Reprint of Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2

Accepted Manuscript Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2 L. Lundström, C. Bissantz, J. Beck, M. Dellenbach, T.J. Woltering, J. Wichmann, S. Gatti PII:

S0028-3908(17)30050-3

DOI:

10.1016/j.neuropharm.2016.08.040

Reference:

NP 6596

To appear in:

Neuropharmacology

Received Date: 5 September 2015 Revised Date:

22 August 2016

Accepted Date: 24 August 2016

Please cite this article as: Lundström, L., Bissantz, C., Beck, J., Dellenbach, M., Woltering, T.J., Wichmann, J., Gatti, S., Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2, Neuropharmacology (2017), doi: 10.1016/ j.neuropharm.2016.08.040. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2

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L. Lundström*, C. Bissantz#, J. Beck*, M. Dellenbach, , T. J. Woltering#, J. Wichmann# and S. Gatti* *

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F. Hoffmann-La Roche AG, pRED, Pharma Research & Early Development, NORD Neuroscience, Discovery

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Chemistry, Roche Innovation Center Basel, Grenzacherstrasse 124, Basel, Switzerland, CH4070,

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Running title: mGlu2 PAMs pharmacological properties

Corresponding author: Silvia Gatti, present address CNS Innovation Unit , R&D Pierre Fabre

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Laboratories , Toulouse, France E-mail: [email protected].

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ACCEPTED MANUSCRIPT Abstract The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2

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PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are

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therefore carried out here using the radiolabeled mGlu2 agonist 3[H]-LY354740 and mGlu2 PAM 3[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action

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shows that mGlu2 PAMs increase the affinity of 3[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of 3[H]-LY354740 binding sites. 3[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr3.40 and

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Asn5.46. Also of remark, in the described experimental conditions S731A (Ser5.42) residue is

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important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the

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information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are

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discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity.

Keywords Metabotropic glutamate receptor, mGlu2, mGlu3, mGluR, negative allosteric modulators, positive allosteric modulators, LY354740, LY487379

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Abbreviations

GPCR; G-protein coupled receptor

NAM; negative allosteric modulator

PAM; positive allosteric modulator

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TM; transmembrane

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mGluR; metabotropic glutamate Receptor

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FLIPR; Fluorometric Imaging Plate Reader

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ECL; extracellular loop

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7TM; Seven transmembrane domain

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VFT; venus flytrap

WT, wild type

Nh, Hill Coefficient

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ACCEPTED MANUSCRIPT Introduction Allosteric modulators of G-protein coupled receptors (GPCRs) are of high interest for the development of new therapeutics (Burford et al., 2011; Keov et al., 2011) and the family of

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noncompetitive ligands for metabotropic glutamate receptors (mGluR1-8 homodimers, group C GPCR) is among the best studied because of the role played by mGluRs in psychiatric and neurological disorders (Ellaithy et al., 2015; Gregory and Conn, 2015; Sheffler et al., 2011a;

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Vinson and Conn, 2012).

Metabotropic glutamate receptors exhibit a large extracellular N-terminal domain, containing

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the orthosteric binding site for glutamate in a Venus flytrap (VFT) module. In presence of the endogenous ligand glutamate the VFT domains of both mGlu2 monomers closes into a transiently stable conformation. This conformational change(s) is then transmitted to the cysteine rich region and to the 7TM part with a unique mechanism that only now starts to be defined in details (Kunishima et al., 2000; Kniazeff et al., 2011; Rondard and Pin, 2015; Wu

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et al., 2014; Zhang and Jang, 2014).

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The 7TM domain of mGluRs share common topology with the 7TM spanning α-helices in

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Class A GPCRs, in particular helices I to IV (Congreve et al., 2011; Wu et al., 2014). Several studies have also shown the relative relevance of homology models for other TM regions,

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which are part of the binding sites of negative allosteric modulators (NAMs) in mGlu1 (Malherbe et al., 2003a; Wu et al., 2014), mGlu5 (Malherbe et al., 2006) and mGlu2 (Lundström et al., 2011), respectively. Crystal data have then finally demonstrated that group A GPCR templates can provide structural information relative to the NAM binding mode within the TM region of group C GPCRs (Jazayeri and Marshall, 2015; Kratochwil et al., 2011). When it comes to the definition of the mode of action of positive allosteric modulators (PAMs) however, the available models are less refined and still not supported by a sufficient amount of structural data. Moreover experimental observation is made mostly with mGlu1/5

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ACCEPTED MANUSCRIPT ligands and possibly less relevant for group II or group III mGluRs (Gregory et al., 2011; Gregory et al. 2014; Hemstapat et al., 2007; Schaffhauser et al., 2003; Rowe et al., 2008, Poutiainen et al., 2015). Only just recently Farinha et al. (2015) have provided an extensive site directed mutagenesis study that, using as many as nine different mGlu2 PAMs, describes

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new key residues relevant for the positive allosteric modulation of the human mGlu2 receptor. These residues, in TM3 (R635, L639, F643), TM5 (L732) and TM6 (W773, F776) are also mapped in the 3D model with the help of the recent X-ray mGlu1 NAM crystallographic data

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(Wu et al., 2014). Of relevance is also the observation made by Farinha et al. (2015) that some of the mutations in TM3-TM6 are impacting in different manner the function of different

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mGlu2 PAMs.

This means that, while in the past decade mGlu2 PAM drug discovery was based mainly on chemical optimization (Sheffler et al., 2011b) based on shape and electrostatic similarities (Tresadern et al., 2010) and affinity studies in absence of the mGlu2 VFT domains (Schann et

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al., 2010), now new molecular data will possibly guide this process, thanks to a better

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structural understanding of PAMs effects on the mGluII effector systems (Dhanya et al., 2014). New mGlu2, mGlu3 or mGlu2/3 PAM agents are in fact still needed to solve some of the

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current limitations in drug development, like a better definition of the best experimental settings vs efficacy in native systems (Bertekap et al., 2015) or even a better understanding of

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receptor occupancy in clinical trials (Fuchigami et al., 2015). The study of the molecular determinant of mGlu2 NAM binding has in a way contributed to this progress, since it has partially clarified the relative position of residues important for PAM and NAM binding within the 7TM of mGlu2 and has shown the importance of combining radioligand binding studies at the orthosteric and allosteric sites and multiple functional measures to obtain a complete mechanistic characterization of allosterism at mGlu2 (Lundström et al., 2011). Moreover this approach has highlighted the peculiarities of NAM binding to the mGlu2 TM allosteric binding pocket versus mGlu5 TM allosteric sites 5

ACCEPTED MANUSCRIPT (Malherbe et al., 2006). Hereby a similar multidimensional analysis is applied to the binding features of mGlu2 PAMs, using a 3D homology model that includes the available crystallographic information related to receptor activated state (Nicoletti et al., 2011) and mGlu3 / mGlu5 alignments as previously described (Lundström et al., 2011). PAMs are then

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manually docked into the TM allosteric binding pocket cavity in order to achieve the best complementarily to the binding site in terms of shape and electrostatic properties. The main aim of this new pharmacology and molecular study of mGlu2 PAM ligands is to improve the

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current understanding of PAM effects on cross-talk events between allosteric / ortosteric sites, an element which are still lacking proper structural analysis and could be important for the

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whole group C GPCRs.

Methods

Materials. L-glutamate, (1S,2R,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid

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(LY354740, Figure 1a; Schoepp et al., 1997); (2S)-2-Amino-2-((1S,2S)-2-carboxycycloprop-

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1-yl)-3-(9-xanthyl) propanoic acid disodium salt (LY351495; Kingston et al.,1998); (rac)methyl-4-carboxyphenylglycine (MCPG), (2S,2'R,3'R)-2-(2',3'-Dicarboxycyclopropyl)glycine and

2,2,2-Trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-

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(DCG-IV)

pyridinylmethyl)ethanesulfonamide hydrochloride (LY487379, Figure 1b) are purchased from Cookson

(Bristol,

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Tocris

U.K.).

1-Butyl-4-[4-(2,6-dimethyl-pyridin-3-yloxy)-3-fluoro-

phenyl]-2-oxo-1,2-dihydro-pyridine-3-carbonitrile (PAM-1, Compound 22, Figure 1c) (CidNunez et al., 2008; Fraley, 2009); 2,2,2-Trifluoroethyl [3-(1-methyl-butoxy)-phenyl]pyridine-3-ylmethyl-sulfonamide (2,2,2-TEMPS, Figure 1d) (Barda et al., 2004); 3'-(8Methyl-4-oxo-7-trifluoromethyl-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl)-biphenyl-3sulfonic acid (RO5488608, Figure 1f) (Lundstrom et al., 2011) and the mGlu3 selective NAM [(S)-1-(5-Chloro-pyrimidin-2-yl)-pyrrolidin-3-yl]-(2,4-dichloro-benzyl)-amine (Britton et al., 2011) are synthesized at F. Hoffmann La Roche. The radioligands 2,2,2-Trifluoro6

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acid

[3-(3,4-ditritio-1-methyl-butoxy)-phenyl]-pyridin-3-ylmethyl-amide,

(3[H]-2,2,2-TEMPS, Figure 1e) (specific activity 92 Ci/mmol), 3[H]-LY354740 (specific activity 43 Ci/mmol) (Schaffhauser et al., 1998) and 3[H]-HYDIA (specific activity 21.9 Ci/mmol) (Lundström et al. 2009) and not radiolabeled HYDIA (1S,2R,3R,5R,6S)-2-amino-

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3-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid) are synthesized at F. Hoffmann La Roche by Drs. D. Muri, P. Huguenin, J. Wichmann, T. Woltering and H. Stadler (with a radiochemical purity >95%). With the exception of LY354740 and L-glutamate, all

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compounds are dissolved in DMSO before dilution in assay buffer. Final concentration of DMSO (% v/v) used in each assay is: 2.7 % in 3[H]- 2,2,2-TEMPS, binding and 35[S]-GTPγS

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binding, 2% in 3[H]-LY354740 binding and 0.1% in Ca2+ release measured by FLIPR, respectively.

Plasmids, cell cultures and membrane preparation

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Cell cultures of CHO mGlu2 cells permanently expressing rat mGlu2 (Flp-in T-rex Gα16)

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HEK-293 mGlu3 cells with tetracycline inducible expression of human mGlu3 and of HEK293 and CHO-Gα16 cells are carried out as previously described (Lundström et al., 2011).

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All point mutations are constructed using the QuickChange Lightening Site-Directed Mutagenesis Kit (catalogue no. 210519, Stratagene, La Jolla, CA). For cell membrane

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preparation, ice-cold 20 mM HEPES buffers containing either 10 mM or 0.1 mM EDTA pH 7.4 are used. Membranes are resuspended in ice-cold 20 mM HEPES buffer containing 0.1 mM EDTA pH 7 and frozen at -80 °C before use. Western Blot analysis is carried out for all membrane preparations

(including transient transfections and site directed mutagenesis

studies) using a polyclonal rabbit anti rat mGlu2 antibody (Millipore, 1:1000 dilution in blocking buffer) and a polyclonal rabbit anti human mGlu3 (Genway Biotech, CA- cat n.18461182 dil. 1:500) as previously described (Lundström et al., 2011).

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ACCEPTED MANUSCRIPT Assays 3

[H]-LY354740 mGlu2/3 agonist and 3[H]-2,2,2-TEMPS mGlu2 PAM binding in equilibrium

conditions were also described previously (Lundstöm et al., 2011). In brief 3[H]-LY354740 saturation isothermic studies are performed in a total reaction volume of 1 ml for 3 h

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incubation at RT. Binding inhibition is determined in the presence of 10 nM 3[H]-LY354740, with various concentrations of inhibitory compounds in a total reaction volume of 1 ml for 1 h incubation at RT. Nonspecific binding is measured in the presence of 10 µM DCG-IV.

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(Schaffhauser et al., 1998 ). The radioactivity on the filters is measured by liquid scintillation on a beta counter in the presence of Ultima-gold (Canberra Packard SA, Zürich, Switzerland). [H]-2,2,2-TEMPS binding membrane homogenates are resuspended in buffer (20 mM

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HEPES and 2mM MgCl2 pH 7.4) to a final concentration of 10 µg per well and preincubated with polylysine Coated Yttrium Silicate SPA beads (0.5mg/well) (catalogue no. RPNQ0010, Perkin Elmer) at RT. Displacement studies are carried out in presence of 3 nM 3[H]- 2,2,2-

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TEMPS, with various concentrations of inhibitors in a total reaction volume of 180 µl (1 h

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incubation at RT). Kinetic studies are carried out for both radioligands to make sure equilibrium conditions were obtained. Binding data are analyzed by Prism 5.0 (GraphPad

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Software, San Diego, CA USA), using equations Y= Bmax × X/(Kd + X) and Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*Hill Slope)), respectively. Ki values are calculated from

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the IC50 values using the Cheng Prusoff equation. The 35[S]-GTPγS binding assay is performed using CHO mGlu2 cell membranes according to (Schaffhauser et al., 1998. Briefly, the effect of increasing concentrations of agonist is measured using 10 µg membrane protein in the presence of 2 µM GDP, 1 mg of Weatgerm Agglutinin SPA beads (GE Healthcare, Buckinghamshire, UK) and 0.3 nM

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[S]-GTPγS

(Perkin Elmer, Waltham, MA USA) in assay buffer containing 20 mM HEPES, 100 mM NaCl and 10 mM MgCl2. The reaction is carried out for 1h under shaking and beads were allowed to settle for 1 hour before counting on a Top-count (Packard, Zürich, Switzerland) 8

ACCEPTED MANUSCRIPT with quench correction. The effect of mGlu2 PAMs, LY487379 and PAM-1, is measured on a sub-maximal concentration of LY354740 (2 nM) and expressed as % of maximum response of LY354740 in each experiment. Intracellular Ca2+ release measurements are described in Lundström et al., 2011). CHO-Gα16

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cells grown to 80% confluence were transfected with either WT or mutant mGlu 2 receptor plasmids. The allosteric modulators are applied 10 min prior the application of agonist.

The ability of PAMs to potentiate the agonist response at mGlu2 is determined on a sub-

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maximal concentration of LY354740 agonist which induced approximately 20 % of the maximum response of the agonist. On the mGlu2 mutant receptors the EC20 concentration of

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the agonist was individually determined when the agonist max response showed a deviation larger than 20% .

The intracellular Ca2+ release responses were measured as peak increase in fluorescence minus basal, normalized to the maximal stimulatory effect induced by 10 µM LY354740 in

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each plate. Potentiating curves of agonist and PAMs were fitted using Prism 5.0 (GraphPad

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Software, San Diego CA), equation Y=Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*Hill Slope)). The relative efficacy (Emax) values of LY354740 were calculated as fitted maximum

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of the dose-response of each mutated receptor expressed as a percentage of fitted maximum of

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the WT dose–response curve from cells transfected and assayed on the same day.

Alignment and building of mGlu2 homology model The amino acid sequences of rat mGlu2 (accession number: P31421), rat mGlu3 (accession number: P31422), and rat mGlu5 (accession number: P31424) are aligned to each other and to the bovine rhodopsin receptor (accession number: P02699) in its active conformation (pdb code 3pqr, (Nicoletti et al., 2011)) as also previously described (Lundström et al., 2011). In summary a slow pairwise alignment using the BLOSUM matrix series and a gap opening penalty of 15.0 were chosen for aligning the amino acid sequences. The aligned mGluR 9

ACCEPTED MANUSCRIPT sequences were then manually aligned against the sequence of the human β2-adrenergic receptor (accession number: P07550) such that the alignment of class A and class C receptors was reproduced as previously published (Bissantz, Logean et al. 2004). Using this alignment and the X-ray structure of the human β2-adrenergic receptor (pdb code

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2rh1, (Cherezov, Rosenbaum et al. 2007)) and of the bovine rhodopsin receptor as template, the software package MOE (MOE v.2009.10, Chemical Computing Group, Montreal, Quebec, Canada) was used to generate a three-dimensional model of the rat mGlu2. The structural

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model provided by Farinha is also referred to during the discussion (Farinha et al., 2015).

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Results Affinity studies

[H]-LY354740 is a high affinity ligand (Kd 9.1 nM) and potent full agonist at the rat mGlu2 (EC50 7-15 nM in different cellular assays) (Lundstrom et al. 2011 Schoepp et al. 1998,

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Schwitzer et al. 2000). In 3[H]-LY354740 agonist binding studies, increasing concentrations

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of all mGlu2 PAMs enhance the specific binding of 10 nM 3[H]-LY354740 agonist at the orthosteric site when determined on CHO mGlu2 membranes (Figure 2a). LY487379 has an

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EC50 value of 107 nM and reaches 185 % of the 3[H]-LY354740 specific binding, confirming previous data using 3[H]-DCG-IV (Schaffhauser et al. 2003; Lavreysen et al., 2013). 2,2,2-

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TEMPS has an EC50 value of 9.1 nM also consistent with previous pharmacological studies (EC50 14 nM

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[S]-GTPγS). An increase of 3[H]-LY354740 specific binding to 214 % is

observed with this agent. On the other hand, PAM-1 which is structurally different from LY487379 and 2,2,2-TEMPS increases the specific binding to 363 % at 10 µM, without reaching a plateau at the highest concentration tested in the dose response curve, and the predicted EC50 value in this assay is 791 nM (Figure 2a, Table 1).

