670.67 366136613661

G689VG689VG689V (#2)(#2) (#2) 0.780.780.78 437943794379

R636AR636AR636A (#6)(#6) (#6) 0.880.880.88 319731973197

L639AL639AL639A (#7)(#7) (#7) 0.760.760.76 374837483748

3.2.3.2.3.2. GlutamateGlutamate Glutamate potencypotency potency forfor for WTWT WT andand and mutatedmutated mutated mGlu2mGlu2 mGlu2 receptorsreceptors receptors

InInIn order orderorder to toto further furtherfurther verify verifyverify the thethe effect effecteffect of ofof the thethe mutations mutationsmutations on onon t thethehe glutamate glutamateglutamate binding bindingbinding pocket pocketpocket andandand onon on thethe the signallingsignalling signalling mechanismsmechanisms mechanisms ofof of thethe the receptor,receptor, receptor, thethe the potencypotency potency ofof of glutamateglutamate glutamate inin in WTWT WT andand and mutantmutant mutant hmGlu2hmGlu2hmGlu2 waswas was assessedassessed assessed throughthrough through functionalfunctional functional studiesstudies studies usingusing using aa a [[ 35[3535S]GTPγSS]GTPγSS]GTPγS assayassay assay.. .

pECpECpEC505050 valuesvaluesvalues were were were calculated calculated calculated from from from concentration concentration concentration--response-responseresponse curves curves curves of of of glutamate glutamate glutamate in in in membranesmembranesmembranes of ofof CHO CHOCHO--K1-K1K1 cells cellscells expressing expressingexpressing the thethe WT WTWT (stably (stably(stably and andand transiently transientlytransiently transfected) transfected)transfected) and andand the thethe mutatedmutatedmutated hmGlu2 hmGlu2 hmGlu2 receptors receptors receptors (Figure (Figure (Figure 16, 16, 16, Table Table Table 6). 6). 6). On On On the the the WT WT WT receptor, receptor, receptor, glut glut glutamateamateamate t typically typicallyypically 111 exertsexertsexerts effectseffects effects withwith with aa a pECpEC pEC505050 ofof of ~~ ~ 55 5.05.05.05 andand and ECEC EC505050 valuesvalues values ofof of ~~ ~ 99 9 μMμM μM.. .

FigureFigureFigure 16:16: 16: RepresentativeRepresentative Representative graphgraph graph ofof of thethe the effecteffect effect 120120120 StableStableStable WTWT WT Trans.Trans.Trans. WTWT WT ofofof a a a set set set of of of mGlu2 mGlu2 mGlu2 receptorreceptorreceptor mutationsmutationsmutations on on on glutamateglutamateglutamate--inducedinduced-induced [ [35 [3535S]GTPS]GTPS]GTPγγSγSS binding. binding. binding. 100100100 F643AF643AF643A (#8)(#8) (#8) DataDataData are are are from from from one one one experiment experiment experiment where where where S688LS688LS688L (#1)(#1) (#1) glutamateglutamateglutamate concentration concentration concentration--responseresponse-response curves curves curves 808080 H723VH723VH723V (#16)(#16) (#16) werewerewere determineddetermined determined onon on membranesmembranes membranes fromfrom from CHOCHO CHO---

L732AL732AL732A (#10)(#10) (#10) K1K1K1 cells cells cells expressing expressing expressing WT WT WT (stably (stably (stably and and and S S binding S binding S S binding 606060 W773AW773AW773A #11#11 #11 transientlytransientlytransiently transfected) transfected) transfected) and and and 5 5 5 mutant mutant mutant receptorsreceptorsreceptors (transiently(transiently (transiently transfected)transfected) transfected) inin in parallel.parallel. parallel. ResultsResultsResults aa rearere expressedexpressed expressed asas as aa a percentagepercentage percentage ofof of thethe the S]GTP S]GTP S]GTP 404040 35 35 35 responseresponseresponse toto to 11 1 mMmM mM glutamate.glutamate. glutamate.

% [ % [ % [ (% of 1 mM glutamate)(% of 1 mM glutamate) (% of 1 mM glutamate) 202020

000 -8-8-8 -7-7-7 -6-6-6 -5-5-5 -4-4-4 -3-3-3 -2-2-2 logloglog [glutamate],[glutamate], [glutamate], MM M

11 1 DataData Data fromfrom from Janssen’sJanssen’s Janssen’s centralcentral central datadata data warehousewarehouse warehouse.. .

616161

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Table 7: Effect of mGlu2 receptor mutations on glutamate-induced [35S]GTPγS binding. Concentration-response curves were determined in the presence of increasing concentrations of glutamate on membranes from CHO-K1 cells expressing WT (stably and transiently transfected) and mutant receptors (transiently transfected). Mean pEC50±SD and mean percentage of glutamate stimulation (defined as the ratio between the response obtained under basal conditions i.e. only using assay buffer, corresponding to 100%, and the response obtained with 1 mM glutamate) ±SD are given. Data presented was calculated from at least 3 concentration-response measurements performed in triplicate; except for mutants R636A (#6) and M728A (#19) for which data was calculated from 2 concentration-response measurements performed in triplicate. Mutations are again sorted by receptor region.

Receptor Region Mutation pEC50 Response amplitude (%)

--- Stable WT 5.0 ± 0.12 637% ± 122%

--- Transient WT 5.3 ± 0.15 261% ± 73%

R635A (#18) 4.9 ± 0.24 217% ± 49%

R636A (#6) 5.2 ± 0.15 196% ± 22%

L639A (#7) 5.2 ± 0.12 280% ± 37%

TM3 F643A (#8) 4.9 ± 0.22 222% ± 60%

S644A (#14) 5.2 ± 0.12 237% ± 78%

S688L (#1) 5.2 ± 0.15 232% ± 64%

G689V (#2) 5.2 ± 0.22 237% ± 89%

S688L/G689V (#4) 5.2 ± 0.18 249% ± 76%

TM4 S688L/G689V/N735D (#5) 5.3 ± 0.27 235% ± 69%

V700L (#15) 5.2 ± 0.14 278% ± 32%

S644A/V700L/H723V (#17) 5.0 ± 0.19 313% ± 55%

H723V (#16) 5.1 ± 0.13 282% ± 58%

D725A (#9) 5.3 ± 0.14 250% ± 19%

M728A (#19) 5.1 ± 0.12 274% ± 132%

S731A (#20) 5.2 ± 0.25 331% ± 61%

L732A (#10) 5.4 ± 0.18 346% ± 103% 5 EL2/TM N735D (#3) 5.1 ± 0.04 253% ± 69%

V736A (#21) 5.1 ± 0.07 248% ± 19%

W773A (#11) 5.0 ± 0.48 171% ± 30%

F776A (#12) 5.3 ± 0.12 326% ± 82%

6 TM F780A (#13) 5.2 ± 0.13 303% ± 43%

As shown in Figure 16 and Table 7, mutated receptors elicited concentration-response curves for glutamate with potencies that were similar to those of the WT receptor. The amplitude of the response to glutamate – i.e. the ratio between the response obtained under basal conditions (only using assay buffer), corresponding to 100%, and the response obtained with 1 mM glutamate – was considerably lower in transiently transfected CHO-K1 cells.

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 62 28/06/2012 9:58:44

3.3. Effect of mGlu2 mutations on the activity of positive allosteric modulators

[35S]GTPγS binding was used to assess the effect of each mutation on the activity of 14 mGlu2 receptor PAMs. The results of this analysis are summarized and presented as follows. From each chemical series, one representative compound is chosen to be presented graphically: JNJ-40068782 (1,4-pyridones), JNJ-35814376 (1,5-pyridones), JNJ-42153605 (triazolopyridines) JNJ-40297036 (isoquinolones) and JNJ-41482012 (imidazopyridines). Results for the reference compounds JNJ-52149617 (LY2605740, acetophenone, Fell et al., 2011), JNJ-35815013 (BINA, Galici et al., 2006) and JNJ-35814090 (LY487379, Schaffhauser et al., 2003) are also visualized in the graphs. Mutations are again sorted by receptor region. Hence, the results presented graphically represent a summary of the effects for a particular chemical structure, for a given receptor region. The results for the complete set of compounds are displayed in Table 8. This complete set of data will be discussed elsewhere (Section 4.3), and particular focus will be put on dissimilarities between compounds in a particular chemical series. The [35S]GTPγS experiments were set up as follows. In a first step, to preliminary assess the effect of each mutation on the PAM activity, only two concentrations of PAM were

tested: a PAM concentration that produces 50% stimulation (EC50) and a concentration that 2 produces a maximal stimulation (EC100) . This two-concentration screening was considered

necessary, as testing only the highest concentration (EC100) could mask the true effect of the

compound: as displayed in Figure 17, the maximal response (Emax) of the compound on the mutated receptor is the same as for the WT receptor, but the curve has shifted to the right.

Although the Emax – which would be detected in case of using the compound’s EC100 – is the same, which would indicate that the compound has similar activity in WT and mutated receptor, the potency is lower.

2 These values were taken from previous experiments for which data is stored in Janssen’s central database.

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 63 28/06/2012 9:58:44

180 Trans. WT

160 pEC50 : 7.20 140 G689V

120 pEC50 : 6.31

S binding 100 N735D no pEC 80 50 S]GTP

35 60 % [ (% of 1 mM glutamate) 40 20 0 -10 -9 -8 -7 -6 -5 -4 -3 Log[BINA], M Figure 17: Effect of increasing concentrations of BINA (JNJ 35815013), a reference PAM, on glutamate-induced [35S]GTPγS binding and the importance of performing a screening assay with EC50 and EC100: for EC100 the receptor with the mutation G689V exhibits similar effects as the WT (transiently transfected into CHO-K1); however, since the compound is less potent, this similarity is no

longer observed with lower concentrations (e.g. EC50).

As mentioned in the Methods section, PAMs were tested using 4 μM glutamate 35 (correspondent to the EC20). Per mutation, for each PAM compound, [ S]GTPγS binding in the presence of the PAM was calculated as a percentage of the response to 1 mM glutamate (no PAM added). Effects lower than 75% of the effect seen on the transiently transfected WT receptor were considered significantly different, and hence these mutations were selected for further analysis. Figure 18 displays the results obtained in the screening test for the representative compounds of each chemical series, comparing the effect of the different compounds between WT and mutant mGlu2. For a detailed overview table, showing the exact values obtained for the complete set of compounds and mutations, we refer to Appendix 3.

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 64 28/06/2012 9:58:44 300 300 JNJ-35815013 JNJ-35814090 250 250

200 200 S assay S assay

γ JNJ 35815013 γ JNJ 35814090 150 150 S]GTP S]GTP 35 100 35 100 % [ % [ % (% of of (%1 mM glutamate) of (%1 mM glutamate) 50 50

0 0

6 7 8 9 6 7 8 9 10 11 12 13 10 11 14 15 16 17 18 19 20 21 12 13 14 15 16 17 18 19 20 21 Trans. WTTrans. WTTrans. Stable Stable WT Stable WT 75% of75% T. WT of75% T. WT 300 JNJ-52149617 300 JNJ-40068782 250 250

200 200 S binding

S binding S binding JNJ 52149617 JNJ 40068782 γ γ 150 150 S]GTP

35 100 100 % [ % % [35S]GTP % (% of of 1 glutamate)mM (% (% of of 1 glutamate)mM (% 50 50

0 0

6 7 8 9 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 12 13 14 15 16 17 18 19 20 21 10 11 Trans. WT Trans. WT Stable Stable WT WT Stable 75% of75% T. WT 75% of75% T. WT

250 250 JNJ-35814376 JNJ-41482012

200 200

150 150 S binding S binding JNJ 35814376 S binding JNJ 41482012 γ γ S]GTP

100 S]GTP 100 35 35 % [ % % [ % (% of of 1 glutamate)mM (% (% of of 1 glutamate)mM (% 50 50

0 0

8 9 6 7 8 9 6 7 10 11 12 13 14 15 16 17 18 19 20 21 10 11 12 13 14 15 16 17 18 19 20 21 Trans. WTTrans. WTTrans. Stable Stable WT Stable WT 75% of75% T. WT of75% T. WT 200 JNJ-40297036 400 JNJ-42153605 350

150 300 250 binding JNJ 40297036 JNJ 42153605 S binding S γ γ 100 200 S]GTP S]GTP 150 35 35 % [ % % [ % (% of of 1 glutamate)mM (% (% of 1 1 of mMglutamate) (% 50 100 50 0 0

6 7 8 9 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 10 11 12 13 14 15 16 17 18 19 20 21 Trans. WTTrans. WTTrans. Stable Stable WT Stable WT 75% of75% T. WT of75% T. WT Figure 18: Screening of 8 mGlu2 receptor PAMs, representative of the 8 different chemical classes tested in this study, using the EC50 and EC100 on WT (stably transfected “Stable WT” and transiently transfected “Trans. WT”) and mutated hmGlu2 (mutations #6 to #21). For the mutations S688L (#1), G689V (#2), N735D (#3), S688L/G689V (#4) and S688L/G689V/N735D (#5) this screening was not performed. 75% of Trans. WT (“75% of T. WT”) response is also displayed. Mutations for which the compound effect was lower than “75% of T. WT”, for any of the concentrations (i.e. the mutations for which bars appear below the red dashed line) were further tested. See Appendix 3 for a complete summary of results. 65

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 65 28/06/2012 9:58:45

As shown in Figure 18, this screening assay enabled an early exclusion of several mutations based on the criteria mentioned above: for all compounds, mutations R636A (#6), D725A (#9), V700L (#15), M728A (#19) and V736A (#21) showed an effect higher than

75% of the effect on WT, for both EC50 and EC100 of all compounds, and were therefore considered to have no effect on the activity of any of the compounds tested. For the mutations showing an effect lower than 75% of the effect in WT for a given compound, a concentration-response curve of that compound on membranes of CHO-K1

cells expressing that mutation was generated. From this, potency (EC50) and relative efficacy

(Emax) values were calculated for the 14 PAMs on mGlu2 WT versus mutant receptors. Figures 19-22 graphically display the results obtained with the 8 representative compounds

on the mutations localized in the different receptor regions. The potency (EC50) and relative

Emax calculated from the concentration-response curves for the 14 compounds on WT and mutant receptors are presented in Table 8. In this table, the difference in compound potency

is also calculated as a ratio of EC50 between WT and mutant mGlu2 (for a more detailed table with additional information on individual experiments, see Appendix 4). Figure 19 shows the results obtained from the concentration-dependent enhancement of glutamate-induced [35S]GTPγS binding by the 8 representative compounds on WT mGlu2 and on mGlu2 with mutations localized in transmembrane 3. As seen in Figure 18, all the mutations seem to affect the activity of the tested compounds. Mutation F643A (#8) shows the most prominent results, as it not only causes a decrease in potency (right-ward shift of the

concentration-response curve) but also a decrease in Emax. EC50 values were increased by 6.4 to 157-fold for the different compounds tested (Table 8). Mutation L639A (#7) also affects the activity of all compounds, albeit to a lower extent compared to mutation F643A (#8) (1.3

to 15-fold increase in EC50, as shown in Table 8). Mutation S644A (#14) decreased the potency of all the compounds tested, with the exception of JNJ-39226421 (Figure 19, Table 8). Mutation R635A (#18) showed a decrease in potency of JNJ-35814376 (Figure 19E) and JNJ-40068782 (Figure 19H), but an increased maximal response. Additionally, this mutation caused a 5.6-fold increase in the potency of JNJ-35815013.

