title: Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors.
abstract: 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-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.
authors.affiliation: Unité Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS, Ecole Supérieure de Biotechnologie de Strasbourg, Université de Strasbourg, BP 10413, 67412 Illkirch, France.
authors.forename: Sandrine B
authors.initials: SB
authors.lastname: Daval
authors.forename: Céline
authors.initials: C
authors.lastname: Valant
authors.forename: Dominique
authors.initials: D
authors.lastname: Bonnet
authors.forename: Esther
authors.initials: E
authors.lastname: Kellenberger
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Galzi
authors.forename: Brigitte
authors.initials: B
authors.lastname: Ilien
mesh_terms: D000494:Allosteric Regulation; D002118:Calcium / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D010880:Piperidines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1 / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: C456520:4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)-piperidine hydrogen chloride; D005456:Fluorescent Dyes; D015335:Molecular Probes; D010880:Piperidines; D043643:Receptor, Muscarinic M1; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C029773:rhodamine B; D002118:Calcium