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ACCEPTED MANUSCRIPT None of the tested mGlu2 PAMs or NAMs described in this study affect the specific binding of the competitive antagonist 3[H]-HYDIA (50 nM) at concentrations up to 10 µM (data not shown). The subtype selectivity of the utilized mGlu2 PAMs is confirmed by the lack of effect in the [H]-LY354740 binding assay on HEK-293 mGlu3 cell membranes.

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In order to better understand how mGlu2 PAMs influence agonist binding at the orthosteric site, the 3[H]-LY354740 saturation isotherm is determined in the presence of LY487379 or

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PAM-1. The presence of 1 µM LY487379 increases the affinity (Kd) of 3[H]-LY354740 from 9.1 to 6.8 nM, confirming previous data (Schaffhauser et al. 2003; Lavreysen et al. 2013) but

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in contrast to the same reports the presence of 1µM LY487379 also increases the number of binding sites (Bmax) from 17245 to 29175 fmol/mg (Figure 2b). With similar effect, the presence of 300 nM PAM-1 increases the affinity of 3[H]-LY354740 to 6.5 nM and the Bmax to 24913 fmol/mg (Figure 2b). The effect is concentration dependent (Fig.2c).

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In equilibrium binding studies 3[H]-2,2,2-TEMPS, displays high affinity for the mGlu2

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receptor with a Kd value of 4.2 nM. 3[H]- 2,2,2-TEMPS, binding at mGlu2 is not sensitive to temperature when tested at RT and 4˚C. Complete displacement of 3[H]- 2,2,2-TEMPS can be

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observed with unlabeled 2,2,2-TEMPS, PAM-1 and LY487379 with IC50 values of 5.5, 42.8, and 492.2 nM, respectively (Figure 3b, Table 1). In addition, complete inhibition of the 3[H]-

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2,2,2-TEMPS binding from its allosteric site is obtained with two high affinity mGlu2 NAMs, RO4988546 and RO5488608 (Lundstrom et al. 2011). When comparing the number of binding sites (Bmax) recognized by the orthosteric 3[H]LY354740 agonist and the allosteric 3[H]- 2,2,2-TEMPS ligand on membrane preparations of CHO mGlu2 expressing cells it becomes evident that the two ligands recognize the receptor differently. A ratio of the Bmax values between the 3[H]-LY354740 and the 3[H]- 2,2,2TEMPS ligand of 2.6 ± 0.3 indicate the 3[H]- 2,2,2-TEMPS ligand to only recognize 40 % of

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ACCEPTED MANUSCRIPT the binding sites as does the 3[H]-LY354740 ligand as previously reported (Lundstrom et al. 2011) (see Table S1 supplementary material). The modulatory interaction seen between the orthosteric and allosteric binding sites in the 3

[H]-LY354740 binding assay (Figure 2) is also observed in the 3[H]-2,2,2-TEMPS binding

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assay where a significant increase in specific binding is observed with increasing concentrations of the receptor agonists LY354740 and L-glutamate (Figure 3c). In contrast, mGlu2 competitive antagonists like LY341495 and HYDIA are not able to influence the

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allosteric 3[H]-2,2,2-TEMPS binding. Taken together, this data confirm a modulatory interaction between the orthosteric and the allosteric binding sites in mGlu 2 when occupied

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with agonist and potentiating modulator, respectively.

Functional studies

To characterize and compare allosteric potentiators, their EC50 value is measured as the

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concentration of the potentiator required to achieve 50% of the maximal agonist response in a

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functional assay when determined on a sub-maximal concentration of agonist (Figure 4a). The calculated EC50 value for the dose response curves of LY487379 and PAM-1 on the

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mGlu2 receptor is 395 and 144 nM, respectively showing good correspondence with previous reported data (Fraley, 2009 (Cid-Nunez et al., 2008; Johnson et al., 2003). The presence of

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LY487379 (0.3, 1 or 3 µM) or PAM-1 (0.1, 0.3 or 1 µM) increases the potency of the LY354740 dose-response curve in the Ca2+ release assay (Figure 4b-c, Table 2S Supplementary Material). A slight increase in the maximum LY354740 response could be seen with the lowest concentration of LY487379 (0.3µM) while a more pronounced increase was observed at the two lower concentrations of PAM-1, reaching 126-133 % of the maximum LY354740 agonist response. As previously reported (Barda et al., 2004), 2,2,2TEMPS has an increased PAM potency at the mGlu2 receptor as compared to LY487379 and

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ACCEPTED MANUSCRIPT when determined in the 35[S]-GTPγS binding assay 2,2,2-TEMPS has an EC50 value of 45 nM compared to 368 nM for LY487379 (Figure S3 Supplementary Material, Table 1).

Site directed mutagenesis study

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In order to characterize the allosteric binding site of the two mGlu2 PAMs LY487379 and PAM-1, their ability to potentiate the agonist response is determined on a set of mGlu2 mutants in the intracellular Ca2+ release assay. The EC50 of the PAM potentiation is

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determined on a sub-maximal concentration of the LY354740 agonist and a potentiating effect is considered present when, in a concentration dependent manner, the maximum

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response reaches higher than 50% of the maximal LY354740 effect. On the receptor mutants where the maximal response of LY354740 is at least 20% lower than in WT transfections an effect is consider present when the potentiation is larger than 30%. On five of the mGlu2 mutants both LY487379 and PAM-1 are not able to generate a dose

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dependent potentiation of the LY354740 induced response in intracellular Ca2 release: F643A

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(Phe3.36), Y647V (Tyr3.40), L732A (Leu5.43), N735D (Asp5.46) and W773A (Trp6.48) (Table 2; Figure 5a-e). The LY354740 agonist effect is also slightly reduced on the same mutants, with

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the exception of L732A (Leu5.43) mutant which shows enhanced agonist potency when compared to WT. The lack of PAM effect of these receptor mutants in the intracellular Ca2+

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release assay was also confirmed afterwards in the functional 3[H]-LY354740 agonist binding assay as well as in the 3[H]-PAM affinity binding assays (table 2). While the receptor function of LY487379 and PAM-1 is completely abolished on the W773A (Trp6.48) mutant, the presence of an aromatic residue at this position, W773F (Trp6.48), rescues the ability for PAM potentiation, resulting in a 2.4 and 3.3 fold reduction in potency (Figure 6a, Table 2). Effects of mutations in TM2: both PAMs show reduced potencies on the T620A (Thr2.61) mutant, 2.1 and 3.2-fold on LY487379 and PAM-1 respectively (Figure 6d, Table 2). In both cases the reduced PAM potentiation is accompanied by a reduced LY354740 agonist potency 13

ACCEPTED MANUSCRIPT of 2.2 fold (Table 2) which indicates this residue to be important for the overall receptor activation induced by both agonist and PAM stimulation. Effects of mutations in TM3: F643A (Phe3.36) and Y647V (Tyr3.40) show complete loss of PAM function. LY487379 shows a moderate reduction in PAM potentiation on the L639A

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(Leu3.32), S644A (Ser3.37) whereas no reduction in potency was observed for PAM-1 on these receptor mutants (Table 2). Looking at the molecular model LY487379 is positioned close to these residues, allowing probably interactions that cannot take place with the PAM-1 ligand

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(Figure 7).

Effect of mutations in TM5: on the M728A (Met5.39) receptor mutant the LY354740 agonist

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response is in line with the one of mGlu2 WT, but for both LY487379 and PAM-1 we see an increase in potency of 0.2 and 0.4 fold, respectively (Table 2). On the S731A (Ser5.42) receptor mutant, LY487379 shows a reduction in potency of 6 fold whereas only a minor reduction of 1.4 fold is observed for PAM-1 (Figure 6b, Table 2).