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 66 28/06/2012 9:58:45

120 A WT 220 B WT F643A (#8) 200 L639A (#7) 100 180 F643A (#8) 160 S644A (#14) 80 140 S binding S S binding 120 60 100 S]GTP S]GTP 80 40 35 35 60 (% of mM1 glutamate % [ (% of mM1 glutamate % [ 20 40 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 41482012], M Log [JNJ 35815013], M

180 C WT 140 D L639 (#7) WT 160 L639A (#7) F643A (#8) 120 140 F643A (#8) S644A (#14) 120 100

S S binding 100 S S binding 80

80 60 S]GTP 60 S]GTP 35 35 40 (% of 1 mM glutamate % [ (% of mM1 glutamate 40 % [

20 20

0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 40297036], M Log [JNJ 35814090], M

E F 280 WT 240 WT 260 R635A (#18) 220 F643A (#8) 240 L639A (#7) 200 220 S644A (#14) 180 200 160 180 140 S binding

S S binding 160 140 120 120 100 S]GTP S]GTP 100 80 35 35 80 60 (% of mM1 glutamate % [ (% of mM1 glutamate % [ 60 40 40 20 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -11 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 35814376], M Log [JNJ 42153605], M

G H 300 WT 280 R635A (#18) 240 WT 260 L639A (#7) 220 F634A (#8) 240 200 L639A (#7) 220 F643A (#8) 180 S644A (#14) 200 S644A (#14) 160 180 S S binding 160 140 S S binding 140 120 120 100 S]GTP 100 35 S]GTP 80 35 80 (% of mM1 glutamate 60 % [ 60 (% of mM1 glutamate % [ 40 40 20 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 52149617], M Log [JNJ 40068782], M Figure 19: Effect of PAMs on glutamate-induced [35S]GTPγS binding in WT mGlu2 and mGlu2 with mutations located in transmembrane 3 (TM3). A to H, concentration-dependent enhancement of 4 μM glutamate-induced [35S]GTPγS binding by 8 PAMs, representative of the 8 chemical classes used in this study. Results are expressed as a percentage of the response to 1 mM glutamate, and refer to one experiment performed in triplicate. See Table 7 for a complete summary of results obtained for 14 PAMs on 21 receptor mutants. 67

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 67 28/06/2012 9:58:46

WT 120 200 WT 120 A WT 200 B WT A S688L/G689V (#4) B S688L/G689V (#4) 180 H723V (#16) 180 L732A (#10) S688L/G689V/N735D (#5) 100 S688L/G689V/N735D (#5) 100 160 L732A (#10) 160 N735D (#3) S644A/V700L/H723V (#17) S644A/V700L/H723V (#17) 140 N735D (#3) 80 S688L (#1) S688L (#1) 140 80 G689V (#2) 120 G689V (#2) S binding S S binding S 120 60 100 S binding S binding 60 100 80 S]GTP S]GTP 40 80 35 35

60 S]GTP S]GTP 40 35 35 (% of 1 (%mM glutamate 1 of (% of 1 (%mM glutamate 1 of % [ % [ 40 60 20 (% of mM 1 glutamate (% of mM 1 glutamate % [ % [ 40 20 20 0 0 20 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 0 0 Log [JNJ 41482012], M Log [JNJ 35815013], M -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 41482012], M Log [JNJ 35815013], M

C 140 D WT 180 WT 180 WT 180 WT S731A (#20) G689V/S688L (#4) 160 H723V (#16) 120 C S688L/G689V (#4) 160 D L732A (#10) 160 G689V/S688L/N735D (#5) L732A (#10) S688L/G689V/N735D (#5) 140 140 N735D (#3) 140 S644A/V700L/H723V (#17) N735D (#3) 100 S644A/V700L/H723V (#17) 120 S689L (#1) 120 120 S688L (#1)

S S binding 80

S binding S G689V (#2) G689V (#2) 100 S binding 100 S binding S 100 80 60 80 80 S]GTP S]GTP S]GTP S]GTP 60 35 35 60

60 35 35 40 (% glutamate mM of 1 % % [ (% mM of glutamate 1 % [

(% of 1 (%mM glutamate 1 of 40 % [ (% of 1 mM glutamate 40 % [ 40 20 20 20 20 0 0 0 -10 -9 -8 -7 -6 -5 -4 -3 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 40297036], M Log [JNJ 35814090], M Log [JNJ 40297036], M Log [JNJ 35814090], M

280 WT 220 E WT 260 F H723V (#16) 200 H723V (#16) 240 WT WT 300 L732A (#10) 220 180 S731A (#20) 220 F S688L/G689V (#4) N735D (#3) 200 E S688L/G689V (#4) 200 S688L/G689V/N735D (#5) 160 L732A (#10) S688L/G689V/N735D (#5) 250 180 N735D (#3) 180 S644A/H700L/H723V (#17) S644A/V700L/H723V (#17) 140

160 S binding 160 S688L (#1) 200 S688L (#1) S binding 120 140 140 G689V (#2) G689V (#2) S binding S S binding 120 100 120 150 S]GTP 100 100 S]GTP 80 35 35 80 S]GTP S]GTP 80 60 (% mM of glutamate 1 100 % [ 35 (% of 1 mM glutamate 35 % [ 60 60 40 (% of 1 (%mM glutamate 1 of % [ (% of mM1 glutamate % [ 40 40 50 20 20 20 0 0 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -11 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 -11 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 35814376], M Log [JNJ 42153605], M Log [JNJ 35814376], M Log [JNJ 42153605], M

200 WT G 220 H WT 180 H723V (#16) 200 S731A (#20) H723V (#16) 160 180 L732A (#10) 280 WT 240 WT L732A (#10) 260 G H 140 160 N735D (#3) S688L/G689V (#4) 220 S688L/G689V (#4) N735D (#3) 240 S688L/G689V/N735D (#5) 200 S688L/G689V/N735D (#5) 120 140

220 binding S

180 S644A/V700L/H723V (#17) S binding 200 S644A/V700L/H723V (#17) 100 120 160 S688L (#1) 180 S689L (#1) 100 140 G689V (#2) 80 S binding S 160 binding S

G689V (#2) S]GTP

S]GTP 80

140 120 35 60 35 120 60

100 (% mM of glutamate 1 % [ (% of 1 mM glutamate % [

S]GTP S]GTP 40 100 80 40 35 80 35 60 20 (% of 1 (%mM glutamate 1 of (%mM glutamate 1 of % [ 60 % [ 20 40 40 0 0 20 20 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 0 0 Log [JNJ 52149617], M Log [JNJ 40068782], M -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 52149617], M Log [JNJ 40068782], M Figure 21: Effect of PAMs on glutamate-induced [35S]GTPγS binding in WT mGlu2 and mGlu2 with mutations located in Figure 20: Effect of PAMs on glutamate-induced [35S]GTPγS binding in WT mGlu2 and mGlu2 with mutations located in extracellular loop 2 and transmembrane 5 (EL2/TM5). A to H, concentration-dependent enhancement of 4 μM 35 transmembrane 4 (TM4). A to H, concentration-dependent enhancement of 4 μM glutamate-induced [35S]GTPγS binding by 8 glutamate-induced [ S]GTPγS binding by 8 PAMs, representative of the 8 chemical classes used in this study. Results are PAMs, representative of the 8 chemical classes used in this study. Results are expressed as a percentage of the response to 1 mM expressed as a percentage of the response to 1 mM glutamate, and refer to one experiment performed in triplicate. See Table glutamate, and refer to one experiment performed in triplicate. See Table 7 for a complete summary of results obtained for 14 7 for a complete summary of results obtained for 14 PAMs on 21 receptor mutants. PAMs on 21 receptor mutants.

68 69

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120 A WT 200 B WT H723V (#16) 180 L732A (#10) 100 L732A (#10) 160 N735D (#3) N735D (#3) 140 80 120 S S binding S binding 60 100 80 S]GTP 40 S]GTP 35 35 60 (% of mM 1 glutamate (% of mM 1 glutamate % [ % [ 40 20 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 41482012], M Log [JNJ 35815013], M

C 140 D WT 180 WT S731A (#20) H723V (#16) 120 160 L732A (#10) L732A (#10) 140 N735D (#3) N735D (#3) 100 120

S S binding 80

S S binding 100

80 60 S]GTP S]GTP

60 35 35 40 (% mM of glutamate 1 % [ (% of 1 mM glutamate % [ 40 20 20

0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 40297036], M Log [JNJ 35814090], M

280 WT 220 E WT 260 F H723V (#16) 200 H723V (#16) 240 L732A (#10) 180 S731A (#20) 220 N735D (#3) 160 L732A (#10) 200 180 140 N735D (#3)

S S binding 160 S S binding 120 140 100 120

S]GTP 100 S]GTP 80 35 35 80 60 (% mM of glutamate 1 % [ (% of 1 mM glutamate % [ 60 40 40 20 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -11 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 35814376], M Log [JNJ 42153605], M

200 WT G 220 H WT 180 H723V (#16) 200 S731A (#20) H723V (#16) 160 180 L732A (#10) L732A (#10) 140 160 N735D (#3) N735D (#3) 120 140 S binding S 100 S binding 120 100 80 S]GTP

S]GTP 80 35

60 35 60 (% mM of glutamate 1 % [ (% of 1 mM glutamate 40 % [ 40 20 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 52149617], M Log [JNJ 40068782], M

Figure 21: Effect of PAMs on glutamate-induced [35S]GTPγS binding in WT mGlu2 and mGlu2 with mutations located in extracellular loop 2 and transmembrane 5 (EL2/TM5). A to H, concentration-dependent enhancement of 4 μM glutamate-induced [35S]GTPγS binding by 8 PAMs, representative of the 8 chemical classes used in this study. Results are expressed as a percentage of the response to 1 mM glutamate, and refer to one experiment performed in triplicate. See Table 7 for a complete summary of results obtained for 14 PAMs on 21 receptor mutants.

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120 A WT 200 B WT W773A (#11) 180 W773A (#11) 100 F776A (#12) 160 80 140 120 S S binding S S binding 60 100 80 S]GTP 40 S]GTP 35 35 60 (% of 1 mM glutamate % [ (% of mM1 glutamate % [ 40 20 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 41482012], M Log [JNJ 35815013], M

160 C WT 140 D WT W773A (#11) W773A (#11) 140 120 F776A (#12) 120 100 100

S S binding 80 S S binding 80 60 S]GTP

S]GTP 60 35 35 40 40 (% of 1 mM glutamate % [ (% of 1 mM glutamate % [

20 20

0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 40297036], M Log [JNJ 35814090], M

240 200 E WT F WT 220 180 W773A (#11) W773A (#11) 200 160 F780A (#13) F776A (#12) 180 140 160 120 140 S S binding S S binding 100 120 80 100 S]GTP S]GTP 80 35 35 60 60 (% of 1 mM glutamate % [ (% of mM1 glutamate % [ 40 40 20 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -11 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 35814376], M Log [JNJ 42153605], M

160 WT 220 H WT G W773A (#11) 200 W773A (#11) 140 F776A (#12) 180 F776A (#12) 120 F780A (#13) 160

100 140 S S binding S S binding 120 80 100

60 S]GTP S]GTP 80 35 35 40 60 (% ofmM 1 glutamate (% ofmM 1 glutamate % [ % [ 40 20 20 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log [JNJ 52149617], M Log [JNJ 40068782], M

Figure 22: Effect of PAMs on glutamate-induced [35S]GTPγS binding in WT mGlu2 and mGlu2 with mutations located in transmembrane 6 (TM6). A to H, concentration-dependent enhancement of 4 μM glutamate-induced [35S]GTPγS binding by 8 PAMs, representative of the 8 chemical classes used in this study. Results are expressed as a percentage of the response to 1 mM glutamate, and refer to one experiment performed in triplicate. See Table 7 for a complete summary of results obtained for 14 PAMs on 21 receptor mutants. 70

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Figure 20 displays the results obtained with mutations localized in transmembrane 4. As shown in Figure 20 and Table 8, the entire set of mutations in this receptor region seems to affect the activity of all compounds, except for mutation S688L (#1) which for most cases yielded results similar as for WT mGlu2. Mutation G689V/S688L/N735D (#5) disrupted the

concentration-response curve for all the compounds, and a prominent potency/Emax decrease was seen for the compound representing the triazolopyridines, namely JNJ-42153605. Figure 21 displays the results from mutations localized in the regions of extracellular loop 2 (mutation H723V) and transmembrane 5. All the mutations seem to affect the activity of the compounds, but mutations N735D (#3) and L732A (#10) elicited the biggest shifts in the activity of the tested compounds. Interestingly, the effect of the EL2 mutation, H723V (#16), seems consistent between compounds from different chemical series. Finally, Figure 22 shows the results obtained for mutations localized in transmembrane 6. Mutation W773A (#11) affects the activity of all compounds, disrupting completely their ability to generate a concentration-response curve. This mutation caused a 4

to 55-fold increase in EC50 of the compounds tested (Table 8). Although to a smaller extent, mutation F776A (#12) also consistently affects the activity of the compounds belonging to different chemical series. For the compound JNJ-35814090 (Figure 22D), this mutation also led to the complete loss of the receptor’s ability to respond to increasing concentrations of the compound.

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Sc Sc Sc Sc Sc Sc Sc 0.7 0.9 0.8 0.8 0.8 1.0 0.8 110 139 153 174 196 105 131 Emax

(14) S644A(14)

Sc Sc Sc Sc Sc Sc Sc 1.2 2.5 1.2 2.7 0.7 3.2 1.8 119 625 108 216 180 276 305 EC50

86 36 36 49 84 90 89 84 53 62 Sc

0.4 0.4 0.5 0.4 0.5 0.4 0.7 0.4 0.4 0.4 0. 0.3 0.4 107 137 101 Emax after after transient transfection

(8) F643A

Sc n.v 6.4 n.v. n.v. n.v. 316 447 254 724 27.4 20.0 55.6 84.3 38.7 16.3 6783 5868 2396 1142 115.6 156.8 EC50 1414.1

74 90 Sc Sc Sc Sc Sc 0.7 1.0 0.9 0.5 0.6 1.1 0.7 1.1 1.2 121 244 121 186 204 171 148

Emax

(7) L639A

9 Sc Sc Sc Sc Sc 2.0 2.9 6.5 1.4 1.3 9.1 1.4 4.4 227 572 109 161 692 15.2 1351 1067 1320 EC50

on WT and mutant receptors 35 Transmembrane 3 Table 8: Potency (EC50) and relative Emax of the enhancement of glutamate-induced [ S]GTPγS binding by 14 PAMs on WT and mutant receptors after transient transfection

into CHO-K1 cells. Compound potency is presented as a ratio of EC50 between WT and mutant mGlu2.

PAMs

1 1

NT – not tested; Sc – tested in screening assay; – ratio ECSc mutantSc mGlu2Sc /ECSc WTSc > 3 orSc n.v – noSc valueSc (absence of curve)Sc Sc Sc Sc (absence of curve) of (absence 50 50 Emax 14 Transmembrane 3 by by

Stable WT

(6) R636A

(18) R635A (6) R636A (7) L639A (8) F643A (14) S644A no value no value Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

EC50 EC50– E max EC50 E max EC50 Emax EC50 Emax EC50 Emax EC50 Emax EC50 Emax

n.v

S binding

JNJ-40068782 139 206 87 201 378 246 Sc Sc 572 186 2396 89 216 174

1 92 Sc Sc Sc Sc Sc Sc Sc Sc Sc 0.7 1.2 1.2 1.1 1,4-Pyridones 246 1.0 1.0225 4.3 1.2 6.5 0.9 27.4 178 0.4 2.5 0.9 Emax JNJ-41329782 68 215 57 202 Sc Sc Sc Sc 109 204 1142 84 180 196 S]GTP

WT > 3> or WT

[

1.0 1.0 2.9 1.0 20.0 0.4 3.2 1.0

(18) R635A(18)

1 50 Sc Sc Sc Sc Sc Sc Sc Sc Sc 0.7 1.6 4.3 5.6 378 853 117 490

JNJ-35814376 637 196 EC50 523 186 853 225 Sc Sc 1067 121 Sc Sc 625 139 1,5-Pyridone 1.0 1.0 1.6 1.2 2.0 0.7 1.2 0.7

induced -

JNJ-46281222 8 214 6 211 Sc Sc Sc Sc Sc Sc 316 107 Sc Sc 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 201 202 186 211 214 204 200 227 141 147 101 159 168 128 E max

between WT and WT mGlu2 mutant between

1.0 1.0 55.6 0.5

50 JNJ-42153605 6 WT 224 5 214 Sc Sc Sc Sc Sc Sc 447 90 Sc Sc

glutamate

mutant mGlu2 /EC mGlu2 mutant

1.0 1.0 6 5 7 84.3 0.4 87 57 59 37 87 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

523 172 148 175 114 156 50 JNJ-46356479 91 227 EC50 59 204 Sc Sc Sc Sc Sc Sc 6783 86 Sc Sc Triazolopyridines 1.0 1.0 115.6 0.4

97 ratio EC ratio 173 215 227 218 224 224 214 166 130 139 116 206 JNJ-42329001 49 218 37 200196 Sc Sc Sc Sc Sc Sc 5868 137 Sc Sc

E max – 1.0 1.0 156.8 0.7

Stable Stable

6 224 7 227 Sc Sc Sc Sc 9 244 254 101 Sc Sc

JNJ-43245046

6 6 8 97 68 91 49 113 178 172 143 173 139 637

EC50 1.0 1.0 1.4 1.1 38.7 0.4 of the enhancement of

JNJ-39226421 178 130 172 141 117 92 Sc 1 227 74 n.v. 36 119 110 max

E 1.0 1.0 0.7 0.7 1.3 0.5 0.3 0.7 0.8 Isoquinolones JNJ-40297036 eening assay; 143 139 148 147 1 1 Sc 1 1351 90 n.v. 49 Sc Sc

35815013 52149617 39226421 41482012 41329782 46356479 42329001 43245046 40297036 42153605 35814090 40068782 46281222 1.0 1.035814376 9.1 0.6 0.3 ------

172 97 175 101 Sc NJ Sc Sc Sc Sc Sc n.v 36 Sc Sc JNJ JNJ JNJ JNJ JNJ J JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ-41482012 JNJ Imidazopyridine ) and relative

tested in tested scr 1.0 1.0 0.4

50 – ompound potency is presented a ratio EC as of potency C ompound

113 166 114 159 Sc Sc Sc Sc 161 171 724 62 305 131

THIIC JNJ-52149617 1.0 1.0 1.4 1.1 6.4 0.4 2.7 0.8

97 173 87 168 490 178 Sc Sc 1320 121 1414.1 84 108 153

BINA JNJ-35815013 otency (EC BINA P Pyridone THIIC Pyridones 1.0 1.0 - 5.6 1.1 15.2 0.7 16.3 0.5 1.2 0.8 - LY487379 nottested; Sc 1,5

1,4 Isoquinolones Imidazopyridine

– 173 116 156 128 Sc Sc Sc Sc 692 148 n.v. 53 276 105 LY487379 JNJ-35814090 Triazolopyridines 1.0 1.0 4.4 1.2 0.4 1.8 0.8

NT NT Table Table 8: into CHO - K1 cells.

