An optical dynamic mass redistribution assay reveals biased signaling of dualsteric GPCR activators

Abstract

Increasing attention is paid in basic science and in drug discovery to pathway selective intracellular signaling as a novel approach to achieve precise control of cell function via G protein-coupled receptors (GPCRs). With respect to signaling, GPCRs are often promiscuous in that more than one intracellular biochemical pathway is activated upon receptor stimulation by the endogenous transmitter or by exogenous drugs. We studied signaling by a novel class of GPCR activators that were designed to bind simultaneously to the orthosteric transmitter-binding site and the allosteric site of muscarinic acetylcholine receptors. An optical biosensor technique was applied to measure activation-induced dynamic mass redistribution (DMR) in CHO cells stably expressing the muscarinic receptor subtype of interest. The use of tools to modulate signaling and measuring G protein activation directly proved that DMR is a valid and comfortable approach to gain real-time insight into intracellular signaling pathway activation and to identify signaling pathway-selective drugs.

Keywords::

• Dynamic mass redistribution
• functional selectivity
• dualsteric ligands
• GPCR
• muscarinic receptor

Acknowledgments

A.K. is member of the Research Training School GRK 677, which is supported by the Deutsche Forschungsgemeinschaft (DFG). E.K. and K.M. are members of Bonn University Center for Innovative Drugs and Therapies. Their work on dualsteric agents is supported by the DFG (KO1582/3-1, MO821/2-1). We thank Corning Life Sciences for their support on the Epic^® system. We thank Prof. Dr. Marco De Amici (University of Milan) and Prof. Dr. Ulrike Holzgrabe (University of Würzburg) for generously providing iperoxo and the dualsteric hybrid.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.