The interplay of conformational states and dynamics in the signaling complexes tune GPCR-mediated functional outcomes. We develop an integrated pharmacology strategy that is based on the combined use of biochemical, biophysical and cell biology methods to illuminate how GPCRs transmit their signal depending on the molecules in their surrounding. The data that will emerge will be crucial for advancing our current understanding of basic mechanisms governing GPCR function. This is of more than academic interest since stabilization of receptor states is also the key to modulating GPCR function in drug design.
Membrane protein biochemistry
Stabilization of the native fold of GPCRs out of their membrane environment is key to the analysis of the molecular bases underlying their functioning. In this context, we develop original methods to assemble the purified receptors into membrane-like model systems that preserve their pharmacological properties. These systems actually include amphipols, nanodiscs, lipodisqs and liposomes.
We apply to the isolated receptors a palette of biophysical method aimed at delineating the conformational landscape a GPCR can explore. These include fluorescence transfer (FRET, LRET, smFRET) and solution state-NMR. We use in parallel structural biology methods (NMR, cryoEM, scattering techniques) to provide a “three-dimensional” framework to our dynamics observations.
We use transfected cell-lines expressing the target receptor(s) to decipher how signaling occurs in a more integrated system. This includes the analysis of both the signaling outputs (second messenger, arrestin recruitment, G protein activation) and the protein-protein interactions that occur at the membrane. These analyses rely on the concerted use of fluorescence (FRET, HTRF..) and bioluminescence (BRET) transfer techniques in living cells.
Pharmacological Characterization of Selected Ligands on Metabolic Processes: from the cellular target to animal models
Appropriate control of metabolic processes and feeding behavior are essential to achieve energy homeostasis. Our aim is to characterize the pharmacological mechanism by which ghrelin receptor ligands and polyphenols or their metabolites regulate key metabolic processes.
Compounds are characterized and selected using cell-based assays predictive of their potency and efficacy.
Concentration-response curve for the polyphenol metabolite, urolithin C, on glucose-stimulated insulin secretion in INS-1 β-cell line.
Technical approaches: binding (Ki, Kd) and functional assays (EC50), western blot, insulin secretion assays, molecular docking, intracellular calcium monitoring and electrophysiology
The pharmacological activity of the compounds and their mechanisms of action are investigated in models expressing the endogenous targets.
The inverse agonist JMV-6480 blocks the insulinostatic action of ghrelin in rat pancreatic islets of Langerhans.
Models : rodent pancreatic islets and perfused pancreas
Lead compounds are evaluated in healthy and pathological animal models.
Models and technical approaches: DIO, ob/ob and db/db mice, glucose tolerance test, glycemia, insulinemia, food intake
Ghrelin affects glucose tolerance (left) and stimulates food intake (right) in C57BL/6J mice.