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 72 28/06/2012 9:58:54

Table(Cont.) 8 Triazolopyridines Imidazopyridine Isoquinolones 1,4 1,5 LY487379 - - Pyridones THIIC Pyridone BINA A4 -06094 -Ana Farinha - thesis binnenwerk.indd 73

J JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ NJ ------52149617 35814090 35815013 41482012 40297036 39226421 43245046 42329001 46356479 42153605 46281222 35814376 41329782 40068782

Table 8 (Cont.)

EC50 178 637 139 173 113 172 143 97 49 91 68 6 6 8

Stable

Transmembrane 4

Emax 116 173 166 139 130 224 218 227 224 214 196 215 206

97 Stable WT

(4) G689V / (5) N735D /

(1) S688L (2) G689V (15) V700L (17) S644A/V700L/H723V

S688L G689V / S688L

EC50 Emax EC50 Emax EC50 Emax EC50 Emax EC50 Emax EC50 Emax EC50 Emax EC50 Emax

EC50 156 114 175 148 172 523 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 87 37 59 57 87 7 5 6

JNJ-40068782 139 206 87 201 160 201 603 187 635 201 n.v. 42 Sc Sc 1262 163

1,4-Pyridones 1.0 1.0 1.8 1.0 6.9 0.9WT 7.3 1.0 0.2 14.4 0.8

Emax 141 227 200 204 214 211 186 202 201 128 168 159 101 147

1.0 1.0 1.0 1.0 JNJ-413297821.0 1.0 681.0 1.0 215 1.0 57 1.0 2021.0 NT1.0 1.0 NT 1.0 NT NT 1349.0 124 NT NT Sc Sc 621 186

1.0 1.0 23.6 0.6 10.9 0.9

JNJ-35814376 637 196 523 186 520 206 2512 218 1445 125 n.v. 49 Sc Sc 2225 135 130.0 EC50

114 1,5-Pyridone145 154 2 520 160 0.7 1.7 1.3 0.7 1.4 1.3 3.6 1.0 1.8 NT NT NT NT NT 20 09 7 (1) S688L (1)

1.0 1.0 1.0 1.1 4.8 1.2 2.8 0.7 0.3 4.3 0.7

JNJ -46281222 8 214 6 211 20 159 83 229 61 215 767.2 203 Sc Sc 72 213

1.0 1.0 3.6 0.8 14.6Emax 1.1 10.8 1.0 134.9 1.0 12.7 1.0 133 167 181 104 147 250 159 206 201 1.0 1.0 1.1 1.0 1.0 1.2 0.8 1.1 1.0 NT NT NT NT NT

6 224 5 214 7 250 25 215 22 223 212 180 Sc Sc 47 209

JNJ -42153605

1.0 1.0 1.3 1.2 4.7 1.0 4.2 1.0 40.0 0.8 8.8 1.0

73 EC50 1349 2512 ~0.1 23.0 14.6 ~24 327 832 646 537 170 603

2.1 9.6 5.7 3.6 5.9 4.5 4.7 4.8 6.9 NT NT JNJ-46356479 91 39 227 59 25 204 83 NT NT 1349 269 309 197 3162.3 174 Sc Sc 692 215 (2) G689V (2)

Triazolopyridines

1.0 1.0 23.0 1.3 5.3 1.0 53.9 0.9 11.8 1.1

JNJ-42329001 49 218 37 200 NT NT 170 178 577 186 489.8 208 Sc Sc 115 230 Ema 132 171 177 117 203 178 269 215 229 218 187 1.0 1.0 1.1 0.6 0.8 0.9 0.9 1.3 1.0 1.1 1.2 0.9 NT NT 61

1.0 1.0 4.5 0.9 15.4 0.9 13.1 1.0 3.1 1.1

JNJ-43245046 6 224 7 227 NT NT 39 203 41 216 n.v. 142 Sc Sc 42 289 1349.0 EC50

1.0 1.0 1445 5.9 0.9 6.2 1.0 0.6 6.4 1.3 15.4 10.8 23.6 345 575 635 223 264 577 309 635 n.v. 2.2 6.6 5.6 1.3 1.8 6.2 5.3 4.2 2.8 7.3

41 22 61 (4) G689V / G689V (4)

S688L

JNJ -39226421 178 130 172 141 NT NT NT NT n.v. 40 NT NT Sc Sc 539 95

1.0 1.0 0.3 3.1 0.7

Emax

Isoquinolones Transmembrane 4 Transmembrane 110 186 175 114 216 186 197 223 215 125 124 201 0.9 1.1 1.1 0.6 0.8 0.3 1.0 0.9 1.0 1.0 1.0 0.7 0.6 1.0 JNJ58 -40297036 40 143 139 148 147 209 147 537 117 264 114 n.v. 40 Sc Sc 389 101

1.0 1.0 1.4 1.0 3.6 0.8 1.8 0.8 0.3 2.6 0.7

21379.6 2896.3 3162.3 G689V / S688L G689V

2 172 97 489.8 175 101134.9 767.2 130.0 104 ~24 EC50 61 223 58 n.v 37 Sc Sc 274 73 _n.v. 25.4 13.1 53.9 40.0 212 n.v. n.v. n.v. n.v. 46.6 n.v NT NT Imidazopyridine JNJ-41482012 / N735D (5)

1.0 1.0 0.7 1.0 ~0.1 0.6 1.3 0.6 0.4 1.6 0.7

113 166 114 159 154 181 646 177 635 175 2896.3 56 Sc Sc 620 151

THIIC JNJ-52149617 Emax 142 208 174 180 203 0.3 0.3 0.4 0.4 0.3 0.6 1.0 0.9 0.8 1.0 0.3 0.2 NT NT 33 44 56 37 40 1.0 1.0 1.3 49 1.1 42 5.7 1.1 5.6 1.1 25.4 0.4 5.4 1.0

97 173 87 168 145 167 832 171 575 186 21379.6 44 Sc Sc 78 206 BINA JNJ-35815013 1.0 1.0 1.7 1.0 9.6 EC50 1.0 6.6 1.1 246.6 0.3 0.9 1.2 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (15) V700L

173 116 156 128 114 133 327 132 345 110 _n.v. 33 Sc Sc 670 111 LY487379 JNJ-35814090 Emax 28/06/2012 9:58:56 1.0 1.0 0.7 1.0 2.1 1.0 2.2 0.9 0.3 4.3 0.9 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

(17) S644A/V700L/H723V EC50 2225 1262 11.8 12.7 10.9 14.4 670 620 274 389 539 115 692 62 4.3 0.9 5.4 1.6 2.6 3.1 6.4 3.1 8.8 4.3 78 42 47 72

Emax 111 206 151 101 289 230 215 209 213 135 186 163 0.9 1.2 1.0 0.7 0.7 0.7 1.3 1.1 1.1 1.0 1.0 0.7 0.9 0.8 73 95

Table(Cont.) 8

Triazolopyridines Imidazopyridine Isoquinolones 1,4 1,5 LY487379 - - Pyridones THIIC Pyridone BINA

JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ A4 -06094 -Ana Farinha - thesis binnenwerk.indd 74 ------35814090 41482012 35815013 52149617 40297036 39226421 43245046 42329001 46356479 42153605 46281222 35814376 41329782 40068782

Table 8 (Cont.)

EC50 113 172 143 178 637 139 173 97 49 91 68 6 6 8

Stable

E max E Extracellular loop 2/Transmembrane 5

116 173 166 139 130 224 218 227 224 214 196 215 206

97 Stable WT

(16) H723V (9) D725A (19) M728A (20) S731A (10) L732A (3) N735D (21) V736A

E EC50 E max EC50 EC50 Emax EC50 Emax EC50 Emax EC50 Emax EC50 Emax EC50 Emax EC50 Emax

max EC50

156 114 175 148 172 523 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 37 59 57 87 87 7 5 6

139 206 87 201 549 189 Sc Sc Sc Sc 1 1 95 130 3019.95 70 Sc Sc

JNJ-40068782

WT 1,4-Pyridones 1.0 1.0 6.3 0.9 1.1 0.6 34.5 0.3

max 227 200 204 214 211 186 202 201 128 168 159 101 147 141 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

JNJ-41329782 68 215 57 202 260 217 E Sc Sc Sc Sc 245 247 63 140 NT NT Sc Sc

1.0 1.0 4.6 1.1 4.3 1.2 1.1 0.7

EC50 1625 195 260 549 281 175 JNJ351 -35814376253 637 196 523 186 1625 152 Sc Sc Sc Sc 1190 189 633 129 n.v. 81 Sc Sc 2.5 1.0 2.4 1.5 2.3 3.3 5.2 3.1 4.6 6.3 Sc Sc Sc Sc 15 28 (16) H723V

1,5-Pyridone

1.0 1.0 3.1 0.8 2.3 1.0 1.2 0.7 0.4

JNJ-46281222 8 214 6 211 Sc Sc Sc Sc Sc Sc Sc Sc 58 142 ~617 221 Sc Sc Emax 174 116 115 281 220 226 152 217 189 1.1 0.8 0.8 0.8 1.2 1.1 1.1 0.8 1.1 0.9 Sc Sc Sc Sc 78

1.0 1.0 10.3 0.7 102.0 1.0

JNJ-42153605 6 224 5 214 28 226 Sc Sc Sc Sc Sc Sc 49 159 27 220 Sc Sc

1.0 1.0 5.2 1.1 EC50 9.2 0.7 5.2 1.0 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

D725A (9) 74

JNJ-46356479 91 227 59 204 195 220 Sc Sc Sc Sc Sc Sc 1015 131 NT NT Sc Sc

Triazolopyridines 1.0 1.0 3.3 1.1 17.3 0.6 Emax Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

JNJ-42329001 49 218 37 200 Sc Sc Sc Sc Sc Sc Sc Sc 576 141 NT NT Sc Sc

1.0 1.0 15.4 0.7

EC50 (19) M728A Sc Sc Sc Sc JNJSc -43245046Sc Sc 6 Sc 224Sc Sc 7 Sc 227 Sc 15Sc 281Sc Sc Sc Sc Sc Sc Sc 82.5697 130 NT NT Sc Sc

1.0 1.0 2.3 1.2 12.6 0.6

Emax Extracellular loop 2/Transmembrane 5 2/Transmembrane loop Extracellular

Sc Sc Sc Sc JNJSc -39226421Sc Sc 178Sc 130Sc Sc 172 Sc 141 Sc 253Sc 115Sc Sc Sc Sc Sc Sc Sc 57 73 NT NT Sc Sc

1.0 1.0 1.5 0.8 0.3 0.5 Isoquinolones JNJ-40297036 143 139 148 147 351 116 Sc Sc Sc Sc Sc Sc 329 78 n.v. 41 Sc Sc EC50 1190 834 285 245 5.3 2.5 2.3 4.3 Sc Sc Sc S Sc Sc Sc Sc Sc 1 (2

1.0 1.0 2.4 0.8 2.2 0.5 0.3

0) S731A 0)

172 97 175 101 175 78 Sc Sc Sc Sc Sc Sc n.v 52 n.v 39 Sc Sc Imidazopyridine JNJ-41482012 Emax 109 169 189 247 0.9 1.1 1.0 1.2

Sc Sc Sc Sc Sc Sc Sc Sc 1.0 Sc 1.0 1.0 0.8 0.5 0.4 1

113 166 114 159 281 174 Sc Sc Sc Sc 285 169 357 91 2790.7 96 Sc Sc THIIC JNJ-52149617 1.0 1.0 2.5 1.1 2.5 1.1 3.1 0.6 24.5 0.6 82.5697 EC50 1015 12.6 15.4 17.3 10.3 357 329 57 633 n.v. 0.2 3.1 2.2 0.3 9.2 1.2 1.1 1.1 n.v 15 57 49 58 63 95

97 173 87 168 Sc Sc ScL732A (10) Sc Sc Sc Sc Sc 15 110 7413.1 74 Sc Sc

BINA JNJ-35815013

1.0 1.0 0.2 0.7 85.5 0.4

173 116 156 128 Sc Sc Emax Sc Sc Sc Sc 834 109 n.v. 62 n.v. 40 Sc Sc 110 130 141 131 159 142 129 140 130 0.5 0.7 0.6 0.5 0.5 0.5 0.6 0.7 0.6 0.7 0.7 0.7 0.7 0.6 62 91 52 78 73

LY487379 JNJ-35814090

1.0 1.0 5.3 0.9 0.5 0.3

3019.95 7413.1 2790.7 102.0 EC50 ~617 85.5 24.5 34.5 n.v. n.v. n.v. 5.2 n.v NT NT NT NT NT 27

(3) N735D (3) 74

28/06/2012 9:58:59

Emax 220 221 0.3 0.4 0.6 0.4 0.3 1.0 1.0 0.4 0.3 NT NT NT NT NT 40 74 96 39 41 81 70

EC50 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (21) V736A

Emax

Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

Triazolopyridines Table(Cont.) 8 Imidazopyridine Isoquinolones 1,4 1,5 LY487379 - - Pyridones THIIC Pyridone BINA

JNJ JNJ JNJ J JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ NJ ------35814090 35815013 52149617 41482012 40297036 39226421 43245046 42329001 46356479 4215 46281222 35814376 41329782 40068782

3605

EC50 173 113 172 143 178 637 139 97 49 91 68 6 6 8

Stable

Emax 116 173 166 139 130 224 218 227 224 214 196 215 206 97

EC50 156 114 175 148 172 523 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 87 37 59 57 87 7 5 6

Emax 128 168 159 101 147 141 227 200 204 214 211 186 202 201 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

1258.9 2344.2 631.0 EC50 1059 55.0 33.6 40.0 13.3 18.5 361 n.v. n.v. n.v. n.v. n.v. n.v. n.v. 4.0 n.v 76 (11) W773A

Emax 0.5 0.3 0.3 0.5 0.5 0.3 0.4 0.4 0.4 0.4 0.4 0.2 0.4 0.4 63 44 51 48 68 49 92 82 72 76 95 46 80 89

Transmembrane6 EC50 199 125 192 440 n.v. 2.3 1.1 1.1 1.9 4.6 3.4 5.0 Sc Sc Sc Sc Sc Sc 70 24 (12)F776A 7

Emax 106 118 158 163 178 152 132 0.4 0.6 0.7 0.7 0.8 0.8 0.8 0.7 Sc Sc Sc Sc Sc Sc 48

218.8 398.1 EC50 1.9 0.8 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (13)F780A

Emax 156 148 1.0 0.8 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

Table(Cont.) 8

Triazolopyridines Imidazopyridine Isoquinolones 1,4 1,5 LY487379 - - Pyridones THIIC Pyridone BINA

JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ ------35814090 41482012 35815013 52149617 40297036 39226421 43245046 42329001 46356479 42153605 46281222 35814376 41329782 40068782

EC50 173 113 172 143 178 637 139 97 49 91 68 6 6 8

Stable

E max E 116 173 166 139 130 224 218 227 224 214 196 215 206

EC50

156 114 175 148 172 523 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 87 37 59 57 87 7 5 6

max 128 168 159 101 147 141 227 200 204 214 211 186 202 201 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 E E

EC50 1625 281 175 351 253 195 260 549 2.5 1.0 2.4 1.5 2.3 3.3 5.2 3.1 4.6 6.3 Sc Sc Sc Sc 15 28 (16) H723V

Emax 174 116 115 281 220 226 152 217 189 1.1 0.8 0.8 0.8 1.2 1.1 1.1 0.8 1.1 0.9 Sc Sc Sc Sc 78

EC50 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (9) D725A (9) 74

Emax Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

EC50 (19) M728A Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

Emax Extracellular loop 2/Transmembrane 5 2/Transmembrane loop Extracellular Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

EC50 1190 834 285 245 2.3 4.3 5.3 2.5 Sc Sc Sc S Sc Sc Sc Sc Sc 1 (2

0) S731A 0)

Emax 109 169 189 247 1.0 1.2 0.9 1.1 Sc Sc Sc Sc Sc Sc Sc Sc Sc 1

82.5697 EC50 1015 12.6 15.4 17.3 10.3 357 329 57 633 n.v. 0.2 3.1 2.2 0.3 9.2 1.2 1.1 1.1 n.v 15 57 49 58 63 95 L732A (10)

Emax 110 130 141 131 159 142 129 140 130 0.7 0.7 0.6 0.5 0.7 0.6 0.5 0.5 0.5 0.6 0.7 0.6 0.7 0.7 62 91 52 78 73

3019.95 7413.1 2790.7 102.0 EC50 ~617 85.5 24.5 34.5 n.v. n.v. n.v. 5.2 n.v NT NT NT NT NT 27

(3) N735D (3)

Emax 220 221 1.0 1.0 0.4 0.3 0.3 0.4 0.6 0.4 0.3 NT NT NT NT NT 40 74 96 39 41 81 70

EC50 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (21) V736A

A4 -06094 -Ana Farinha - thesis binnenwerk.indd 75 Emax Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

Triazolopyridines Table(Cont.) 8 Imidazopyridine Isoquinolones 1,4 1,5

LY487379 - - Pyridones THIIC Pyridone BINA

Table 8 (Cont.)

