Contribution à l'étude de la modulation allostérique du récepteur muscarinique M1 (Recherche de modulateurs positifs originaux. Caractérisation de l'interaction de divers ligands fluorescents)

Les récepteurs muscariniques appartiennent à la vaste famille des RCPGs, des protéines membranaires assurant la communication entre les cellules de l'organisme. Le récepteur muscarinique M1 est principalement exprimé au niveau central, où il constitue une cible thérapeutique privilégiés pour des pathologies telles que la maladie d'Alzheimer et la schizophrénie.Dans un premier temps, j'ai recherché de nouveaux modulateurs allostériques positifs du récepteur muscarinique M1 par criblage fonctionnel au sein de la chimiothèque Prestwick Chemical. Dans un deuxième temps, j'ai étudié le mode d'interaction des ligands fluorescents Bodipy-pirenzépine avec le récepteur M1, mettant ainsi en évidence la nature bitopique orthostérique/allostérique de tous ces dérivés quelques soit la longueur de leur espaceur. La troisième partie de mon travail est consacré au développement et à la caractérisation d'un dérivé fluorescent d'AC-42 comme traceur allostérique fluorescent du récepteur M1.Muscarinic receptors belong to the large GPCRs family. They are involved in a number of physio-pathological processes and are potential therapeutic targets. During my PhD thesis, I have much interest in the muscarinic M1 sub-type which is essentially expressed in the CNS and is of particular interest for the treatment of Alzheimer Disease or schizophrenia. First, the prestwick chemical library was screened in order to find novels positives allosteric modulators. Calcium mobilization was used as a functional read out, but only leads to identification of signaling pathway modulators. In a second time, the orthosteric/allosteric nature of Bodipy-pirenzepine derivatives/muscarinic M1 receptor interaction was studied. All bodipy-pirenzepine derivatives are bitopic ligands whatever the nature and the length of their linker.Finally, AC-42 fluorescent derivatives were developed and characterized as fluorescent allosteric tracer of the muscarinic M1 receptor.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Les récepteurs couplés aux protéines G: Des régulateurs allostériques du métabolisme cellulaire

International audience> Il y a environ cinquante ans, l'isolement des premières enzymes et l'analyse de leur méca-nisme réactionnel ont mis les biochimistes au défi de comprendre le fonctionnement des enzymes régulatrices. Différents modèles fondés sur des informations pharmacologiques, enzymatiques et structurales ont été proposés. Parmi ceux-ci, le modèle dit allostérique de Monod, Wyman et Changeux décrit des protéines régulatrices qui peuvent adopter plusieurs conformations, inter-convertibles et différemment stabilisées par les substrats, les produits et les effecteurs allosté-riques. Ces derniers interagissent au niveau de sites régulateurs topographiquement distincts du site enzymatique. Les divers états conformation-nels associent des propriétés fonctionnelles et structurales distinctes. Enfin, la nature oligomé-rique des protéines qui ont servi à construire ce modèle a permis de décrire un phénomène important , la coopérativité. Celle-ci reflète la capacité d'une molécule liée à une sous-unité de la pro-téine à faciliter ou à défavoriser la liaison d'une deuxième molécule sur une sous-unité voisine. Ce concept d'allostérie a évolué. Il est à présent étendu aux phénomènes de modulation allosté-rique qui mettent en jeu des molécules distinctes, se liant à leurs sites propres sur une protéine monomérique, comme par exemple un récepteur couplé aux protéines G. Cet article a pour objectif de discuter la manière dont les récepteurs cou-plés aux protéines G, d'une part, s'inscrivent dans un modèle d'architecture fonctionnelle répondant aux règles de l'allostérie et, d'autre part, sont soumis à une modulation de leurs propriétés phar-macologiques et fonctionnelles par des effecteurs allostériques, des petites molécules ou des pro-téines cellulaires, avec lesquels ils établissent des contacts stables ou transitoires. < À la croisée des chemins entre RCPG et allostérie Les récepteurs couplés aux protéines G (RCPG) forment une super-famille de protéines membranaires monomériques de près de 900 membres [1]. Globalement impliqués dans les phénomènes de communication et d'homéostasie cellulaire, ils interviennent dans de nom-breuses fonctions physiologiques et physiopathologiques et représen-tent des cibles thérapeutiques importantes. Des efforts de recherche soutenus ont permis d'enrichir un arsenal thérapeutique considérable composé d'agonistes (stabilisant un état actif) et d'agonistes inverses (stabilisant un état inactif) de ces récepteurs [2, 31, 32] (➜). Notons que la majorité des molécules décrites et utilisées en tant qu'antagonistes sont pro-bablement des agonistes inverses. La plupart de ces ligands ont été sélectionnés sur la base d'une interaction compétitive avec la liaison de l'agoniste endogène au niveau de son site récepteur (site orthos-térique). Ces molécules orthostériques ont souvent des affinités très élevées mais ne sont pas dénuées d'effets secondaires liés en par-tie à leur manque de sélectivité. En effet, de nombreux récepteurs coexistent sous forme de sous-types distincts qui présentent de fortes homologies de séquence au niveau de leur site orthostérique [1]. D'autre part, il est à présent bien reconnu qu'un même agoniste (et a fortiori des agonistes de structures chimiques différentes) peut sélectionner des voies de signalisation intracellulaires variées dépen-dantes du contexte cellulaire ou de la réponse biologique considérée [3]. Département biotechnologie et signalisation cellulaire

Functional Characterization and Potential Applications for Enhanced Green Fluorescent Protein-and Epitope-Fused Human M1 Muscarinic Receptors

International audienceFour recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential gly-cosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal micros-copy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their functional properties were examined through McN-A-343-evoked Ca 2ϩ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC 50 values) to mediate the Ca 2ϩ response, although reduced amplitudes (E max values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visual-ization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocyto-chemistry. Altogether, this work demonstrates that these EGFP-and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors

ON THE USE OF NON-FLUORESCENT DYE- LABELLED LIGANDS IN FRET-BASED RECEPTOR BINDING STUDIES

The efficiency of Fluorescence Resonance Energy Transfer (FRET) is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. We report here on the design, synthesis and characterization of two novel pirenzepine derivatives that were coupled to Patent Blue VF and Pinacyanol dyes. These non-fluorescent compounds, when added to cells stably expressing EGFP-fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-, and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor, when determined using either FRET or classical radioligand binding conditions. We provide evidence that these compounds behave as potent acceptors of energy from excited EGFP, with quenching efficiencies comparable to those of analogous fluorescent Bodipy- or Rhodamine Red- pirenzepine derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed in terms of reliability,sensitivity and wider applicability of FRET-based receptor binding assays

Fluorescent Derivatives of AC-42 To Probe Bitopic Orthosteric/Allosteric Binding Mechanisms on Muscarinic M1 Receptors

International audienceTwo fluorescent derivatives of the M1 muscarinic selective agonist AC-42 were synthesized by coupling the lissamine rhodamine B fluorophore (in ortho and para positions) to AC42-NH 2. This precursor, prepared according to an original seven-step procedure, was included in the study together with the LRB fluorophore (alone or linked to an alkyl chain). All these compounds are antagonists, but examination of their ability to inhibit or modulate orthosteric [ 3 H]NMS binding revealed that para-LRB-AC42 shared several properties with AC-42. Carefully designed experiments allowed para-LRB-AC42 to be used as a FRET tracer on EGFP-fused M1 receptors. Under equilibrium binding conditions, orthosteric ligands, AC-42, and the allosteric modulator gallamine behaved as competitors of para-LRB-AC42 binding whereas other allosteric compounds such as WIN 51,708 and N-desmethylclozapine were noncompetitive inhibitors. Finally, molecular modeling studies focused on putative orthosteric/allosteric bitopic poses for AC-42 and para-LRB-AC42 in a 3D model of the human M1 receptor

Exploration of the Orthosteric/Allosteric Interface in Human M1 Muscarinic Receptors by Bitopic Fluorescent Ligands

International audienceBitopic binding properties apply to a variety of muscarinic compounds that span and simultaneously bind to both the orthosteric and allosteric receptor sites. We provide evidence that fluorescent pirenzepine derivatives, with the M1 antagonist fused to the boron-dipyrromethene [Bodipy (558/568)] fluoro-phore via spacers of varying lengths, exhibit orthosteric/ allosteric binding properties at muscarinic M1 receptors. This behavior was inferred from a combination of functional, radio-ligand, and fluorescence resonance energy transfer binding experiments performed under equilibrium and kinetic conditions on enhanced green fluorescent protein-fused M1 receptors. Although displaying a common orthosteric component, the fluorescent compounds inherit bitopic properties from a linker-guided positioning of their Bodipy moiety within the M1 allosteric vestibule. Depending on linker length, the fluorophore is allowed to reach neighboring allosteric domains, overlapping or not with the classic gallamine site, but distinct from the allosteric indolocarbazole "WIN" site. Site-directed mutagene-sis, as well as molecular modeling and ligand docking studies based on recently solved muscarinic receptor structures, further support the definition of two groups of Bodipy-pirenzepine derivatives exhibiting distinct allosteric binding poses. Thus, the linker may dictate pharmacological outcomes for bitopic molecules that are hardly predictable from the properties of individual orthosteric and allosteric building blocks. Our findings also demonstrate that the fusion of a fluorophore to an orthosteric ligand is not neutral, as it may confer, unless carefully controlled, unexpected properties to the resultant fluorescent tracer. Altogether, this study illustrates the importance of a "multifacet" experimental approach to unravel and validate bitopic ligand binding mechanisms

Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties.

International audienceHuman M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N-terminal extracellular moiety of the receptor and (iii) to set up a fluorescence-based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N-terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino-acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy-pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor-ligand complexes, relative donor (EGFP)-acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino-terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence-based assay, prone to miniaturization, allows reliable detection of unlabelled competitors

Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties.

International audienceHuman M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N-terminal extracellular moiety of the receptor and (iii) to set up a fluorescence-based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N-terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino-acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy-pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor-ligand complexes, relative donor (EGFP)-acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino-terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence-based assay, prone to miniaturization, allows reliable detection of unlabelled competitors

Fluorescent Derivatives of AC-42 To Probe Bitopic Orthosteric/Allosteric Binding Mechanisms on Muscarinic M1 Receptors

Two fluorescent derivatives of the M1 muscarinic selective agonist AC-42 were synthesized by coupling the lissamine rhodamine B fluorophore (in ortho and para positions) to AC42-NH₂. This precursor, prepared according to an original seven-step procedure, was included in the study together with the LRB fluorophore (alone or linked to an alkyl chain). All these compounds are antagonists, but examination of their ability to inhibit or modulate orthosteric [³H]­NMS binding revealed that para-LRB-AC42 shared several properties with AC-42. Carefully designed experiments allowed para-LRB-AC42 to be used as a FRET tracer on EGFP-fused M1 receptors. Under equilibrium binding conditions, orthosteric ligands, AC-42, and the allosteric modulator gallamine behaved as competitors of para-LRB-AC42 binding whereas other allosteric compounds such as WIN 51,708 and <i>N</i>-desmethylclozapine were noncompetitive inhibitors. Finally, molecular modeling studies focused on putative orthosteric/allosteric bitopic poses for AC-42 and para-LRB-AC42 in a 3D model of the human M1 receptor