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TM6 and TM7: an interesting observation is the slightly enhanced potency of the LY354740

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agonist on the W773F (Trp6.48) mutant (Table 2, Figure 6), a mechanism that does not require direct interaction with the residue but entail the conformational change of the TM domain

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necessary for receptor activation. In the functional potentiation measurements of the agonist induced Ca2+ release, both LY487379 and PAM-1 show 10.4 fold reduced potency when

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mutating Val7.43 (Figure 6c, Table 2). On V798A (Val7.43) receptor mutants the reduced PAM potentiation is accompanied by a reduced LY354740 agonist potency of 3.2 fold (Table 2) which indicates this residue to be important for the overall receptor activation induced by both agonist and PAM stimulation. LY487379 shows a moderate reduction in PAM potentiation on T769S (Thr6.44) and S797A (Ser7.42) receptor mutants ranging between 2 and 3.3-fold, whereas no reduction in potency is observed for PAM-1 on these receptor mutants (Table 2). In contrast, PAM-1 shows an increase in potency of 0.3 fold on the T769S (Thr6.44) receptor mutant, although only reaching 60 % of the maximal agonist response (Table 2). Also on the 14

ACCEPTED MANUSCRIPT L777A (Leu6.52) receptor mutant LY487379 showed a clear enhancement in potency of 0.2 fold compared to WT which is accompanied by a 0.6 fold increase in the LY354740 agonist response (Table 2). The most important mutations characterized in the present study as

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relevant for PAM function are conserved between rat and human species (data not shown).

Discussion

The potent mGlu2 agonist LY354740 (Figure 1a) and the endogenous agonist L-glutamate are

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the reference probes for mGlu2 molecular studies (Farinha et al. 2015; Lundstrom et al. 2011). LY354740 (3[H]-LY354740) is a high affinity ligand (Kd 9.1 nM) and potent full agonist at

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the rat mGlu2 (EC50 7-15 nM in different cellular assays) (Schaffhauser et al.1998; Kratochwil et al. 2005; Lundstrom et al. 2011). The main advantage associated with the use of LY354740, vs the endogenous agonist glutamate, is the linear relationship between receptor occupancy and cellular effect in the described experimental conditions (Schaffhauser et al. 1998). This is

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making possible to compare data obtained in affinity and functional studies and it is possibly

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limiting the impact of variability in receptor reserve across the different preparations. The pyridylmethylsulfonamide LY487379 (EC50 270 nM, compound 3 in Johnson et al., 2003.

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(Figure 1b) (Sheffler et al., 2011b) is selected as representative of a sulphonamide series of mGlu2 PAM with similar pharmacological properties Other mGlu2 PAMs series like

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oxazolidinone (Duplantier et al., 2009), dihydroisoindolinones and pyridones (Cid-Nunez et al., 2008) that seem to share a common pharmacophore (Tresadern et al., 2010) are represented by PAM-1 (Fraley, 2009, compound 22; Figure 1c) from the pyridone series. This compound was chosen for this study because of its high potency as PAM at the mGlu2 receptor (GTPγS EC50 138 nM). 2,2,2-TEMPS (Figure 1d), from the class of pyridylmethylsulfonamides, has been presented as a mGlu2 PAM with higher potency than LY487379 (Barda et al., 2004) and for this reason 2,2,2-TEMPS is utilized as reference and to develop a new high affinity radioligand that binds 15

ACCEPTED MANUSCRIPT to the mGlu2 allosteric site. Several other mGlu2 PAM compounds, beside those mentioned in this study, were extensively characterized in previous publications and are reviewed in Trabanco et al. 2013. The two PAMs chosen for the mutagenesis study were selected based on receptor potency and structural diversity (compare Figure 1b and 1c) with the anticipation that

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these criteria would provide the best possible information about the allosteric binding site. mGlu2 NAMs are also relevant to study the molecular mechanisms of PAM interaction with the mGlu2 receptor. In particular the mGlu2/3 NAM RO5488608 (Figure 1f) has been

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previously characterized as the best pharmacological tool for the determination of nonspecific binding in 3[H]- 2,2,2-TEMPS, binding studies thanks to the high solubility of this

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compound and to the high affinity for the allosteric mGlu2 TM binding site (Ki 1.9 nM)(Lundstrom et al., 2011).

A ratio of the Bmax values between the 3[H]-LY354740 and the 3[H]- 2,2,2-TEMPS, ligand on 2.6 ± 0.3 indicate the 3[H]- 2,2,2-TEMPS ligand to only recognize 40 % of the binding sites as

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does the 3[H]-LY354740 ligand. The same observation was made by Lavreysen et al (2013)

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using different radioligands. In contrast to mGluR NAMs, it has been shown that only one of the allosteric binding sites in the mGluR dimer needs to bind the PAM ligand in order to

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achieve maximal receptor potentiation (Goudet et al., 2005). Hence, only one TM domain is in its active conformation state. A study providing structural insights on the interface between

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mGluRs monomers during receptor activation has been recently published by Xue et al (2015). Some of those considerations in particular the role played by TM4,5 in the inactive state and TM6 in the active state could be relevant to understand cooperativity events between the two homodimers. 3

[H]-HYDIA saturation experiments detected a number of binding sites significantly higher

than those detected by 3[H]-LY354740 in the different membrane preparations (data not shown, observation also previously reported in Lundstrom et al. 2009) consistent with the bona fide antagonist nature of this radioligand and also suggesting that this radioligand could 16

ACCEPTED MANUSCRIPT access a portion of receptors not detected by the other two radioligands.. Moreover specific binding of this radioligand could not be displaced by allosteric agents either PAM or NAM confirming the separation of allosteric and orthosteric binding sites (see also Lunstrom et al., 2011). Of relevance to this study is also the observation that non radiolabeled HYDIA could

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clearly displace 3[H]-LY354740 but not 3[H]- 2,2,2-TEMPS. This means that in the absence of an agonist, the molecular requirements sustaining the affinity of 3[H]- 2,2,2-TEMPS for the receptor are not affected by the presence of a competitive antagonist.

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The situation is however different when 3[H]-LY354740 agonist is used as radioligand and mechanisms of functional binding inhibition or increase can be triggered by allosteric agents

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(Lundstrom et al.,2011).

Affinity studies are overall consistent with functional data when it comes to the general description of the pharmacological properties of the characterized mGlu2 PAMs, and also consistent with literature (Dumboos et al., 2016). With the remarkable difference of the

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observed increase in total number of binding sites in saturation studies with 3[H]-LY354740

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(Fig. 2 b). This difference is possibly partially related to the experimental conditions used in the present study (high level of expression of recombinant mGlu2) and it is reminiscent of a

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similar trend observed with Ro 01-7476 and Ro 01-6128 enhancers at the mGlu1 receptor (Knoflach et al., 2001). Interesting in this sense is also the recent report on mGlu4 PAM of

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Poutiainen et al. (2015) that suggests cooperativity between allosteric and orthosteric sire in this receptor and highlights the importance of affinity studies to characterize this property of specific PAM ligands.

Bmax changes of the magnitude observed in saturation binding studies when in presence of 3

[H]-LY354740 and could be explained suggesting the engagement of a population of

receptors not otherwise accessible for the radioligands

3

[H]- 2,2,2-TEMPS in this

experimental preparations. This study has been carried out at RT for consistency reasons but

17

ACCEPTED MANUSCRIPT temperature dependence of the effect on Bmax should be further investigated to address this point (Dor’ et al., 2014). The mGlu2 PAMs properties were extensively analyzed for the two series across functional assays (Table 1). PAM-1 shows an enhanced potency to increase the agonist maximum

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response compared to LY487379 in both mGlu2 functional agonist binding and intracellular Ca2+ release. This is possibly an interesting pharmacological difference between the two PAMs and an explanation could be found in their specific interaction in the allosteric binding

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cavity of the receptor.

The site directed mutagenesis strategy was defined with the help of previous observations.

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In TM3: Phe3.36 is identified as a crucial residue for PAM-enhanced functional response of the mGlu2 receptor. The same residue is reported to be crucial for NAM inhibition of mGlu2 receptor function (Lundström et al., 2011) indicating this residue to be a central interaction point in the mGlu2 allosteric binding cavity. Interestingly, the corresponding residue is not

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found important for allosteric potentation of mGlu5 but for allosteric inhibition (Lundström et

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al., 2011; Muhlemann et al., 2006). Although not directly interacting with the mGlu2 PAMs, the Tyr3.40 residue is crucial for PAM potentiation of the receptor response, most likely

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because of its important aromatic interaction with Trp6.48. Thus, the loss of function of the Trp6.48 and Tyr3.40 mutations is probably due to an indirect effect by affecting the activation

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mechanism.. Trp6.48 is in fact widely known to be crucial for GPCR activation thus, interactions affecting this residue, directly or indirectly, are important for the functional response that the receptor can produce (Gregory et al., 2011). In the mGlu2 NAM model this residue is located too distant to form a direct interaction with the ligands and its influence on the NAM inhibition of receptor function was minor (Lundström et al., 2011); on the other hand the corresponding residue is found to be of great importance for mGlu5 NAM function (Malherbe et al., 2006).