Transmembrane 6 Stable WT

JNJ JNJ JNJ J JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ JNJ (11) W773A (12) F776A (13) F780A

------EC50- - Emax - EC50- Emax- EC50 Emax EC50 Emax EC50 Emax 40297036 39226421 43245046 42329001 46356479 4215 46281222 35814376 41329782 40068782 35814090 35815013 52149617 41482012

JNJ- 40068782 139 206 87 201 n.v. 89 440 132 Sc Sc 1,4-Pyridones 3605 1.0 1.0 0.4 5.0 0.7

JNJ-41329782 68 215 57 202 1059 80 192 152 Sc Sc 1.0 1.0 18.5 0.4 3.4 0.8 EC50 143 178 637 139 173 113 172 49 91 68 97 6 6 8

JNJ- 35814376 637 196 523 186 n.v. 46 Sc Sc 398.1 148

Stable 1,5-Pyridone 1.0 1.0 0.2 0.8 0.8

Emax

116 173 166 139 130 JNJ224 -46281222218 227 8224 214 214 196 6 215 211206 76 95 Sc Sc Sc Sc 97

1.0 1.0 13.3 0.4

JNJ-42153605 6 224 5 214 n.v. 76 24 178 Sc Sc EC50 156 114 175 148 172 523

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.4 4.6 0.8 87 37 59 57 87 7 5 6

JNJ-46356479 91 227 59 204 2344.2 72 Sc Sc Sc Sc 75

Triazolopyridines

1.0 1.0 40.0 0.4

Emax 128 168 159 101 147 141 227 200 204 214 211 186 202 201 1.0 1.0 1.0 1.0 1.0 1.0 1.0 JNJ-1.0 423290011.0 1.0 49 1.0 2181.0 1.0 37 1.0 200 1258.9 82 70 163 Sc Sc

1.0 1.0 33.6 0.4 1.9 0.8

JNJ-43245046 6 224 7 227 361 92 7 158 Sc Sc 1258.9 2344.2 EC50 631.0 1059 55.0 33.6 40.0 13.3 18.5 361 n.v. n.v. n.v. n.v. n.v. n.v. n.v. 4.0 n.v (11) W773A 76 1.0 1.0 55.0 0.4 1.1 0.7

JNJ-39226421 178 130 172 141 n.v. 49 Sc Sc Sc Sc

1.0 1.0 0.3

Isoquinolones Emax 0.5 0.3 0.3 0.5 0.5 0.3 0.4 0.4 0.4 0.4 0.4 0.2 0.4 0.4 63 44 51 48 68 49 JNJ92 -4029703682 72 14376 95 139 46 14880 14789 n.v. 68 Sc Sc Sc Sc

1.0 1.0 0.5

Transmembrane6

172 97 175 101 EC50 n.v 48 Sc Sc Sc Sc 199 125 192 440 n.v. 2.3 1.1 1.1 1.9 4.6 3.4 5.0 Sc Sc Sc Sc Sc Sc Imidazopyridine JNJ-4148201270 24 (12)F776A 7

1.0 1.0 0.5

113 166 114 159 n.v. 51 125 118 218.8 156 THIIC JNJ-52149617 Emax

106 118 158 163 178 1.0 152 1.0132 0.3 1.1 0.7 1.9 1.0 0.4 0.6 0.7 0.7 0.8 0.8 0.8 0.7 Sc Sc Sc Sc Sc Sc 48

97 173 87 168 n.v. 44 199 106 Sc Sc

BINA JNJ-35815013 1.0 1.0 0.3 2.3 0.6

218.8 398.1 EC50 1.9 0.8 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (13)F780A

173 116 156 128 631.0 63 n.v. 48 Sc Sc

LY487379 JNJ-35814090 1.0 1.0 4.0 0.5 0.4 28/06/2012 9:59:01 Emax 156 148 1.0 0.8

Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 76 28/06/2012 9:59:01

Chapter 4 Discussion

A4 - 06094 - Ana Farinha - thesis binnenwerk.indd 77 28/06/2012 9:59:01

The pursuit for alternative therapeutic approaches devoid of the disadvantages associated with the use of orthosteric agonists has conferred prominence to allosteric modulators, namely PAMs. The recent emergence of PAMs as highly attractive subtype- selective mGlu2 receptor modulators makes it important to investigate the molecular factors mediating the interaction between these compounds and the receptor, as a way not only to understand how these compounds bind and activate the receptor, but also to enable the development of future more optimal compounds. In the past decade, a number of studies have focused on the investigation of the binding mode of allosteric modulators on mGlu receptors. The different studies have suggested that residues in TM 3, 4, 5, 6 and 7, and also in the EL2, constitute the allosteric binding pocket for allosteric modulators. The reported mutations do not necessarily affect the binding of all allosteric ligands, although it has been shown that different compounds may share an overlapping binding site – even if they exhibit opposite effects towards the receptor (PAMs versus NAMs). In the present study, the molecular interaction of 14 PAMs with the mGlu2 receptor was elucidated. For that, the activity of these compounds on WT and point-mutated mGlu2 receptors was compared in order to identify amino acids potentially important for the interaction between PAMs and the mGlu2 receptor. In previous work, the compounds tested in this study were shown not to displace [3H]-LY341495 binding, confirming their binding to an allosteric binding site (data not shown); additionally, previous studies revealed that these compounds exhibit selective positive allosteric modulation at recombinant mGlu2 receptors (data not shown).

4.1. Expression of WT and mutant mGlu2 receptors

Western blot analysis revealed a similar pattern in the expression of WT and mutant receptors, although membranes of CHO-K1 cells stably transfected with WT mGlu2 receptor revealed apparent higher expression levels. In addition to Western blot analysis, saturation binding assays were performed for mutations S688L, G689V, N735D, S688L/G689V and S688L/G689V/N735D which enabled the quantification of the total amount of receptors. Although this quantification was not carried out for all the mutations, it clearly shows that mutant receptors have variable expression levels, displaying lower expression levels when compared to the stable transfection.

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4.2. Comparison between the affinity and potency for glutamate between WT and mutant mGlu2

Despite the confirmation that mutant receptors are being expressed by CHO-K1 cells, their ability to bind the agonist, glutamate, also needed to be confirmed to assure that the receptor could achieve the conformation required for agonist binding. In fact, glutamate was able to displace [3H]-LY341495 in a concentration-dependent manner, both from the WT receptor stably expressed in CHO-K1 cells and from transiently transfected WT as well as

mutant receptors. Accordingly, the IC50 values were overall similar. These results confirm that mutant receptors are not only being expressed by CHO-K1 cells, but they also are able to bind glutamate in a similar way as WT, indicating that they are still able to acquire the conformation needed for agonist binding. Further studies were carried out to assess the functional properties of mutant receptors. Concentration-response curves for glutamate-induced [35S]GTPγS binding were performed in order to compare the functional activity of WT and mutant receptors. Even though glutamate response amplitudes varied (likely as a result of differences in expression levels and/or transfection efficiency), mutated receptors elicited glutamate concentration- response curves with resulting potency values that were similar to those of WT receptor. This result suggests that the mutations did not have a significant effect on the potency of glutamate towards the receptor and confirmed that the mutant receptors were functionally active.

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4.3. Effect of mGlu2 mutations on the activity of positive allosteric modulators

A brief overview of the results presented in Figures 19-22 and Table 4 makes it possible to infer that different mutations have different effects on the activity of the PAMs tested in this study, which suggests that the different compounds may not share the exact same binding mode as expected. Given the diversity of the chemical structures of the compounds, they will form subtly different interactions. Several studies, as the ones performed by Hemstapat et al. (2006, 2007), showed indeed evidence of different allosteric binding pockets for different allosteric modulators.

Effect of mGlu2 mutations on the different chemical classes

When analyzing the effect of mutant mGlu2 receptors on the activity of 1,4- Pyridones, mutations F643A, S688L/G689V/N735D, N735D and W773A were shown to

decrease the enhancing activity of this compound, with high increases in EC50 values (18.5 to 34.7-fold increase) and a reduction in the efficacy of the compounds. Interestingly, mutations H723V, S644A/V700L/H723V and F776A (and also the mutation G689V/S688L in JNJ 41329782) caused a considerable increase in EC50 values (3.4 to 14.4-fold increase), but the efficacy was not severely affected, which can indicate that this mutation affected the affinity of the compound towards the receptor. Figure 23 illustrates the 3D position of the mutated amino acids in the mGlu2 receptor.

Figure 23: 3D representation of the receptor and the mutations that seem to affect the binding of 1,4-Pyridones (the two images represent different angles). 80

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For the 1,5-pyridones tested on this study, mutations G689V, G689V/S688L, H723V

and S644A/V700L/H723V led to an increase in the EC50 (2.76 to 4.8-fold increase) but to small effects on the relative efficacy, whereas mutations N735D, N735D/G689V/S688L and W773A completely disrupted the activity of this compound.

For triazolopyridines, the most potent of the compounds tested (EC50 vary between 6 and 91 nM), the effects of the mutations in the different compounds were overall consistent. Mutations S688L/G689V/N735D (and, even though with smaller effects, each one of the correspondent single mutations), F643A, L732A, W773A and S644A/V700L/H723V show a

prominent increase in EC50 values for the different compounds. The mutations F643A and W773A caused major decreases in the relative efficacy of this class of compounds. Interestingly, mutation L732A consistently and somewhat preferably affects this class of compounds. Figure 24 illustrates the 3D position of the mutations that seem to be important for the activity of this class of compounds. Compounds belonging to triazalopyridine class were, by far, the most affected by the mutations tested: an overview of the ratios between the

EC50 of the compounds on mutant and WT mGlu2 clearly demonstrates that the mutations have a greater impact on the activity of these compounds. Interestingly, this is accompanied by the high potency of this class of molecules at WT receptor. This increased potency arises due to a more optimal interaction with the protein target. Therefore given this highly optimized fit with the mGlu2 receptor it is natural to expect they would be most sensitive to changes in the binding site. Consequently, L732 seems to be important to render that additional interaction, or indeed increase compound’s potencies.

Figure 24: 3D representation of the receptor and the mutations that seem to affect the binding of triazalopyridines (the two images represent different angles). 81

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For isoquinolones, mutations F643A and W773A completely abolished the PAMs activity. The EC50 was impossible to calculate due to a completely disruption of a concentration-response curve. Mutation S644A/V700L/H723V caused a 2.6 to 3.1-fold

increase in EC50 and a reduction in the relative efficacy. Particularly for the compound JNJ 40297036, mutation L639A caused a 9.1-fold increase in EC50 and a relative efficacy of 90%, against the 141% observed for the WT. The lack of data for some mutations of the transmembrane 4 does not enable the assessment of their effect on compounds activity. Nevertheless, mutation G689V and the triple mutation S688L/G689V/N735D seem to affect the binding of JNJ-40297036. Interestingly, mutant L732A increased five times the activity of the isoquinolone compound, JNJ-39226421. The enhancing property of the imidazopyridine was abolished in mutants N735D, N735D/G689V/S688L, F643A, L732A and W773A. These mutations completely abolished the receptor capacity of responding to increasing PAM concentrations. For the reference compounds acetophenone, BINA and LY487379, mutations N735D, N735D/G689V/S688L (Figure 25), F643A and W773A show relatively consistent results.

These mutations caused an increase in EC50 values (4 to 246.6-fold increase), or a complete

obliteration of the PAM effect. BINA showed a ~ 250-fold increase in EC50 for the mutation N735D/G689V/S688L, suggesting the importance of this binding pocket for this compound. As for the isoquinone compound JNJ-39226421, also for BINA the mutation L732A increased its activity three times. Interestingly, mutations G689V, G689V/S688L, L639A, L732A, F776A, S644A/V700L/H723V, R635A and S731A differently affected these three

compounds, causing an increase in the EC50 for one of them, but having no effect on the others.

Figure 25: 3D representation of the receptor and the mutations that seem to affect the binding of reference compounds (the two images represent different angles). 82

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Mutation W773A consistently affected the binding off all the compounds, without exception. W773 is located on transmembrane 6 and is conserved in class C GPCRs. Previous mutagenesis studies have shown that the amino acid located at the same position at other mGlu receptors (namely, mGlu1 and mGlu5) is important for the binding of allosteric modulators (Malherbe et al., 2003a,b; Malherbe et al., 2006; Muhlemann et al., 2006). Accordingly, the modeling studies performed for this work suggest that this amino acid is part of a hydrophobic cluster within the expected transmembrane allosteric binding site, formed also by the amino acids F643, L732, W773, and F776. This hydrophobic cluster interacts with the ligand in its predicted binding mode, as Figure 26 displays. Therefore, the high impact of this mutation on the binding of all the compounds tested is consistent with the predictions of the homology model. As mentioned above, mutation L732A increased the potency of two compounds: JNJ- 35815013 (BINA) and JNJ-39226421 (isoquinolone). Although the reasons of this potency increase are unknown, it is interesting to notice that this effect was seen for only one of the compounds belonging to isoquinolone chemical class.

Figure 26: Modelled mGlu2 receptor structure showing bound pose for Janssen mGlu2 PAM in surface representation and the cluster of hydrophobic amino acids, Phe643, Leu732, Trp773, and Phe776.

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mGlu2/3 sequences comparison

When assessing the impact of the mutations selected for this study based on the differences between mGlu2 and mGlu3 receptor sequences (S644A, V700L and H723V), only the triple mutation caused major shifts in the activity of PAMs. This large effect did not occur when using the single amino acid mutations (only the mutation H723V led to a loss of effect in several compounds when applied alone) (Table 8). This suggests that these three amino acids may form an important binding pocket for the activity of PAMs and may be responsible for their subtype-specificity. However, these mutations represent only a small part of the mutations that were selected based on this criteria (Table 4, Methods section). Therefore, the testing of the other mutations is needed in order to have more conclusive results. The amino acids S688, Gly689 and Asn735, previously identified in literature, were also selected for mutagenesis based on the sequence divergence between mGlu2 and mGlu3 receptor (Rowe et al., 2008) and the high impact of G689V, N735D and the double and triple mutations of these three amino acids confirms their importance in the mGlu2 subtype- selective properties of PAMs.

Overall important amino acids

The overall observation of the ratios between the EC50 of mutant and WT mGlu2 receptors clearly highlights some trends in the effects of the different mutation on PAM performance: mutations F643A, G689V, W773A, S688L/G689V, S688L/G689V/N735D (but not the individual mutations) and S644A/V700L/H723V (but not the individual mutations) have a general impact on the activity of all the structurally distinct compounds (Figure 27). Therefore, these amino acids seem to participate in the structure of a common binding pocket for PAM. The differences in the effects observed for other mutations are possibly a consequence of the features specific for each structure.

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Figure 27: 3D representation of the receptor and the mutations that seem to affect the binding of the PAMs tested in these study (the two images represent different angles).

When assessing the effects of mutations on PAMs activity (in our case using [35S]GTPγS binding assay), it is important to clarify the possible effects that a mutation can have in the receptor function: 1) the mutation can prevent the receptor to acquire a functionally active conformation in the membrane, regardless of the activity of the compound (it can cause, for example, an increase in the internalization of the receptor, disabling the receptor to localize in the membrane); 2) the mutations may enable the binding of the compound, but lead to a conformation that does not allow the interaction with G protein and, therefore, no functional response is detected; 3) the mutated receptor does not allow the binding of the compound or decreases the affinity of the compound for binding to the receptor. In the first and second situations, we hypothesize that the receptor would not be able to be activated even by glutamate, in which case this would be picked up in the glutamate

concentration-response curve. Still, the fact that not only potency but also efficacy (Emax) for some compounds is decreased, may suggest that certain mutations affect the receptor signaling machinery in some way. In the last situation, although the effects of each mutation are assessed through the impact on a functional response (efficacy), this is in fact the result of an upstream event – the binding of the compound (affinity). As a next step, it would hence be important to assure that the compound is actually able to bind the mutant receptor which could be investigated through radioligand binding studies using a tritiated form of an allosteric modulator or using a tritiated agonist and assessing the effects of the PAM on its binding properties.

