POSTER ABSTRACT POSTER 01J1

Membrane proteins gene-to-structure pipeline at leadXpro: highlight on the structural design of selective antagonists for CCR2A

R. CHENG, W. ABDUL RAHMAN, N. BOCQUET, M. BOTTE, D. BUCHER, J. EBNER, S. HUBER, E. PLICHTA, S. SCHENK, M. HENNIG

leadXpro AG, PARK INNOVAARE, 5234 Villigen, Switzerland corresponding author : [email protected]

Today, structure-based drug discovery is well implemented in the drug discovery engine of many pharmaceutical companies. Despite their high importance as drug targets, transmembrane proteins such as GPCRs, ion-channels and transporters remain to be challenging for structure determination. LeadXpro combines excellence in production of membrane proteins and investigation of the interaction of drug candidates with biophysical binding assays and high resolution structure determination, including the use of most advanced methods like X-ray Free Electron Laser and cryo-EM. Cutting edge computational data analysis methods convert the knowledge into the design of novel drug candidates with unprecedented efficacy and specificity generating greatest benefit for the patient. For example, in a collaboration with Boehringer Ingelheim, we generated CCR2A protein, crystallized it in LCP with the drug candidate MK-0812 and determined the structure using serial X-ray crystallography. Analysis of the interaction of MK-0812 in the binding site facilitates the design of CCR2 specific antagonists for treating inflammatory diseases.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 02J1

Mapping GPCR contacts and their stability – a web-based analysis tool of the GPCRmd database

D. ARANDA-GARCIA

GPCR Drug Discovery group, Research Programme on Biomedical Informatics (GRIB), Universitat Pompeu Fabra (UPF)-Hospital del Mar Medical Research Institute (IMIM), Doctor Aiguader, 88 E-08003 Barcelona, Spain.

corresponding author: [email protected]

Molecular dynamics (MD) techniques are increasingly gaining importance in drug discovery programs (Borhani & Shaw, 2012),​ as they provide a way to analyze the behavior of macromolecules such as GPCRs through time. For this reason, we are developing GPCRmd (www.gpcrmd.org): a database to store and display MD simulations of GPCRs from all over the world. In particular, this project is centered on the creation of ContactMaps (https://submission.gpcrmd.org/contmaps/), an online tool for the analysis of intra- and intermolecular interactions and their stability derived from GPCR dynamics. The analysis tool is based on GetContacts (https://getcontacts.github.io/) scripts, which offers a way to precisely calculate different types of interactions (hydrogen bonds, water bridges, …) formed between the residues of a molecular system in an MD simulation. It has been designed as an easy, user-friendly web tool allowing researchers with different expertise (e.g. biologists, medicinal chemists, pharmacologists) to browse and exploit simulation data. A strength of the developed tool is to detect how inter- and intramolecular interaction networks are affected by (i) evolution and (ii) polymorphism across different GPCRs, which could ultimately be useful for drug design studies.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 03J1

Sucrose bingeing modifies alcohol reward in mice

G. AWAD1, LA. ROECKEL1, MC. OLMSTEAD2, K. BEFORT1

1 Laboratoire de Neurosciences Cognitives et Adaptatives (LNCA), UMR 7364, CNRS/Université de Strasbourg, Faculté de psychologie, 12 rue Goethe, 67000 Strasbourg, France 2 Department of Psychology, Center for Neuroscience Studies, Queen's University, Kingston, Ontario K7L 3N6, Canada corresponding author: [email protected]

Eating disorders and substance abuse are highly comorbid although the relationship between the expression of these two maladaptive behaviours is unclear. Obese and subjects with alcohol use disorder show similar characteristics: continuous use despite adverse consequences, persistent craving, and repeated unsuccessful attempts to quit, suggesting that they are linked by a common etiology. To understand the nature of this relationship, we induced sucrose bingeing in C57Bl6/J male and female mice using 14-day limited access protocol (17.1% w/v). Binge intake was measured as increased solution consumption during the first hour. All mice given limited access to sucrose developed binge-like intake. We then examined the impact of sucrose bingeing on alcohol reward using a conditioned place preference paradigm with ethanol (i.p. ; 3%). Interestingly, all mice developed a conditioned place preference to 3% ethanol with the exception of female and male mice given limited access to sucrose. Our findings suggest that binge-like sucrose intake affects reward processing, at least to alcohol-related cues. We will examine this further by assessing ethanol consumption and preference across a range of concentrations (3%, 6%, 9%, 12%, 15%). Our study will provide insights into the relationship between eating disorders and vulnerability to alcohol consumption.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 04J1

Effects of mechanical stress on the angiotensin II AT1 receptor: Implications for sodium binding and water permeation

R. BEN BOUBAKER1, A. TISS1,2, D. HENRION1, and M. CHABBERT1

1 Angers University, MITOVASC, UMR CNRS 6015 – INSERM 1083, Angers, France. 2 Faculty of Sciences of Tunis, University of Tunis El Manar, GIPH Laboratory, Tunis, Tunisia.

Corresponding author: [email protected]

The angiotensin II receptor 1 (AT1) belongs to the superfamily of G-protein coupled receptors (GPCRs). AT1 signaling mediates the major physiological effects of angiotensin II including vasoconstriction, cardiac contractility and hypertrophy. This receptor is a putative mechanosensor that can be activated by various mechanical stimuli, albeit the signaling pathways are controversial. The mechanisms leading to the activation of AT1 in response to mechanical stress are not understood and need further investigations.

In order to investigate mechanical stresses that can be sensed by AT1, we carried out molecular dynamics simulations of the receptor embedded within a hydrated POPC bilayer under NPγT conditions, using the molecular dynamics simulation program NAMD. Positive and negative values of the surface tension γ led to membrane stretching and compression, respectively. We investigated the effect of both membrane stretching and compression on lipid and receptor properties.

Mechanical stress affected the physico-chemical properties of POPC, including area per lipid, membrane width and order parameters of lipid aliphatic chain. Stretching did not significantly alter receptor properties in the presence of allosteric sodium. However, in the absence of sodium, we could observe a transition towards an intermediate state of activation accompanied by the augmentation of water molecules in the transmembrane domain. By contrast, compression reduced water molecules in the transmembrane domain and altered sodium binding mode favoring the interaction with Asn7.49.

This study suggests that the molecular properties and responses of AT1 could depend on the mechanical stress present in its environment and might be altered upon hypertension.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 05J1

Molecular Mechanisms of Action of Photoswitchable mGlu5 Negative Allosteric Modulators

A.E. BERIZZI1, M. RICART-ORTEGA1,2, V. PEREIRA1, F. MALHAIRE1, X. GÓMEZ- SANTACANA1, J. FONT1, J. CATENA2, L. MUÑOZ2, C. SERRA2, X. ROVIRA3, J.P. PIN1, A. LLEBARIA2 and C. GOUDET1

1IGF, CNRS, INSERM, Univ. de Montpellier, F-34094 Montpellier, France 2MCS, Laboratory of Medicinal Chemistry & Synthesis, Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain 3Molecular Photopharmacology Research Group, University of Vic – Central Univ. of Catalonia, 08500 Vic, Spain corresponding author: [email protected]

The metabotropic glutamate 5 receptor (mGlu5) has emerged as an important therapeutic target for a number of diseases. A major hurdle for drug discovery endeavours though, is the development of candidate drugs whose therapeutic efficacy can be maximised whilst avoiding adverse effects. The rise in the discovery of mGlu5 allosteric ligands has therefore been an important advancement since they offer numerous advantages over orthosteric-targeting ligands, including the opportunity for improved selectivity for a receptor. Despite this, the systemic administration of allosteric drugs can cause on-target adverse effects in unwanted tissues. Thus, controlling an allosteric ligand’s activity with light offers the opportunity for enhancing the functional selectivity of a ligand in a more spatiotemporal manner. To date, few mGlu5 photoswitchable allosteric ligands have been described and an in-depth characterisation of their mechanisms of action is lacking. The aim of the current study was to therefore characterise the mechanisms of two photoswitchable negative allosteric modulators (NAM) at the mGlu5, under different light conditions, in IPone accumulation and radioligand binding assays. Our studies confirm that under dark and green light both photoswitchable NAMs, alloswitch-1 and XGS-023, negatively modulate agonist signalling at the mGlu5 at a site that is shared or overlaps with the MPEP-binding site. While under violet light, NAMs have reduced negative cooperativity with agonist signalling and decreased affinity for the MPEP- binding site, which may be due to each NAMs’ photoisomeric forms making differential contacts with the receptor. Our findings will be important to the development of improved photoswitchable mGlu5 NAMs.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 06J1

Cannabinoid receptor 1 regulation by GRK3 and SGIP1

M. GAZDARICA1, J. N. MAYOR1, M. DVORAKOVA1, L. PREZEAU2, J.-P.. PIN2, J. BLAHOS1

1 Institute of Molecular Genetics Academy of Science of Czech Republic, Videnska 1083 142 20 Prague 4, Czech Republic

2 Institute of Functional and CBS, University of Montpellier, French National Center for Scientific Research (CNRS), French National Institute of Health and Medicine (INSERM), Montpellier, France

Cannabinoid receptor 1 (CB1R) is a target for endocannabinoids, as well as exocannabinoids, such as THC. Besides canonical regulation by G-protein coupled receptor kinases (GRKs), CB1R signaling is also modulated by protein SGIP1. Phosphorylation of intracellularly positioned carboxyterminal end of CB1R results in desensitization and down-regulation. The aim of our study is description of the action of GRK3 and SGIP1 protein on CB1R signaling in relation to its phosphorylation-dependent modulation. The CB1R desensitization is, at least partly, governed by GRK3.. We prepared a set of CB1R mutants within the C-terminus, defective in phosphorylation, or mimicking phosphorylated states of the Serine and Threonine residues. These mutants were employed in transiently transfected HEK293 cells to study association with GRK3 and β-arrestin2 and modification of the signaling properties in the presence, or absence of SGIP1. Our data suggest that the phosphorylation of CB1R carboxyterminal end at the S426 and S430 residues, and those within the extreme C-tail, play important role in regulation of CB1R signalling in a pathway specific manner. The presence of SGIP1 further adjusts this regulation of CB1R activity, ass well a the receptor association with regulatory and signaling molecules. This project was supported by Grant Agency of Czech Republic (16-24210S, 18-10591S and 19-24172S) and MSMT.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 07J1

Fluorogenic Probes for Background-Free Live-Cell Imaging of GPCRs

I.A. KARPENKO,1 L. ESTEOULLE,1 M. COLLOT,2 F. DAUBEUF,1 A.S. KLYMCHENKO,2 D. BONNET1

1Laboratoire d’Innovation Thérapeutique, LabEx MEDALIS, UMR 7200 CNRS/Université de Strasbourg, 74 route du Rhin, 67401 Illkirch, France. 2Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS/Université de Strasbourg, 74 route du Rhin, 67401 Illkirch, France. corresponding authors : [email protected]

Fluorogenic (or turn-on) probes, which increase their fluorescence in response to a certain change in the microenvironment, are powerful tools in bioimaging. They allow for direct detection of analytes without removal of the unbound probe and thus for in situ monitoring of biomolecular interactions. In particular, turn-on probes are of high interest to study G protein- coupled receptors (GPCRs), the largest and the most diverse group of transmembrane receptors in higher eukaryotes.

We describe the first ligands that turn on their fluorescence in the red and far-red spectral region after binding to the target GPCR in situ in living cells. We also demonstrate that squaraines, in contrary to common belief, are stable in cell culture and imaging medium and therefore are well suited for the imaging of GPCRs. In order to refine the turn-on properties of the dye, we introduced the approach of fluorogenic dimers with polarity-sensitive folding. The squaraine dimers can be considered as the brightest turn-on molecules reported to date, having extinction coefficient of ∼660,000 M−1 cm−1 and quantum yield up to 0.4, whereas far- red emission enables the detection of GPCRs in living cells with minimum background.

Finally, by applying the dimeric approach to a near-infrared dye, we created a fluorogenic molecular imaging agent which allowed for the first time the detection of the native oxytocin receptor in living mouse.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 08J1

Diet-induced obesity differentially regulates endocannabinoid transcripts in reward circuitry depending on sucrose vs fat overconsumption

R. BOURDY1, A. HERTZ1, D. FILLIOL1, V. CHAVANT2, Y. GOUMON2, J. MENDOZA2, M.C. OLMSTEAD3, and K. BEFORT1

1 Laboratoire de Neurosciences Cognitives et Adaptatives (LNCA), UMR 7364, CNRS/Université de Strasbourg, Faculté de psychologie, 12 rue Goethe, 67000 Strasbourg, France 2 Institut des Neurosciences Cellulaires et Intégratives (INCI), UPR 3212, CNRS,5 rue Blaise Pascal, 67084 Strasbourg, France 3 Department of Psychology, Center for Neuroscience Studies, Queen's University, Kingston, Ontario K7L 3N6, Canada

Increased availability of high-calorie palatable food in most Western countries has led to overconsumption, suggesting that this behavior is driven by pleasure, rather than metabolic need. This has led to a dramatic rise in eating disorders, obesity, and associated pathologies (diabetes, cardiac diseases, cancers...). Brain activation and behavioral adaptations linked to palatable food overconsumption overlap closely with those induced by drugs of abuse, giving rise to the debated concept of «food addiction». The reward system plays a central role in the switch from controlled to compulsive intake of both drugs of abuse and palatable food. The endocannabinoid system controls feeding behavior and energy balance and regulates palatable food intake by acting on the reward system. Both systems have been involved in obesity. In our study, we used a RT-qPCR approach to investigate the effects of a 6 weeks high fat high sugar diet (fcHFHS) on gene expression of key components of the endocannabinoid system in reward brain regions (PFC, NAc, VTA). Km cluster analysis highlighted separate groups of animals based on individual palatable food intake. We also measured endocannabinoid levels in the PFC and NAc using mass spectrometry. Our results show that the fcHFHS diet induced hyperphagia and obesity in rats. Cluster analysis revealed that excessive sucrose or fat intake is differently associated with endocannabinoid system gene expression in reward brain regions. Moreover, transcriptional data pointed to cannabinoid receptors as GPCR targets involved in neuroplasticity mechanisms associated with maladaptive responding for palatable food.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 09J1

Cryo-EM structure of the active state of the antidiuretic hormone V2 receptor in complex with the signaling Gs protein

J. BOUS 1,2, H. ORCEL2, J. LAI-KEE-HIM1, G. GAIBELET2, A. ANCELIN1, S. TRAPANI1, J. SAINT-PAUL2, R. SOUNIER2, C. LEYRAT2, N. FLOQUET2, S. GRANIER2, P. BRON1*, B. MOUILLAC2*

1Centre de Biochimie Structurale, CNRS-5048, INSERM-U1054, Université de Montpellier, Montpellier, France 2 Institut de Génomique Fonctionnelle (IGF), CNRS, UMR 5203, INSERM U1191, Université de Montpellier, Montpellier, France. 3 Institut des Biomolecules Max Mousseron, CNRS-5048, INSERM-U1054, Université de Montpellier, Ecole nationale supérieure de chimie de Montpellier corresponding author: [email protected] [email protected] [email protected]

The neurohypophysial hormone arginine-vasopressin (AVP) is known as the antidiuretic hormone. It controls body water balance through binding to the renal V2 receptor (V2R) subtype, a prototypical class A G protein-coupled receptor (GPCR). The V2R is coupled to the adenylyl cyclase stimulatory Gs protein and its activation gives rise to a cAMP signal upon AVP binding. The V2R is an important therapeutic target, however, our understanding of its mechanism of action is limited since no structure has ever been described in the AVP receptor family. Here we report the near-atomic resolution cryo-electron microscopy structure of the hormone-activated V2R-Gs complex. The AVP interacts with the V2R through an interface spanning from the receptor core to its extracellular domains, as demonstrated for other small peptide ligands, such as the hormone angiotensin II or the opioid DAMGO. At the intracellular interface, the Gs protein engages the V2R core primarily through amino acid residues of the Gαs-α5helix, as observed for the β2-adrenergic or the adenosine A2A receptors in complex with the heterotrimeric Gs protein, but with distinct orientations. This structure is the first 3D structure ever described for a GPCR from the vasopressin family.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 10J1

Evaluation of a new method to monitor the endogenous Gi protein activation by a GPCR

A. BOUYSSOU1, 2, M. DA SILVA1, 2, S. TRIA1, J. SOULE1, L. FABRE1, L. LAMARQUE2, S. GRANIER1, T. DURROUX1, E. DUPUIS2, T. ROUX2 and E. TRINQUET2, P. RONDARD1 and J-P. PIN1

1 Institut de Génomique Fonctionnelle (IGF), CNRS UMR5203, INSERM U1191, Université de Montpellier, Montpellier France. 2 Cisbio, Codolet France ;

G proteins are the main transducers of the G protein-coupled receptors (GPCRs). GPCRs are able to activate different G protein families, thus governing the activation of several different signaling pathways. Nowadays GPCRs are the target for approximatively 30% of the drugs approved by the FDA, so it is important to develop new technologies to investigate on GPCR ligands. Currently, several assays are available to detect G protein activation. While some of these assays are indirect, based on the measurement of second messengers, others rely on recombinant proteins. Finally some G protein activation assays are compatible with endogenous proteins which require the incorporation of radioactive non hydrolysable GTP analog by G proteins. This radioactive assay does not allow to discern the different G protein families. Here, the aim of our project is to investigate on new method to measure G protein activation, distinguishing different G protein families and compatible with native systems. We then investigate on using the homogeneous time-resolved FRET (HTRF®) technology to measure the activation of endogenous Gi proteins by a GPCR. Our approach is based on Gi protein conformational changes detection by developing a pair of fluorescent tools specific of the endogenous Gai subunits. The Ga subunit adopts three different structural states during the G protein activation cycle: a GDP-bound state (inactive state), a GTP-bound state (active state) and an empty state (transient GPCR bound conformation). Our tools enable to discriminate these states. We have performed this assay on membrane preparations from mammalian cells expressing recombinant GPCRs. Our results show it can discriminate between agonists and antagonists of different efficacies and potencies. This method has been used on different GPCRs indicating that this new approach may help the investigation of GPCR ligands and the development of future drugs.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 11J1

Molecular dissection of a natural analgesia mechanism: the bacterial toxin mycolactone acting on the angiotensin 2 receptor (AT2R) and TRAAK channel system.