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ACCEPTED MANUSCRIPT In TM5: the highly conserved Leu5.43 residue (referred to as Leu5.47 in previously published mGluR alignments) is identified as a strategic gating residue for positive and negative allosteric modulation of mGluRs, being important for both PAM and NAM function in mGlu1 and mGlu5 and for NAM function in mGlu2 (Lundström et al., 2011 ; Malherbe et al., 2003a;

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Malherbe et al., 2003b; Muhlemann et al., 2006). This important function of the Leu5.43 is confirmed also for the modulation of PAM function of mGlu2. The Asn5.46 residue in TM5 is important for LY487379 PAM function in the study by Schaffhauser et al. (2003), where it

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was targeted because of its lack of conservation within the Group II mGluR family (See Figure 3S). In Schaffhauser et al. 2003 the authors show that Ser

4.44

and/or Gly 4.45 in TM4

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and Asn5.46 (D735) in TM5 are important for LY487379 potentiation of the mGlu2 receptor. However, when building the homology model of mGlu2 it is obvious that the adjacent residues Ser4.44 and Gly4.45 are located outside the characterized binding cavity of class A GPCRs while the Asn5.46 in TM5 is positioned inside the cavity. In the β2-adrenergic receptor

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three serine residues in TM5 (located at position 5.42, 5.43 and 5.46) are completely

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conserved and have been identified as crucial interaction points for agonist function (Liapakis et al., 2000). The same positions in TM5 are of importance for mGlu2 PAM function, Ser5.42,

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Leu5.43 and Asn5.46. In addition, Leu5.43 and Asn5.46 have previously been shown important for mGlu2 NAM function (Lundström et al., 2011).

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The impact of two mutations in TM5: S371A (Ser5.42) and TM6 W773F (Trp6.48) on the functional response when in presence of the lower range of concentrations of the 2 PAMs is shown in Figure 6. Slight agonistic effect of the PAM compounds alone was detected (data not shown), but the time course of the determination of the PAM effect was sufficient to separate the event. Interestingly in the case of both mutations an increase in threshold receptor activation could potentially be envisaged (De Vree et al., 2016) but no data where obtained in the context of the present study to address this point.f

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ACCEPTED MANUSCRIPT A total of 28 mutants are used for the present analysis of 2 mGlu2 PAMs. This strategy is generally in agreement to the one used by Farinha et al. (2015) with quite a few complementary elements (Table 2). The results of the study are also generally in agreement with the observation made by Farinha et al (2015) with possibly one exception: the mutation

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R635A in TM3.

In details, in TM3: when docking LY487379 and PAM-1 into the binding cavity, residue F643A (Phe3.36) is located within Van der Waals distance to the core phenyl ring of the

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diphenylether moiety present in both PAMs, possibly having a direct interaction with both aromatic rings of this substructure of the PAMs (Figure 7) (Farinha et al. 2015). Although not

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directly interacting with the mGlu2 PAMs, the Tyr3.40 residue is instead crucial for PAM potentiation of the receptor response, because of its important aromatic interaction with Trp6.48. Thus, the loss of function of the Trp6.48 and Tyr3.40 mutations is probably due to an indirect effect by affecting the activation mechanism rather than a direct protein-ligand

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interaction. L639A (Leu3.32), S644A (Ser3.37) and S797A (Ser7.42) are located close to

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LY487379 but not to PAM-1, explaining why these mutations impact only LY487379 potentiation (Figure 7a). The mutation R635A is not affecting the response of the two PAMs

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studied here while Farinha et al. 2015 have reported a specific effect of this mutation on the function of 3 PAMs namely JNJ40068782; JNJ35814376 and BINA (Farinha et al. 2015).

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This difference clearly highlight the specific compound related elements that define the vectors defining structure activity relationship for mGlu2 PAMs. Mutations in TM4 and ECL2 regions exert no impact on mGlu2 PAM effects. Coming to TM5, 3 mutants exert a major effect on mGlu2 PAMs function. Asn5.46: both LY487379 and PAM-1 are located close to the Asn5.46 in the deeper part of the binding cavity (Figure 7) and introduction of a negatively charged aspartate residue at this position is not tolerated for functional potentiation by either PAM. The reason lies in the high desolvation costs of a negatively charged carboxylic acid which, when located in a binding site, must 20

ACCEPTED MANUSCRIPT either stay solvated or form a salt bridge or hydrogen bonds with another group to avoid high desolvation costs. However, neither can the PAMs form hydrogen bonds with the Asp5.46 residue nor can it stay solvated since the PAMs come close to it when entering the binding cavity (Figure 7). In the binding cavity the methoxy group at the phenyl ring of LY487379 is

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located in the close proximity to Ser5.42 to form an H-bond with this residue while no equivalent interaction can be observed for PAM-1 (Figure 7a). Specific for LY487379, a Hbond interaction is observed with the serine residue at position 5.42 and reduced potency was

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seen on its alanine mutant. In the binding cavity, Leu5.43 is situated above the terminal phenyl (Figure 7a) and pyridine (Figure 7b) ring of the PAMs in a distance of approximately 4.3 Å

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from both PAMs, the optimal geometry for a CH- interaction. With both PAMs we observe a direct interaction with Asn5.46 which could not be formed when the Asp residue is located at this position and this interaction is therefore likely to account for the subtype specificity that the studied PAMs exhibit for mGlu2 versus mGlu3. No other mGlu2 specific residue located

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within the allosteric cavity is found to be crucial for PAM function. Asn5.46 is not found to be

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directly involved in the function of mGlu2 NAMs as these ligands are unable to reach down to the deeper part of the binding pocket where Asn5.46 is located (Lundström et al., 2011). The

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mGlu2 selective NAM RO645229 (Kolczewski et al., 1999) when positioned in the mGlu2 allosteric pocket reaches down close to Asn5.46 and will not accept the presence of a negative

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charge at this position as is the case in mGlu3. In conclusion, interaction with the Asn5.46 residue, located in the deeper part of the binding cavity, is of high importance for mGlu 2 selectivity, independent of functional response (PAM or NAM). TM6: Since the threonine residue can influence the conformation of a helix by breaking its hydrogen bond pattern, its mutation to alanine can indirectly affect PAM binding by changing the helix conformation and thus the shape of the binding site. In the homology model T769S (Thr6.44) is too far away for a direct interaction with the PAMs and its effect can thus only be explained by an indirect effect via its importance in the receptor activation mechanism (Figure 21

ACCEPTED MANUSCRIPT 7). L777A (Leu6.52) is located so that it can make a direct CH-interaction with both ligands, sitting on the side of the phenyl (Figure 7a) and pyridine (Figure 7b) ring of the PAMs. TM7: S797A (Ser7.42) are located close to the 3-pyridyl ring of LY487379 with Ser7.42 being in hydrogen bond distance to the pyridine nitrogen while S644A (Ser3.37) is located close to

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the Methoxy group at the Phenyl- ring (Figure 7a). Both substituents have no equivalent in the PAM-1 compound (compare Figure 7a and b). When docking the two mGlu2 PAMs in the binding cavity the Val7.43 residue comes in close proximity of the pyridine ring of LY487379

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to form a strong interaction (Figure 7a) while the pyridine ring of PAM-1 as well as is n-butyl side chain are located further away (Figure 7b) and can only explain a weak interaction. On

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mutant L777A (Leu) an enhanced potency is seen for both PAMs but also for the LY354740 agonist activation. The Leu6.52 residue is positioned so that it can make a direct CH- interaction with both PAMs in the allosteric site. Removal of the Leu residue is gaining space in the allosteric binding pocket which could result in an easier switch from inactive to active

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state for the receptor. This would then also explain the increasing potency of the LY354740

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agonist acting at the orthosteric binding site. The Leu6.52 residue is positioned at the beginning of TM6 and close to TM5, a region that is highly involved in the movements associated with

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GPCR activation. Despite the large number of mGlu2 receptor mutants studied no residues with specific importance for PAM-1 activity is identified. The LY487379 specific interaction

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points identified (Ser5.42, Leu3.32, Ser3.37 and Ser7.42) are furthermore of weak nature with only 2-6 fold reduction in potency. In contrast, the major five interactions in the allosteric binding pocket (Phe3.36, Tyr3.40, Leu5.43, Asn5.46 and Trp6.48) which are shared by the two PAMs are completely crucial for receptor potentiation. When studying the impact of mGlu2 NAMs on 3[H]-LY354740 agonist binding we could conclude on an increased contribution from interactions in the extracellular regions of the allosteric binding pocket (Lundstrom et al., 2011). As the mGlu2 PAMs bind in the deeper region of the pocket, with little or no interactions in the extracellular area, this mechanism of 22

