Dualsteric compounds modulate the signaling pattern of muscarinic M1 acetylcholine receptors

G protein-coupled receptors (GPCRs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric G proteins [1]. GPCR-induced cell signaling can be rather complex as several GPCRs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other “allosteric” binding sites [2]. In the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (mAChRs). To this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). Binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [2]. Therefore, application of different linker length may control the signaling outcome. Here, we applied the human M1 mAChR which preferentially activates G proteins of the Gq/11 type but can also promiscuously stimulate Gs proteins. Gq/11- and Gs- dependent signaling pathways were analyzed by application of CHO cells stably transfected with the human M1 mAChR in IP1 and cAMP accumulation assays, respectively. In comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both Gq-mediated and Gs-mediated signaling. Our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. Particularly, the compound iper-6-naph completely lost intrinsic activity for both Gq/11 and Gs activation at the M1 mAChR. Moreover, Gs-mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than Gq-signaling. Interestingly, longer linker length led to improved signaling for both pathways (Gq and Gs) in both hybrid series. Iper-7-phth seems to be an exception as it had a higher intrinsic efficacy for Gs-dependent signaling than the other phth hybrids with longer linker chains. Strikingly, only iper-fri-phth, which corresponds to iper-8-phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on M1 mAChR for both signaling pathways under investigation. Taken together, these data strongly suggest that, in comparison to Gq/11-mediated signaling, activation of the Gs protein in M1 mAChR is more sensitive to spatial restriction in the allosteric vestibule. Thus, it appears to be possible to control signaling of the M1 mAChR by allosteric constraint of the receptor’s conformational flexibility. [1] Magalhaes, A. et al.: Br. J. Pharmacol. 2011, 165(6): 1717-36 [2] Bock, A. et al.: Nat. Commun. 2012, 3:1044 doi: 10.1038/ncomms2028