J. BRIOT1,6, P. AVALOS PRADO2,6, M. REYNAERT3,6, D. MAUREL1, C. VOL1, O.K. SONG3,6, N. DEBOOSERE3,6, E. YERAMIAN4,6, L. MARSOLLIER5,6, S. HAFNER2,6, Y. COMOGLIO2,6, B. WDZIEKONSI2,6, P. ROYAL2,6, P. BRODIN3,6, G. SANDOZ2,6, G. LEBON1,6. 1 Institut de Génomqiue Fonctionnelle, CNR, INSERM, Université, 141 rue de la cardonille, 34094 Montpellier 2 Université Côte d'Azur, CNRS, Inserm, Institut de Biologie Valrose, Nice, France; Laboratories of Excellence, Ion Channel Science and Therapeutics, Nice, France. 3 France Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France. 4 Unité de Microbiologie Structurale, Institut Pasteur, CNRS, Univ. Paris, F-75015 Paris, France. 5 Institut de Cancérologie de l'Ouest Paul Papin, 15 rue André Boquel-49055 Angers, France. 6 Consortium ANR AT2R Traak Bioanalgesics. corresponding author: [email protected]

Pain represents the most common symptom for which medical attention is sought by patients. Efficient handling of pain remains a major societal and humanitarian objective and challenge. In regards to resolving pain, the available pharmacopoeia is very limited, with compounds displaying variable effectiveness and usually associated with side effects. The absence of pain in Buruli ulcer disease has been attributed to the powerful long-lasting analgesic effect of the mycolactone polyketide toxin secreted by Mycobacterium ulcerans. Mycolactone-dependent analgesia is mediated by a peculiar signalling system involving two integral membrane proteins, the angiotensin type-2 receptor (AT2R) and TRAAK potassium channel. We show that in presence of mycolactone, AT2R triggers the release of potassium through TRAAK channel inducing a sustained hyperpolarization of the plasma membrane limiting excitability and therefore reducing pain. We investigate the molecular mechanism by which mycolactone induces AT2R-dependent TRAAK activation. We first show that activation of TRAAK channel is independent from G-protein and b-arrestin pathway. Ligand stimulation of AT2R does not engaged Gai/o proteins and the receptor failed to internalize in HEK cells, remaining at the cell surface. This is in agreement with the long-standing effect of mycolactone-induce analgesia that remains after several hours. We hypothesized a direct interaction between AT2R, which was demonstrated by pull-down assays coupled to mass spectrometry analysis and single molecule pull-down assay, strongly in favour of a physical interaction between the two membrane proteins. The project will unravel the molecular mechanism underlying a natural analgesia system allowing further tools developed to identify new analgesic drug.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 12J1

Long time-scale molecular dynamic simulations of mGlu receptors

A. CABAYEa,b, F. ACHERa, A. GOUPILb, H.-O. BERTRANDb

a Université Paris Descartes, UMR8601 – CNRS, 45 rue des Saint-Pères 75270, Paris, France b Dassault Systèmes, BIOVIA, 10 rue Marcel Dassault 78946, Vélizy-Villacoublay, France corresponding author: [email protected]

Simulating the activation motions of large protein receptors can be challenging. Indeed, it is known that mGlu receptors’ activation motion occurs on a milliseconds time-scale [Rondard & Pin, Curr. Opin. Pharmacol. 2015], when actual computer simulations can produce a few hundred nanoseconds up to few microseconds. To overcome this issue, we investigated a new technic called Gaussian accelerated Molecular Dynamics (GaMD) [Miao et al., J. Chem. Theory Comput. 2015], which consists in boosting the potential energy of the system along the simulation to smoothen the energy potential landscape. The main advantage of this technic is that it does not require to predefined reaction coordinates, allowing an un-biased exploration of the conformational space.

Moreover, we tackled the problem of modelling for the first time, a full-length model of mGlu2 receptor, based on the recently published structure of mGlu5 receptor [Koehl et al., Nature 2019]. We chose to make, on the extracellular side, an active closed-closed (Acc) dimer, bound with a well-known active ligand, 3Me-CCG [Collado et al., J. Med. Chem., 2002].

As all available structures for the transmembrane domain were in an inactive state, so was our model. Therefore, our goal for the simulation was to explore a transition towards a fully active state (both extracellular and transmembrane domains) and potentially the key residues controlling it.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 13J1

NanoLuX: a network-wide Nanoluciferase-based platform to monitor activation, modulation and bias of classical and atypical chemokine receptors

M. MEYRATH, N. REYNDERS, M. COUNSON, N. BEAUPAIN, M. OLLERT, M. SZPAKOWSKA and A. CHEVIGNÉ

Luxembourg Institute of Health, Department of Infection and Immunity, 29, rue Henri Koch, L- 4354 Esch-sur-Alzette corresponding author: [email protected]

The activity of chemokine receptors is dependent on G proteins that, upon chemokine binding, trigger various intracellular signaling cascades, as well as on β-arrestins that, following receptor phosphorylation by GRK, orchestrate its desensitization, endocytosis and trafficking. In addition to the 19 classical chemokine receptors, 4 receptors form a subfamily of atypical chemokine receptors (ACKR1-4) with ligand-scavenging functions. These receptors are unable to couple to G proteins but their activity can be monitored via β-arrestin recruitment.

Here, we present the NanoLux platform, a network-wide profiling platform for chemokines and chemokines receptors based on the complementation of the Nanoluciferase (NanoBiT). This platform allows to monitor the activation, modulation or bias of receptors or ligands by measuring the binding or the recruitment of effectors, regulators or partners such as G proteins, GRK or β-arrestin isoforms to the classical and atypical chemokine receptors. For that purpose, the LgBiT portion of Nanoluciferase was N-terminally fused to the miniG proteins, GRK2 or human β-arrestin 1 and 2, while the SmBiT was fused to the C terminus of all of the 23 human chemokine receptors. This approach enables to assess and compare the activity of molecules at the chemokine-receptor network level but it can also be applied to investigate the interactions of ligands and intracellular effectors of many other membrane proteins, including GPCRs, receptor tyrosine kinases or ion channels.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 14J1

Donecopride: a Multi-Target-Directed Ligand with potential interest against Alzheimer’s disease

S. CLAEYSEN

IGF, Univ. Montpellier, CNRS, INSERM, 141 rue de la Cardonille, Montpellier, France corresponding author: [email protected]

Alzheimer’s disease (AD) is currently one of the most socially devastating neurodegenerative disorders. Due to the complexity of AD pathogenesis, Multi-Target-Directed Ligands (MTDLs) seem of high relevance for the treatment of AD. We have described donecopride, a novel preclinical drug candidate exhibiting two activities of interest regarding AD pathology: –an acetylcholinesterase (AChE) dual-binding site inhibition, to restore cholinergic concentrations in the brain and –a partial agonism towards serotonergic 5-HT4 receptor, to promote the non- amyloidogenic cleavage of the amyloid precursor protein (APP) and the release of the neuroprotective sAPPa protein.

In the present study, we show that, under chronic administration, donecopride displays potent anti-amnesic properties in two different animal models of AD (transgenic 5XFAD mice and mice exposed to soluble amyloid-b peptides). Donecopride preserved learning capacities, including working and long-term spatial memories, which were evaluated with various behavioral tests (novel object recognition, Y-maze, Morris water maze). These behavioral observations found molecular correlation with the interactions of donecopride related to the two main types of molecular damage in AD. Indeed, a decrease in amyloid aggregation was observed in the brain of the treated transgenic mice, as well as a reduction in tau hyperphosphorylation in rat primary cultures of hippocampal neurons. At the cellular level, donecopride exerted neuroprotective effects toward neurons and the neurite network, as well as neurotrophic benefits, expressed as the formation of new synapses. This neurotrophic effect is not observed with donepezil.

Clinical trials will soon be undertaken to confirm the promising therapeutic potential of donecopride against AD.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 15J1

Extracellular allosteric modulators of metabotropic glutamate receptor 7 to elucidate the inhibitory mechanism of XAP044

N. CRISTIANO1, A. CABAYE1,2. BRABET3, J.-P. PIN3, F. C. ACHER1, I. MCCORT1

1 Laboratoire de Chimie & Biochimie Pharmacologiques et Toxicologiques, UMR CNRS 8601, Université Paris Descartes, Sorbonne Paris Cité, 45, rue des Saints-Pères 75270, Paris cedex 06, France 2 Dassault Systèmes, 10 Rue Marcel Dassault, 78140 Vélizy-Villacoublay 3 Institut de Génomique Fonctionnelle, CNRS UMR 5203, Université de Montpellier, 141 Rue de la Cardonille, 34094 Montpellier cedex 5, France.

Glutamate is the major excitatory neurotransmitter of the mammalian central nervous system (CNS) and is able to bind two different families of receptors: ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). mGluRs are class C G-protein coupled receptors (GPCRs) dimers responsible for slower neuromodulatory actions of glutamate. Depending of sequence homology, signal transduction and pharmacology, mGluRs are subdivided into three groups: Group-I mGluRs (mGlu1,5) are mainly coupled to Gq that activate PLC and generate intracellular calcium signals. Both group-II (mGlu2,3) and group-III (mGlu4,6,7,8) mGluRs are mainly coupled to Gi/o and can therefore inhibit adenylate cyclase and regulate the activity of various ion channels. Among the 8 subtypes of mGlu receptors, mGlu7 has been shown to be related to autism, drug abuse, anxiety, depression, pain and epilepsy. A negative allosteric modulator (NAM) of mGlu7R XAP044 has been disclosed by Novartis scientists. They found that XAP044 is not binding as classical NAMs but they could not carry out further investigations. The aim of this project is to understand the binding mode of XAP044, elucidating its inhibition mechanism and developing new mGlu7 ligands. For this purpose, several analogs have been designed, synthesized and tested:constrained-, open- and photoswitch analogs.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 16J1

Heterodimerization of melatonin and cannabinoid type 1 receptors

Y. CUI, O. LAHUNA, R. JOCKERS

1 Inserm U1016, CNRS UMR 8104, Univ. Paris Descartes, Sorbonne Paris Cité, Institut Cochin, 22 rue Méchain,75014 Paris, France corresponding author: [email protected]

Membrane receptors belonging to the G proteins coupled receptors (GPCRs) form the largest family of proteins in the human genome with more than 800 members. Until recently GPCRs functions were thought to occur only at the plasma membrane after activation upon binding of their cognate ligand. However evidences show that many functional GPCRs are found in intracellular compartments opening new direction of research to understand their roles in a cellular context. Among these intracellular compartments mitochondria are the latest organelle in which some GPCRs were identified. Melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were identified in mouse neuronal mitochondria where they were shown to exert an inhibitory action on cytochrome c release (MT1) or on the respiratory chain (CB1). Using several techniques my current results describe a new cross- talk between MT1 and CB1 receptors. Confocal analysis of immunofluorescence experiments of cells coexpressing both receptors showed a high degree of colocalization. A combination of coimmunoprecipitation experiments performed on extracts of transfected HEK293T or HeLa cell lines and immunodetection of receptors by Western-blot revealed that MT1 and CB1 receptors can physically interact to form heterodimers in absence of ligand. Heterodimers formation was also confirmed by Proximity Ligation Assay (PLA) experiments in live HEK293T and HeLa cells. Confocal analysis revealed a colocalization of PLA staining with the mitochondrial marker TOMM20. Experiments in relation with the functional role of MT1 and CB1 in mitochondria are ongoing.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 17J1

Membrane-mimicking systems: different clothes to dress GPCRs for each season

M. DAMIAN, M. CASIRAGHI, S. ESTARAN, C. M’KADMI, M. LOUET, G. FERRE, J. LAI-KEE- HIM, S. CANTEL, S. DENOYELLE, S. MARY, J.-A. FEHRENTZ, E. LESCOP, P. BRON, A. CHAVANIEU, J. MARIE, A. MILON, N. FLOQUET, L. CATOIRE, J.-L. BANERES.

UMR5247-IBMM Université Montpellier CNRS ENSCM, UFR pharamcie, bat K 2ème étage, 15 avenue Charles Flahault, 34070 MONTPELLIER, FRANCE corresponding author: [email protected]

G protein coupled receptors (GPCRs) compose the largest family of cell surface receptors and are involved in regulating virtually every aspect of physiological processes. Understanding how signaling is regulated requires molecular information to be obtained, usually through a combination of stat-of-the-art biophysical methods. In many cases, these methods rely on the use of isolated receptors stabilized in their native state into a membrane-mimicking environment. Different such membrane-like model systems have been developed so far, ranging from simple polymeric species (e.g. amphipols) to more complex lipid vesicles (liposomes). We have used these different systems to isolate two model class A GPCRS in vitro, i.e. BLT2R and GHSR. BLT2R is the low affinity GPCR for leukotriene B4 (LTB4), a lipid that acts as the main mediator of inflammation. GHSR is the membrane receptor for a peptide hormone, ghrelin, which exerts a wide range of biological effects including control of food intake, of reward, or of glucose metabolism. We assembled these two receptors into model membrane systems of increasing complexity, i.e. amphipols, nanodiscs, SMALPs, Salipro particles and liposomes (see Figure below). We show that these different environments preserve the native function of the receptors and provide examples of specific applications of each of these systems for biophysical analyses aimed at illuminating the molecular functioning of the receptors considered.

Amphipols Nanodiscs Salipros SMALPS Liposomes

increasing complexity

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 18J1

Towards the understanding of V2R signalisation by NMR

H. ORCEL1, G. GAIBELET1, S. RICHE2, R. SOUNIER1, S. GRANIER1, D. BONNET2, B. MOUILLAC1, H. DEMENE3

1Institut de Génomique Fonctionnelle (IGF), CNRS, UMR 5203, INSERM U1191, Université de Montpellier, Montpellier, France. 2Laboratoire d’Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, 74 route du Rhin, Illkirch, France 3Centre de Biochimie Structurale, UMR5048 CNRS-INSERM-Univ Montpellier, Montpellier, France corresponding author: [email protected]

The V2 receptor (V2R) is a class A GPCR whose endogenous peptide is the 9 amino acid vasopressin peptide. It is mainly expressed in kidney membranes, where it governs water reabsorption and thus water homeostasis in vertebrate organisms. The 3D dimensional structure of V2R is still unknown. Much remains to be learned about the mechanisms by which different agonists can induce distinct levels of G protein activation and/or arrestin recruitment upon activation. Pharmacological and biophysical studies suggest that this versatility can be achieved through the structural plasticity of GPCRs.

In this work, we analyze the conformational landscape of the V2R in distinct pharmacological conditions using liquid-state NMR spectroscopy, by monitoring signals from methyl-labelled lysines. We investigate the structure and dynamics changes upon binding to different ligands ranging from agonist to antagonists. Our results outline common features with other GPCRs as well as distinct particularities. In particular, we retrieve the weak allosteric coupling between the agonist binding pocket and G protein coupling interface seen for the µ receptor to opioids, the b2 adrenergic receptor or the muscarinic M2 receptor. Of note, the different signaling states of V2R are characterized by an equilibrium between at least 2 different states at the level of TM6, which are differently affected according to the pharmacological nature of the ligand. We also show how NMR restraints also help in deciphering the pose of the endogenous vasopressin ligand.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 19J1

Chronic sodium bromide relieves autistic-like deficits in the Oprm1 mouse model of autism and modulates the activity of serotonin receptors in vitro

C. DERIEUX1, S. ROUX1, A. LEAUTE1, T. PLOUVIER2, J.A.J. BECKER1, J. LE MERRER1

1 Déficits de Récompense, GPCRs et Sociabilité, Physiologie de la Reproduction et des Comportements, INRA UMR0085, CNRS UMR7247, Université de Tours, Inserm ; 33380 Nouzilly, France 2 Térali Innov, 37230 Fondettes, France corresponding author : [email protected]

Autism spectrum disorders (ASD) are complex neurodevelopmental diseases whose diagnosis lies on the detection of impaired social skills together with restricted and repetitive behavior and interests (DSM-5). Although the etiology of ASD remains mostly unknown, impaired excitation/inhibition ratio appears as a common mechanistic feature. Consistent with this, molecules that decrease neuronal excitability, such as NKKC1 antagonists, were shown to ameliorate autistic symptoms in animal models as well as in patients with ASD. Bromide ions can reduce neuronal hyperexcitability, possibly by competing with chloride ions at channels and transporters. Here we first evaluated the therapeutic potential of chronically administered sodium bromide to relieve autistic-like behavioral deficits in a mouse model of ASD. Sodium bromide (125, 250 and 500 mg/Kg) dose-dependently improved social interaction and preference, reduced stereotypies and decreased anxiety. Bromide administration was significantly more effective than bumetanide (0.5 and 2 mg/Kg). Then, we tried to shed light on the molecular mechanisms involved in bromide effects. Bromide administration impact on gene expression was evaluated using q-PCR in the CPu and the NAc, known to play a key role in reward and social behavior. Finally, we evaluated the influence of bromide ions on the activity of serotonin (5-HT) receptors. Bromide behaves as a positive allosteric modulator of 5-HT6 and

5-HT7 receptors. The beneficial effects of bromide administration in a genetic murine model of ASD and its impact both on gene expression and 5-HT receptor pharmacology predicts high translational potential in patients with autism, despite high heterogeneity in etiology and symptoms.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 20J1

Reversible activation of Βeta-Adrenoceptors using synthetic light-regulated molecules

A.DURAN-CORBERA

MCS, Laboratory of Medicinal Chemistry & Synthesis, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain corresponding authors : [email protected] [email protected]

Beta-Adrenoceptors (β-AR) are important pharmacological targets within the superfamily of G protein-coupled receptors (GPCRs). Indeed, a number of approved drugs target these receptors due to their key role on many important physiological functions. Among other examples, we encounter β-AR antagonists (known as β-Blockers), which constitute the first- line therapy for the treatment of heart failure and other cardiac conditions. Therefore and considering the therapeutic value of these receptors, achieving a reversible and localised control of their activity would provide a powerful tool, both for its research applications and its clinical potential. In this context, photopharmacology arises as a potent approach.

