Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors

Sandrine B Daval; Céline Valant; Dominique Bonnet; Esther Kellenberger; Marcel Hibert; Jean-Luc Galzi; Brigitte Ilien

doi:10.1021/jm201348t

Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors

J Med Chem. 2012 Mar 8;55(5):2125-43. doi: 10.1021/jm201348t. Epub 2012 Feb 27.

Authors

Sandrine B Daval¹, Céline Valant, Dominique Bonnet, Esther Kellenberger, Marcel Hibert, Jean-Luc Galzi, Brigitte Ilien

Affiliation

¹ Unité Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS, Ecole Supérieure de Biotechnologie de Strasbourg, Université de Strasbourg, BP 10413, 67412 Illkirch, France.

PMID: 22329602
DOI: 10.1021/jm201348t

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Allosteric Regulation
Calcium / metabolism
Fluorescence Resonance Energy Transfer
Fluorescent Dyes / chemical synthesis*
Fluorescent Dyes / chemistry
Fluorescent Dyes / pharmacology
Green Fluorescent Proteins / genetics
HEK293 Cells
Humans
Models, Molecular
Molecular Probes / chemical synthesis*
Molecular Probes / chemistry
Molecular Probes / pharmacology
Piperidines / chemical synthesis*
Piperidines / chemistry
Piperidines / pharmacology
Radioligand Assay
Receptor, Muscarinic M1 / agonists*
Receptor, Muscarinic M1 / antagonists & inhibitors
Receptor, Muscarinic M1 / genetics
Receptor, Muscarinic M1 / metabolism*
Recombinant Fusion Proteins / agonists
Recombinant Fusion Proteins / antagonists & inhibitors
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism
Rhodamines / chemical synthesis*
Rhodamines / chemistry
Rhodamines / pharmacology
Solubility
Structure-Activity Relationship

Substances

4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)-piperidine hydrogen chloride
Fluorescent Dyes
Molecular Probes
Piperidines
Receptor, Muscarinic M1
Recombinant Fusion Proteins
Rhodamines
enhanced green fluorescent protein
Green Fluorescent Proteins
rhodamine B
Calcium