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Chapter 5 Conclusions

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The proven success of GPCRs as drug targets for the treatment of CNS disorders renders them attractive targets of research. Specifically, the mGlu2 receptor is currently being pursued as the target for the treatment of several neurological and psychiatric diseases. The quest for increasingly potent and selective ligands for this receptor is leading to the identification of novel ligands, with improved physicochemical and pharmacokinetic properties. Accordingly, mGlu2 positive allosteric modulators constitute an attractive approach for the selective activation of this receptor. Therefore, the identification of the molecular determinants of mGlu2 receptor-PAMs binding is highly attractive. The present study provides further additional insight on the interactions that drive the activity of PAMs towards mGlu2 receptor. Surprisingly, not only did double or triple mutations affect the activity of the PAMs tested, but a strong and consistent effect was also observed for single amino acid mutations, which enabled the identification of specific molecular determinants that might be important for the activity of these compounds. Mutations F643A, G689V, W773A, S688L/G689V, S688L/G689V/N735D and S644A/V700L/H723V were shown to have a general impact on the activity of all the structurally distinct compounds. Interestingly, the mutations found by Lundström et al (2011) to be important for NAMs binding show different results for the PAMs tested in this study: R636A and F780A, two mutations with a high effect on the binding of NAMs, have no effect on the activity of the PAMs tested. Still, mutations F643A, H723V, L732A, N735D and R635A were shown by Lundström et al. to have an effect on NAMs, also seem to affect the activity of PAMs which may suggest common binding sites for NAMs and PAMs. The knowledge obtained from the mapping of new allosteric modulators is helping to understand the regions of the mGlu receptors that are critical for modulation of positive allosteric modulators activity. Ultimately, this study will contribute to guide research towards the achievement of more selective and potent drugs.

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Appendix

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Appendix 1: Complete names of the compounds mentioned in the text

Abbreviation Name (+)-ACPT-III (3RS,4RS)-1-aminocyclopentane-1,3,4-tricarboxylic acid (1S, 3R)- 1S,3R-1-aminocyclopentane-trans-1,3-dicarboxylic acid ACPD (2R, 4R)- (2R, 4R)-4-Aminopyrrolidine-2,4-dicarboxylic acid APDC (RS)-PPG (RS)-4-Phosphonophenylglycine (S)-3,4-DCPG (S)-3,4-Dicarboxyphenylglycine (S)-3,5-DHPG 3,5-Dihydroxyphenylglycine 1S,3S-ACPD 1S,3S-1-aminocyclopentane-1,3-dicarboxylic acid 2S,4S-4MG 2S,4S-4-methylglutamic acid ABH x D-I (1S,2S,4S,5S)-2-aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid ACPT-I 1-aminocyclopentane-1,3,4-tricarboxylic acid (S)-(4-fluorophenyl)-(3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5- ADX47273 yl]piperidin-1-yl)methanone AMN082 N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (3aS6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro- Bay 36-7620 cyclopental[c]furan-1-on 3'-[[(2-Cyclopentyl-2,3-dihydro-6,7- dimethyl-1-oxo-1H-inden-5- BINA yl)oxy]methyl]-[1,1'-biphenyl]-4-carboxylic acid CDPPB 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide CHPG 2-chloro-5-hydroxyphenylglycine CPCCOEt 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester CPPG (RS)-a-cyclopropyl-4-phosphonophenylglycine N-[4-Chloro-2-(phthalimidomethyl)phenyl]salicylamide N-[4-Chloro-2- CPPHA [(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2- hydroxybenzamide CTZ Cyclothiazide DCG-IV (2S,2’ R,3’R)-2-(2’,3’)-Dicarboxycyclopropyl glycine DFB 3,3'-Difluorobenzaldazine

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E-CBQA 1-amino-3-[2’-3’,5’-dioxo-1’,2’,4’-oxadiazo-lidinyl)] cyclobutane-1- carboxylic acidcyclobutane-1-carboxylic acid 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6- EM-TBPC dihydro-pyrimidine-5-carbonitrile 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2, 3-triazol-4-yl]-N-isopropyl- FTIDC N-methyl-3,6-dihydropyridine-1(2H)-carboxamide (1S,2R,3R,5R,6S)-2-amino-3-hydroxybicyclo[3.1.0]hexane-2,6- HYDIA dicarboxylic acid (3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)- JNJ16259685 methanone L-AP4 (2S)-2-amino-4-phosphonobutanoic acid L-CBG-I (2S,1’S,2’S)-2-(2-carboxycyclobutyl)glycine LCCG-I (2S,1’S,2’S)-2-(carboxycyclopropyl)glycine L-F2CCG-I (2S,1’S,2’S)-3’3’-difluoro-2-(carboxycyclopropyl)glycine 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic LY341495 acid LY354740 (1S,2S,5R,6S)-(+)-2-aminobicylco[3.1.0]hexane-2,6-dicarboxylic acid LY367385 (S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid LY379268 (–)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid LY38979 (–)-2-thia-4-aminobicylco[3.1.0]hexane-4,6-dicarboxylic acid 2,2,2-Trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3- LY487379 pyridinylmethyl)ethanesulfonamidehydrochloride MAP4 (S)-a-methyl-2-amino-4-phosphonobutanoic acid MCPG α-Methyl-4-carboxyphenylglycine (1R,2S,5S,6S)-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6- MGS0028 dicarboxylic acid (1R,2R,3R,5R,6R)-2-amino-3-(3,4-dichlorobenzyloxy)-6- MGS0039 fluorobicyclo[3.1.0] hexane-2,6-dicarboxylic acid 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin- MMPIP 4(5H)-one MNI-135 [3-(7-iodo-4-oxo-4,5-dihydro-3H-benzo[1,4]diazepin-2-yl)-benzonitrile] [7-bromo-4-(3-pyridin-3-yl-phenyl)-1,3-dihydro-benzo [1,4] diazepin-2- MNI-136 one]

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[4-(7-bromo-4-oxo-4,5-dihydro-3H-benzo[1,4]diazepin-2-yl)-pyridine-2- MNI-137 carbonitrile] MPEP 2-methyl-6-(phenylethynyl)pyridine MTEP 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine NAAG N-acetylaspartylglutamate NPS 2390 2-quinoxaline-carboxamide-N-adamantan-1-yl

PHCCC N-phenyl-7-(hydroxyhydroxy-imino) cyclopropa[b]chromen-1a- carboxamide R214127 [3H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone Ro 01-6128 (Diphenylacetyl)-carbamic acid ethy ester Ro 67-4853 (9H-Xanthen-9-ylcarbonyl)-carbamic acid butyl ester Ro 67-7476 (2S)-2-(4-Fluorophenyl)-1-[(4-methylphenyl)sulfonyl]-pyrrolidine 5-[7-trifluoromethyl-5- (4 - trifluoromethyl-phenyl)-pyrazolo[1,5- RO4988546 a]pyrimidin-3-ylethynyl]-pyridine-3-sulphonic acid 3′-(8-methyl-4-oxo-7- trifluoromethyl -4,5-dihydro-3H- RO5488608 benzo[b][1,4]diazepin-2-yl)-biphenyl-3-sulphonic acid S-4MeGlu S-4-methyleneglutamic acid S-AP4 S-4-Phosphono-2-aminobutyric acid S-Homo- 2-Amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid AMPA SIB-1757 6-methyl-2-(phenylazo)pyridin-3-ol SIB-1893 (E)-2-methyl-6-styrylpyridine S-SOP S-serine-O-phosphate

VU0080421 pyrazolo[3,4-d]pyrimidine VU0155041 cis-2-[[(3,5-Dichlorophenyl)amino]carbonyl]cyclohexanecarboxylic acid VU29 N-(1,3-Diphenyl-1H-pyrazolo-5-yl)-4-nitrobenzamide

VU71 4-nitro-N-(1,4-diphenyl-1Hpyrazol-5-yl)benzamide 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2- YM-298198 carboxamide (Z)-1-Amino-3-[2’-(3’,5’-dioxo-1’,2’,4’-oxadiazolidinyl-cyclobutane-1- Z-CBQA carboxylic acid Z-cyclopentyl cis-(±)-1-Amino-3-phosphonocyclopentane carboxylic acid AP4

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Appendix 2: mGlu2 receptor positive allosteric modulators tested in this study

1,4-Pyridones JNJ-40068782 JNJ-41329782

1,5-Pyridones JNJ-35814376

Triazolopyridines JNJ-46281222 JNJ-42153605 JNJ-46356479 JNJ-42329001 JNJ-43245046

Isoquinolones JNJ-39226421 JNJ-40297036

Imidazopyridine JNJ-41482012

THIIC JNJ-52149617

BINA JNJ-35815013

LY487379 JNJ-35814090

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Appendix 3: Results from the screening assay performed for all the mutations and PAMs. The EC and EC of each PAM are indicated. Appendix 3: Results from the screening assay performed for all the mutations and50 PAMs.100 The EC50 and EC100 of each PAM are indicated. PAM JNJ 40068782-AAA JNJ 46281222 - AAA JNJ 42153605 - AAA JNJ 46356479-AAA JNJ 42329001-AAA JNJ 39226421 - AAA JNJ 35814376 - AAA [PAM]PAM (M) 1.E-07 JNJ1.E-05 40068782-AAAn 3.E-07 3.E-06JNJ 46281222n 3.E-08 - AAA 3.E-06 nJNJ 421536051.E-07 -3.E-06 AAA n 3.E-08JNJ 46356479-AAA3.E-06 n 1.E-07 JNJ3.E-06 42329001-AAAn 3.E-07 3.E-06JNJ 39226421n - AAA JNJ 35814376 - AAA [PAM]Stable (M) 117%1.E-07205% 1.E-0510 207%n 223%3.E-07 103.E-06149% n221% 3.E-0811 138%3.E-06 212% n 11 1.E-07122% 3.E-06206% 11n 62%3.E-08 122%3.E-0610 n122% 1.E-07206% 3.E-0611 n 3.E-07 3.E-06 n WTStable 116%117%180% 205%8 179%10 194%207% 8 223%136% 10197% 149%8 140%221% 187%11 8 138%114% 212%181% 118 87%122% 153%206% 8 11114% 62%181% 122%8 10 122% 206% 11 75% ofWT WT 87% 116%135% 180% 134%8 146%179% 194%102% 8148% 136% 105%197% 141% 8 140%85% 187%136% 8 66%114% 114%181% 885% 87%136% 153% 8 114% 181% 8 R636A 96% 204% 1 222% 286% 1 143% 228% 1 146% 252% 1 184% 237% 1 93% 160% 1 184% 237% 1 75%L639A of WT 90% 87%183% 135%1 173% 163%134% 1 146%121% 227% 102%1 118%148% 195% 1 105%168% 141%220% 1 50%85% 93%136% 1 168% 66%220% 114%1 85% 136% F643AR636A 35% 96%113% 204%1 59%1 121%222% 1 286%32% 1 89% 143%1 24%228% 68% 1 1 146%24% 252%80% 11 31%184% 32%237% 1 124% 93%80% 160%1 1 184% 237% 1 D725AL639A 100% 90%166% 183%1 166%1 207%173% 1 163%138% 1195% 121%1 119%227% 200% 1 1 118%164% 195%183% 11 93%168% 143%220% 1 1164% 50%183% 93%1 1 168% 220% 1 L732AF643A 74% 35%109% 113%1 88%1 111%59% 1 121%64% 1127% 32%1 59%89% 78% 1 1 24%47% 68%106% 11 65%24% 79% 80% 1 147% 31%106% 32%1 1 24% 80% 1 W773AD725A 30% 100%52% 166%1 37%1 65%166% 1 207%25% 1 48% 138%1 41%195% 85% 1 1 119%30% 200%62% 11 14%164% 23%183% 1 130% 93%62% 143%1 1 164% 183% 1 F776A 63% 161% 1 191% 218% 1 99% 219% 1 146% 237% 1 83% 178% 1 98% 141% 1 83% 178% 1 F780AL732A 115% 74%219% 109%1 230%1 228%88% 1 111%161% 1248% 64%1 172%127% 243% 1 1 59%155% 78%211% 11 83%47% 147%106% 1 1155% 65%211% 79%1 1 47% 106% 1 S644AW773A 80% 30%254% 52%1 261%1 350%37% 1 65%138% 1325% 25%1 151%48% 319% 1 1 41%98% 85%307% 11 49%30% 129%62% 1 198% 14%307% 23%1 1 30% 62% 1 V700LF776A 127% 63%161% 161%1 192%1 193%191% 1 218%139% 1181% 99%1 133%219% 198% 1 1 146%127% 237%182% 11 81%83% 133%178% 1 1127% 98%182% 141%1 1 83% 178% 1 H732VF780A 64% 115%165% 219%1 156%1 202%230% 1 228%81% 1176% 161%1 93%248% 179% 1 1 172%88% 243%204% 11 56%155% 120%211% 1 188% 83%204% 147%1 1 155% 211% 1 S644A/V700L/H723V 33% 153% 1 111% 196% 1 50% 197% 1 48% 175% 1 69% 232% 1 38% 109% 1 69% 232% 1 R635AS644A 74% 80%269% 254%1 267%1 331%261% 1 350%144% 1365% 138%1 130%325% 277% 1 1 151%131% 319%370% 11 56%98% 142%307% 1 1131% 49%370% 129%1 1 98% 307% 1 M728AV700L 161%127%181% 161%1 173%1 170%192% 1 193%179% 1184% 139%1 153%181% 172% 1 1 133%144% 198%197% 11 123%127% 123%182% 1 1144% 81%197% 133%1 1 127% 182% 1 S731AH732V 99% 64%254% 165%1 245%1 315%156% 1 202%125% 1292% 81%1 152%176% 257% 1 1 93%132% 179%271% 11 72%88% 143%204% 1 1132% 56%271% 120%1 1 88% 204% 1 S644A/V700L/H723VV736A 128% 33%223% 153%1 237%1 273%111% 1 196%170% 1266% 50%1 143%197% 259% 1 1 48%129% 175%241% 11 70%69% 135%232% 1 1129% 38%241% 109%1 1 69% 232% 1 R635A 74% 269% 1 267% 331% 1 144% 365% 1 130% 277% 1 131% 370% 1 56% 142% 1 131% 370% 1 M728A 161% 181% 1 173% 170% 1 179% 184% 1 153% 172% 1 144% 197% 1 123% 123% 1 144% 197% 1 PAMS731A JNJ99% 41482012 - 254%AAA 1JNJ 40297036245% - AAA 315% JNJ 358150131 - AAA125% 292%JNJ 358140901 - AAC 152% JNJ 52149617257% - AAC 1 132%JNJ 41329782271% - AAA 1 JNJ 4324504672% - AAA143% 1 132% 271% 1 [PAM]V736A (M) 3.E-07128%3.E-06 223%n 3.E-071 3.E-06237% n 273%1.E-08 13.E-06 170%n 3.E-07266% 3.E-061 n 143%1.E-07 259%3.E-06 n1 1.E-07129% 3.E-06241% n 13.E-09 70%3.E-07 135%n 1 129% 241% 1 Stable 76% 99% 10 110% 139% 10 59% 169% 10 95% 134% 10 91% 185% 9 142% 225% 9 104% 226% 9 WT 82% 113% 8 130% 158% 8 71% 170% 8 102% 129% 8 88% 159% 7 142% 190% 7 107% 204% 7 75% of WT 61% 84% 97% 119% 53% 127% 77% 97% 66% 119% 107% 143% 80% 153% R636A 109% 141% 1 118% 146% 1 81% 201% 1 113% 100% 1 106% 178% 1 130% 231% 1 163% 258% 1 L639APAM 110% JNJ129% 414820121 - AAA62% 83% JNJ 402970361 33% - AAA 116% 1JNJ 3581501372% - 128%AAA 1 JNJ 35814090 - AAC1 JNJ 52149617 1- AAC JNJ 413297821 - AAA JNJ 43245046 - AAA [PAM]F643A (M) 24%3.E-0725% 3.E-061 27%n 45%3.E-07 1 3.E-0616% n117% 1.E-081 3.E-0621% 33% n 1 3.E-0734% 3.E-0668% 1n 35%1.E-07 84%3.E-06 1 n46% 1.E-07119% 3.E-061 n 3.E-09 3.E-07 n D725AStable 130% 76%140% 99%1 118%10 153%110% 1 139%74% 10137% 59%1 87%169% 119%10 1 95%112% 134%171% 101 117%91% 159%185% 1 9 142% 225%1 9 104% 226% 9 L732A 50% 56% 1 75% 88% 1 73% 100% 1 51% 59% 1 55% 86% 1 92% 145% 1 47% 111% 1 W773AWT 25% 82%34% 113%1 39%8 52%130% 1 158%22% 8 26% 71%1 39%170% 67% 8 1 102%33% 129%11% 18 21%88% 76%159% 1 742% 142%77% 190%1 7 107% 204% 7 75%F776A of WT 69% 61%102% 84%1 151% 184%97% 1 119%48% 102% 53%1 34%127% 42% 1 77%52% 97%100% 1 88%66% 177%119% 1 80% 107%203% 143%1 80% 153% F780AR636A 108%109%141% 141%1 123%1 171%118% 1 146%59% 1165% 81%1 80%201% 113% 1 1 113%56% 100%162% 11 119%106% 199%178% 1 1113% 130%228% 231%1 1 163% 258% 1 S644AL639A 63% 110%93% 129%1 98%1 168%62% 1 83% 48% 1223% 33%1 63%116% 111% 1 1 72%54% 128%182% 11 83% 277% 1 186% 304% 1 1 1 V700L 74% 89% 1 109% 148% 1 56% 148% 1 107% 115% 1 76% 158% 1 156% 192% 1 119% 195% 1 H732VF643A 66% 24%84% 25%1 88%1 115%27% 1 45% 70% 1196% 16%1 89%117% 116% 1 1 21%58% 33%156% 11 92%34% 141%68% 1 175% 35%186% 84%1 1 46% 119% 1 S644A/V700L/H723VD725A 46% 130%60% 140%1 55%1 98%118% 1 153%61% 1258% 74%1 41%137% 91% 1 1 87%32% 119%124% 11 40%112% 164%171% 1 161% 117%266% 159%1 1 1 R635AL732A 96% 50%138% 56%1 135%1 184%75% 1 88% 13% 1137% 73%1 78%100% 171% 1 1 51%83% 59%233% 11 145%55% 371%86% 1 1166% 92%444% 145%1 1 47% 111% 1 M728AW773A 136% 25%152% 34%1 157%1 167%39% 1 52% 81% 1153% 22%1 105%26% 98% 1 1 39%109% 67%151% 11 184%33% 185%11% 1 1161% 21%212% 76%1 1 42% 77% 1 S731A 84% 98% 1 99% 159% 1 72% 214% 1 55% 92% 1 61% 186% 1 97% 242% 1 91% 240% 1 V736AF776A 67% 69%87% 102%1 104%1 153%151% 1 184%57% 1197% 48%1 79%102% 146% 1 1 34%80% 42%191% 11 188%52% 252%100% 1 199% 88%225% 177%1 1 80% 203% 1 F780A 108% 141% 1 123% 171% 1 59% 165% 1 80% 113% 1 56% 162% 1 119% 199% 1 113% 228% 1 S644A 63% 93% 1 98% 168% 1 48% 223% 1 63% 111% 1 54% 182% 1 83% 277% 1 86% 304% 1