This emerging methodology is based on the use of synthetic light-regulated molecules to allow reversible spatiotemporal control of target receptors in native tissues. In consequence, photopharmacology can provide precise and controllable delivery of therapeutics yielding molecules with increased efficiency and reduced side effects. The present project intends to provide the first proof of concept for Beta Adrenergic photopharmacology.

We have designed and synthesised a library of photoswitchable compounds in order to inhibit β-AR in a light-dependent manner. Subsequent biological testing has highlighted the successful development of a series of compounds with a variety of promising pharmacological properties in vitro which can be reversibly controlled by light. This can potentially open new avenues towards personalised and efficient treatments for a number of diseases.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 21J1

Identification of new ligands of 5-HT7 receptor with anti-nociceptive properties: Molecular bases of their action

C. EL KHAMLICHI1, F. REVERCHON1, N. HERVOUET-COSTE1, E. ROBIN1, N. CHOPIN2, E. DEAU2, A. BOJARSKI3, G. GUILLAUMET2, F. SUZENET2, E. REITER4, S. MORISSET- LOPEZ1

1 Centre de Biophysique Moléculaire, CNRS UPR 4301, Université d’Orléans et INSERM, rue Charles Sadron, 45071 Orléans Cedex 2, France. 2 Institut de Chimie Organique et Analytique, Université d’Orléans, CNRS, UMR 7311, rue de Chartres, 45067 Orléans, France. 3 Institute of Pharmacology, Polish Academy of Sciences, 12 Smętna Street, Kraków 31-343, Poland 4 UMR Physiologie de la Reproduction et des comportements, UMR 7247 INRA- CNRS- Univ. Tours, 37380 Nouzilly, France corresponding author: [email protected]

The serotonin 5-HT7R receptor (5-HT7R) is the most recently cloned member of serotonin receptors family which comprises at least 15 subtypes. The 5-HT7R belongs to the superfamily of the G proteins coupled receptors (GPCRs) and is coupled to the heterotrimeric G protein Gs which in turn leads to AC activation and initiate specific signaling cascade including activation of MAP/ERK kinases. In the last decade, 5-HT7 receptor has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders and of neuropathic pain. Agonists, inverses agonists or antagonists have been developed to target the receptor. Our main objective was to develop 5-HT7 ligands with new pharmacological properties. Considering the role of 5-HT7R in various disorders, development of 5-HT7R biased ligands may offer interesting therapeutic issue. Indeed, biased ligands are molecules capable to stabilize subsets of receptor conformations, leading to modifications in the balance of pathways activated compared to the reference agonist. This concept of functional selectivity of a ligand has recently emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side-effects by regulating biological functions of GPCRs in a more precise way.

In this study, we characterized the pharmacological profile of new 5-HT7R ligands derived from pharmacomodulation studies. For that purpose, we studied their capacity to activate specific signaling pathways and defined their agonist, inverse agonist or antagonist activity by measurement of cAMP levels using BRET or TR-FRET methods in HEK cells stably expressing the 5-HT7R. We also investigated their ability to activate others 5-HT7R signaling pathways (ERK activation, calcium mobilization, recruitment of G proteins and β-arrestins). Our results demonstrate that some molecules can engage different transducer–effector systems compared to classical agonists. Considering the 5-HT7R implication in pain, we also investigated the anti-nociceptive properties of our compound in three different pain behavioral tests. Therefore, we showed that our new 5-HT7 ligand clearly decreases the hyperalgesia and pain sensation in response to thermal, mechanical and inflammatory stimulation. These anti- nociceptive effects are fully blocked by administration of SB269-970, a specific 5-HT7 antagonist, demonstrating the specificity of action of our compound. In conclusion, by using various functional assays and behavioral assays in mice, we identified a new 5-HT7R- ligand able to reduce pain behavior through a Gs-independent signaling mechanism.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 22J1

Novel regulation of GIRK potassium channels by δ-opioid receptors

A.S. ERIA OLIVEIRA, J. REVILLOUD, M. FOLACCI, M. VIVAUDOU

Channels Group, Institut de Biologie Structurale, Grenoble, France corresponding author: [email protected]

Opioid GPCRs (OR) and G protein-gated potassium channels (GIRK or Kir3 channels) interact to modulate pain perception. That is, GIRK channels are activated by direct binding of the Gβγ subunits released upon activation of OR, ultimately leading to pain relief. We have studied the regulation of GIRK channels by OR by coexpressing them in Xenopus oocytes and measuring channel activity using electrophysiological techniques. We discovered that agonists acting through δ-opioid receptors (δOR) activate GIRK channels at nM concentrations but inhibit them at higher concentrations. Notably, inhibition of the GIRK channels was only observed at high levels of expression of δOR. This observation suggests that inhibition is dependent on receptor density, possibly by impacting receptor oligomerization. Unlike channel activation, inhibition appears not to be mediated by G proteins, nor arrestins. Control experiments performed with the closely related µ-opioid receptor (µOR) did not reveal any sign of inhibition. Inhibition appears to be specific to δOR. Using chimeras, we were able to confer inhibitory capability to µOR by substituting its intracellular loops with those of δOR. The intracellular regions of both receptors have only small differences. By further mutagenesis, we expect to pinpoint the residues of δOR that are involved in the inhibition, which in turn should allow us to investigate and identify the pathway involved. Our data therefore disclose an unreported phenomenon specific to δOR where receptor monomers and oligomers potentially activate different signalling pathways. These observations uncover a novel, complex regulation with mechanistic and physiological implications that remain to be fully elucidated.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 23J1

Pharmacological characterization of the VHH DN45 for functional and binding studies on mGlu4 receptor.

J. FONT1*, J. HAUBRICH1*, J. KNIAZEFF1, L. PRÉZEAU1, J.P.PIN1

1Institut de Génomique Fonctionnelle, CNRS, INSERM, University of Montpellier. corresponding authors : [email protected], [email protected].

The metabotropic glutamate receptor 4 (mGluR4) is a GPCR involved in glutamatergic and GABAergic neurotransmission and therefore an interesting target not only for treating diseases in the neurological system, but also for functional and binding studies. mGluR4 forms homodimers, but also heterodimers with mGluR2. VHHs, also knonw as nanobodies, could be used to detect these receptor complexes due their high selectivity. We developed a camelid VHH, DN45, for the recognition of wild type human mGluR4. Human mGluR4 with a N-terminal SNAP-tag was overexpressed in HEK293 cells. With HTRF®-compatible methods we determined the specificity of DN45 nanobody and we pharmacologically characterized it. Interestingly, DN45 was specific for the human mGluR4 orthologue vs rat. The epitope of the nanobody has been determined by introducing amino acids for the human receptor into the rat receptor. In addition, the nanobody is clearly specific for the active conformation of the human mGluR4. Furthermore, DN45 acts as agonist and has the same potency as L-AP4, the orthosteric agonist, in two pharmacological assays. These data form the first characterization of DN45 nanobody for the detection and activation of the human mGluR4.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 24J1

Serpentine receptors in human malaria parasites

P. SCARPELLI 1,2, M. SINGH 1, M. BOUVIER 2. C. GARCIA 1,

1 Department of Clinical and Toxicological Analyses, University of São Paulo – Brazil. 2 Institut de recherche en immunologie et en cancérologie, Université de Montréal - Canada corresponding author: [email protected]

Malaria causes millions of deaths worldwide and is considered a huge public health problem for underdeveloped countries. The increasing number of cases of multi-resistant parasites makes understanding the physiology of Plasmodium falciparum a powerful tool for identifying new molecules to fight the disease. PfSR12 and PfSR25 are seven-transmembrane proteins with GPCR characteristics expressed by P. falciparum during infection of human cells. The knock-out of the gene PfSR25 reveals that the receptor is a monovalent cation sensor. PfSR25 can modulate Ca2+ signaling in the parasites upon changing from high to low KCl concentration in a process that depends on phospholipase C or internal Ca2+ pools. This response persists even in the absence of free extracellular Ca2+ and cannot be elicited by addition of Na+, Mg2+ or Ca2+. Also, parasites lacking PfSR25 show decreased survival in stress condition, such as SNP or antimalarials exposure and albumax deprived media. Another plasmodial receptor, PfSR12, showed a calcium response due to thrombin addition to PfSR12 transfected HEK293 cells, increasing cytoplasmatic Ca2+. Using BRET based biosensors, we further explored the pathways involved in PfSR12 signaling and found out that the Ca2+ release is a consequence of Gq/11 pathway activation. It was not possible to detect β-arestin recruitment or cAMP formation. Surprisingly, despite PfSR12 expression being 40x lower in HEK293 cells, it boosted the expression of co-transfected human GPCRs. These functional evidence of plasmodial receptors with GPCR characteristics add a new level of complexity to Plasmodium-host interaction.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 25J1

Controlling the GPCR’s stoichiometry with ICCR technology

M.D. GARCIA-FERNANDEZ

Institut de Biologie Structurale, 71 Avenue des Martyrs, 38000, Grenoble, France corresponding author : [email protected]

Ion Channel-Coupled Receptors (ICCRs) are artificial ligand-gated ion channels created by genetic fusion of G protein-coupled receptors to a potassium ion channel. The conformational changes in the GPCR, evoked by the ligand binding, generate an electrical signal that can be detected by electrophysiological techniques such as the two-electrode voltage-clamp in Xenopus laevis oocytes.

The ICCR technology is an original tool for functional GPCR studies as it is independent of any intracellular pathways, the measurement is in real-time and no labeling is required. Moreover, ICCRs detect GPCR ligand binding of agonists and antagonists in a concentration-dependent manner.

Initially, ICCRs were designed by fusing GPCRs to the inward-rectifier potassium channel Kir6.2. This channel is formed by four monomers, consequently, there are four GPCRs surrounding the pore in the functional and surface-expressed complex.

In the present study, with the aim of modifying and controlling the stoichiometry of GPCRs within the ICCR complex, the GPCRs were fused to other ion channels with dimeric and trimeric order of oligomerization and we assessed the role of the GPCR stoichiometry in both the ICCR function and the G protein activation.

The current results show a new generation of ICCR which is expressed at the surface of Xenopus oocytes and can be functionally characterized by electrophysiological techniques. In addition, the properties of the new ion channels fused give rise to a higher conductance, regulation by different effectors and better expression of these ICCRs. Altogether, it expands the possibilities for functional and structural GPCRs studies.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 26J1

1st and 2nd generation of photoswitchable ligands for GPCRS: shifting potency or switching efficacy?

X. GÓMEZ-SANTACANA1-5, S. de MUNNIK2, S, PITTOLO4, X. ROVIRA1, C. ZUSSY1, M. LOPEZ6,7, N. HAUWERT2, T. MOCKING2, K. ECKELT4,8, A. FAUCHERRE1, C. JOPLING1, F. CIRUELA6,7, C. GOUDET1, J. P. PIN1, H. VISCHER2, I. de ESCH2, J. GIRALDO4, M. WIJTMANS2, P. GOROSTIZA4,8,9, A. LLEBARIA3, R. LEURS2

1Institut de Génomique Fonctionnelle, CNRS, UMR-5203; INSERM, U661; Université de Montpellier; France 2 Amsterdam Institute for Molecules Medicines and Systems (AIMMS), Department of Medicinal Chemistry, VU University Amsterdam, The Netherlands; 3MCS, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain; 4 Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain; 5Institut de Neurociències, Universitat Autònoma de Barcelona (INc-UAB), Spain; 6Bellvitge Biomedical Research Institute (IDIBELL), Universitat de Barcelona, Spain; 7Institut de Neurociències, Universitat de Barcelona, Spain; 8Network Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain; 9Catalan Institution for Research and Advanced Studies (ICREA), Spain.

Corresponding autor: [email protected]

Photopharmacology is a discipline located on the boundaries of chemistry, biology and physics and makes use of photoswitchable ligands as pharmacological tool compounds to yield temporal and spatial control of receptor signalling with light. However, the development of photopharmacology to target G protein-coupled receptors (GPCRs) is still in its infancy. In the last years, alloswitch-1 and analogues were reported as the first GPCR photoswitchable allosteric modulators with activity in vivo. They selectively exert potent NAM activity in mGlu5 in the trans-isomer, while the potency in the cis disposition was decreased significantly. This behaviour was easily controlled by illumination with 380 nm and 500 nm of wavelength and was reversible in transfected cells and native cultures. Moreover, they allowed to control the nantatorial behaviour, of native zebrafish larvae or tadpoles with light and control their analgesic effect in rodents with light. An approach based on photoswitchable compounds having a different efficacy profile is advantageous because of the direct photoswitching of target GPCR functional activity. We developed functionally photoswitchable CXCR3 ligands by azologization of a biaryl-type series, which harbour a subtle functional efficacy switch. With this approach, a set of 27 azocompounds was synthesised and most showed antagonist activity. However, some display a photoinduced switch in the efficacy, with VUF16216 reaching an antagonist to full agonist switch. This represents the first full-efficacy photoswitchable ligand for GPCRs and the birth of a second generation of photoswitchable ligands.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 27J1

Not all 7TMs are GPCRs

R. HEALEY, S. GRANIER

1 IGF, INSERM, CNRS, Univ of Montpellier, France corresponding author: [email protected]

A new class of 7TM enzymes: intracellular ceramidases, have been structurally and functionally characterised. These enzymes are somewhat structurally similar to GPCRs: both possessing 7-transmembrane alpha helices. Intriguingly however, the enzymes display an inverted topology with extracellular C-termini and intracellular N-termini.

This poster will showcase three intracellular ceramidases; adiponectin receptors 1 and 2 (ADIPOR1/2)1 and alkaline ceramidase 3 (ACER3)2. An integrated structural biology approach has been applied to understand structurally the molecular mechanism of enzymatic function. Ongoing experiments utilise this understanding to develop tool compounds and therapeutics which specifically control enzymatic function in disease models including leukodystrophy, hepatocellular carcinoma, acute myeloid leukemia and type 2 diabetes.

References 1. Vasiliauskaité-Brooks, I. and Sounier, R., et al. Structural insights into adiponectin receptors suggest ceramidase activity. Nature. 544, 120 (2017). 2. Vasiliauskaité-Brooks I. and Healey R.D., et al. Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy. Nat. Comm. 9, 5437 (2018).

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 28J1

Interrogating the therapeutic window of novel opioids

R.HILL

University of Nottingham, COMPARE, School of Life Sciences, QMC, Nottingham, UK Corresponding author : [email protected]

Several recent novel opioid agonists, such as TRV130, have been reported to have a decreased propensity to induce severe opioidergic side-effects such as respiratory depression. Recently the design of novel opioid agonists has shifted from pursuing opioids devoid of respiratory depression towards developing opioids with an increased therapeutic window between the desired analgesic effect and the undesired side-effects profile of opioids, therefore decreasing the likelihood of overdose fatalities.

In order to assess the relative therapeutic window of two novel opioids, TRV130 and tianeptine, full dose response curves examining morphine, TRV130 and tianeptine induced respiratory depression were performed using whole body plethysmography in mice (administered i.p.). Equi-respiratory depressant doses calculated by regression were then used to determine the relative induction of antinociception in the hot plate assay at these doses in mice.

Morphine, TRV130 and tianeptine all dose dependently depressed mouse respiration when compared to their own baseline as well as saline administered control with the relative order of potency being TRV130>morphine>tianeptine. Calculated equi-effective doses to induce respiratory depression were examined and confirmed as being equi-respiratory depressant. TRV130 and tianeptine returned to baseline ~ 1.5 hours after administration compared to morphine which maintained significant respiratory depression.

