Activation of the G protein-coupled glutamate receptors : a single molecule study. – NanoGluR

The G protein-coupled receptor (GPCR) family represents more than 30% of all therapeutic drug targets and hence pharmaceutical companies are developing programs to identify new modulators of these receptors. The metabotropic glutamate receptors (mGluRs), are GPCRs activated by glutamate, the major excitatory neurotransmitter in the central nervous system. As such, mGluRs are particularly interesting as drug targets for the treatment a whole range of neurological and psychiatric disorders (anxiety, depression, migraine, Parkinson’s disease and pain). However, despite intense researches and clinical proof of concepts, still no drug targeting mGluRs are on the market. A better understanding of the multiple conformational states of the mGluRs and their dynamics could enable a better view of the mode of action of drugs for these receptors, and then to design new compounds. mGluRs are 250 kDa homodimeric multidomain proteins stabilized by a disulfide bridge. This multidomain structure offers many possibilities to regulate mGluR activity and different types of drugs have recently be developed : (i) orthosteric ligands (agonists or antagonists) that bind to the glutamate binding site located in the extracellular domain (ECD) ; (ii) allosteric drugs, that act mainly by binding to the heptahelical transmembrane domain (7TM) common to all GPCRs and responsible for G-protein activation; (iii) bioprobes (“nanobodies”). One of the major and exciting challenges in mGluRs pharmacology is to understand the molecular bases of the ligand efficacy on these receptors.
The aims of this proposal is to study, at the structural level, the mode of action of: (i) the partial agonists in the ECD ; (ii) the positive and negative allosteric modulators ; (iii) potential biotherapeutics such as llama antibodies. To analyze ligand efficacy, we propose for the first time to develop innovative biochemical and biophysical approaches, based on single molecule fluorescence (fluorescence lifetime, polarization, and FRET).
Although classical structural biology methods (such as X-ray crystallography and nuclear magnetic resonance) have been useful to generate high resolution information on GPCR structures and mechanisms, they are less suitable to study large, multidomain and dynamic biomolecular complexes such as full-length class C GPCR. Conversely, single molecule fluorescence requires minute amounts of biological material, and allows to investigate fine details such as molecular variability or structural dynamics, by overcoming the averaging effect of bulk biochemical methods. The combination of single molecules fluorescence approaches we propose to use will enable the determination of the conformational dynamics of mGluRs, in terms of distances (at angstrom-resolution), and time-scales (from nanoseconds to second). Our scientific research project requires five major steps to be accomplished: (i) to purify functional mGluRs; (ii) to reconstitute the purified receptors in a native-like environnement (lipid nanodiscs); (iii) to perform site-specific labeling of the purified receptors with fluorophores, using a combination of cysteine residues and un-natural amino acid approaches; (iv) to determine the distance changes between the two fluorophores by single molecule FRET (smFRET) upon ligands binding; (v) to analyze the kinetics of these conformational changes. We will use mGluR2 and mGluR5 as models since they are important drug targets in the pharmaceutical industry and different classes of well-characterized partial agonists and allosteric modulators, as well as antibodies, are available for these receptors. This program will be developed by two partners: 1/ The group of Dr P. Rondard (Institut de Génomique Fonctionnelle, Montpellier) for the mGluRs functional purification, reconstitution, and pharmacology; 2/ The group of Dr E. Margeat (Centre de Biochimie Structurale, Montpellier) for fluorescence labeling and biophysical analysis on single molecules.