ACCEPTED MANUSCRIPT action cannot be supported here. With no specific interactions identified for PAM-1 in the mutagenesis study its pronounced effect on the 3[H]-LY354740 specific binding must either come from the combination of minor interactions within the allosteric pocket, that are difficult to detect in studies like this one, or from interactions with residues that were not included in

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our study. Limitations in our current understanding of the PAM interaction with the receptor are also obviously coming from the limited predictivity of the 3D model used in this study. In particular several elements possibly impacting on PAM function are underrepresented in the

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model like: presence of cholesterol molecules (Byrne et al., 2016); presence of multiple active conformational states (Staus et al. 2016), effect of the interaction with the G protein (De Vree

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et al.,2016) and the overall impact of the above in thermodynamic terms (Guarnera and Berezowsky, 2016)

Conclusions

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The late arrival of refined molecular information has somehow limited the possibility to

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differentiate chemical series / clinical candidates in terms of relationship between receptor occupancy and functional effects for quite a number of mGluR positive modulators. Since the

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allosteric binding pocket of the mGlu2 receptor is an attractive drug target, both for NAMs and PAMs, molecular studies on PAM affinity and function are still important to improve the

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development of potent and selective ligands. While summarizing the available information this contribution supports the case of mGlu2 receptors as reference for more general considerations on the presence of some form of cooperativity across allosteric and orthosteric binding sites. The vectors controlling it and the associated SAR are however not completely solved, even using data obtained with different high affinity radioligands. The determinants of PAMs and NAMs interaction in within the mGlu2 transmembrane region are instead more clearly defined as well as the determinants of selectivity vs mGlu3.

23

ACCEPTED MANUSCRIPT Without the ambition of being exhaustive, this study is providing further elements to describe the complexity of mGluR PAM function. These considerations can be relevant for the development of new PET ligands and possibly even for the still incomplete understanding of

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the functional impact of mGluR PAMs on heterodimeric interactions.

24

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Acknowledgements

The authors would like to thanks Dr. P. Huguenin and T. Hartung for the synthesis of tritiated

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radioligands.

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Conflicts of interest: All authors work at F.Hoffmann-La Roche Ltd., Basel, Switzerland

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ACCEPTED MANUSCRIPT Johnson, M. P., Baez, M., Jagdmann, G. E., Jr., Britton, T. C., Large, T. H., Callagaro, D. O., Tizzano, J. P., Monn, J. A., Schoepp, D. D., 2003b. Discovery of allosteric potentiators for the metabotropic glutamate 2 receptor: synthesis and subtype selectivity of N-(4-(2methoxyphenoxy)phenyl)-N-(2,2,2- trifluoroethylsulfonyl)pyrid-3-ylmethylamine. J. Med. Chem. 46, 3189-3192.

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Kunishima, N., Tsuji ,Y., Sato, T., Yamamoto M., Kumasaka, T., Nakanishi,S., Morikawa, H. J. K., 2000. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407, 971-977. Lavreysen, H., Langlois, X., Ahnaou, A., Drinkenburg, W., te Riele, P., Biesmans, I., Van der Linden, I., Peeters, L., Megens, A., Wintmolders, C., Cid, J. M., Trabanco, A. A., Andres, J. I., Dautzenberg, F. M., Lutjens, R., Macdonald, G., Atack, J. R., 2013. Pharmacological characterization of JNJ-40068782, a new potent, selective, and systemically active positive allosteric modulator of the mGlu2 receptor and its radioligand [3H]JNJ-40068782. JPET346, 514-527. Liapakis, G., Ballesteros, J. A., Papachristou, S., Chan, W. C., Chen, X., Javitch, J. A., 2000. The forgotten serine. A critical role for Ser-2035.42 in ligand binding to and activation of the beta 2-adrenergic receptor. J. Biol. Chem. 275, 37779-37788. Lundström, L., Kuhn, B., Beck, J., Borroni, E., Wettstein, J.G., Woltering, T.J., Gatti, S. 28

ACCEPTED MANUSCRIPT 2009. Mutagenesis and molecular modeling of the orthosteric binding site of the mGlu2 receptor determining interactions of the group II receptor antagonist (3)H-HYDIA. ChemMedChem. 4(7):1086-94.

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Lundström, L., Bissantz, C., Beck, J., Wettstein, J., Woltering, T., Wichmann, J., Gatti, S., 2011. Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mechanistic studies with new potent negative allosteric modulators. Br. J. Pharmacol. 164, 521-537. Malherbe, P., Kratochwil, N., Knoflach, F., Zenner, M. T., Kew, J. N., Kratzeisen, C., Maerki, H. P., Adam, G., Mutel, V., 2003a. Mutational analysis and molecular modeling of the allosteric binding site of a novel, selective, noncompetitive antagonist of the metabotropic glutamate 1 receptor. J. Biol. Chem. 278, 8340-8347.

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Malherbe, P., Kratochwil, N., Zenner, M. T., Piussi, J., Diener, C., Kratzeisen, C., Fischer, C., Porter, R. H., 2003b. Mutational analysis and molecular modeling of the binding pocket of the metabotropic glutamate 5 receptor negative modulator 2-methyl-6-(phenylethynyl)-pyridine. Mol. Pharmacol. 64, 823-832. Malherbe, P., Kratochwil, N., Muhlemann, A., Zenner, M. T., Fischer, C., Stahl, M., Gerber, P. R., Jaeschke, G., Porter, R. H., 2006. Comparison of the binding pockets of two chemically unrelated allosteric antagonists of the mGlu5 receptor and identification of crucial residues involved in the inverse agonism of MPEP. J. Neurochem. 98, 601-615.

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Nicoletti, F., Bockaert, J., Collingridge, G. L., Conn, P. J., Ferraguti, F., Schoepp, D. D., Wroblewski, J. T., Pin, J. P., 2011. Metabotropic glutamate receptors: from the workbench to the bedside. Neuropharmacol. 60, 1017-1041.

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Poutiainen, P.; Kil, K.; Zhang, Z.; Kuruppu, D.; Tannous, B.; Brownell, A. 2015 Co-operative binding assay for the characterization of mGlu4 allosteric modulators. Neuropharmacol. 97, 142-148. Rondard, P., Pin, J. P., 2015. Dynamics and modulation of metabotropic glutamate receptors. Curr. Opin. Pharmacol. 20, 95-101. Rowe, B. A., Schaffhauser, H., Morales, S., Lubbers, L. S., Bonnefous, C., Kamenecka, T. M., McQuiston, J., Daggett, L. P., 2008. Transposition of three amino acids transforms the human metabotropic glutamate receptor (mGluR)-3-positive allosteric modulation site to mGluR2, and additional characterization of the mGluR2-positive allosteric modulation site. JPET. 326, 240-251. Schann, S., Mayer, S., Franchet, C., Frauli, M., Steinberg, E., Thomas, M., Baron, L., Neuville, P., 2010. Chemical switch of a metabotropic glutamate receptor 2 silent allosteric modulator into dual metabotropic glutamate receptor 2/3 negative/positive allosteric modulators. J. Med. Chem. 53, 8775-8779.

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ACCEPTED MANUSCRIPT Schaffhauser, H., Richards, J. G., Cartmell, J., Chaboz, S., Kemp, J.A., Klingelschmidt, A., Stadler H., Woltering T., and Mutel V., 1998. In Vitro Binding Characteristics of a New Selective Group IIMetabotropic Glutamate Receptor Radioligand, [3H]LY354740,in Rat Brain. Mol. Pharmacol. 52, 228-233.

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Schaffhauser, H., Morales, S., Chavez-Noriega, L. E., Yin R., Jachec C., Bain G., Pinkerton A.B., Vernier, J., Bristow L.J., and Daggett, L.P. 2003. Pharmacological Characterization and Identification of AminoAcids Involved in the Positive Modulation of MetabotropicGlutamate Receptor Subtype 2. Mol. Pharmacol. 5, 798-810.

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Schoepp, D. D., Johnson, B.G., Wright, R.A., Salhoff, C.R., Mayne, N.G., Wu, S., Cockerman , S.L., Burnett ,J.P,. Belegaje, R., Bleakman, D., Monn ,J.A. 1997. LY354740 is a potent and highly selective group II metabotropic glutamate receptor agonist in cells expressing human glutamate receptors. Neuropharmacol. 36,1-11.