In the hot plate assay these doses produced equivalent levels of antinociception with morphine antinociception maintained compared to the short duration of action of TRV130 and tianeptine. These data suggest that for tianeptine and TRV130 there is a similar therapeutic window to morphine, albeit with a reduced drug half-life preventing extended respiratory depression.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 29J1

Inhibitory mechanisms of adenosine on the Ob-R receptor mediated leptin signaling

T. HOSOI1,2, A. SILVA1, T. MATSUURA2, N. SUGIYAMA2, K. SHIMIZU2, K. OZAWA2, R. JOCKERS1, J. DAM1

1 Inserm U1016, Institut Cochin, Dept Endocrinology, Metabolism and Diabetes, Paris, France. 2 Department of Pharmacotherapy, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan. corresponding author: [email protected]

Obesity, characterized by chronic low grade inflammation, is a worldwide health problem associated with metabolic diseases. Inefficient action of the anti-obesity hormone, leptin, known as leptin resistance, has been suggested to be one of the mechanisms underlying obesity development. The nucleoside adenosine, produced in almost all mammalian cells, is generated by the degradation of ATP, ADP and AMP, and can be released in the extracellular milieu. Adenosine, being suggested to participate in the development of obesity, was investigated for its implication in promoting leptin resistance.

Leptin induces anti-obesity action through binding to its cognate receptor, ObR, and activating JAK2-STAT3 pathway in neuronal cells. Treatment with adenosine dose- dependently inhibited leptin-induced STAT3 phosphorylation in ObR-expressing SH-SY5Y neuroblastoma cells. The antagonist of the adenosine receptor, a G-protein coupled receptor, failed to reverse the inhibitory action of adenosine on leptin signaling. Conversely, the inhibition of adenosine transport into the cell rescued the deleterious effect of cell treatment with adenosine, suggesting that intracellular adenosine is responsible for interfering with leptin signaling. Intracellular adenosine did not affect leptin-induced ObR conformational change / oligomerization as assessed using ObR BRET biosensor, suggesting intracellular downstream effects of adenosine.

Overall, these results suggest that adenosine enters the cell and inhibits leptin signaling by affecting subsequent mechanisms downstream of ObR activation. The molecular events underlying those effects are still under investigation.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 30J1

The rearrangement of a class B GPCR dimer/oligomer

G.HOU, M. YUAN, L. XUE, J. LIU, Q. SUN, P. RONDARD, J.-P. PIN, J. LIU

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.

Corresponding authorl: [email protected]

G protein-coupled receptors (GPCRs) can form dimers and higher-order oligomers to integrate extracellular signals. In the previous work, we have investigated the interface of class C GPCR during activation, which are constitutive dimers. First, we found mGlu2 dimer interface is formed by transmembrane helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state (2015, Nat. Chem. Biol.). This major change in the dimer interface is required for receptor activity. Then, in mGlu2-4 heterodimers, we find G protein activation is mediated by mGlu4 heptahelical domain (HD) exclusively, though both mGlu2 and mGlu4 can couple to G proteins. G proteins activation can also be selectively changed by mGlu2 positive allosteric modulator or mGlu4 negative modulator (2017, elife). Further, taking GABAB receptor as a model of heterodimer and oligomer, we identify the transmembrane interfaces involved in GABAB1-

GABAB2, as well as GABAB1-GABAB1 interactions. While the GB1-GB2 interface is proposed to involve TM5 in the inactive state, cross-linking of TM6s lead to constitutive activity (2019, Nat Commun.). Based on the findings in Class C GPCR, we explored the structural basis of GPCR dimer and oligomer in class A and B. We found that GLP-1R, a class B GPCR, can form dimer and oligomer in basal condition and the interface is not regulated by agonist up to now. We expect this series of studies will bring new sight for our understanding of structural and functional basis in GPCR dimerization and/or oligomerization.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 31J1

A novel multi-target-directed ligand with triple activity alleviates Alzheimer’s disease pathology in vivo

C. ISMEURT, B. HATAT

University of Montpellier, Institute of Functional Genomic, 141 rue de la cardonille, 34090 Montpellier, FRANCE

Corresponding author: [email protected]

Face to the complexity and multi-factorial etiology of Alzheimer's disease (AD), a combination therapy could be the key to achieve clinical efficiency and stop the progression of the disease. Our approach is to combine in a sole molecule, several activities targeting molecular agents involved in AD pathology.

The 5-hydroxytryptamine type 4 and type 6 receptors (5-HT4R and 5-HT6R) and the acetylcholinesterase (AChE) are three targets with potential interest against AD. Based on structure activity relationships between these targets, duals compounds with either 5-HT4R agonist and 5-HT6R antagonist activities, or 5-HT4R agonist and AChE inhibition activities have been synthesized and have showed their efficacy in vivo in mouse models of AD and in preclinical studies. We synthesized new molecules aiming to act on these three targets at the same time. A pharmacological screen identified MR 35602 as a promising compound bearing the requested three activities. A chronic treatment with this triple-activity compound alleviated the pathology of 5XFAD mice, a transgenic murine model of AD. The results of MR 35602 ability to reduce the amyloid pathology and to prevent the cognitive deficits in several behavioural tests will be presented.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 32J1

Modelling of the conformational response of CCR5

to the binding of HIV-1 gp120 variants

C. JACQUEMARD

Université de Strasbourg/Laboratoire d’Innovation Thérapeutique – UMR7200, 74 route du Rhin, Illkirch-Graffenstaden, France corresponding author: [email protected]

CCR5 is the main co-receptor for entry of HIV-1 into cells. CCR5 binds the CD4-bound form of the viral envelope glycoprotein gp120, hence triggering fusion between the viral and cell membranes. Recently, we have shown that gp120s from different HIV-1 strains do not recognize the same CCR5 conformations, a process that regulates the virus biological properties (Colin, PLoS Pathog, 2018). However, the underlying molecular mechanisms, as well as the nature of CCR5 conformations, are unclear.

We used molecular dynamics to compare the binding modes of two gp120s (#25 and #34), which interact with different CCR5 conformations. We modelled the complexes between CCR5, CD4 and either of the two gp120s by homology to an experimental structure (PDB code: 6MEO). We simulated the two complexes in explicit water, after insertion into a lipid bilayer containing cholesterol. The systems were prepared using CHARMM-GUI in an all-atom representation. For each of the two systems, three 100ns-long trajectories have been produced using AMBER16.

Structural analyses reveal common, but also specific, CCR5 residues for binding of gp120#25 and gp120#34. Differences in the binding modes correspond to distinct conformations of TM5, TM6 and TM7, which are involved in CCR5 homodimerization (Jin, Sci Signal, 2018), and suggest an explanation for the functional selectivity of HIV-1 gp120s.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 33J1

Phosphorylation-deficient G-protein-biased μ-opioid receptors improve analgesia and diminish tolerance but worsen opioid side effects

A. KLIEWER1, F. SCHMIEDEL1, S. SIANATI2, A. BAILEY3, J. T. BATEMAN4, E. S. LEVITT4, J. T. WILLIAMS5, M. J. CHRISTIE2 AND S. SCHULZ1*

1Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller- University, 07747 Jena, Germany 2Discipline of Pharmacology, School of Medical Sciences, University of Sydney, NSW 2006, Australia. 3Institute of Medical and Biomedical Education, St George’s University of London, London SW17 ORE, UK 4Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32608 5The Vollum Institute, Oregon Health and Science University, 3181S.W. Sam Jackson Pk. Rd., Portland, OR, 97239, USA. corresponding author: [email protected]

Opioid analgesics are powerful pain relievers; however, over time, pain control diminishes as analgesic tolerance develops. The molecular mechanisms initiating tolerance have remained unresolved to date. We have previously shown that desensitization of the μ-opioid receptor and interaction with β-arrestins is controlled by carboxyl-terminal phosphorylation. Here we created knockin mice with a serief s o serine- and threonine-to-alanine mutations that render the receptor increasingly unable to recruit β-arrestins. Desensitization is inhibited in locus coeruleus neurons of mutant mice. Opioid-induced analgesia is strongly enhanced and analgesic tolerance is greatly diminished. Surprisingly, respiratory depression, constipation, and opioid withdrawal signs are unchanged or exacerbated, indicating that β-arrestin recruitment does not contribute to the severityf o opioid side effects and, hence, predicting that G-protein-biased µ-agonists are still likely to elicit severe adverse effects. In conclusion, our findings identify carboxyl-terminal multisite phosphorylation as key step that drives acute μ-opioid receptor desensitization and long-term tolerance.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 34J1

The orphan receptor GPR88 blunts the signaling of opioid receptors and multiple striatal GPCRs

T.LABOUTE1§, J. GANDIA1§, L. P. PELLISSIER1,2, Y. CORDE1, F. REBEILLARD3, M. GALLO4, C. GAUTHIER2, A. LEAUTE1, J. DIAZ3, A. POUPON2, B. L. KIEFFER5,6, J. LE MERRER1,6*, J. A.J. BECKER1,6*

1 Deficits of Reward, GPCRs and Sociability, Physiologie de la Reproduction et des Comportements, INRA UMR-0085, CNRS UMR-7247, Université de Tours, Inserm, 37380 Nouzilly, France 2 Biology and Bioinformatics of Signalling Systems, Physiologie de la Reproduction et des Comportements, INRA UMR-0085, CNRS UMR-7247, Université de Tours, 37380 Nouzilly, France. 3 Cellular Biology and Molecular Pharmacology of central Receptors, Centre de Psychiatrie et Neurosciences, Inserm UMR_S894 - Université Paris Descartes, Sorbonne Paris Cité, 75013 Paris, France. Current affiliation: CNRS (Integrative Neuroscience and Cognition Center, UMR 8002), Paris, France - Université Paris Descartes, Sorbonne Paris Cité, 75013 Paris, France 4 Department of Experimental & Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, 08003 Barcelona, Spain 5 Department of Psychiatry, Douglas Mental Health University Institute and McGill University, H4H 1R3 Montreal, Canada 6 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, Inserm U1258, Université de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch, France §* Authors contributed equally to this work. corresponding author: [email protected] [email protected]

GPR88 is an orphan G protein coupled receptor (GPCR) considered as a promising therapeutic target for neuropsychiatric disorders. The pharmacology of this receptor, however, remains scarcely understood. Based on our previous report of increased delta opioid receptor activity in Gpr88 null mice, we investigated the impact of GPR88 coexpression on the signaling of opioid receptors in vitro and revealed that GPR88 inhibits the activation of both their G protein- and b-arrestin-dependent signaling pathways. In Gpr88 knockout mice, morphine-induced locomotor sensitization, withdrawal and supraspinal analgesia were facilitated, consistent with a tonic inhibitory action of GPR88 on μOR signaling. We then explored GPR88 interactions with more striatal versus non-neuronal GPCRs, and revealed that GPR88 can bias the G protein-dependent signaling of most receptors in close proximity, but impedes b-arrestin recruitment by all receptors tested. Our study unravels an unsuspected buffering role of GPR88 expression on GPCR signaling, with intriguing consequences for opioid and striatal functions.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 35J1

Biased agonism and conformational heterogeneity of CCR5 in HIV infection

B. LAGANE

INSERM U1043/Centre de Physiopathologie de Toulouse-Purpan (CPTP), BP 3028, 31024 Toulouse Cedex 3, France corresponding autor: [email protected]

CCR5 is a receptor for chemokines and the major coreceptor for HIV-1 entry into CD4 T cells. As a GPCR, it exists in diverse conformations and oligomeric arrangements. The molecular mechanisms and the functional consequences of CCR5 conformational heterogeneity have just begun to be evaluated in recent years. CCR5 chemokines can produce biased agonism, a phenomenon whereby distinct chemokines have different signaling outcomes owing to stabilization of distinct CCR5 conformations. The role of coreceptor heterogeneity in HIV-1 pathogenesis and AIDS development is however far less understood. In previous works, we showed that HIV-1 strains take advantage of low- chemokine affinity conformations of CCR5 to escape inhibition by chemokines (Colin, PNAS, 2013; Jin, JBC, 2014). More recently, we described that distinct HIV-1 envelope glycoproteins (Envs) may differ in the nature of the CCR5 conformations with which they interact, a process that critically regulates the virus biological properties (Colin, PLoS Pathog, 2018). In light of these studies, we then speculated that selection of particular CCR5 conformations might provide distinct HIV-1 Envs and chemokines with distinct signaling properties. By using a method based on Bioluminescence Resonance Energy Transfer, we analyzed the capacity of diverse viral and native CCR5 ligands to activate distinct G protein isoforms. Results showed that differences exist between chemokines and HIV-1 Envs, but also between distinct Envs, in the nature of the CCR5-G protein complexes targeted. These results represent a step forward towards understanding the role of Env-mediated signaling in the pathophysiology of HIV infection.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 36J1

Structural and molecular basis of Class C GCPRs signalling

C. NASRALLAH1, G. CANNONE2, K. ROTTIER1., P. GUILLET3, G. DURAND3, J.-P. PIN1, J.- L. BANÈRES3, K. R. VINOTHKUMAR4, R. HENDERSON2 & G.LEBON1.

1 Institut de Génomique Fonctionnelle, UMR CNRS 5203, INSERM 1191, Université de Montpellier, 141 rue de la cardonille 34094 Montpellier, cedex 05, France. 2 MRC Laboratory of Molecular Biology, Francis Crick avenue, CB2 0QH Cambridge, UK. 3 Institut des Biomolécules Max Mousseron, UMR CNRS-UM-ENSCM 5247, Université d'Avignon, 301 rue Baruch de Spinoza, 84916 AVIGNON cedex 9, France. 4 National Centre for Biological Sciences – TIFR, GKVK Post, Bellary Road, Bangalore 560065 India. corresponding author: [email protected]

G protein-coupled receptors (GPCRs) mediate signal transduction from the extracellular to the intracellular compartment and play an important role in different physiological and pathological processes. GPCRs are classified into five main distinct classes, A, B and C, Frizzled and adhesion. Class C GPCRs that include metabotropic glutamate receptors (mGlu), are unusual in terms of their molecular architecture and allosteric regulation. They all form obligate dimer and dimerization being fundamental for their function. Class C mGlu receptors are activated by the binding of the main excitatory neurotransmitter glutamate, within a large extracellular domain (ECD). Conformational changes induced by glutamate- binding are then transmitted to the transmembrane domain composed of 7 transmembrane helices (7TM) that allows signal transduction. Class C GPCR activity can also be allosterically modulated by the binding of Positive Allosteric Modulators (PAM) or Negative Allosteric Modulators (NAM) to the 7TM domain. As a first step toward structure determination of mGlu receptors, we have thermostabilised the mGlu5 receptor bound to negative allosteric modulator (NAM). Thermostabilised mGlu receptor is fully functional and displays an increased thermal stability compared to the WT mGlu5 receptor. Using single particle electron cryomicroscopy (cryoEM), we obtained high quality images of mGlu5 receptor dimer in ice that allowed us to visualise isolated single particle of the receptor dimer. The 2D-class averages revealed the presence of dimeric single particles of the receptor. Understanding the structural basis of mGlu receptor dimer signalling will represent a landmark achievement and pave the way for structural investigation of GPCR dimer signalling in general.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 37J1

A novel in vivo anti-amnesic agent, specially designed to express both AChE inhibitory, 5-HT4R agonist and 5-HT6R inverse agonist activities, with a potential interest against Alzheimer’s Disease

B. HATAT1,2, S. YAHIAOUI1, C. LECOUTEY1, A. DAVIS1, T. FRERET3, M. BOULOUARD MICHEL3, S. CLAEYSEN2, C. ROCHAIS1, P. DALLEMAGNE1

1 Normandie Université, UNICAEN, CERMN, Caen, France. 2 IGF, University of Montpellier, CNRS, INSERM, Montpellier, France 3 Normandie Université, UNICAEN, INSERM, U1075, GIP CYCERON, COMETE, Caen, France corresponding author: [email protected]

This work describes the conception, synthesis, in vitro and in vivo biological evaluation of novel Multi-Target Directed Ligands able to both activate 5-HT4 receptors, block 5-HT6 receptors and inhibit acetylcholinesterase activity, in order to exert a synergistic anti-amnesic effect, potentially useful in the treatment of Alzheimer’s disease (AD). Indeed, both activation 1,2 of 5-HT4 and blockage of 5-HT6 receptors led to an enhanced acetylcholine release, suggesting it could lead to efficiently restoring the cholinergic neurotransmission deficit observed in AD. Furthermore, 5-HT4 receptor agonists are able to promote the non- amyloidogenic cleavage of the amyloid precursor protein (APP) and to favor the production of the neurotrophic protein sAPPα. Finally, we identified a pleiotropic compound, [1-(4-amino- 5-chloro-2-methoxyphenyl)-3-(1-(3-methylbenzyl)piperidin-4-yl)propan-1-one fumaric acid salt, which displayed in vivo an anti-amnesic effect in a model of scopolamine- induced deficit of working memory at a dose of 0.3 mg/kg. This work was supported by funding from the Fondation Vaincre Alzheimer (#FR-15072) and the Fondation Plan Alzheimer (AAP2015 Project TRIAD 016).

References 1. Kilbinger, H., Wolf, D. Naunyn Schmiedebergs Arch. Pharmacol. 1992, 345, 270–275; Consolo, S., Arnaboldi, S., Giorgi, S., Russi, G., and Ladinsky, H. Neuroreport 1994, 5, 1230–1232. 2. Shirazi-Southall, S., Rodriguez, D. E., Nomikos, G. G. Neuropsychopharmacology 2002, 26, 583–594; Riemer, C., et al. J. Med. Chem. 2003, 46, 1273–1276.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 38J1

Serotonin 5-HT6 receptors expression pattern: new insight into their neurodevelopmental roles

V. DUPUY

Université de Montpellier/Institut de Génomique Fonctionnelle/Département Neurosciences, 141 rue de la Cardonille, Montpellier, France corresponding author: [email protected]

The serotonin 6 receptors (5-HT6Rs) are Gs-coupled receptors that hold special promise as targets for the treatment of cognitive deficits associated with neurodevelopmental and psychiatric disorders. However, the mechanisms underlying their impact upon neurodevelopment and cognition remain partially characterized. A major limitation is the absence of specific antibodies allowing precise assessment of cellular localization of 5-HT6Rs in the brain.