28/06/2012 9:59:04 V700L 74% 89% 1 109% 148% 1 56% 148% 1 107% 115% 1 76% 158% 1 156% 192% 1 119% 195% 1 H732V 66% 84% 1 88% 115% 1 70% 196% 1 89% 116% 1 58% 156% 1 92% 141% 1 75% 186% 1 S644A/V700L/H723V 46% 60% 1 55% 98% 1 61% 258% 1 41% 91% 1 32% 124% 1 40% 164% 1 61% 266% 1 R635A 96% 138% 1 135% 184% 1 13% 137% 1 78% 171% 1 83% 233% 1 145% 371% 1 166% 444% 1 M728A 136% 152% 1 157% 167% 1 81% 153% 1 105% 98% 1 109% 151% 1 184% 185% 1 161% 212% 1 S731A 84% 98% 1 99% 159% 1 72% 214% 1 55% 92% 1 61% 186% 1 97% 242% 1 91% 240% 1 V736A 67% 87% 1 104% 153% 1 57% 197% 1 79% 146% 1 80% 191% 1 188% 252% 1 99% 225% 1

94 Appendix4 JNJ-46281222 JNJ-35814376 JNJ-41329782 JNJ-40068782 JNJ-35814090 JNJ-35815013 JNJ-52149617 JNJ-41482012 JNJ-40297036 JNJ-39226421 JNJ-43245046 JNJ-42329001 JNJ-46356479 JNJ-42153605 JNJ-46281222 JNJ-35814376 JNJ-41329782 JNJ-40068782 JNJ-35814090 JNJ-35815013 JNJ-52149617 JNJ-41482012 JNJ-40297036 JNJ-39226421 JNJ-43245046 JNJ-42329001 JNJ-46356479 JNJ-42153605 n.v. NT Sc : : No value (absence curve) Novalue of 3orn.v > ratio If Testedscreening in assay Nottested Compoundpotency is ofEC presented ratio as a Potency(EC mean EC50 520 160 173 113 172 143 178 637 139 114 145 154 130 209 0.7 1.7 1.3 0.7 1.4 1.3 3.6 1.0 1.8 NT NT NT NT NT 20 97 49 91 68 6 6 8 7 145 147 118 120 SD SD 34 18 28 15 59 65 23 27 31 36 47 19 46 ------1 4 4 2 50 ) and ) relative Emax enhancementofthe ofglutamate-induced [ 12 10 16 17 10 12 15 9 9 9 9 6 2 1 2 3 8 9 3 3 3 2 1 n n (1)S688L Stable mean mean 173 224 218 227 224 214 215 250 159 206 201 116 166 139 130 196 206 133 167 181 104 147 1.0 1.0 1.1 1.0 1.0 1.2 0.8 1.1 1.0 NT NT NT NT NT 97 SD SD 11 16 10 12 14 25 22 16 16 16 13 20 13 38 ------9 6 2 9 7 8 9 12 10 16 17 10 12 15 9 9 9 9 6 2 1 2 3 8 9 3 3 3 2 1 n n mean mean 2512 1349 ~0.1 23.0 14.6 ~24 603 156 114 175 148 172 523 327 832 646 537 170 9.6 5.7 3.6 5.9 4.5 4.7 4.8 6.9 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 2.1 NT NT 87 37 59 57 25 83 87 39 7 5 6 50 between WTand mutant mGlu2. standarddeviation) (SD: A4 -06094 -Ana Farinha - thesis binnenwerk.indd 95 212 102 163 257 720 324 SD SD 35 42 28 25 37 27 15 69 27 ------1 9 3 5 4 (2)G689V 4 8 7 8 3 4 4 3 4 4 3 3 5 4 3 3 3 2 1 2 2 1 7 1 1 1 n n WT mean mean 187 128 168 159 101 147 141 227 200 204 214 211 186 202 132 171 177 117 215 229 218 201 269 203 178 1.0 1.1 0.8 0.9 0.9 1.0 1.2 0.9 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.3 1.0 1.1 0.6 NT NT 61 35 35 AppendixAppendix 4: 4: PotencyPotency (EC ) and(EC relative) and relative Emax of Emax the enhancement of the enhancement of35 glutamate-induced of glutamate-induced [ S]GTPγS [ S]GTPγS binding binding by 14 PAMs by 14 onPAMs WT onand WT mutant and mutantreceptors receptors after transient after transient transfection transfection into CHO-K1 into CHO-K1 cells. cells. Appendix 4: Potency (EC50) and50 relative 50Emax of the enhancement of glutamate-induced [ S]GTPγS binding by 14 PAMs on WT and mutant receptors after transient transfection into CHO-K1 cells. CompoundCompound potency potency is presented potency is presented as a ratio is presented of ECas50 a between ratio as ofaWT ratio EC and50 mutant of between EC mGlu2.50 between WT(SD: standardand WT mutant deviation) and mutantmGlu2. mGlu2. (SD: standard (SD: standard deviation) deviation) SD SD 23 12 22 26 20 11 30 13 30 25 14 28 32 43 14 23 ------2 9 9 7 Transmembrane 3 Stable WT (18) R635A (6) R636A (7) L639A (8) F643A Transmembrane(14) S644ATransmembrane 3 3 35 EC50 SD n meanStableSD Stablen mean SD n mean SD n meanWTSD WTn mean SD n mean mean mean SD n mean SD n mean SD n mean SD n mean SD n mean SD n S]GTPγS binding S]GTPγS by PAMs14 after transientWT and on mutant receptors transfection into CHO-K1 cells. JNJ-40068782 139 47 15 206 13 15 87 27 7 201 23 7 378 27 3 246 21 3 Sc Sc (18)572 R635A(18)225 R635A3 186 20 3 (6)2395.6 R636A(6)862 R636A3 89 25 3(7) 216L639A(7)111 L639A3 174 32 3 (8) F643A(8) F643A (14) S644A(14) S644A 4 8 7 8 3 4 4 3 4 4 3 3 5 4 3 3 3 1 2 2 1 7 2 1 1 1 n n EC50 EC50SD SDn meann meanSD1.0 SDn mean1.0n meanSD SDn4.3 meann meanSD1.2 SDn meann meanSD SDn 6.5 meann meanSD 0.9 SDn meann 27.4meanmean meanmean0.4 meanSD SDn 2.5meann meanSD 0.9 SDn meann meanSD SDn meann meanSD SDn meann meanSD SDn meann meanSD SDn n JNJ-41329782 68 15 10 215 14 10 57 15 4 202 9 4 Sc Sc Sc Sc 109 16 2 204 15 2 1142 256 3 84 10 3 180 144 3 196 51 3 JNJ-40068782JNJ-40068782 139 47139 1547 15206 2061.013 1513 871.015 2787 727 2017 23201 723 3787 27378 3272.9 2463 212461.0 321 Sc3 20.0 ScSc 572Sc 0.4 225572 2253 3.2 1863 20186 1.0 320 2395.63 2395.6862 8623 893 2589 325 2163 111216 1113 1743 32174 332 3 mean mean 1445 1349 15.4 23.6 JNJ-35814376 637 147 12 196 16 12 523 163 5 186 14 5 853 83 2 225 86 3 Sc10.8 Sc 1067 822 3 121 8 3 Sc Sc 625 165 3 139 14 3

1.0 1.0 n.v. 1.0 1.0 1.2 1.2 0.9 0.9 0.4 0.4 2.5 2.5 0.9 0.9 635 490 117 853 345 575 635 264 378 223 309 577 4.3 4.3 6.5 6.5 27.4 27.4 6.6 5.6 1.8 6.2 4.2 7.3 0.7 1.6 5.3 5.6 4.3 2.2 1.3 Sc Sc Sc Sc Sc Sc Sc Sc 22 61 41 Sc JNJ-41329782JNJ-41329782 68 1568 1015 10215 2151.014 1014 571.010 1557 4151.6 2024 2029 1.2 49 Sc4 Sc 2.0 Sc Sc0.7 Sc ScSc Sc 109Sc 16109 2161.2 2042 15204 0.7 215 11422 1142256 2563 843 1084 310 1803 144180 1443 1963 51196 351 3 JNJ-46281222 8 4 6 214 6 6 6 5 3 211 25 3 Sc Sc Sc Sc Sc Sc 316 --- 1 107 12 2 Sc Sc 1.0 1.01.0 1.0 1.0 1.0 55.6 2.90.5 2.9 1.0 1.0 20.0 20.0 0.4 0.4 3.2 3.2 1.0 1.0 JNJ-35814376JNJ-42153605JNJ-35814376 6376 4 1476379 12147224 1219612 9 196165 3 12163 52321412 30163523 3 1635Sc 1865 14186Sc 514 8535 Sc 83853Sc 283Sc 2252 86225Sc 386 Sc3 447 ScSc323 21067Sc 90 82210671 2 8223 Sc 1213 1218 Sc 38 Sc3 Sc Sc Sc 625 165625 1653 1393 14139 314 3 416 106 270 280 142 1.0 1.0 598 84.3 0.4 SD SD 83 34 11 52 27 ------JNJ-46356479 91 28 9 227 10 9 59 27 4 1.0204 131.0 4 Sc --- 1.0 1.0Sc 1.6 Sc 1.6 Sc Sc 1.2 1.2Sc 6782.9 2171 2 2.086 2.09 3 Sc 0.7 0.7 Sc 1.2 1.2 0.7 0.7

JNJ-46281222JNJ-46281222 8 48 64 6214 2141.06 66 61.06 56 35 2113 25211 325 Sc3 Sc Sc Sc S688L(4)G689V / Sc 115.6 ScSc ScSc0.4 Sc Sc Sc 316 316--- 1--- 1071 12107 212 Sc2 Sc Sc Sc JNJ-42329001 49 18 9 218 16 9 37 9 4 1.0200 301.0 4 Sc 1.0 1.0Sc Sc Sc Sc Sc 5867.8 1045 2 137 33 3 Sc Sc 55.6 55.6 0.5 0.5 1.0 1.0 156.8 0.7 JNJ-42153605JNJ-43245046JNJ-42153605 66 1 46 9 94224 922411 9 224127 1 9123 22759 1135 3 33Sc 2143 30214Sc 330 Sc3 Sc Sc Sc 9 Sc1 2 Sc244 26 2Sc 254 ScSc49 2 ScSc101 Sc4 3 Sc Sc Sc Sc 447 323447 3232 902 190 21 Sc2 Sc (18)R635A Sc Sc 3 1 1 2 3 3 3 2 2 2 1 3 2 1 1 1 1 2 1.0 1.01.0 1.0 1.0 1.0 1.4 1.1 n 38.7 0.4 84.3 84.3 0.4 0.4 n

JNJ-39226421 178 36 9 130 16 9 172 37 4 141 20 4 117 --- 1 92 30 2 Sc Sc 227 97 2 74 8 2 Transmembrane4 n.v. 2 36 11 2 119 10 2 110 20 2 JNJ-46356479JNJ-46356479 91 2891 928 9227 2271.010 910 591.09 2759 4270.7 2044 132040.7 413 Sc4 Sc 1.3 Sc Sc0.5 Sc ScSc ScSc0.3 Sc 0.7 Sc Sc 0.8 6782.9 6782.92171 21712 862 986 39 Sc3 Sc Sc Sc

JNJ-40297036 143 31 10 139 8 10 148 25 4 1.0147 9 1.0 4 Sc 1.0 1.0Sc Sc Sc 1351 1073 3 90 mean 15 3 n.v. 3 49 3 Sc Sc 115.6 115.6 0.4 0.4 mean 201 178 225 110 186 175 114 223 215 125 246 197 124 216 186 1.1 1.1 0.8 1.0 1.0 1.0 0.7 1.2 0.9 0.7 1.0 1.1 1.2 0.6 0.3 0.9 1.0 1.0 0.6 1.0 0.6 0.3 92 58 40 9.1 Sc Sc JNJ-42329001JNJ-42329001 49 1849 918 9218 21816 916 379 937 49 2004 30200 430 Sc4 Sc Sc Sc Sc ScSc ScSc Sc Sc ScSc ScSc 5867.8Sc 5867.81045Sc 10452 Sc 1372 Sc 33137Sc 333 Sc3 Sc Sc Sc JNJ-41482012 172 27 8 97 7 8 175 28 3 101 2 3 Sc Sc Sc Sc Sc Sc n.v 2 36 2 Sc Sc 1.0 1.01.0 1.0 1.0 1.0 0.4 156.8 156.8 0.7 0.7 JNJ-43245046JNJ-52149617JNJ-43245046 1136 23 1617 91166 922416 17 22411411 42 9118 15979 2617 8 31Sc 2273 11227Sc 311 Sc3 Sc Sc Sc 161 Sc20 4 Sc171 20 4Sc 724 ScSc--- 1 Sc9 62 1019 3 21 305 244269 326244131 22616 25432 25449 249 1012 1014 34 Sc3 Sc Sc Sc 1.0 1.0 1.4 1.1 6.4 0.4 2.7 0.8

1.0 1.0 1.0 1.0 SD 1.4 1.4 1.1 1.1 38.7 38.7 0.4 0.4 SD 16 79 30 86 20 49 71 21 11 33 35 ------JNJ-35815013 97 34 12 173 9 12 87 35 7 168 22 7 490 ------1 178 79 3 Sc Sc 1320 86 2 121 6 3 1414.1 231 3 84 42 4 108 39 3 153 17 3 JNJ-39226421JNJ-39226421 178 36178 936 97 130 1301.016 916 1721.09 0 37172 4375.6 1414 201411.1 420 1174 4 ---1170 1---15.2 921 30920.7 230 Sc2 16.3 ScSc 227Sc 0.5 97227 2971.2 742 874 0.8 28 n.v.2 n.v. 2 362 1136 211 1192 10119 210 1102 20110 220 2 JNJ-35814090 173 65 16 116 9 16 156 102 8 1.0128 121.0 8 Sc 1.0 1.0Sc 0.7 Sc 0.7 Sc 692 0.7--- 1 0.7148 35 3 n.v. 2 1.353 1.319 3 276 0.588 30.5 105 10 3 0.3 0.3 0.7 0.7 0.8 0.8