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Schweitzer, C., Kratzeisen, C., Adam, G., Lundstrom, K., Malherbe, P., Ohresser, S., Stadler, H., Wichmann, J., Woltering, T. Mutel, V. 2000Characterization of [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using Semliki Forest virus vectors. Neuropharmacol. 39, 1700-1706. Sheffler, D. J., Gregory, K. J., Rook, J. M., Conn, P. J., 2011a. Allosteric modulation of metabotropic glutamate receptors. Adv. Pharmacol. 62, 37-77. Sheffler, D. J., Pinkerton, A. B., Dahl, R., Markou, A., Cosford, N. D., 2011b. Recent progress in the synthesis and characterization of group II metabotropic glutamate receptor allosteric modulators. ACS Chem Neurosci. 2, 382-393.

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ACCEPTED MANUSCRIPT Xue, L., Rovira, X., Scholler, P., Zhao, H., Liu, J., Pin, J. P., Rondard, P., 2015. Major ligandinduced rearrangement of the heptahelical domain interface in a GPCR dimer. Nature Chem. Biol. 11, 134-140.

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Zhang X.C., Jiang D., 2014. Why is dimerization essential for class-CGPCR function? New insights from mGluR1crystal structure analysis. Protein Cell 5, 492-495.

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ACCEPTED MANUSCRIPT Figure 1. Chemical structures of mGlu2 ligands, agonists and PAM / NAMs characterized in this study. a. LY354740, (1S,2R,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6dicarboxylic acid (Schoepp et al., 1997) mGlu2/3 selective agonist binding in the glutamate binding

pocket

of

the

VFT

domains.

b.

LY487379,

hydrochloride

mGlu2

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methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)ethanesulfonamide

2,2,2-Trifluoro-N-[4-(2-

PAM (Johnson et al, 2003). c. PAM-1, 1-Butyl-4-[4-(2,6-dimethyl-pyridin-3-yloxy)-3-fluorophenyl]-2-oxo-1,2-dihydro-pyridine-3-carbonitrile (Cid-Nunez et al., 2008). Presented as

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compound 22 in Fraley, 2009. d. 2,2,2-TEMPS. 2,2,2-Trifluoroethyl [3-(1-methyl-butoxy)phenyl]-pyridine-3-ylmethyl-sulfonamide Presented as compound 18k in Barda et al., 2004. e. [H]-2,2,2-TEMPS. Specific activity 92 Ci/mmol, radiochemical purity >95%, f. RO5488608.

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3'-(8-Methyl-4-oxo-7-trifluoromethyl-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl)-biphenyl-

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3-sulfonic acid from Lundstrom et al., 2011.

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Figure 2. Effect of mGlu2 PAMs on 3[H]-LY354740 agonist binding (% increase and saturation isotherms). a. By binding to an allosteric binding site LY487379, PAM-1 and 2,2,2-TEMPS concentration dependently increase the 3[H]-LY354740 specific binding (10

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nM) at the orthosteric site of mGlu2 (60 min incubation at RT). The calculated potencies

(EC50), Hill coefficients (nH) and Relative maximum responses (Emax) are shown in Table 1. Data is mean ± SEM of 4 - 5 dose response curves, performed in duplicate using CHO mGlu2

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membranes. b. The isothermal curve of 3[H]-LY354740 (Kd = 9.1 nM and Bmax = 17245 ± 1447 fmol/mg, 3h incubation at RT) show an increased affinity for the orthosteric binding

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site as well as an increased number of binding sites in the presence of a mGlu2 PAM. In the presence of 1µM LY487379 the Kd of the 3[H]-LY354740 ligand is reduced to 6.8 ± 1.0 nM and the Bmax is 29175 ± 1063 fmol/mg. In the presence of 300 nM PAM-1 the Kd of the 3[H]LY354740 ligand is 6.5 ± 0.7 nM and the Bmax is 24913 ± 860 fmol/mg. 3[H]-LY354740

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saturation binding was performed on CHO mGlu2 membranes and non-linear regression is the

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mean ± SEM from 3 separate dose response curves, performed in quadruplicate. c.

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Displacement studies with LY354740 in presence of increasing concentrations of PAM1.

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ACCEPTED MANUSCRIPT Figure 3. Effect of mGlu2 PAMs on 3[H]- 2,2,2-TEMPS, binding at the allosteric binding site of mGlu2. a. The saturation binding isotherm at the allosteric site of mGlu2 shows the 3[H]PAM to be a high affinity ligand, having a Kd of 4.23 ± 1.8 nM and a Bmax of 7828 ± 1757 fmol / mg protein. Data of non-linear regression is mean ± SEM of 6 measurements,

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performed in duplicate, from three separate experiments. b. The mGluR2 PAMs; LY487379, PAM-1 and 2,2,2-TEMPS completely displace 3[H]- 2,2,2-TEMPS, from the allosteric

binding site and the calculated EC50 and nH values are presented in Table 1. Data represents

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the mean ± SEM of 6-10 dose-response curves, performed in duplicate. C. Agonists binding at the orthosteric site of mGluR2 significantly (two-tailed t-test) increase 3[H]- 2,2,2-TEMPS,

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binding at the allosteric site, * p<0.05, ** p< 0.01 compared to the lowest concentration of agonist (100%). Data is expressed as % increase of 3[H]- 2,2,2-TEMPS, specific binding. Data is the mean ± SEM of 3 experiments, each including three measurements performed in

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duplicate. All measurements are made on CHO mGlu2 membranes.

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Table 1. Effect of mGlu2 PAMs on 3[H]-LY354740 agonist binding, LY354740 induced intracellular Ca2+ release, LY354740 induced 35[S]-

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GTPγS binding and 3[H]- 2,2,2-TEMPS, affinity binding. The graphical illustration of the data is shown in Figure 2a, 3a, 4 and 5b, respectively.

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*; The Ki value of LY354740 in the 3[H]-LY354740 binding assay is 19.6 nM, Fig.2c (Lundström et al., 2011), N.d ; Not determine

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Figure 4. Effect of mGlu2 PAMs on agonist induced intracellular Ca2+ release at the mGlu2

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WT receptor. a. When determined on a sub-maximal concentration of the LY354740 agonist, LY487379 and PAM-1 dose-dependently potentiate the functional response, reaching 71-89% of the maximum agonist response. Calculated EC50, nH and relative Emax values are presented in

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Table 1. Data is normalized to the maximum response of LY354740 in each experiment and each curve represents the mean ± SEM of 5-6 dose response curves, performed in duplicate, from 4-6

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independent transfections. b-c. Increasing concentrations of mGlu2 PAMs causes a leftward shift of the LY354740 dose-response curve, increasing the potency and efficacy of the agonist. Calculated EC50, nH and relative Emax values are presented in Table 2. Data is normalized to the maximum response of LY354740 in each experiment and the plotted curves represents the mean ± SEM; LY487379 from 6-7 curves from 5-7 independent transfections, PAM-1 from 4 curves

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from 4 independent transfections and each curve is performed in duplicate. All data is obtained

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from CHO-Gα16 cells transiently transfected with the rat mGlu2 WT receptor.

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Table 2. Impact of mGlu2 mutations on agonist induced intracellular Ca2+ release and on mGlu2 PAM potentiation of agonist induced Ca2+ release. Potency (EC50), and relative maximum response (Emax) for LY487379 and PAM-1 potentiation of LY354740 agonist induced

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Ca2+ release on mGlu2 WT and mutant receptors transiently transfected into CHO-Gα16 cells. Mutations on which the PAM potentiation was markedly reduced compared to mGlu2 WT are indicated in bold. Data are presented as mean ± SEM. Values for LY354740 agonist curves are

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calculated from 4 to 10 dose-response measurements, from 3 to 10 different transfections. Values for PAM potentiation of agonist response for LY487379 are calculated from 4 to 10 dose-

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response measurements, from 3 to 10 different transfections and for PAM-1, 4 to 10 dose-

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response measurements, from 2 to 10 different transfections.

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Figure 5. mGlu2 mutations on which PAM potentiation of the agonist induced intracellular Ca2+ release was completely abolished. The ability of LY487379 and PAM-1 to potentiate a sub-maximal concentration of LY354740 agonist was completely abolished on the F643A3.36,

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Y647V3.40, L732A5.43, N735D5.46 and W773A6.48 mutants while the full dose response curve of the LY354740 agonist only showed minor changed compared to the mGlu2 WT receptor.

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Potencies (EC50) of the LY354740 agonist dose response curve are given in Table S2.