We used a genetically modified knock-in mouse strain expressing 5-HT6Rs fused to a green fluorescent protein. We explored 5-HT6Rs expression pattern in several brain regions at different developmental stages.

We show that 5-HT6Rs are expressed as early as E15, in a wide variety of areas related to high cognitive functions including the striatum, the hippocampus and the cortex. In all structures examined, 5-HT6Rs are mostly expressed in projection neurons and, to a lesser extent, in interneurons & astrocytes.

In embryonic and adult mice, 5-HT6Rs are mostly localized in the primary cilium, a non-motile cellular structure extending from the soma of almost every cell type. Intriguingly, 5-HT6Rs are also detected in the somato-dendritic compartment of neurons specifically at the postnatal stage, from P1 to P10. This suggests a peculiar role of 5-HT6Rs in events related to postnatal development such as neuronal circuit wiring.

To test this, we pharmacologically inhibited 5-HT6Rs between P1 and P10. Our preliminary results obtained with the Crawley’s sociability test show that inhibiting 5-HT6Rs at this postnatal stage impairs mice social cognition in the long term.

Overall, our data indicate that 5-HT6Rs are key players in neurodevelopment.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 01J2

Sequestration of critical cholesterol molecules by ligand-bound oxytocin GPCR

L. LEMEL

Institut de Biologie Structurale, 71 avenue des Martyrs, Grenoble, France

Corresponding autor: [email protected]

Cholesterol is a major component of mammalian plasma membranes that affects the physical properties of the lipid bilayer but also the function of many membrane proteins including G protein-coupled receptors (GPCRs). For the oxytocin receptor (OXTR), cholesterol acts as an allosteric ligand, inducing a high-affinity state of the receptor for its orthosteric ligands.

Ion channel-coupled receptors (ICCRs) were created as reporters of GPCR conformational changes by functionally linking an ion channel to a GPCR. The channel generates an electrical signal upon conformational changes of the GPCR, allowing for detection by electrophysiological techniques or miniaturized electronic systems.

In the present study, we investigated the ability of ICCR to report the cholesterol dependence of OXTR. Currently, standard methods for testing cholesterol-dependence of GPCRs are based on radiolabeled ligand binding or G-protein activation assays. We experimented a protocol for depletion/restoration of cholesterol level in Xenopus oocytes and functionally characterized the ICCR by the two-electrode voltage-clamp method in those conditions. Furthermore, we demonstrated that both cholesterol and ligand are needed to preserve the high-affinity state of the OXTR.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 02J2

A G protein-coupled receptor micro-switch controls allosteric agonism

L. LIU1,2*, Z FAN1*, X. ROVIRA3*, L. XUE2, M. XIN1, J.-P. PIN2, P. RONDARD2, J. LIU1

1 Cellular Signaling laboratory, International Research Center for Sensory Biology and Technology of MOST, Key Laboratory of Molecular Biophysics of Ministry of Education, School of Life Science and Technology, Huazhong University of Science and Technology, 430074 Wuhan, China; 2 Institut de Génomique Fonctionnelle (IGF), CNRS, INSERM, Université de Montpellier, Montpellier, France; 3 Molecular Photopharmacology Research Group, The Tissue Repair and Regeneration Laboratory, University of Vic - Central University of Catalonia, C. de la Laura,13, 08500 Vic, Spain * Contributed equally to this work. corresponding authors: [email protected] or [email protected]

Activating G protein-coupled receptors represents a way for therapeutic interventions. Hope came with the discovery of positive allosteric modulators that enhance the action of endogenous agonists. Such modulators often display agonist activity. Here, we used the heterodimeric GABAB receptor as a model to investigate the molecular basis of allosteric agonism. Our data reveal a key region in the center of the hydrophobic core of the receptor critical for both constitutive activity and allosteric agonism, these being correlated. We show that this region, found to play a similar role in another class C GPCR, is a functionally conserved micro-switch controlling the conformational landscape of most GPCRs, and corresponds structurally to the Na+ site found in most class A receptors. Mutations in this micro- switch including some identified in human genetic diseases tuned both constitutive activity and allosteric agonism. Our results bring new information on the molecular basis of allosteric agonism in GPCRs, with important consequences for the future development of allosteric modulators.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 03J2

Coarse-grained simulations to predict the dynamical interactions of GPCRs with their partners

M. LOUET, B. DELORT, P. RENAULT & N. FLOQUET

Institut des Biomolécules Max Mousseron, CNRS, Ecole Nationale Superieure de Chimie de Montpellier, Université Montpellier, 34093 Montpellier, France corresponding author: [email protected]

The prediction of protein-protein or peptide-protein interaction constitutes one of the most interesting modeling challenge as it requires a proper exploration of all conformations of the isolated partners and the resulting complexes. In a pioneer work, Dror et al. demonstrated that Molecular Dynamics (MD) simulations performed with classical all-atoms force fields could achieve such a goal and predict at the molecular scale the binding of a drug to its receptor. Unfortunately, the size of these systems and the time-scale required to observe such a binding strongly limit the use of this approach to laboratories having access to huge computational facilities. These last years we have tested and validated a new unbiased method that combines the MARTINI coarse-grained model and Replica-Exchange MD (REMD) simulations to predict the binding of peptides to proteins. The main advantage of this approach is to be much less computationally expensive and yet accurate. We will briefly describe how we validated our protocol on Neurotensin and CXCR4 receptors. We will then newly propose a model of the ghrelin peptide bound to its receptor, helped by a cross validation with NMR experiments. We were able to suggest a dynamical view of the conformation ensemble of ghrelin in the context of the receptor. We will also show that the method was able to qualitatively reproduce the binding propensity of both acylated and unacelytaed ghrelin. To conclude, and as perspective, we will propose the possible extension of our protocol to the prediction of protein-protein interactions.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 04J2

Fine-tuning the wavelengths and irradiance to photoswitch the activity of photochromic ligands

F. MALHAIRE1, A.E. BERIZZI1, M. RICART-ORTEGA1,2, V. PEREIRA1, X. GÓMEZ- SANTACANA1, J. FONT1, J. CATENA2, L. MUÑOZ2, C. SERRA2, X. ROVIRA3, J.-P. PIN1, A. LLEBARIA2, C. GOUDET1

1IGF, CNRS, INSERM, Univ. de Montpellier, F-34094 Montpellier, France 2MCS, Laboratory of Medicinal Chemistry & Synthesis, Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain 3Molecular Photopharmacology Research Group, University of Vic – Central Univ. of Catalonia, 08500 Vic, Spain corresponding authors: [email protected]

Photopharmacology enables the photocontrol of endogenous receptors by light through the use of photochromic ligands (PCLs). Most PCLs are azobenzene-containing molecules that can rapidly and reversibly photo-isomerise under specific light conditions, modifying the ability of the ligand to interact with its target. In the present study, we determined the optimal wavelenghts and irradiance to photoswitch the activity of two PCLs (namely Alloswitch-1 and XGS-023) targeting the mGlu5 receptor in cell-based assays. To that aim, we measured the pharmacological activity of these ligands on HEK293 cells expressing the human mGlu5 receptor and compared the photoinduced potency shift (pPPS) under different light conditions. We first applied different light colours, ranging from 365 to 530 nm, at a fixed irradiance of 12 mW/cm². In the dark or under green light, both ligands act as potent mGlu5 negative allosteric modulators (NAM). Under violet or blue light, their potencies are significantly reduced (by 20- times for Alloswitch-1 and 13-times for XGS-023), validating that the trans- photoisomers of these two PCLs are the most active. Comparing the pPPS, we determined that the optimal wavelenghts are 380-400 and 500-530 nm for Alloswitch-1, and 365-420 and 470-530 nm for XGS-023. We then applied a fixed wavelenght of 380 nm with variable irradiances ranging from 1.5 to 14.7 mW/cm² to determine that the pPPS was maximum for an irradiance equal or superior to 10 mW/cm² for both Alloswitch-1 and XGS-023. These parameters will be important for further in vitro and in vivo studies using these ligands.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 05J2

Dynamics of Mu-Delta Neuronal Co-expression in Morphine Tolerant and Abstinent Mice.

D. MASSOTTE

Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR3212, 8 Allée du Général Rouvillois, 67000 Strasbourg, France corresponding author: [email protected]

Opiate addiction develops as a chronic relapsing disorder upon drug recreational use or following misuse of analgesic prescription. Mu opioid (MOP) receptors are the primary molecular target of opiates but increasing evidence support in vivo functional heteromerization with the delta opioid (DOP) receptor, which may be part of the neurobiological processes underlying opiate addiction. Here, we used double knock-in mice co-expressing fluorescent versions of the MOP and DOP receptors to examine the impact of chronic morphine administration on the distribution of neurons co-expressing the two receptors. Our data show that MOP/DOP neuronal co-expression is broader in morphine-dependent mice and is detected in novel brain areas located in circuits related to drug reward, motor activity, visceral control and emotional processing underlying withdrawal. After four weeks of abstinence, MOP/DOP neuronal co-expression is still detectable in a large number of these brain areas except in the motor circuit. Importantly, chronic morphine administration increased the proportion of MOP/DOP neurons in the brainstem of morphine-dependent and abstinent mice. These findings establish persistent changes in the abstinent state that may modulate relapse and opiate-induced hyperalgesia and also point to the therapeutic potential of MOP/DOP targeting.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 06J2

Development of clover-mRuby2 FRET to image

intermolecular GPCR signaling.

A. MAZARS

Université Toulouse III, INSERM U1048, Institut des Maladies Métaboliques et Cardiovasculaires, 1 Avenue Jean Poulhès, BP 84225, 31432, Toulouse, FRANCE. corresponding author: [email protected]

This last decade, our knowledge of GPCR signaling dynamics in living cells has been largely improved by the use of BRET sensors. Even if BRET allows an accurate sensing of signaling events, it is highly limited in imaging signaling dynamics. In that sense, FRET sensors represent more suitable tools. FRET has been originally used to monitor intermolecular events. To date, only FRET ratiometric intramolecular sensors have been developed (cAMP, Ca2+, cGMP sensors…). This mainly relies on the high spectral overlap between the CFP energy donor and the YFP energy acceptor, the commonly used FRET pair. Recently, this problem has been resolved thanks to the development of a new clover-mRuby2 FRET pair, yet mainly developed for intramolecular sensors. Here, we report the use of the new Clover/mRuby2 FRET pair to monitor and image intermolecular GPCR signaling events that combine the spectral resolution of BRET2 with the photonic intensity of FRET, and, has the considerable advantage of a complete lack of background that is a major pitfall of conventional CFP/YFP pair. We first validated the new Clover/mRuby2 pair and compared it to the common CFP/YFP pair. Finally we imaged and quantified some GPCR signaling events (b-arrestin and G protein activation). In the future, these new GPCR FRET probes should allow a more accurate visualization of compartmentalized GPCR signaling; therefore improving our knowledge of GPCR pharmacology.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 07J2

The repertoire of family A-peptide GPCRs in archaic hominins

C. MOLLEREAU, X. MATA, G. RENAUD

Laboratoire AMIS (Anthropobiologie Moléculaire et Imagerie de Synthèse) CNRS UMR5288, Université de Toulouse, UPS Faculté de Médecine, 37 Allées Jules Guesde 31073 Toulouse, France corresponding author: [email protected]

Given the importance of G-protein coupled receptors in the regulation of many physiological functions, deciphering the relationships between genotype and phenotype in past and present hominin GPCRs is of main interest to understand the evolutionary process that contributed to the present-day variability in human traits and health. Here, we carefully examined the publicly available genomic and protein sequence databases of the archaic hominins (Neanderthal and Denisova) to draw up the catalog of coding variations in GPCRs for peptide ligands, in comparison with living humans. We then searched in the literature the functional changes, phenotypes and risk of disease possibly associated with the detected variants. Our survey suggests that Neanderthal and Denisovan hominins were likely prone to lower risk of obesity, to enhanced platelet aggregation in response to thrombin, to better response to infection, to less anxiety and aggressiveness and to favorable sociability. While some archaic variants were likely advantageous in the past, they might be responsible for maladaptive disorders today in the context of modern life and/or specific regional distribution. For example, an archaic haplotype in the neuromedin receptor 2 is susceptible to confer risk of diabetic nephropathy in type 1 diabetes in present-day Europeans. Paying attention to the pharmacological properties of some of the archaic variants described in this study may be helpful to understand the variability of therapeutic efficacy between individuals or ethnic groups.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 08J2

Development and implementation of tools to study allosteric communication networks in GPCRs

A. MORALES-PASTOR

Universitat Pompeu Fabra / Institut municipal d’investigacions mèdiques / Research programme on biomedical informatics, Dr. Aiguader 88, 8003, Barcelona, Spain corresponding author: [email protected]

G-protein coupled receptors (GPCR) are the largest family of human cell-surface receptors. They are very important drug targets as they can be modulated to treat many diseases. The set of signalling pathways that a GPCR activates when interacting with a drug is decisive for a successful treatment. Drugs that specifically activate certain pathways, are called biased ligands and are of great interest as they can avoid causing side effects. However, how these ligands work is not fully understood and there is not a standardized protocol to identify them.

Here, we show that mutual information (MI) between χ1 dihedral angles of certain GPCR residues can be tightly related to receptor activity. In total, 16 residues were highlighted by the analysis, some of them overlap with critical regions for GPCR activation. We found that the values of MI in a receptor can be used to classify with excellent accuracy active and inactive GPCR structures. We also see that this pattern of MI may be related to biased signalling. Our results open the path to the development of computational tools to identify biased ligands, as we would be able to identify if a ligand induces the engagement of a biased structure in the targeted receptor.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 09J2

β-arrestin-2 BRET biosensors detect different β-arrestin-2 conformations in interaction with GPCRs

A.OISHI, J. DAM, R. JOCKERS

Université de Paris, Institut Cochin, CNRS, INSERM, F-75014 Paris, France corresponding author: [email protected]

β-arrestins are critical regulators of G protein-coupled receptors (GPCRs) that desensitize G protein signaling, promote receptor internalization and initiate signaling on their own. Recent structural findings indicate that β-arrestins adopt different conformations upon interaction with agonist-activated GPCRs. Here, we established a β-arrestin-2 conformational BRET sensor composed of the bright Nanoluc BRET donor and the red shifted CyOFP1 BRET acceptor. The sensor monitors early intramolecular conformational changes of β-arrestin-2 in complex with a wide panel of different class A and class B GPCRs upon agonist activation, and with orphan GPCRs known to spontaneously recruit β-arrestin-2. The introduction of the R170E mutant in the β-arrestin-2 sensor allowed the detection of a partially active β-arrestin conformation, which is not dependent on receptor phosphorylation, while the deletion of the β-arrestin-2 finger-loop region detected the “tail-conformation” corresponding to the interaction of β-arrestin with the carboxyl-terminal domain of GPCRs. The new sensors combine the advantages of the BRET technique in terms of sensitivity, robustness and suitability for real-time measurements with a high responsiveness towards early conformational changes to help to elucidate the different conformational states of β-arrestins associated with GPCR activation in living cells.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 10J2

Development of Novel Photoswitchable PAM for the Metabotropic

Glutamate Receptor Subtype 1 (mGlu1)

S. PANARELLO1-2, X. GÓMEZ-SANTACANA2, C. SERRA 1, F. MALHAIRE2, L. PRÉZEAU2, C. GOUDET2, J-P. PIN2, A. LLEBARIA1 1MCS, Medicinal Chemistry & Synthesis, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain 2IGF, Institute for Functional Genomics, CNRS, Montpellier, France corresponding authors : [email protected] [email protected]

The therapeutic potential of selective metabotropic glutamate receptor subtype1 (mGlu1) activation is still vastly unexplored, in comparison with the other group I mGluR (mGlu5), although mGlu1 dysfunction can be associated with high incidence neuropsychiatric diseases. That is why the interest in mGlu1 positive allosteric modulators (PAM) has increased.

Besides the potential of mGlu1 PAMs, obtaining a reversible and efficient spatiotemporal control of mGlu1 activity would be advantageous, for both research and clinical applications. Photopharmacology may provide a solution on this topic, since it based on the use of light to modulate a protein activity with photoswitchable ligands. This approach offers new perspectives for drug discovery and therapeutics and promises a better control of drug action reducing side effects to unattained levels.

We have focused on developing photoswitchable mGlu1 PAMs in order to precisely switch on/off the activity of the mGlu1 receptor by means of different wavelengths of illumination. In this proposal, we have explored the azologization approach of known mGlu1 PAMs by introducing an azo-bond, instead of an phthalimide moiety.