95 1.0 1.0 4.4 1.2 0.4 1.8 0.8 JNJ-40297036JNJ-40297036 143 31143 1031 10139 1398 108 14810 25148 425 1474 1479 49 Sc4 Sc Sc Sc Sc ScSc 1351Sc 10731351 10733 903 1590 315 n.v.3 n.v. 3 493 49 3 Sc3 Sc Sc Sc 3 3 2 3 3 3 3 2 2 2 2 3 2 2 1 1 1 2 1.0 1.0 1.0 1.0 n 9.1 9.1 0.6 0.6 0.3 0.3 n Transmembrane 4 Sc Sc Sc ScSc ScSc Sc Sc Sc Sc Sc Sc Sc JNJ-41482012JNJ-41482012 172 27172 (1) S688L827 897 977 87(2) G689V1758 28175 328 1013(4) G689V1012 / S688L32 Sc3 Sc(5) N735D / G689V / S688L (15) V700L (17) S644A/V700L/H723V n.v n.v 2 362 36 2 2 mean SD n mean SD n mean SD n 1.0mean SD1.0 n mean SD1.0 n 1.0mean SD n mean SD n mean SD n mean mean mean SD n mean SD n 0.4 0.4 2896.3 767.17 3162.3 489.78 21380 mean mean 246.6 JNJ-40068782 160 145 3 201 22 3 603 212 4 187 23 4 635 416 3 201 16 3 n.v.134.9 0 2 42 18 3 Sc Sc 1262 123 2 163 12 3 _n.v. Sc Sc Sc ScSc Sc 25.4 13.1 40.0 JNJ-52149617JNJ-52149617 113 23113 1723 17166 16616 1716 11417 42114 842 1598 2615953.9 826 Sc8 Sc 161 20161 420 1714 20171 420 7244 724--- 1--- 621 1062 310 3053 69305 369 1313 16131 316 3 n.v. n.v. 212 n.v. n.v. n.v NT 1.8 1.0 0.9 NT 1.0 0.2 0.8 Sc Sc Sc Sc Sc Sc 6.9 7.3 14.4 Sc Sc Sc Sc Sc Sc Sc Sc JNJ-41329782 NT NT NT 1.0NT 1.0 1349 1.0--- 1 1.0124 --- 1 NT NT Sc Sc 621 170 2 1.4186 1.42 2 1.1 1.1 6.4 6.4 0.4 0.4 2.7 2.7 0.8(6)R636A 0.8 JNJ-35815013JNJ-35815013 97 3497 1234 12173 1739 129 8712 3587 73523.6 1687 221680.6 722 4907 ---490 1--- 1781 79178 379 Sc3 10.9 ScSc 1320Sc 0.9 132086 286 1212 1216 36 1414.13 1414.1231 2313 843 4284 442 1084 39108 339 1533 17153 317 3 JNJ-35814376 520 59 2 206 2 2 2512 --- 1 1.0218 ---1.0 1 1445 1.0--- 1 1.0125 71 25.6 n.v. 5.6 --- 1 1.149 ---1.1 1 Sc Sc 2225 573 2 15.2135 15.223 3 0.7 0.7 16.3 16.3 0.5 0.5 1.2 1.2 0.8 0.8 1.0 1.1 4.8 1.2 0.7 0.3 4.3 0.7 (5)N735D S688LG689V / / JNJ-35814090JNJ-46281222JNJ-35814090 17320 --- 651731 1665159 16116--- 1 116839 69 169 2 15622916 7102156 2 102861 128528 2 12128215 8120 2Sc8 767.17Sc 695 2 Sc203 8 Sc 3 Sc ScSc 72 ScSc43 2 692Sc213 ---6926 2 1--- 1481 35148 335 n.v.3 n.v. 2 532 1953 319 2763 88276 388 1053 10105 310 3 mean 376 695 0.8 1.1 1.0 240 1.0 1.0 3.6 14.6 10.8 134.9 SD 12.7 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc ------1.0 1.0 1.0 1.0 --- 4.4 4.4 1.2 1.2 0.4 0.4 1.8 1.8 0.8 0.8 0 0 JNJ-42153605 7 2 2 250 25 2 25 4 2 215 14 2 22 11 2 0 223 4 2 212 240 3 180 22 3 Sc Sc 47 4 3 209 15 3 1.3 1.2 4.7 1.0 4.2 1.0 40.0 0.8 8.8 1.0 JNJ-46356479 NT NT 1349 --- 1 269 --- 1 309 --- 1 197 33 2 3162.3 --- 1 174 3 2 Sc Sc 692 0 1 215 4 2 23.0 1.3 5.3 1.0 53.9 Transmembrane0.9 Transmembrane 4 4 11.8 1.1 NT NT Sc Sc mean JNJ-42329001 170 --- 1 178 --- 1 577 598 2 186 35 2 489.78 0 1 208 7 2 115 42 2 230 1320 8 2 1351 1067 15.2 692 161 227 109 572 1.3 2.0 4.4 1.4 1.4 2.9 9.1 6.5 (1) S688L(1) S688L4.5 0.9 (2)15.4 G689V(2) G689V0.9 13.1 (4) G689V(4) /G689V 1.0S688L / S688L 3.1(5) N735D(5) /N735D G689V1.1 / /G689V S688L / S688LSc (15) V700L(15) V700L (17)Sc S644A/V700L/H723V(17)Sc S644A/V700L/H723VSc Sc 2 2 2 1 2 3 2 1 3 1 1 9 1 n JNJ-43245046 meanNT meanSD SDn NT meann meanSD39 --- SDn 1 mean203n ---meanSD 1 SDn41 mean---n 1meanSD216 SDn--- mean1 n n.v.meanSD --- SDn 1 mean142n ---meanSD 1 SDnSc meanScn 42 meanSD34 2 SDn289 mean74n 2meanSD SDn meann meanmean mean meanSD SDn meann meanSD SDn n 5.9 0.9 6.2 1.0 0.6 6.4 1.3 JNJ-40068782JNJ-39226421JNJ-40068782 160NT 145160 1453 NT 3201 201NT22 322 603NT3 212603 2124n.v. 187---4 1 2318740 423--- 16354 NT416635 4163 201NT3 16201 316Sc Scn.v.3 539 n.v.1300 2 02 95 4212 21842 318 Sc3 ScSc 1262Sc 1262123 1232 1632 12163 312 3 mean

0.3 3.1 0.7 1073 203 180 142 174 208 225 822 0.3 0.3 0.6 0.2 0.4 1.0 0.8 0.3 0.9 0.3 1.0 0.4 1.8 1.8 1.0 1.0 6.9 6.9 0.9 0.9 7.3 7.3 1.0 1.0 0.2 0.2 14.4 14.4 0.8 0.8 SD NT NT 42 56 49 40 37 33 44 20 97 16 86 JNJ-40297036 209 --- 1 147 --- 1 537 --- 1 117 --- 1 264 34 2 114 0 2 n.v. 2 40 1 2 Sc Sc 389 16 3 --- 101 12 3 1 JNJ-41329782JNJ-41329782 1.4NT NT 1.0 NT NT3.6 NT0.8 NT 1.8 NT NT0.8 1349 1349--- 1--- 1240.31 ---124 1--- NT1 2.6 NT 0.7 NT NT Sc ScSc 621Sc 170621 1702 1862 1862 22 2 JNJ-41482012 130 46 2 104 9 2 ~24 --- 2 61 --- 2 223 142 2 58 11 23.62 n.v 23.6 2 0.637 1 0.6 2 Sc Sc 274 50 3 73 3 3 10.9 10.9 0.9 0.9 0.7 1.0 ~0.1 0.6 1.3 0.6 0.4 1.6 0.7 JNJ-35814376JNJ-52149617JNJ-35814376 154520 120595203 259181 220638 3 2066462 324 22 3 25121772 432512--- 3 1---635 2182801 3 ---218175 149--- 14453 1 2896.31445--- 376 1---2 125561 22711253 271Sc Scn.v.2 620 n.v.---78 3 ---1151 49121 3 ---49 1--- Sc1 ScSc 2225Sc 2225573 5732 1352 23135 323 3 SD (7)L639A 18 22 22 23 ------8 1 1 9 3 3 2 7 1 3 4 2 2 3 2 1.31.0 1.0 1.1 1.1 1.15.7 4.81.1 4.8 5.6 1.2 1.21.1 25.4 0.70.4 0.7 5.4 1.0 0.3 0.3 4.3 4.3 0.7 0.7 n JNJ-46281222JNJ-35815013JNJ-46281222 14520 118---203 1---167 115913 3 159832--- 720 1--- 3 831711 326983 3 269575 2292702 3 2297 186 2207 3612 213805261 --- 2521 215442 9 2150 3 20Sc 767.17Sc2 78767.1769526 36952206 203322 3 2038 38 Sc3 ScSc 72Sc 4372 243 2132 2136 26 2 1.7 1.0 9.6 1.0 6.6 1.1 246.6 0.3 0.9 1.2 JNJ-35814090 1143.6 19 3.63 133 0.820 3 0.8327 257 3 14.6132 2814.63 345 1061.1 3 1.1110 7 10.83 _n.v.10.80 3 1.033 231.0 3 Sc 134.9Sc 670134.985 3 111 1.014 3 1.0 12.7 12.7 1.0 1.0 mean 244 148 171 204 186 0.7 1.0 2.1 1.0 2.2 0.9 0.3 4.3 0.9 121 121 0.5 0.7 1.2 1.1 1.1 1.0 0.6 7 27 22 2250 25025 225 252 425 24 2152 14215 214 222 1122 211 2232 2234 24 2122 212240 2403 0.7 1803 22180 322 Sc3 ScSc 47Sc 474 34 2093 15209 315 3 0.9 28/06/2012 9:59:06 90 JNJ-42153605JNJ-42153605 74 Sc Sc Sc Sc Sc 3 3 1 3 3 2 2 1 3 3 2 2 n 1.3 1.3 1.2 1.2 4.7 4.7 1.0 1.0 4.2 4.2 1.0 1.0 40.0 40.0 0.8 0.8 8.8 8.8 1.0 1.0 Transmembrane3 JNJ-46356479JNJ-46356479NT NotNT tested NT NT NT 1349 1349--- 1--- 2691 ---269 1--- 3091 ---309 1--- 1971 33197 233 3162.32 3162.3------1 1741 1743 23 Sc2 ScSc 692Sc 6920 10 2151 2154 24 2 Sc Tested in screening assay 23.0 23.0 1.3 1.3 5.3 5.3 1.0 1.0 mean 53.9 53.9 0.9 0.9 11.8 11.8 1.1 1.1 (15)V700L

If ratio > 3 or n.v SD 15 26 35 20 15 20 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 8 JNJ-42329001JNJ-42329001n.v. NoNT value (absenceNT of curve) NT NT 170 ---170 1--- 1781 ---178 1--- 5771 598577 5982 1862 35186 235 489.782 489.780 01 2086 1 2087 27 Sc2 ScSc 115Sc 11542 242 2302 2308 28 8 2 4.5 4.5 0.9 0.9 15.4 15.4 0.9 0.9 13.1 13.1 1.0 1.0 3.1 3.1 1.1 1.1 JNJ-43245046JNJ-43245046 NT NT NT NT 39 ---39 1--- 2031 ---203 1--- 411 ---41 1--- 2161 ---216 1--- n.v.1 n.v.------1 1421 ---142 1--- Sc1 ScSc 42Sc 3442 234 2892 74289 274 2 mean Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 5.9 5.9 Sc 0.9 0.9 6.2 6.2 1.0 1.0 0.6 0.6 6.4 6.4 1.3 1.3 3 2 3 4 2 3 2 3 3 JNJ-39226421JNJ-39226421 NT NT NT NT NT NT NT NT n.v. ---n.v. 1--- 401 ---40 1--- NT1 NT NT NT Sc ScSc 539Sc 130539 1302 952 195 21 2 n 95 0.3 0.3 3.1 3.1 0.7 0.7 2395.6

1414.1 Sc ScSc Sc 5867.8 6782.9 mean mean JNJ-40297036JNJ-40297036 209 ---209 1--- 1147 147--- 1--- 5371 ---537 1--- 1171 ---117 1--- 2641 34264 234 1142 1140 20 n.v.2 n.v. 2 402 140 21 2 389 156.8 38916 115.6 316 1013 12101 312 3 2225 1262 1142 14.4 20.0 38.7 84.3 55.6 11.8 16.3 27.4 10.9 12.7 n.v. 254 670 620 274 389 539 115 692 621 447 316 n.v. 724 n.v. 3.1 6.4 n.v 0.9 2.6 6.4 5.4 3.1 8.8 4.3 4.3 1.6 78 42 47 72 1.4 1.4 1.0 1.0 3.6 3.6 0.8 0.8 1.8 1.8 0.8 0.8 0.3 0.3 2.6 2.6 0.7 0.7 Sc JNJ-41482012JNJ-41482012 130 46130 246 2104 1049 29 ~242 ---~24 2--- 612 ---61 2--- 2232 142223 1422 582 1158 211 n.v2 n.v 2 372 137 21 Sc2 ScSc 274Sc 27450 350 733 373 33 3 0.7 0.7 1.0 1.0 ~0.1 ~0.1 0.6 0.6 1.3 1.3 0.6 0.6 0.4 0.4 1.6 1.6 0.7 0.7 (17)S644A/V700L/H723V

JNJ-52149617JNJ-52149617 154 120154 1203 3181 18138 338 6463 324646 3243 1773 43177 343 6353 280635 2803 1753 49175 349 2896.33 2896.3376 3762 562 2256 322 Sc3 ScSc 620Sc 620781045 3782171 1513 12151 312 3 130 170 323 573 123 256 862 231 SD SD 49 85 78 50 16 42 26 34 43 ------4 1.3 1.3 1.1 1.1 5.7 5.7 1.1 1.1 0 5.6 5.6 1.1 1.1 25.4 25.4 0.4 0.4 5.4 5.4 1.0 1.0 JNJ-35815013JNJ-35815013 145 118145 1183 3167 16713 313 8323 720832 7203 1713 32171 332 5753 270575 2703 1863 20186 320 213803 21380------1 441 944 39 Sc3 ScSc 78Sc 2678 326 2063 32206 332 3 1.7 1.7 1.0 1.0 9.6 9.6 1.0 1.0 6.6 6.6 1.1 1.1 246.6 246.6 0.3 0.3 0.9 0.9 1.2 1.2 JNJ-35814090JNJ-35814090 114 19114 319 3133 13320 320 3273 257327 2573 1323 28132 328 3453 106345 1063 1103 1107 37 _n.v.3 _n.v.0 03 333 2333 323 Sc3 ScSc 670Sc 67085 385 1113 14111 314 3 (8)F643A 3 2 2 3 2 2 3 3 3 3 2 2 1 2 2 1 2 2 2 1 2 3 3 3 3 2 2 0.7 0.7 1.0 1.0 2.1 2.1 1.0 1.0 2.2 2.2 0.9 0.9 n 0.3 0.3 4.3 4.3 0.9 0.9 n mean mean 137 209 101 111 151 101 230 215 186 107 135 163 206 289 213 0.8 0.3 0.4 0.4 0.4 0.4 0.4 0.5 1.1 0.7 0.4 0.3 0.5 0.4 0.9 1.0 0.7 0.4 1.2 0.7 1.3 1.0 1.1 1.0 0.7 0.9 0.7 49 86 36 73 95 90 53 62 36 84 89 84 NT NT Not testedNot tested Sc Sc Sc Tested inTested screening in screening assay assay If ratio >If 3 ratio or n.v > 3 or n.v SD SD 33 15 14 12 12 12 23 12 19 10 11 10 25 42 n.v. n.v. No valueNo (absence value (absence of curve) of curve)32 74 9 4 3 1 8 4 2 1 6 3 3 3 3 3 2 3 3 3 3 2 2 2 2 2 2 3 3 3 3 2 3 3 4 3 2 2 n n mean 276 305 119 180 216 108 625 0.7 1.2 1.8 3.2 1.2 2.5 2.7 95 95 Sc Sc Sc Sc Sc Sc Sc 144 111 165 SD 88 69 10 39 (14)S644A 3 3 2 3 3 3 3 n mean 105 131 110 196 174 153 139 0.8 0.8 0.7 1.0 0.8 0.9 0.8 Sc Sc Sc Sc Sc Sc Sc SD 10 16 20 51 32 17 14 3 3 2 3 3 3 3 n Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc mean Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (21) V736A (21) mean n 3 3 3 1 2 1 1 2 3 Appendix 4: (cont.) 3 5 6 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc ------15 31 21 SD mean 81 39 41 70 74 96 40 NT NT NT NT NT 1.0 0.3 0.4 0.3 0.4 0.4 0.6 0.3 1.0 221 220 mean Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (21) V736A (21) mean n 0 2 1 1 1 1 2 2 3

(3) N735D (3) Extracellular loop 2/Transmembrane 5 n 3 3 3 2 1 1 1 2 3 5 ------SD

(16) H723V (9) D725A (19) M728A (20) S731A 227 (10) L732A (3) N735D (21) V736A mean SD n mean SD n mean mean mean mean mean SD n mean SD n mean SD n mean SD n mean SD n mean SD n mean mean 3 5 6 27 ------NT NT NT NT NT 15 31 21 5.2 n.v n.v. n.v. SD 85.5 24.5 ~617 _n.v. 102.0 mean 7413.1 3019.95 JNJ-40068782 549 77 3 189 25 3 Sc Sc Sc Sc Sc Sc 95 2790.74 12 3 130 12 3 3019.95 1 70 15 3 Sc Sc n 3 4 3 3 3 3 3 2 3 3 3 3 6.3 0.9 3 1.1 4 0.6 0.3 JNJ-41329782 260 135.6 3 217 72 3 Sc Sc Sc Sc 245 --- 1 247 19 2 63 7 3 140 5 3 NT NT Sc Sc 39 81 41 70 74 96 40 NT NT NT NT NT 1.0 0.4 0.6 0.3 0.4 1.0 0.3 0.4 0.3 221 220 6 9 6 7 3 7 4 5 6 5 4 24 12 16 SD 4.6 1.1 4.3 1.2 mean 1.1 0.7 52 78 73 91 62 0.6 0.6 0.7 0.5 0.5 0.5 0.7 0.7 0.7 0.7 0.6 0.6 0.7 0.5 130 131 129 141 130 142 159 110 JNJ-35814376 1625 368 3 152 31 3 Sc Sc Sc Sc 1190 97 2 140 189 12 2 633 229 3 129 9 3 n.v. --- 0 81 --- 1 Sc Sc mean n 2 0 1 1 1 2 1 2 3 96