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Figure 6. mGlu2 mutations on which PAM potentiation of the agonist induced intracellular Ca2+ release was affected compared to the mGlu2 WT receptor. The ability of LY487379 and PAM-1 to potentiate a sub-maximal concentration of LY354740 agonist is reduced on the

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W773F6.48, S731A5.42, V798A7.43 and T620A2.61 receptor mutants. The potencies (EC50) for the LY354740 agonist dose response curve and the reduced PAM potentiation by LY487375 and

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PAM-1 are given in Table S2.

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Figure 7. Molecular modeling of mGlu2 PAMs in the characterized allosteric binding pocket of mGlu2. LY487379 (A) and PAM-1 (B) are binding to the allosteric binding pocket of mGlu2, located in between TM2, TM3, TM5, TM6 and TM7. Critical residues identified from PAM

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potentiation of intracellular Ca2+ release are indicated and labeled with Ballesteros-Weinstein

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numbering. Further details on the identified interactions are given in the text.

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Table S1. (Supplementary Material) Bmax values for 3[H]-LY354740 and 3[H]-PAM binding on CHO mGlu2 membranes. Bmax values are calculated from non-linear regression (performed

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in GraphPad Prism) of dose-response curves, performed in quadruplicate, from one homogenous membrane preparation freshly made on the day of experiment, performed on three different batches of membranes. The average ratio between the obtained Bmax values are presented as

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Mean ± SEM.

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Table S2 (Supplementary Material). Effect of the presence of LY487379 and PAM-1 on the LY354740 agonist dose response curve on intracellular Ca2+ release at the mGlu2 WT receptor

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Data represents a set of intracellular Ca2+ release experiments in which the EC50 value for

LY354740 was determined to 6.7 nM when measured from 9 dose response curves, performed in duplicate, from 6 independent transfections. * The increase in maximum response was significant

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in the presence of 0.1µM PAM-1 (p=0.0407) and 0.3 µM PAM-1 (p=0.0368), when determined

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Figure S3. Effect of mGlu2 PAMs on agonist induced 35[S]-GTPγS binding. (Supplementary Material) The LY354740 agonist concentration dependently increase the 35[S]-GTPγS binding

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with an EC50 value of 19.4 nM. Measured on a sub-maximal concentration of LY354740,

LY487379, PAM-1 and 2,2,2-TEMPS concentration dependently increase the agonist response. The calculated EC50 values, hill coefficient (nH) and relative maximum response (Emax) are

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presented in Table 1. The competitive antagonist LY341495 did not affect 35[S]-GTPγS binding in the absence of agonist. Data is normalized to the maximum response of LY354740 in each

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experiment. Data is mean ± SEM of 3 - 4 dose response curves, performed in duplicate, from 3

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Figure S4. .(Supplementary Material) Sequence alignment of the amino acid residues in TM and ECL2 of rat mGlu2 relative to mGlu3 and mGlu5. Residues highlighted in grey are located in the binding pocket of the mGlu2 homology model constructed from the crystal structure of the

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β2-adrenergic receptor (pdb code 2rh1, (Cherezov et al., 2007)). Amino acids targeted by

mutagenesis in this study are underlined and residues identified to be important for PAM binding are shown in bold and tagged with its amino acid number. Residues important for interaction and .

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function of mGlu2 NAMs are indicated with + (Lundström et al., 2011), ; residues conserved

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within Group II mGluRs, * ; residues identical between mGlu2 and Glu5. Critical residues for DFB binding to the allosteric site of mGlu5 are shown in bold (Muhlemann et al., 2006).

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Ballesteros-Weinstein numbering scheme of the mGlu2 residues are shown at the bottom.

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Figure S5 Further details Molecular modeling of two mGlu2 PAMs in the characterized allosteric binding pocket of mGlu2.

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3

Ca2+ release (Figure 3a)

[H]-LY354740 binding (Figure 2a) nH

LY354740

*

-

Specific binding (%) -

EC50 (nM)

nH

9.8 ± 0.6

1.5 ± 0.1

% of max LY354740 response 100

LY487379

107.2 ± 25.6

0.7 ± 0.07

185 ± 11

395 ± 58

1.1 ± 0.16

PAM-1

791 ± 391

1.4 ± 0.1

363 ± 33

144.3 ± 42

2,2,2-TEMPS

9.1 ± 1.2

1.2 ± 0.09

214 ± 12

N.d

nH

19.4 ± 0.7

1.2 ± 0.03

% of max LY354740 response 100

89.3 ± 2.6

367.7 ± 3.9

1.5 ± 0.04

1.7 ± 0.30

79.6 ± 5.9

113.9 ± 32

-

-

44.6 ± 9.4

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[H]-PAM binding (Figure 5b)

EC50 (nM)

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EC50 (nM)

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[S]-GTPγS binding (Figure 4)

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Ki(nM)

nH

N.d

-

86.3 ± 12.4

492.2 ± 74.2

-0.58 ± 0.02

1.1 ± 0.04

86.8 ± 9.8

42.8 ± 13.1

-0.55 ± 0.04

0.8 ± 0.06

106.0 ± 5.7

5.5 ± 0.9

-0.81 ± 0.03

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PAM-1 Fold Emax vs WT 80.3 0.7 69.6 0.7 99.5 3.2 84.2 1.0 77.9 0.5 82.6 1.0 71.0 0.8 77.8 1.6 91.0 1.7 50.0

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EC50 (nM) 144.3 98.7 106.8 464.3 140.4 66.4 148.5 118.4 N.R 224.3 N.R 251.6

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0.99 0.99 0.93 0.78 0.34 1.24 0.80 1.10 0.56 1.33 0.60 1.26 0.84 1.08 1.36 0.86 3.15

218.9 550.1 173.5 2300.8 N.R N.R 692.0 917.6 945.0 N.R 66.6 589.0 473.9 368.4 288 1316 3240

0.6 1.4 0.4 5.8 1.8 2.3 2.4 0.2 1.5 1.2 0.9 0.7 3.3 8.2

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LY487379 EC50 Fold Emax (nM) vs WT 395.1 89.3 487.2 1.2 83.1 396.7 1.0 103.7 822.5 2.1 120.2 366.5 0.9 77.6 99.3 0.3 81.5 1099.8 2.8 70.2 316.1 0.8 75.1 N.R 786.2 2.0 75.0 N.R 603.2 1.5 69.9

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E2.52 E2.52 5.39 5.42 5.43 5.46 5.47 6.44 6.48 6.48 6.52 6.55 7.38 7.39 7.40 7.42 7.43

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WT C616S Y617F T620A R635A R636A L639A A642S F643A S644A Y647V S688LG689V H723F H723V M728A S731A L732A N735D V736A T769S W773F W773A L777A F780A T793A M794A C795A S797S V798A

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LY354740 Position EC50 Fold (nM) vs WT 9.8 2.57 7.8 0.8 2.58 9.2 0.94 2.61 21.8 2.22 3.28 15.5 1.58 3.29 17.6 1.80 3.32 8.1 0.83 3.35 8.6 0.88 3.36 16.4 1.67 3.37 7.5 0.77 3. 40 37.4 3.82 4.44-45 17.3 1.77

82.3 80.9 71.8 91.9 84.4 91.5 79.8 83.6 94.6 78.4 85.3 89.9 88.1 99.4

61.2 192.0 36.03 198.3 N.R N.R 74.1 38.1 473.7 N.R 126.4 73.20 158.3 110.9 290.3 138.1 1499.2

0.4 1.3 0.2 1.4 0.5 0.3 3.3 0.9 0.5 1.1 0.8 2.0 1.0 10.4

71.2 75.0 73.9 85.1 74.3 60.2 86.8 79.2 89.6 79.1 78.1 94.6 127.5 82.8

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A F6.51 T2.61

L6.52

S7.42

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V7.43

W6.48

R3.28

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L3.32

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R3.29

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V7.43

S7.42 F6.51 L6.52 W6.48

R3.28

L5.43

L3.32

R3.29

F3.36

Y3.40

S5.42 N5.46

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ACCEPTED MANUSCRIPT Conflict of interest statement

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All authors were employees of F.Hoffmann La Roche when the study was carried out and at the time of the preparation of this document

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Highlights

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The characterization of the mechanism of action of two positive allosteric modulators of metabotropic receptor 2, using both functional and affinity assays , shows the presence of some level of cooperativity between allosteric and orthosteric sites. A large mutagenesis study completes the current understanding of the key residues relevant for positive allosterism also showing the presence of elements which are compound and/or chemical series related. The combination of affinity and functional studies is proposed as a relevant strategy for the definition of the structural changes associated with receptor activation in presence of a positive allosteric modulators, a more general consideration possibly of relevance for all mGluRs.

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