Subsequent biological in vitro assays revealed that MCS-0242 is a mGlu1 PAM ligand in the dark, whereas under 380-nm light it loses activity. The obtained reversible control of mGlu1 activity with light may be advantageous to study the pharmacological and physiological implications of mGlu1 with an unprecedented precision, which may lead to unexpected findings and new avenues for optimization.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 11J2

Impact of FAK inhibition and co-targeting resistance pathways in Gaq-driven uveal melanoma

J. PARADIS, M. ACOSTA, R. SADDAWI-KONEFKA, X. FENG, N. ARANG, K. WOOD, S. GUTKIND Moores Cancer Center, University of California San Diego (UCSD) corresponding author: [email protected]

Uveal melanoma (UM) is characterized by gain-of-function mutations in GNAQ or GNA11, which encode Ga proteins from the Gq/11 family. UM is the most common primary cancer of the eye in adults and approximately 50% of UM patients develop liver metastasis within 5-10 years after diagnosis1, independent of the successful treatment of the primary lesions. There are effective therapeutic strategies for primary UM lesions, but UM metastatic disease is refractory to current chemotherapies and immune checkpoint inhibitors, thus most patients with UM metastasis die within a year. Mechanistically, Gaq activation stimulates the PKC/ERK canonical pathway and the MEK inhibitor ‘selumetinib’ has recently been approved for UM treatment. However, a new phase III trial demonstrated that MEK inhibition has nearly no impact on UM patient overall survival2. Therefore, there is still no effective treatment options for metastatic UM. We recently found that focal adhesion kinase (FAK) is overactivated in UM by the non- canonical Gαq/Trio/RhoA pathway, thus activating cell growth3. My project aims to establish the therapeutic potential of targeting both the canonical Gaq/PKC/ERK and noncanonical Gaq/Trio/FAK pathways by combining MEK and FAK inhibitors. Our results show that FAKi first decreases ERK activity while subsequently causing ERK compensatory reactivation. Additionally, oncogenes triggering ERK stimulation rescue UM cells from FAKi growth inhibition. Aligned with ERK activation representing a FAKi-resistance pathway, ERK pathway inhibition by various MEKi, combined with FAK inhibition (VS-4718), shows remarkable synergistic growth inhibitory effects in UM cells. The ‘Trametinib+VS-4718’ combination emerges in our tests as the strongest drug combination in vitro. Furthermore, this combination shows cytotoxic effects in UM xenografts leading to tumor shrinkage, whereas the single drugs are cytostatic. Our immunohistochemistry analysis confirms a decrease of cell proliferation (BrdU), phospho-ERK and nuclear YAP, as well as an increase in cellular apoptosis (cleaved Caspase3). Finally, we are implementing a new animal model for liver metastatic uveal melanoma, performing a splenic implantation of luciferase- expressing UM cells followed by hematogenous dissemination to the liver. Altogether, our data provide significant preclinical evidence of the effectiveness of blocking FAK in combination with MEK to prevent the growth in human UM xenograft models. Given the fact that ‘Trametinib’ is an FDA approved drug, we hope that our discoveries will be rapidly translated to the clinic.

1. Thomas S, et al., Br J Cancer (2012) 106(6), 1171-1176 2. Carvajal R., et al., Journal of Clinical Oncology (2018), 12, 1232-1239 3. Feng X., Arang N., et al., Cancer Cell (2019), 35(3), 457-472

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 12J2

Serotonin Receptors-Associated Networks: role of the insulin signalling pathway in APP processing

H. PAYAN1, R. DONNEGER1, E. CASSIER1, O. VIGY1, M. SEVENO1, P. MARIN1; S. CLAEYSEN1 1 IGF, CNRS, INSERM, Univ. Montpellier, France corresponding author: [email protected]

Alzheimer’s Disease (AD) is the most common form of dementia worldwide and has become a major public health problem. This pathology is characterized by the presence of two main features in the brain: neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau and amyloid plaques, dense aggregates of hydrophobic β-amyloid peptide (Aβ). Aβ peptide formation results from the amyloidogenic degradation of the Amyloid Precursor Protein (APP) by β- and γ-secretases mainly in early/sorting and late endosomes. On the other hand, the non-amyloidogenic proteolysis of APP, within the Aβ sequence by α-secretase, releases the extracellular fragment of APP (sAPPα), which is neurotrophic and neuroprotective. Non- amyloidogenic APP processing mainly occurs at the plasma membrane level. The most active α-secretase in the brain is ADAM10. We have demonstrated that the serotonin type 4 receptor (5-HT4R) physically associates directly or indirectly with ADAM10 and APP, modulates their trafficking and maturation, and thereby constitutively promotes the non- amyloidogenic cleavage of APP1,2. Our objectives are to decipher the protein networks underlying the trafficking of the 5-HT4R/ADAM10/APP complex to the plasma membrane in order to propose innovative targets to reduce amyloid production in the context of AD. Using an unbiased proteomic approach, we identified candidate proteins able to interact with this complex. By co-immunoprecipitation, we validated the interaction of selected proteins of interest with 5-HT4 receptors. Interestingly, a series of 5-HT4R-interacting partners belongs to the insulin signalling pathway. These proteins could be of particular relevance because insulin signalling is strongly dysregulated in Alzheimer’s disease3. We decrypt their role in the transduction pathways activated by the 5-HT4R and we explore their ability to promote the non-amyloidogenic processing of APP.

References 1. Claeysen S., et al. (2015) ACS Chem Neurosci. 2. Cochet M., et al. (2013) ACS Chem Neurosci, 4, 130-40. 3. Puglielli L. (2008) Neurobiol Aging, 29, 795-811.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 13J2

The urotensin II receptor relays key neurobiological mechanisms in subarachnoid hemorrhage: Efficacy of pathway-targeted drugs on long-term brain deficits

M. PEDARD1, M. DUBOIS1, R. GRUEL1, L. DESRUES1, D. GODEFROY1, O. WURTZ1 and H. CASTEL1

1Normandie University, UNIROUEN, INSERM, DC2N, IRIB, 25 rue Lucien Tesniere, Mont Saint Aignan, France corresponding author: [email protected]

Cerebral vasospasm (CVS) is a severe complication of aneurysmal subarachnoid hemorrhage (SAH). Urotensin II (UII) is a potent vasoactive peptide activating the UT receptor involved in brain vascular pathologies. Our clinical work established that high plasma level of UII in SAH patients is a predictive factor of the occurrence of CVS. Here we studied the role of the urotensinergic system in the occurrence of SAH-associated complications requiring various contributions of Gq and/or Gi pathways in a SAH mouse model. To seek for the best therapeutic molecule rapidly transferable to the patient, we evaluated the impact of a UT biased ligand mainly targeting the Gi/o pathway (urantide), on in vivo consequences of SAH. We used a double intracisternal blood injection SAH procedure in wild-type (UT+/+), UT knock-down (UT-/-) or humanized UT (UTh+/h+) C57Bl/6 mice to investigate the impact of UT, urantide and the Gq- and/or Gi-signaling pathways on SAH-associated complications. In the SAH UT+/+ or UTh+/h+ mice, UT was overexpressed in the endothelial vasospasmed arteries, cortical capillaries and the meningeal compartment from day1 to day7 post-SAH. In UT-/- mice, SAH-evoked CVS, neuroinflammation, hippocampal apoptosis and cognitive dysfunctions observed in UT+/+ and UTh+/h+, were not detected. Moreover, intracisternal administration of the Gq (YM-254890) and PLC (U73122) inhibitors or urantide, prevented CVS and fine motor coordination impairment consecutive to SAH. This study suggests that UT partial antagonism prevents CVS and protect brain functions via inactivation of a Gq/PLC pathway after SAH, positioning UT as a key therapeutic target in SAH.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 14J2

Multiple CCR7 Receptor Assays to enable Better Drug Discovery

G.WARNER1, R. OSMOND2, H. MARTIN-HARRIS2, V. DUPRIEZ1

1 PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com 2 TGR BioSciences, 31 Dalgleish Street, Thebarton, SA 5031, AUSTRALIA +61 (8) 8354 6170 www.tgr-biosciences.com.au

A main role of CCR7 is to organise the meeting of dendritic and T cells into the lymph nodes, increasing antigen presentation efficiency. In addition to naïve T Cells, TReg are also attracted in a CCR7-dependent mechanism and regulate the naïve T Cells maturation process. CCR7 also participates in the migration of activated B cells at the T-B cell border, where they can further stimulate the follicular B helper T cells differentiation and the B-cell selection and affinity maturation process.

CCR7 can be assayed at multiple levels: modulation of the binding of its natural ligand can be monitored using [125I] MIP-3ß binding displacement. Activation of G-proteins can be directly recorded by the measurement of binding of 35S-labelled GTP. Inhibition of adenylate cyclase by Gαi proteins can be tracked using a cAMP dosage assay. CCR7 can be coupled to phospholipase C activation using co-expression of the promiscuous Gα16 protein, and the subsequent calcium flux can be recorded using the AequoScreen® photoprotein technology, or as well the fluorescent calcium-sensitive dye method. MAP Kinase or Akt phosphorylation that occur following CCR7 activation can be tracked in a cell-based assay using the AlphaScreen® SureFire® corresponding assays. All these assays can now be performed with FroZen cells available from the shelve.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 15J2

Endosomal G protein signalling by US28 as a new target for glioblastoma multiforme

B. PLOUFFE1, S. HOUSTON1, E. HEULET2, C. ROBERTS3

1Queen’s University Belfast/Wellcome-Wolfson Institute for Experimental Medicine/School of Medicine, Dentistry and Biomedical Sciences, 97 Lisburn Road, Belfast, UK 2Université de Nantes/Faculté des Sciences et des Techniques, 2 rue de la Houssinière, Nantes, France 3University of Dundee/School of Medicine, Ninewells Hospital, Dundee, UK corresponding author: [email protected]

Glioblastoma Multiforme (GBM) is the most common and aggressive type of brain cancer. The human cytomegalovirus is detected in most GBM patient samples (50-90%) and encodes US28, a viral chemokine G protein-coupled receptor (GPCR). The constitutive activation

(agonist-independent) of Gαq by US28 is particularly important as it induces important oncomodulatory properties such as cell inflammation, proliferation, and angiogenesis. Using bioluminescence resonance energy transfer (BRET)-based biosensors, we show that US28 constitutively activates all the Gα protein isoforms tested, but most importantly Gαq. Unlike typical viral GPCRs which are found in the plasma membrane, US28 is mainly localised in endosomes due to chronic internalisation. This poor expression at the plasma membrane is somehow surprising given the high constitutive activity associated to US28. However, since the last decade a growing number of GPCRs have been reported to activate Gα proteins from intracellular compartments such as endosomes upon GPCR internalisation. Using enhanced bystander BRET (EbBRET)-based biosensors, we show a gradual redistribution of Gα proteins, in particular Gαq, from the plasma membrane to early, fast-recycling and slow- recycling endosomes concomitant with a gradual increase of Gαq constitutive activity when US28 is progressively overexpressed. These results suggest that the important endosomal pool of US28 may be actively stimulating Gαq and the associated oncomodulatory signalling. Consequently, directly targeting a US28 blocker (inverse agonist or nanobody) to the endosomes may represent a promising therapeutic approach to successfully block GBM malignancy.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 16J2

Platelet P2Y1 receptor exhibits physiological constitutive activity that is enhanced during aging

V. PONS

INSERM U1048, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), 1 avenue Jean Poulhès, Toulouse, France. corresponding author: [email protected]

P2Y1-R and P2Y12-R are ADP receptors that mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y1-R is responsible for platelet shape change and reversible platelet aggregation through Gq-dependent IP3 production and subsequent calcium release, P2Y12-R activation results in amplification and stabilization of the aggregation, through Gi-dependent adenylyl cyclase inhibition and subsequent cAMP decrease. While P2Y12-R is the major target of antiplatelet drugs, it is still puzzling that no

P2Y1-R selective blocker was yet developed for clinical use. Indeed, accumulating data suggest that P2Y1-R inhibition would ensure efficient inhibition of platelet activation with a minimal effect on bleeding, thereby displaying potential advantages over aggressive antiplatelet therapies targeting P2Y12-R. Here, using BRET/HRTF-based assay, we accurately characterized P2Y1-R pharmacology and demonstrated that P2Y1-R constitutively activates the Gq protein signaling at basal state both in living HEK293T cells and human platelets, highlighting the importance of P2Y1-R basal activity in physiological conditions. MRS2179 inhibited P2Y1-R-dependent constitutive signaling, thus behaving as an inverse agonist.

Interestingly, this constitutive P2Y1-R signaling was enhanced in platelets from old mice compared to young mice, in agreement with the age-associated platelet hyperactivity and increased thrombotic risk. Altogether, these results shed new light on the molecular mechanisms of P2Y1-R activation and suggest that the level of agonist-independent P2Y1-R basal signaling could represent a valuable readout to evaluate the potential thrombotic risk in the management of thrombotic disorders in the elderly population.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 17J2

Development and optimization of novel radiolabelled neurotensin analogues for NTS1-positive tumours theranostic

S. PREVITI, A. CHASTEL, R. FANELLI, E. HINDIÉ, E. RÉMOND, C. MORGAT‡, F. CAVELIER‡

Institut des Biomolécules Max Mousseron, IBMM, UMR-5247, CNRS, Université de Montpellier, ENSCM, Place Eugène Bataillon, 34095 Montpellier, France

‡These authors contribute equally corresponding author: [email protected]

Neurotensin (NT) is an endogenous 13-amino acids peptide distributed in the central nervous system and in peripheral tissues, where it is responsible for a wide range of biological activities. NT effects occur via the activation of the neurotensin receptors NTS1 and NTS2, belonging to the class A GPCR family, and NTS3, a single transmembrane receptor. Several independent studies demonstrated that NTS1 is overexpressed in a panel of tumours, such as invasive ductal breast carcinoma, pancreatic adenocarcinoma, prostate and lung cancer. With this in mind, we focused our efforts on developing a library of radiolabelled NT analogues, as potential agents for cancer theranostic. Following a rational approach based on our results previously obtained, the designed novel radiopharmaceuticals were synthesized through SPPS. More in detail, we synthesized a series of analogues in which the minimal bioactive sequence, known as NT[8-13], was modified through the introduction of backbone modifications and silicon-containing amino acids. Subsequently, further backbone modifications were inserted and several linkers between the chelating macrocycle agent and peptide sequence were evaluated. All the conjugates were radiolabelled with 68Ga and fully characterized. The obtained results clearly showed as the incorporation of silicon-containing amino acids, backbone modifications and an appropriate linker anchoring the chelating agent and peptide sequence, improve the binding affinity towards NTS1, plasmatic stability and radiopharmaceutical properties. The most promising NT-analogue is a good candidate to perform PET scan detecting NTS1- expressing tumours, as well as for a targeted radionuclide therapy after radiolabelling with 177Lu.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 18J2

Non-classical ligand-independent regulation of Go protein by an orphan Class C GPCR

M. HAJJ1, T. DE VITA1, C. VOL1, C. RENASSIA1, J.-C. BOLOGNA1, I. BRABET1, M. CAZADE1, M. PASTORE1, J. BLAHOS2, G. LABESSE3, J.-P. PIN1, L. PREZEAU1

1: IGF, Univ. Montpellier, CNRS, INSERM, Montpellier, France 2: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic and Department of Pharmacology, 2nd Medical School, Charles University, Prague, Czech Republic: JB 3: CBS, Univ. Montpellier, CNRS, INSERM, Montpellier, France: GL corresponding author: [email protected]

The orphan G protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-b5 leading to the regulation of neighboring GPCR-induced Go protein activity. Intriguingly, GPR158 also interacts with ao although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain putatively involved in G protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-b5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in ao binding. Using a Gao-bg BRET sensor we found that GPR158 decreases the BRET signal as observed upon G protein activation. However, no constitutive activity of GPR158 could be detected through the measurement of various G protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of Gao by the VCPWE motifs possibly leading to its dissociation from bg. Such action of GPR158 is expected to prolong the bg activity as also observed with some Activators of G protein Signaling (AGS). Taken together our data revealed a complex functional scaffolding/signaling role for GPR158 controlling Go through an original mechanism.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 19J2

Selective Quantitation of GPCR’s Ligand Affinity and Binding Kinetics in Living Cells

R. FRIEDMAN OHANA, M. BOURSIER, K. ZIMMERMAN, E. LACKNER, S. LEVIN, T. MACHLEIDT, K. WOOD

Promega Corporation, 2800 Woods Hollow Rd, Madison; 2Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA

G-protein coupled receptors (GPCR) continue to be prominent candidates for new therapeutics. While binding affinity and selectivity of prospective GPCR ligands are often assessed by applying radioactive ligands to membrane preparations, these non-homogenous assays can be costly, time consuming, cumbersome for kinetic measurements, and undesirable for safety issues. To provide a sensitive, homogenous method suitable for living cells, we developed a bioluminescence resonance energy transfer (BRET) assay for quantifying dynamic binding interactions to selective GPCRs on the cell surface. The assay detects specific interactions between fluorescent ligands and their cognate GPCRs that are genetically fused to a very small N-terminal HiBiT peptide (1.3 kDa), which is capable of producing bright luminescence upon high affinity complementation with an 18 kDa subunit derived from NanoLuc (LgBiT). The cell impermeability of LgBiT enables selective detection of binding interactions on the cell surface. Furthermore, it provides the means to monitor ligand-induce internalization through changes in cell surface density of HiBiT-tagged GPCRs. Notably, the sensitivity afforded by the NanoBiT complex allows for such analyses at endogenously relevant expression levels. Here, using the beta-adrenergic receptor (β-AR) family as model systems, we applied those assays to study various aspects of GPCR pharmacology including ligand binding and internalization in both endpoint and kinetic formats. We anticipate that these assays, which are readily adaptable to laboratory automation, will be immensely useful in the study of other GPCR families.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 20J2

Structural dynamics of single metabotropic glutamate receptor dimers

R. QUAST1, A.-M. CAO1, L. OLOFSSON1, P. RONDARD2, C. SEIDEL3, J.-P. PIN2, E. MARGEAT1

1 Centre de Biochimie Structurale, CNRS UMR 5048, INSERM U 1054, Université de Montpellier, Montpellier, France 2 Institut de Génomique Fonctionnelle, CNRS, INSERM, Université de Montpellier, Montpellier, France 3 Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany corresponding author: [email protected]

The activation mechanisms of GPCRs, where an external signal is propagated across the membrane through conformational rearrangements, have been extensively studied over the last decades by various biochemical and biophysical methods. These have led to the conclusion that GPCR activation cannot be sufficiently explained by a simple on/off transition from an inactive to a distinct active state. Instead, it is rather a highly dynamic process where the equilibrium between multiple coexisting conformational states is altered by interacting molecules such as proteins, lipids, ions and others. Therefore, methods to monitor these conformational changes, preferentially at the single molecule level, are needed. Here, using single molecule Förster resonance energy transfer (smFRET) we are studying the structural dynamics that occur during activation of metabotropic glutamate receptors (mGluRs) in response to ligands. We have previously shown that isolated ligand binding domains oscillate between an inactive/open and an active/closed state in a time range of 50-100 µs and that orthosteric ligands shift the equilibrium depending on their efficacy. Currently, we are extending these observations to detergent-solubilized full-length receptor dimers. We use allosteric modulators to decipher the role of the 7 transmembrane domain, and explore the allosteric communication between the various mGluR functional domains.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 21J2

RFamide related peptide-3/Neuropeptide FF1 receptor is a pronociceptive system involved in the development of hyperalgesia induced by opiates and inflammatory pain.

