Fluorescent pirenzepine derivatives as potential bitopic ligands of the human M1 muscarinic receptor.
Following a recent description of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP)-fused human muscarinic M1 receptors and Bodipy-labeled pirenzepine, we synthesized seven fluorescent derivatives of this antagonist in order to further characterize ligand-receptor interactions. These compounds carry Bodipy [558/568], Rhodamine Red-X [560/580], or Fluorolink Cy3 [550/570] fluorophores connected to pirenzepine through various linkers. All molecules reversibly bind with high affinity to M1 receptors (radioligand and energy transfer binding experiments) provided that the linker contains more than six atoms. The energy transfer efficiency exhibits modest variations among ligands, indicating that the distance separating EGFP from the fluorophores remains almost constant. This also supports the notion that the fluorophores may bind to the receptor protein. Kinetic analyses reveal that the dissociation of two Bodipy derivatives (10 or 12 atom long linkers) is sensitive to the presence of the allosteric modulator brucine, while that of all other molecules (15-24 atom long linkers) is not. The data favor the idea that these analogues might interact with both the acetylcholine and the brucine binding domains.
Laboratoire de Pharmacochimie de la Communication Cellulaire, Faculté de Pharmacie, UMR CNRS/ULP 7081, IFR 85, 74 route du Rhin, BP 10413, 67412 Illkirch, France.
Chouaib
C
Tahtaoui
Isabelle
I
Parrot
Philippe
P
Klotz
Fabrice
F
Guillier
Jean-Luc
JL
Galzi
Marcel
M
Hibert
Brigitte
B
Ilien
D000494:Allosteric Regulation; D001665:Binding Sites; D001667:Binding, Competitive; D001896:Boron Compounds / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D010890:Pirenzepine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000494:pharmacology; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D013329:Structure-Activity Relationship; D013331:Strychnine / Q000031:analogs & derivatives* / Q000494:pharmacology
D001896:Boron Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D008164:Luminescent Proteins; D043643:Receptor, Muscarinic M1; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; C103220:rhodamine red-X succinimidyl ester; D049452:Green Fluorescent Proteins; D010890:Pirenzepine; C083806:brucine; D013331:Strychnine