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* Pharmacology and Toxicology Section, Institute of Pharmacy, and
Institute of Pharmaceutical Biology, Rheinische Friedrich-Wilhelms-University, Bonn, Germany;
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Julius-Maximilians-University, Würzburg, Germany;
Institute of Medicinal and Toxicological Chemistry "Pietro Pratesi," University of Milan, Milan, Italy;
|| Department of Pharmacological, Biological and Applied Chemical Sciences, University of Parma, Parma, Italy;
¶ Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Düsseldorf, Germany; and
# Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Victoria, Australia
2 Correspondence: Pharmacology and Toxicology Section, Institute of Pharmacy, Gerhard-Domagk-Str.3, D-53121 Bonn, Germany. E-mail: k.mohr{at}uni-bonn.de
Selective modulation of cell function by G protein-coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype-selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine-like orthosteric activator with M2-selective bis(ammonio)alkane-type allosteric fragments. Radioligand binding in wild-type and mutant receptors supplemented by receptor docking simulations proved M2 selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid-induced dynamic mass redistribution in CHO-hM2 cells disclosed pathway-specific signaling. Selective receptor activation (M2>M1>M3) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation.—Antony, J., Kellershohn, K., Mohr-Andrä, M., Kebig, A., Prilla, S., Muth, M., Heller, E., Disingrini, T., Dallanoce, C., Bertoni, S., Schrobang, J., Tränkle, C., Kostenis, E., Christopoulos, A., Höltje, H.-D., Barocelli, E., De Amici, M., Holzgrabe, U., Mohr, K. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity.
Key Words: agonism • allosterism • muscarinic acetylcholine receptor • signal trafficking • subtype selectivity • dynamic mass redistribution
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