R. QUILLET1, S. SCHNEIDER2, M. GERUM1, V. UTARD1, A. DRIEU LA ROCHELLE1, K. ELHABAZI1, J. B. HENNINGSEN3, M. KREMER3, P. GIZZI4, M. SCHMITT2, V.KUGLER1, T. SORG-GUSS5, M.-F. CHAMPY5, H. MEZIANE5, B. PETIT-DEMOULIERE5, V. SIMONNEAUX3, B. ILIEN1, F. BIHEL2, F. SIMONIN1

1 Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS, Université de Strasbourg, Illkirch, France 2 Laboratoire Innovation Thérapeutique, UMR 7200 CNRS, Université de Strasbourg, Illkirch, France 3 Institut des Neurosciences Cellulaires et Intégratives, UPR 3212 CNRS, Strasbourg, France 4 TechMedIll, UMS 3286 CNRS, Université de Strasbourg, Illkirch, France 5 Institut Clinique de la Souris, PHENOMIN, IGBMC/ICS-MCI, Université de Strasbourg-CNRS-INSERM, Illkirch, France corresponding author: [email protected]

RF-amide-relatide peptide-3 (RFRP-3) and Neuropeptide FF (NPFF), belong to the family of so-called RF-amide peptides. In mammals, they are involved in the modulation of several functions including metabolism, reproduction and nociception (Quillet et al., 20161). They target two different G protein- coupled receptor subtypes called Neuropeptide FF1 receptor (NPFF1R alias GPR147 or GnIH receptor) and Neuropeptide FF2 receptor (NPFF2R or GPR74), respectively. However, the respective role of these two receptors is unclear, and the study of their function in vivo is severely limited by the lack of highly selective antagonists. In this work, we describe the identification of small compounds that display high affinity and selectivity for NPFF1R as well as potent antagonist activity in vitro. We then showed that one of them -RF3286- efficiently and selectively blocks RFRP-3 induced hyperalgesia in mouse and LH release in hamster, indicating that this compound is a useful pharmacological tool to study the in vivo functions of NPFF1R and its endogenous ligand RFRP-3. Pharmacological blockade of NPFF1R with RF3286 prevented the development of pain hypersensitivity and analgesic tolerance induced by chronic administration of morphine revealing that NPFF1R/RFRP-3 system is critically involved in neuroadaptation associated with administration of opiates. These results were further confirmed in NPFF1R knockout animals. Moreover, we observed the expression of NPFF1R and RFRP-3 transcripts by fluorescent in situ hybridization in the dorsal horn of spinal cord, indicating that this receptor/peptide system can modulate nociception in part by spinal mechanism. We further observed that cells expressing NPFF1R transcripts were also MOP positives (50%) and DOP positives (20%), suggesting a direct modulatory role of NPFF1R on the action of opioid. Finally, we observed an increase of NPFF1R positive cells in dorsal horn of spinal cord of CFA-treated animals compared to saline controls suggesting a potential role of this system in inflammatory pain. In agreement with these data, we further showed that pharmacological blockade of NPFF1R with RF3286 can efficiently reverse hyperalgesia induced by CFA injection. Altogether, our data allowed us to identify NPFF1R/RFRP-3 as a pronociceptive anti-opioid system and further suggest that antagonists of this receptor might represent interesting therapeutic tools to limit the development of OIH and analgesic tolerance associated with chronic opioid administration as well as hyperalgesia induced by inflammatory pain.

1. Quillet, R., Ayachi, S., Bihel, F., Elhabazi, K., Ilien, B., Simonin, F. (2016) RF-amide neuropeptides and their receptors in Mammals: Pharmacological properties, drug development and main physiological functions. Pharmacol Ther. 160:84-132.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 22J2

Deciphering the influence of applied light on photoswitchable mGlu5 negative allosteric modulators binding

M. RICART-ORTEGA1,2, A.E. BERIZZI2, V. PEREIRA2, F. MALHAIRE2, X. GÓMEZ-SANTACANA2, J. FONT2, J. CATENA1, L. MUÑOZ1, C. SERRA1, X. ROVIRA3, J.P. PIN2, C. GOUDET2, A. LLEBARIA1

1MCS, Laboratory of Medicinal Chemistry & Synthesis, Institute of Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain 2IGF, CNRS, INSERM, Univ. de Montpellier, F-34094 Montpellier, France 3Molecular Photopharmacology Research Group, University of Vic – Central Univ. of Catalonia, 08500 Vic, Spain corresponding authors: [email protected] [email protected] [email protected]

Photopharmacology offers the possibility of controlling the protein activity in a precise spatiotemporal manner by light using photochromic ligands (PCLs). In turn, applying light remotely and non-invasively enables highly localised therapeutic effects and accurate dosing patterns. In the current study, we have expanded these tools to metabotropic glutamate 5 receptor (mGlu5) since the molecular mechanisms that control their activity and kinetics are still not fully understood. Therefore, we have determined the kinetic parameters of two PCLs (namely Alloswitch-1 and XGS-023), under different light conditions through MS binding assays. Alloswitch-1 and XGS-023 are potent and selective negative allosteric modulators (NAMs) of mGlu5 receptor presenting photoswitchable properties, while maintaining its drug-like characteristics. Illumination by green light leads to the trans-configuration while irradiation with violet light leads to the cis isomer of the ligands. In vitro studies have shown significant differences on the pharmacological properties of the compounds before and after violet light illumination with the compounds being more active on the trans state. Moreover, we have corroborated that the affinity of both photoswitchable NAMs decreases under violet light. In addition, our MS binding studies suggested that both XGS-023 isomers can bind to the mGlu5 allosteric pocket being the cis-XGS023 dissociation process upon violet light illumination faster than the trans-XGS023 dissociation process under dark condition. In conclusion, our findings will be important to the development of improved photoswitchable mGlu5 NAMs.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 23J2

GPCRmd: molecular dynamics online repository for GPCRs and its analysis tools

I. RODRÍGUEZ-ESPIGARES1, M. TORRENS-FONTANALS1, A. VARELA-RIAL1, J. M. RAMÍREZ-ANGUITA1, GPCRMD consortium2 & J. SELENT1

1 GPCR Drug Discovery group, Research Programme on Biomedical Informatics (GRIB), Universitat Pompeu Fabra (UPF)-Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain. 2. https://gpcrmd.wordpress.com/home/community/ corresponding author: [email protected]

We present GPCRmd (www.gpcrmd.org), an online collection of molecular dynamics (MD) data linked to a platform for submission and visualization of GPCR MD. This platform is not only a repository of MD trajectories, but it also includes a database with the necessary metadata (e. g. force-field simulation, software used, integration time-step) for ensuring data reproducibility and integrity and making possible analysis after publication. For this reason, information regarding MD simulation molecular components is stored and indexed. These components are categorized as either protein or small- molecule (non-protein). Protein details such as name, sequence, references to UniprotKB1 entries and mutations are included. In the other hand, small-molecules’ information is stored at two levels of description: first as chemical substance and second as molecular entity. Molecules are described with cheminformatics annotations (SMILES and InChI) and linked to PubChem2 and CHEMBL3 identifiers. These two elements ease the browsing for the desired simulations. As an online visualization tool, the platform uses NGL4. Figure 1 Na+ occupancy (%) in D2.50 (blue) and This software allows playing MD trajectories using a D3.32 (red) across different GPCR types (PDB IDs modern internet browser through WebGL technology, and UniprotKB entry names). hence external add-ons are not needed. Furthermore, this visualization tool is provided with a set of analysis tools that allow computing Root Mean Square Deviation, interatomic distances, the hydrogen bond network and receptor-ligand interactions. Finally, a case study, based on the MD data of numerous GPCR types in our platform, is presented showing the phylogenetic evolution of sodium binding across class A, B, C and F GPCRs.

References 1. UniProt Consortium, Nucleic Acids Research, 2017, 45, D158–D169. 2. S. Kim, P. A. Thiessen, E. E. Bolton, J. Chen, et al., Nucleic Acids Research, 2016, 44(D1), D1202–D1213. 3. A. P. Bento, A. Gaulton, A. Hersey, L. J. Bellis, et al., Nucleic Acids Research, 2014, 42(D1), D1083–D1090. 4. A. S. Rose, P. W. Hildebrand, Nucleic Acids Research, 2015, 43(W1), W576–W579.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 24J2

Plasmodium falciparum Serpentine Receptor 12 (PfSR12) increases surface expression of mammalian GPCRs in HEK293 cells

P. SCARPELLI 1,2, C. GARCIA1, M. BOUVIER2

1 Department of Clinical and Toxicological Analyses, University of São Paulo - Brazil 2 Institut de recherche en immunologie et en cancérologie, Université de Montréal - Canada corresponding author: [email protected]

Malaria causes millions of deaths worldwide and is considered a huge public health problem for underdeveloped countries. The increasing number of cases of multi-resistant parasites makes understanding the physiology of Plasmodium falciparum a powerful tool for identifying new molecules to fight disease. The most severe cases of malaria present complications of the host circulatory system, which may cause clogging and rupture of blood vessels, leading to death or formation of sequelae. We have identified a possible role for P. falciparum serpentine receptor (PfSR12) as a receptor for thrombin, an essential coagulation cascade component, possibly acting as a GPCR. In this work, our objective was to unravel classical GPCR signaling pathways in mammalian cells using BRET based biosensors to elucidate PfSR12 mechanism of action. Using this system, it was not possible to detect GRK recruitment, or EPAC and G protein activation. On the other hand, our data suggests that there is indeed a response due to thrombin addition in PfSR12 transfected cells, activating Gq/11 family pathway, since we observed DAG formation and PKC activation. However, the presence of the cellular response observed is not due to direct action of PfSR12 itself, but a consequence of the increase in the number of endogenous thrombin receptors present in the cells transfected with PfSR12. In conclusion, it is unlikely that PfSR12 is a thrombin receptor, but it can act as a chaperone and increase the expression of other GPCRs in HEK293 cells.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 25J2

Super-resolution imaging of metabotropic glutamate receptor 4 at presynaptic active zones

S. SIDDIG1, S. AUFMKOLK2, S. DOOSE2, M.-L. JOBIN1, M. SAUER2, D. CALEBIRO1,3,4

1Institute of Pharmacology and Toxicology and Bio-Imaging Center, University of Würzburg, Würzburg, Germany; 2Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Würzburg, Germany; 3Institute of Metabolism and Systems Research, University of Birmingham, UK; 4Centre of Membrane Proteins and Receptors (COMPARE), Universities of Nottingham and Birmingham, UK corresponding author: [email protected] [email protected]

Despite the large number of G protein-coupled receptors (GPCRs) expressed in the central nervous system, little is known about their spatial organization within synapses. mGluR4, a prototypical presynaptic family C GPCR, modulates presynaptic glutamate release mainly via the inhibition of P/Q type voltage-dependent calcium channels (CaV2.1). However, the underlying mechanisms are insufficiently understood. In this study, we analyzed the nanoscale organization of mGluR4 at the synapses between parallel fibers and Purkinje cells in the mouse cerebellum with near-molecular resolution (10-20 nm) using two-color direct stochastic optical reconstruction microscopy (dSTORM). Quantitative analyses revealed a four-fold mGluR4 enrichment at parallel fiber active zones (AZs). We found that an AZ contains 25 mGluR4 nanoclusters on average. Each nanocluster contains approximately one or two mGluR4s, with few nanoclusters containing three or more receptors. To assess the spatial distribution of mGluR4 relative to functional AZ components such as CaV2.1 and Munc 18-1 (an essential element of the synaptic secretory machinery), a distance-based colocalization analysis was used. The analysis revealed a positive correlation between mGluR4 and both proteins at distances as short as 40 nm. These results indicate that mGluR4 forms highly organized nanodomains with CaV2.1 and Munc 18-1 at AZs, which provides a new ultrastructural basis to explain the presynaptic inhibition of glutamate release by mGluR4.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 26J2

Design and chemical synthesis of photo-regulated melatonin receptor ligands in type 2 diabetes and neurodegenerative diseases

G. SOMALO

Institut Cochin (Inserm U1016), 22 rue Méchain, 75014 Paris, France. corresponding author: [email protected]

Melatonin regulates a big variety of physiological and neuroendocrine functions, and it has well-established neuroprotective properties. Consequently, its deregulation is associated with many metabolic, autoimmune and neurodegenerative diseases, although its specific molecular mechanism still remains elusive. Emerging evidence shows that GPCR signaling can be prolonged or initiated not only in the plasma membrane but also in intracellular compartments. Therefore, the interplay between cell surface and intracellular melatonin receptors needs to be characterized. Innovative pharmacological tools based on photo-regulated melatonin ligands will be used to accomplish this aim. Different melatonin-caged and azobenzene-containing melatonin derivatives have been synthesized. These structures have been designed to be inactive at the level of receptor, but able to be activated by using specific light conditions. For caged-type derivatives, light triggers a photolytic reaction that separates the caging moiety from active melatonin, which is released at a specific intracellular location. For azobenzene-type derivatives, light switches reversibly the structure of the ligand derivative, in such a way that the new conformation is ideally recognized by the receptor.

Functional and affinity assays have been performed on the first family of melatonin-caged compounds. According to the obtained results, all tested ligands show a notable loss of activity before light irradiation, as well as lower affinity to the receptors in comparison to melatonin (at least 2-log shift decrease). Likewise, they present the same properties as melatonin after applying proper light conditions. In conclusion, these promising results are being useful for the design of improved structures that will provide new insights about intracellular GPCRs.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 27J2

Molecular determinants of biased signaling within the σ-opioid receptor

T.M. STEPNIEWSKI, I. RODRIGUEZ-ESPIGARES, M MARTI-SOLANO, M. TORRENS- FONTANALS, A TROYA-BRUGER, G. DE FABRITIIS, S. FILIPEK, P. GIGUERE, J. SELENT

Research Programme on Biomedical Informatics, Hospital del Mar Medical Research Institute & Department of Experimental and Health Sciences, Pompeu Fabra University Dr. Aiguader 88, Barcelona, E 08003 Spain corresponding author: [email protected]

G protein-coupled receptors (GPCRs) are a family of receptor proteins that consist of seven transmembrane helices connected by extracellular and intracellular loops. GPCRs in response to extracellular chemical or physical ligands can initiate a wide range of intracellular signalling cascades, modifying the cell function. A single GPCR can govern multiple signalling pathways, certain ligands show preference in the engaged signalling pathways.

Here we study the σ-opioid receptor (σ-OR). This receptor is primarily expressed in the central nervous system, and involved in the pain mitigation. Opioid receptors are targets of analgesic drugs like morphine or fentanyl. It is believed that signalling through G-protein within this receptors is responsible for the therapeutic effect, while through β-arrestin (β-arr) for side-effects like addiction. As such understanding the molecular determinants of bias within this receptor could contribute to the design of more efficient drugs.

A study showed that certain mutations within σ-OR receptor transform its ligand naltrindole into a β-arr biased ligand.4 By using molecular dynamic simulations, we were able to catch a glimpse into how studied mutations change the signalling response of the σ-OR, and generate a model of β-arr bias within the naltrindole/σ-OR complex. The generated model has been successfully verified by additional mutations within the σ-OR.

The results of this study provide insight into biased signalling within GPCRs, as well as demonstrate how by combining experimental and mutational data it is possible to engineer a GPCR into a desired signalling state.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 28J2

Development of LIH383 a novel small subnanomolar highly selective of ACKR3/CXCR7 full agonist peptide

M. SZPAKOWSKA, M. MEYRATH, A. CHEVIGNÉ

Luxembourg Institute of Health, Department of Infection and Immunity, 29, rue Henri Koch, L- 4354 Esch-sur-Alzette corresponding author: [email protected]

ACKR3, the atypical chemokine receptor 3, formerly known as CXCR7, plays a crucial role in many processes including cardiovascular and neuronal development as well as in migration and homing of hematopoietic stem/progenitor cells. ACKR3 is also present in many cancer cell types and on tumour-associated vasculature and accumulating evidence demonstrates its involvement in metastasis development, making it an interesting drug target. The activity of ACKR3 mainly relies on β-arrestin recruitment, while its ability to trigger G protein- dependent or -independent signalling upon activation is highly debated. So far, there is a clear lack of small, pharmacologically well-characterized and easily available ACKR3 modulators that can be used in vitro and in vivo to investigate the functions and signalling of ACKR3. Therefore, there is an urgent need to develop robust and efficient pharmacological and imaging tools to specifically detect and modulate the activity of ACKR3.

