Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein–coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein–coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2 acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein–coupled receptor.
- Received October 24, 2016.
- Accepted February 2, 2017.
↵1 Current affiliation: Institute for Bioengineering of Catalonia, Parc Cientific de Barcelona, Barcelona, Spain.
↵2 Current affiliation: CNS Discovery Research, UCB Pharma, Braine-l'Alleud, Belgium.
This work was supported by Deutsche Forschungsgemeinschaft (DFG) [Grants HO1368/12-1 and MO821/2-1 to K.M. and U.H.]. A.D.M. was supported by the DFG-funded Research Training Group 1873. The University of Milan financed a 4-year postdoctoral position for C.M.
- Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics