TY - JOUR T1 - Exploration of the Orthosteric/Allosteric Interface in Human M1 Muscarinic Receptors by Bitopic Fluorescent Ligands JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.113.085670 SP - mol.113.085670 AU - Sandrine B. Daval AU - Esther Kellenberger AU - Dominique Bonnet AU - Valerie Utard AU - Jean-Luc Galzi AU - Brigitte Ilien Y1 - 2013/01/01 UR - http://molpharm.aspetjournals.org/content/early/2013/04/19/mol.113.085670.abstract N2 - Bitopic 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 Bodipy [558/568] fluorophore via spacers of varying lengths, exhibit orthosteric/allosteric binding properties at muscarinic M1 receptors. This behaviour was inferred from a combination of functional, radioligand and FRET binding experiments, performed under equilibrium and kinetic conditions on EGFP-fused M1 receptors. Though 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 neighbouring allosteric domains, overlapping or not with the classical gallamine site, but distinct from the allosteric indocarbazolole 'WIN' site. Site-directed mutagenesis, 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 'multifacette' experimental approach to unravel and validate bitopic ligand binding mechanisms. ER -