In this study, we describe the properties of an octapeptide LIH383, a novel highly selective full agonist of ACKR3/CXCR7 with subnanomolar potency (EC50 = 0.4 nM). LIH383 is more potent in inducing β-arrestin recruitment to ACKR3 than the full-length chemokine ligands CXCL12 or CXCL11 (EC50 = 1.2 nM and 2.2 nM, respectively). Importantly, it does not activate or inhibit CXCR4 or CXCR3, with which it shares ligands, nor any other chemokine receptor, even at concentrations as high as 3 µM. LIH383 has also equivalent activity on human and mouse ACKR3. LIH383 directly competes with CXCL12 for ACKR3 binding at low nanomolar concentrations, indicating that it targets the orthosteric pocket of ACKR3. Moreover, fluorescently labelled LIH383 binds to ACKR3-expressing cells, but not to native or CXCR4-expressing cells. The strong and highly selective binding and receptor activation profile makes LIH383 a potentially attractive and versatile tool for specific ACKR3 modulation, for structural biology or for the detection of ACKR3-expressing cells in different mouse and human models.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 29J2

Crosstalk between PAFR and mGluR2 receptors reveals new insight into GPCR signal integration

J. LIU, Z. FAN, H. TANG, L. LIN, S. MA, J. LIU

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.

Correspondence authors: [email protected]

G protein–coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Although a wide range of GPCRs have been identified in the same cells, the mechanism by how signals (Gi/o signal and Gq signal) are integrated remains elusive. Here, we have found novel signaling cross talking between Gq-coupled Platelet-activating factor receptor (PAFR) and Gi/o-coupled metabotropic glutamate 2 (mGluR2) receptor. We showed that stimulation of HEK cells co- expression PAFR-mGluR2 heteromers or Hippocampus neuron (Our Western blot result show that they are co-express in hippocampus neuron. ) with an mGluR2 agonist led to activation of Gq protein by PAFR, however stimulation of cells only expression mGluR2 can not because it is Gi/o-coupled. Further, we showed this cross talking phenomenon have disappeared when co-transfected PAFR and mGluR2 loss-function mutants (ligand no-binding mutants and G protein un-coupled mutants) or co-tranfected PAFR loss-function mutants and mGluR2. Also this phenomenon could be inhibited by PTX (Gi/o inhibitor), βγ inhibitor and PLC inhibitor. In the future, we will lucubrate physiology effect of these signal cross talking. These findings offer insights into this important target in molecular psychiatry.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 30J2

Molecular evolution of the sodium binding site of the angiotensin II receptors AT1 and AT2

A. TISS1,2, R. BEN BOUBAKER1, L. GRIMAUD1, D. HENRION1, L. MARSOLLIER3, H. GUISSOUMA2, M. CHABBERT1

(1) Laboratoire MITOVASC, UMR CNRS 6015 – INSERM 1083, Université d’Angers, Angers, France ; (2) Laboratoire GIPH, Faculté des Sciences de Tunis, Université de Tunis El Manar, Tunisie ; (3) Centre de recherche en Cancérologie et Immunologie Nantes-Angers (CRCNA), INSERM U 1232, Angers, France. corresponding author: [email protected]

The renin-angiotensin system has a key role in cardiovascular homeostasis. The octopeptide angiotensin II (Ang II) activates two G protein-coupled receptors, AT1 and AT2 that share around 35% sequence identity. Most known effects of Ang II on the cardiovascular system are mediated by AT1. The effects of AT2 on this system usually counteract those of AT1, resulting in cardioprotection. Thus, AT2 may represent a valuable drug target. Developing specific AT2 ligands requires a better understanding of the structural differences between AT1 and AT2.

Sodium is a negative allosteric modulator of GPCRs. Difference in the sodium binding modes of AT1 and AT2 was suggested to explain, at least in part, the unusual mechanism of action of AT2. The evolutionary history of their sodium binding site indicates that the divergence between these sites is specific of vertebrates, with a Ser to Asn mutation at position 7.46 in AT1. Molecular dynamics simulations were carried out to determine the mechanism of sodium binding to these receptors. This study reveals that the sodium binding mode is dynamical. The sodium ion moves between different sub-sites within the binding cage on the sub-microsecond timescale. In AT2, the decreased contacts with residue 7.46 are compensated in part by increased contacts with Ser3.39, as compared to AT1.The AT2 receptor shows fewer contacts with protein residues but more contacts with water molecules compared to AT1 receptor. The implications for the stability of the sodium binding site are discussed.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 31J2

GPCRmd workbench: a GPCR-specialized platform for sharing, visualization and analysis of GPCR dynamics

M. TORRENS-FONTANALS1, I. RODRIGUEZ-ESPIGARES1, J.K.S. TIEMANN2, GPCRMD community3, R. GUIXA-GONZALEZ4 AND J. SELENT1

1 Laboratoire de Neurosciences Cognitives et Adaptatives (LNCA), UMR 7364, CNRS/Université de Strasbourg, Faculté de psychologie, 12 rue Goethe, 67000 Strasbourg, France 2 Department of Psychology, Center for Neuroscience Studies, Queen's University, Kingston, Ontario K7L 3N6, Canada 1 Research Programme on Biomedical Informatics, Hospital del Mar Medical Research Institute – Universitat Pompeu Fabra, Dr. Aiguader 88, 08003, Barcelona, Spain 2 Institute of Medical Physics and Biophysics, Medical University Leipzig, Leipzig, Sachsen 04107, Germany 3 https://welcome.gpcrmd.org/home/community/ 4 Laboratory of Computational Medicine, Biostatistics Unit, Faculty of Medicine, Autonomous University of Barcelona, Av. de Can Domènech 08193 Bellaterra, Spain corresponding author: [email protected]

The functionality of G protein-coupled receptors (GPCRs) is highly determined by their ability to transition between distinct conformations. Because of this, understanding the structural dynamics of these receptors is essential for advancing our knowledge on their physiology. With this goal in mind, we have created the GPCRmd database, the first open-access research resource hosting molecular dynamics (MD) simulations of most GPCR crystal structures solved to date. GPCRmd is designed to facilitate the access to MD data for all scientists interested in GPCRs, including structural and evolutionary biologists, protein engineers and medicinal chemists among others. As such, a fundamental feature of the database is the GPCRmd workbench, a web-based platform that provides direct and interactive visualization and analysis of each MD trajectory stored in the database from any modern browser. The GPCRmd workbench is equipped with a comprehensive set of easy-to-use tools to analyze molecular interactions and protein motions involving conserved, pharmacologically relevant, or diseased-related residues/motifs potentially involved in GPCR function. Such tools include, for example, a dynamic interaction network of multiple non-covalent contacts, RMSD calculations, and occupancy maps. All in all, being an interactive and user-friendly platform for the study of the extensive set of GPCR simulations collected in the database, the GPCRmd workbench has the potential to boost both multidisciplinary research and the study of GPCRs from a dynamic perspective by putting GPCR MD data within reach of the whole scientific community.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 32J2

Interactions between dopamine and other neuromodulators in the striatum.

C. YAPO1, E. MOTA1, A. NAIR2, D. BETOLNGAR1,J. KOTALESKI3, L.R.V. CASTRO1, P. VINCENT1

1 Sorbonne Université - CNRS UMR8256, Paris. 2 KTH Royal Institute of Technology - Stockholm (Sweden) 3 Karolinska Institutet - Solna (Sweden), corresponding author: [email protected]

Striatal neurons integrate incoming dopaminergic inputs together with several neuromodulators such as adenosine, acetylcholine, •NO, glutamate… To study how these neuromodulatory cues interact dynamically in MSNs, we used genetically-encoded biosensors to image the cAMP/PKA pathway with a high spatial and temporal resolution in live striatal brain slices. These data were used to constrain models of intracellular signaling, and simulations were used to test mechanistic hypotheses.

Surprisingly, we observed that the cAMP response to D1 and D2 receptor stimulation was obtained with dopamine concentrations in a similar sub-micromolar range1, with only a ~2 fold difference in EC50. This observation is contrasting with the commonly accepted notion 2 that D2 receptors exhibit a much higher sensitivity, but consistent with another publication . We also observed that D1 striatal neurons responded non-linearly to transient dopamine, switching-on PKA-dependent phosphorylation in an all-or-none manner, an effect particularly prominent in the nucleus. This effect most likely involves DARPP-32 and a positive feedforward regulatory loop controlling the phosphorylation level of PKA targets. This effect mechanism may work as a “binarizing” filter to selectively detect phasic dopamine while rejecting background cAMP “noise”3. Finally, we report that very low levels of acetylcholine, through M4 muscarinic receptors, efficiently prevented D1 response in MSNs4. Overall, our results highlight the complexity of the dynamic aspects of signal integration in the striatum, with several distinct mechanisms that either potentiate or down-regulate the intracellular response to dopamine.

1. C Yapo, AG Nair, L Clement, LR Castro, J Hellgren Kotaleski, P Vincent. J Physiol (2017); 595:7451. 2. S Weiss, M Sebben, JA Garcia-Sainz, J Bockaert. Mol Pharmacol (1985); 27:595. 3. C Yapo, AG Nair, J Hellgren Kotaleski, P Vincent, LRV Castro. J Cell Sci (2018); 131:jcs.216556. 4. AG Nair, LRV Castro, M El Khoury, V Gorgievski, B Giros, ET Tzavara, J Hellgren-Kotaleski, P Vincent. Neuropharmacology (2019); 146:74.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 33J2

eeFit: Excel-embedded program for interactive analysis and fitting of experimental dose-response data

M. VIVAUDOU

Channels group, Institut de Biologie Structurale, Grenoble, France corresponding author: [email protected]

We present a software program dedicated to the fitting of experimental dose-response data, which integrates seamlessly with Excel and allows curve fitting plots and results to reside alongside data within Excel spreadsheets. The program, named eeFit for Excel-Embedded FITting software, requires no advanced knowledge of Excel or non-linear least-squares fitting. Any experimental data present in an Excel file, such as dose-effect data obtained with membrane receptor or ion channel ligands, can be graphed and fitted interactively with standard Hill models for activation or inhibition, or with more complex models for mixed effects resulting from combinations of activation and inhibition. When benchmarked against commercial programs, eeFit yielded equivalent or better results, in terms of accuracy and convergence, and proved much easier to learn and use because of its advanced user interface and its complete integration within Excel. The software eeFit is available free of charge. A complete installation package for Windows computers can be downloaded from the IBS web site (http://www.ibs.fr/research/scientific- output/software/eeFit).

Reference Vivaudou M (2019) eeFit: A Microsoft Excel-embedded program for interactive analysis and fitting of experimental dose-response data. Biotechniques. 66:186-193. doi: 10.2144/btn-2018-0136.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 34J2

Splice variant R1e of GABAB receptor promotes tumorigenesis in breast cancer

B. WEI, Y. ZHU, S. WANG, X. JIANG, J. LIU

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China.

Corresponding authors: [email protected] [email protected]

GABABR, the receptor for neurotransmitter γ-aminobutyric acid (GABA), belongs to G protein-coupled receptors (GPCRs). GABABR is composed of subunits GABABR1 and

GABABR2. GABABR1 has been reported at least 14 splice variants. Among of these, the truncated isoform R1e lack of the transmembrane and cytosolic domains only contains the extracellular ligand binding domain and 9 amino acids of the transmembrane domain. And it has previously determined that GBR1e was the predominant isoform in the peripherial tissues. Our study has found that GBR1e was widely expressed in breast cancer cell lines, and the expression level was correlated with the malignancy of breast cancer cells. To figure out the role of GBR1e in breast cancer progression, GBR1e was overexpressed in breast cancer MCF-7 cells. The results showed that GBR1e significantly promoted breast cancer malignancy, including enhancing cell proliferation, migration, clonogenicity and spheroid. Conversely, when we knocked down GBR1e in MDA-MB-231 cells, the malignancy was dramatically alleviative. Interestingly, we demonstrated that breast cancer cells can release GBR1e into conditioned media and the secreted GBR1e could be uptaken by neighboring cancer cells. Surprising, the recipient tumour cells can gain of the same function inducing by GBR1e.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 35J2

GABAB receptors couple to G13 to control specific neuronal function

C. XU, Y. WANG, J. LIU

School of Life Science and Technology, Huazhong University of Science and Technology, 430074 Wuhan, China, corresponding author: [email protected] [email protected]

G protein-coupled receptors (GPCRs) activate MAPK pathways to regulate cell function through multiple mechanisms that remain unclear. Although β-arrestins play an important role in this process, several G protein-activated pathways are involved. Here we investigated the

GABAB receptor, a GPCR known to phosphorylate ERK1/2 through a Gi/o-mediated pathway, but does not directly activate β-arrestins. Here we showed that the GABAB receptor can also phosphorylate JNK through Rac1 activation in primary neurons. Although GABAB receptors are assumed to couple exclusively to Gi/o proteins, we reported here that Rac1-JNK pathway is activated by G13. This is best illustrated by the absence of GABAB receptor-mediated phospho-JNK in G12/13 knock-out cells, and by the recovery of this signaling pathway after G13 complementation. GABAB receptor agonists also nicely activate a G13 BRET sensor, though with a slow kinetic and with a low potency. The G13/JNK pathway is shown to promote PSD-

95 phosphorylation and accumulation. This pathway is also shown to be necessary for GABAB receptor-mediated neuroprotection in granule neurons, acting then in synergy with the

Gi/o/insulin-like growth factor-1 receptor pathway that we previously reported. These data revealed that different MAPK can be activated by different G protein-mediated pathways to regulate important cellular functions.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 36J2

Kinetic biased signaling: towards a system biology definition of drugs selectivity. Illustration on the Follicle Stimulating Hormone Receptor

R. YVINEC

PRC, INRA, CNRS, IFCE, Université de Tours, Nouzilly, France corresponding author: romain.yvinec@inra

An active area of research in pharmacology and drug discovery applies to functional selectivity (or biased agonism, biased signaling): the ability of a ligand to selectively activate some signal transduction pathways as compared to the native ligand acting at the same receptor. At the theoretical level, biased signaling is supported by the concept of conformational selectivity: a given receptor may adopt several conformations, that can be stabilized by its interaction with a ligand, and each of these conformations potentially activates the downstream signaling pathways with different efficacies. At the practical level, experimentalists seek to quantify ligand bias in order to classify ligands according to their selectivity. One popular method uses the so-called operational model to fit dose-response curves. In this presentation, I will review the main limitations of this methodology, recently pointed out by our group and others. Our objective is then to design a method that fully take into account the kinetic nature of signaling pathways and as well as their possible cross-talks. Kinetic experiments, that measure the activty of several downstream effectors of a receptor after ligand binding with respect to time, are now widely available. I will explain how one can exploit such data and dynamical reaction network modeling with suitable statistical framework to provide a complete “bias map” of a ligand, compared to the native ligand, that successfully answer to our objective. The methodology is illustrated with kinetic BRET measurements of effectors of the FSHR, stimulated by either the FSH or low molecular weight allosteric ligands.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 37J2

The orphan GPCR GPR50 and its involvement in the development of obesity/type2 diabetes

Q. ZHANG1, A. ROCA-RIVADA1, R. AHMAD1, R. JOCKERS1, J. DAM1

1 Inserm U1016, CNRS UMR 8104, Univ. Paris Descartes, Sorbonne Paris Cité, Institut Cochin, 22 rue Méchain,75014 Paris, France corresponding author: [email protected]

Obesity is regarded as the fifth leading risk factor for the development of type 2 diabetes, cardiovascular diseases and cancer. Obesity is related to a misbalance in energy intake relative to expenditure that is translated into an excess of energy stored as lipids in white adipose tissue (WAT). Enlarged WAT is associated with increased body weight, while brown adipose tissue (BAT) has the ability to dissipate the energy excess as heat production and protects against obesity. Converting some specific cells (called «beige» or «brite» adipocytes) resident in the WAT into brown-like state (“browning”) is associated with weight loss and improvement of metabolic profile. Much of the ‘‘brown fat’’ of adult humans has the molecular characteristics of “beige” fat cells, indicating that “browning” of WAT constitutes an attractive therapeutic approach in humans. GPR50 is an orphan receptor with no known ligand, belonging to G protein coupled receptor (GPCR) family. Our unpublished results reveal that the orphan receptor, GPR50, is important in triggering the “browning” of the white adipose tissue and could constitutes a novel therapeutic target to tackle obesity and associated metabolic disorders.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER POSTER 38J2

MICROFLUIDIC DIFFUSIONAL SIZING (MDS) — A NOVEL METHOD FOR CHARACTERIZING PROTEIN INTERACTIONS

M. Butz1, S. Devenish1, L.. Groß1, S. Fiedler1, T. Barnes1, M. A. Czekalska2, G. Meisl2

1: Fluidic Analytics Ltd, Cambridge, UK

2: University of Cambridge, UK3 corresponding autor : [email protected]

Microfluidic diffusional sizing (MDS)is an in-solution technique that measures the hydrodynamic radius of a protein complex, this technique can be used to determine the binding affinity of protein interactions. MDS requires minimal sample preparation, no surfaces and can be performed in biological mixtures. Here we show current applications of MDS from the literature and our own work.

8th GDR3545-GPCR INTERNATIONAL MEETING IN MONTPELLIER