3.1 0.8 2.3 1.0 N735D (3) 1.2 0.7 0.4 n 3 2 3 3 3 3 3 3 3 2 2 3 3 4 JNJ-46281222 1 1 Sc Sc Sc Sc Sc L732A (10) Sc 58 47 3 142 7 3 ~617 --- 1 221 --- 1 Sc Sc 5 n 1 2 7 1 21 12 47 20 10 ------SD 387 229 131 136 225 SD

10.3 0.7 227 102.0 1.0 JNJ-42153605 28 1 2 226 36 2 Sc Sc Sc Sc Sc Sc 49 20 3 159 16 3 27 5 2 220 5 2 Sc Sc ------95 58 49 57 15 63 SD 1.1 9.2 0.3 2.2 1.1 1.2 n.v 3.1 0.2 633 576 329 357 n.v. 17.3 10.3 15.4 12.6 1015 mean 5.2 1.1 82.57 9.2 0.7 5.2 1.0 n 2 2 3 3 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 27 NT NT NT NT NT 0.8 1.0 156 148 n.v 5.2 n.v. n.v. mean 85.5 24.5 ~617 _n.v. 102.0 JNJ-46356479 195 --- 1 220 25 2 Sc Sc Sc Sc Sc Sc mean 1015 387 2 131 24 4 NT NT Sc Sc 7413.1 3019.95 2790.74 n 1 1 12 19 11 13 SD

3.3 1.1 17.3 F780A (13) 0.6 n 3 4 3 3 3 3 3 2 3 3 3 3 JNJ-42329001 1 1 Sc Sc Sc Sc Sc Sc 576 131 3 141 7 3 3 NT 4 NT Sc Sc ------Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc SD 1.2 1.0 1.1 0.9 189 247 169 109 mean 15.4 0.7 n 2 1 3 3 Extracellular 5 2/Transmembrane loop Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 0.8 1.9 (20) S731A (20) mean 218.8 JNJ-43245046 15 6 3 281 80 3 Sc Sc Sc Sc Sc Sc 82.57 21 3 130 398.1 6 3 NT NT Sc Sc 6 6 9 7 3 7 4 5 5 6 4 24 12 16 SD n 3 3 3 4 3 3 2 3 --- 97 84 68 2.3 1.2 SD 12.6 0.6 JNJ-39226421 253 100 2 115 15 2 Sc Sc Sc Sc Sc Sc 57 10 2 73 4 2 NT NT Sc Sc 9 7 0 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 15 11 36 25 18 SD 4.3 2.3 2.5 5.3 245 285 834 52 78 73 91 62 1190 mean 0.7 0.7 0.6 0.6 0.7 0.6 0.7 0.7 0.7 0.6 0.5 0.5 0.5 0.5 130 131 129 141 130 142 159 110 140

1.5 0.8 mean 0.3 0.5 96 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 48 0.8 0.8 0.7 0.4 0.7 0.6 0.7 0.8 132 152 118 106 178 163 158 mean JNJ-40297036 351 84 3 116 8 3 Sc Sc Sc Sc Sc mean Sc 329 136 3 78 3 3 n.v. --- 1 41 --- 1 Sc Sc n 3 2 3 3 3 3 3 3 3 2 2 3 3 4 n 3 3 4 3 2 3 2 3 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 2.4 0.8 2.2 Sc Sc 0.5 0.3 (10) L732A (10) (12) F776A (12) (19) M728A (19) mean JNJ-41482012 175 77.89 3 78 2 3 Sc Sc Sc Sc Sc Sc n.v 3 52 6 3 n.v 2 39 3 2 Sc Sc 7 1 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 34 34 26 16 SD 233 Transmembrane 6 Transmembrane Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc mean mean n 1 2 7 1.0 0.8 0.5 0.4 1 21 12 47 20 10 SD 387 229 131 136 225 7 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 24 70 3.4 1.9 1.1 1.1 2.3 5.0 4.6 (9) D725A (9) 440 192 199 125 n.v. Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc mean mean JNJ-52149617 281 71 3 174 17 3 Sc Sc Sc Sc 285 84 3 V736A (21) 169 11 3 357 225 3 91 6 3 2790.74 227 2 96 6 3 Sc Sc mean 3 3 3 3 2 3 4 3 3 n n 3 2 2 3 3 3 3 2 3 2 3 3 3 3 3 n 3 3 3 1 2 1 1 2 2.5 1.1 2.5 1.1 3.1 3 0.6 24.5 0.6 ------95 58 49 57 15 63 SD n.v 3.1 0.2 1.1 1.1 1.2 9.2 0.3 2.2 633 576 329 357 n.v. 10.3 15.4 12.6 17.3 1015 mean JNJ-35815013 Sc Sc Sc Sc Sc Sc Sc Sc 15 2 2 82.57 110 5 3 7413.1 --- 1 74 31 3 Sc Sc 6 8 8 8 8 6 8 6 8 2 31 17 12 13 25 25 15 17 36 80 10 17 14 72 3 5 6 SD SD ------15 31 21 SD 0.2 0.7 85.5 0.4 n 2 2 3 3 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 0.8 1.0 156 148

JNJ-35814090 Sc Sc Sc Sc Sc Sc 834 68 3 109 13 3 n.v. 4 62 4 4 _n.v. 3 mean 40 21 3 Sc Sc 81 39 41 70 74 96 40 NT NT NT NT NT 1 1 0.3 0.4 1.0 0.3 0.4 1.0 0.6 0.4 0.3 221 220 Sc Sc 80 89 46 76 49 68 51 44 95 72 82 92 48 63 78 0.4 0.4 0.4 0.4 0.4 0.9 1.1 0.8 1.1 1.1 1.2 0.8 0.8 0.8 1.1 0.5 0.4 0.2 0.4 0.3 0.5 0.5 0.3 0.3 152 189 220 115 116 174 226 281 217 mean 5.3 mean 0.9 mean 0.5 0.3 n 1 1 12 19 11 13 SD n 0 2 1 1 1 1 2 2 3 (13) F780A (13) (16) H723V (16) (11) W773A (11) (3) N735D (3) 2 3 3 3 3 2 3 3 4 n n 3 1 2 3 3 1 1 1 2 3 3 2 3 3 3 5 ------SD 227 1 6 ------77 84 71 93 ------Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc SD SD 368 100 SD 1.2 1.0 1.1 0.9 17.2 189 247 169 109 Transmembrane 6 135.6 77.89 mean 27 NT NT NT NT NT 1 1 5.2 n.v n.v. n.v. Sc Sc 28 15 76 24.5 85.5 6.3 4.6 3.1 5.2 3.3 2.3 1.5 2.4 1.0 2.5 4.0 n.v ~617 _n.v. 549 260 195 253 351 175 281 361 631 n.v. n.v. n.v. n.v. n.v. n.v. n.v. 102.0 mean (11) W773A (12) F776A (13) F780A 18.5 13.3 40.0 33.6 55.0 1625 1059 2344 1259 7413.1 mean mean Not tested Not assay in screening Tested If ratio > n.v or 3 of valueNo curve) (absence 3019.95 2790.74 n 2 1 3 3 Extracellular 5 2/Transmembrane loop Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 0.8 1.9 mean SD n mean SD n mean SD n mean SD n mean SD S731A (20) n mean SD n mean 218.8 398.1 n 3 4 3 3 3 3 3 2 3 3 3 3 3 4 JNJ-40068782 n.v. 3 89 6 3 440 233 3 132 15 3 Sc Sc 6 9 6 7 3 7 4 5 6 5 4 n 3 3 3 3 4 3 2 3 24 12 16 SD --- 97 84 68 Sc NT SD 0.4 5.0 0.7 n.v. JNJ-40068782 JNJ-41329782 JNJ-35814376 JNJ-46281222 JNJ-42153605 JNJ-46356479 JNJ-42329001 JNJ-43245046 JNJ-39226421 JNJ-40297036 JNJ-41482012 JNJ-52149617 JNJ-35815013 JNJ-35814090 JNJ-40068782 JNJ-41329782 JNJ-35814376 JNJ-46281222 JNJ-42153605 JNJ-46356479 JNJ-42329001 JNJ-43245046 JNJ-39226421 JNJ-40297036 JNJ-41482012 JNJ-52149617 JNJ-35815013 JNJ-35814090 52 78 73 91 Sc Sc 62 0.6 0.7 0.7 0.7 0.6 0.5 0.5 0.5 0.7 0.7 0.6 0.6 0.7 1059 17.2 2 80 17 3 192 34 3 152 11 3 0.5 130 131 129 141 130 142 159 110 JNJ-41329782 140 mean 96 7 9 0 Appendix 4: (cont.) Appendix Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 15 11 25 36 18 SD 4.3 2.3 2.5 0.4 0.8 5.3 18.5 3.4 245 285 834 1190 n 3 2 3 3 3 3 3 3 3 2 2 3 3 4 mean JNJ-35814376 n.v. 3 46 8 3 Sc Sc 398.1 L732A (10) --- 1 148 --- 1 n 1 2 7 1 21 12 47 20 10 SD Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 387 229 131 136 225 0.2 0.8 0.8 48 0.8 0.8 0.7 0.4 0.7 0.6 0.7 0.8 132 152 178 118 106 163 158 mean JNJ-46281222 76 --- 1 95 10 2 Sc Sc Sc mean Sc ------95 58 49 57 15 63 SD 1.1 1.1 1.2 9.2 0.3 2.2 n.v 3.1 0.2 633 576 329 357 n.v. 17.3 10.3 15.4 12.6 1015 mean 82.57 n 3 3 3 4 3 2 13.3 0.4 2 3 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc (12) F776A (12) n 2 2 3 3 (19) M728A (19) Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc mean 0.8 1.0 156 148 JNJ-42153605 n.v. 3 76 12 3 24 7 3 178 25 3 Sc Sc mean n 1 1 12 19 11 13 0.4 4.6 0.8 SD 7 1 (13) F780A (13) Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 34 34 26 16 SD 233 Transmembrane 6 Transmembrane JNJ-46356479 2344 --- 1 72 17 3 Sc Sc Sc mean Sc ------Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc SD 1.2 1.0 1.1 0.9 189 247 169 109 40.0 0.4 mean 7 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 24 70 n 2 1 3 3 3.4 1.9 1.1 1.1 2.3 5.0 4.6 (9) D725A (9) Extracellular 5 2/Transmembrane loop 440 192 125 199 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc n.v. 0.8 1.9 (20) S731A (20) mean mean mean 218.8 JNJ-42329001 1259 --- 1 82 6 3 70 16 2 163 0 2 Sc Sc 398.1 n 3 3 3 4 3 3 3 2 --- 97 0.4 1.9 0.8 84 68 33.6 SD 3 3 3 3 3 2 3 3 4 n n 3 2 3 2 3 3 3 3 3 2 2 3 3 3 JNJ-43245046 361 93 2 92 14 3 7 1 3 158 18 3 Sc 3 Sc 9 7 0 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 15 11 25 36 18 SD 4.3 2.3 2.5 5.3 245 285 834 1190 55.0 0.4 1.1 0.7 mean 6 8 8 8 8 6 8 6 8 2 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 48 31 17 12 13 25 15 17 10 17 14 36 25 80 72 0.8 0.8 0.7 0.4 0.7 0.6 0.7 0.8 132 152 178 118 106 158 163 SD SD mean JNJ-39226421 n.v. 2 49 8 2 Sc Sc Sc mean Sc n 3 3 3 4 3 2 3 2 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 0.3 Sc Sc (12) F776A (12) (19) M728A (19) JNJ-40297036 n.v. 3 68 8 3 Sc Sc Sc mean Sc 7 1 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 34 34 26 16 SD 233 Transmembrane 6 Transmembrane 1 1 mean Sc Sc 89 80 46 76 49 68 51 44 95 72 82 92 48 63 78 0.4 0.4 0.4 0.4 0.4 0.9 1.1 0.8 1.1 1.1 1.2 0.8 0.8 0.8 1.1 0.5 0.4 0.2 0.4 0.3 0.5 0.5 0.3 0.3 152 189 115 116 174 226 220 281 0.5 217 mean mean 7 Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc Sc 24 70 3.4 1.9 1.1 1.1 2.3 5.0 4.6 (9) D725A (9) 192 440 125 199 Sc Sc Sc Sc n.v. JNJ-41482012 n.v 3 48 8 3 mean mean 3 3 3 3 3 3 2 3 3 4 n n 3 3 2 2 3 2 2 3 3 3 3 0.5 3 3 3 (16) H723V (16) (11) W773A (11) 3 3 2 3 3 2 3 3 4 n n 3 2 3 3 2 3 3 2 1 3 1 1 1 JNJ-52149617 n.v. 3 51 13 3 125 34 4 118 36 4 218.8 --- 1 1563 --- 1 3 6 8 8 8 8 6 6 8 8 2 25 31 17 12 13 17 36 25 80 15 10 17 14 72 0.3 1.1 0.7 1.9 SD 1.0 SD

n.v. 4 44 8 4 199 26 3 106 9 3 Sc Sc 1 6 JNJ-35815013 ------77 84 71 93 SD SD 368 100 1 1 17.2 Sc Sc 80 89 46 76 49 68 51 44 63 95 72 82 92 48 78 0.4 0.4 0.4 0.4 0.4 0.9 1.1 0.8 1.1 1.1 1.2 0.8 0.8 0.8 1.1 0.5 0.4 0.2 0.4 0.3 0.5 0.5 0.3 0.3 189 152 174 226 220 281 115 116 217 135.6 77.89 0.3 2.3 0.6 mean mean (16) H723V (16)

Sc Sc W773A (11) JNJ-35814090 631 3 63 6 3 n.v. 2 48 7 3 1 1 Sc Sc 28 15 76 3 3 2 3 3 3 2 3 3 4 n n 3 3 2 3 3 2 1 2 1 1 1 3 3 3 6.3 4.6 3.1 5.2 3.3 2.3 1.5 2.4 1.0 2.5 4.0 n.v 549 260 195 253 351 175 281 361 631 n.v. n.v. n.v. n.v. n.v. n.v. n.v. 18.5 13.3 40.0 33.6 55.0 1625 1059 2344 1259 mean mean 4.0 0.5 0.4 tested Not assay in screening Tested If ratio > n.v or 3 of valueNo curve) (absence 1 6 ------77 71 84 93 SD SD 368 100 17.2 135.6 77.89 1 1 Sc Sc 28 15 76 6.3 4.6 3.1 5.2 3.3 2.3 1.5 2.4 1.0 2.5 4.0 n.v 549 281 631 260 195 253 351 175 361 n.v. n.v. n.v. n.v. n.v. n.v. n.v. 18.5 13.3 40.0 33.6 55.0 1625 1059 2344 1259 mean mean NT Not tested tested Not assay in screening Tested If ratio > n.v or 3 of valueNo curve) (absence

Sc Tested in screening assay Sc NT n.v.

If ratio > 3 or n.v Sc NT n.v.

n.v. No value (absence of curve) JNJ-40068782 JNJ-41329782 JNJ-35814376 JNJ-46281222 JNJ-42153605 JNJ-46356479 JNJ-42329001 JNJ-43245046 JNJ-39226421 JNJ-40297036 JNJ-41482012 JNJ-52149617 JNJ-35815013 JNJ-35814090 JNJ-40068782 JNJ-41329782 JNJ-35814376 JNJ-46281222 JNJ-42153605 JNJ-46356479 JNJ-42329001 JNJ-43245046 JNJ-39226421 JNJ-40297036 JNJ-41482012 JNJ-52149617 JNJ-35815013 JNJ-35814090 JNJ-40068782 JNJ-52149617 JNJ-35814090 JNJ-41329782 JNJ-35814376 JNJ-46281222 JNJ-42153605 JNJ-46356479 JNJ-42329001 JNJ-43245046 JNJ-39226421 JNJ-40297036 JNJ-41482012 JNJ-35815013 JNJ-35814090 JNJ-40068782 JNJ-41329782 JNJ-35814376 JNJ-46281222 JNJ-42153605 JNJ-46356479 JNJ-42329001 JNJ-43245046 JNJ-39226421 JNJ-40297036 JNJ-41482012 JNJ-52149617 JNJ-35815013 Appendix 4: (cont.) Appendix Appendix 4: (cont.) Appendix

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