Selective activation of different pathways by dualsteric compounds in muscarinic M1 acetylcholine receptors

G protein-coupled receptors (GPCRs) respond to a broad range of different extracellular stimuli, thereby evoking intracellular signaling. Dualsteric compounds bind simultaneously to the receptor’s orthosteric transmitter binding site and its allosteric vestibule [1]. In the muscarinic acetylcholine M2 receptor (M2 mAChR) these dualsteric compounds were shown to impair the activation-related conformational change of the receptor protein by spatial restriction. This can eventually control the signaling pattern of a GPCR that promiscuously activates a variety of different signaling pathways [2]. In the present work, we checked whether this concept could be transferred to the M1-subtype of muscarinic receptors (M1 mAChR) which preferentially signals into Gq/11-dependent pathways, but can also activate Gs and Gi proteins [3]. Therefore, M1 receptor-mediated signaling induced by the orthosteric full agonist iperoxo and several dualsteric compounds were investigated. The latter consist of iperoxo as the orthosteric building block linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety via alkyl chains of different length. To distinguish between Gq/11- and Gs-dependent signaling pathways, CHO cells stably transfected with the human M1 muscarinic receptor were applied in IP1 and cAMP accumulation assays, respectively. Our findings show that the bulky allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. In particular, the hybrid iper-6-naph completely lost intrinsic activity at the M1 mAChR, although M2 mAChR activation by this compound had been demonstrated in a previous study [2]. In contrast to iper-6-naph, the less spacious congener iper-6-phth only lost intrinsic activity for Gs-activation, while it was still a partial agonist for Gq/11-dependent signaling. All other hybrids tested were able to activate both the Gq/11 protein and the Gs protein. Remarkably, iper-7-phth had a significantly higher efficacy for Gs protein activation than all the other compounds under investigation. However, there was no significant difference between iper-7-phth and iper-8-phth for Gq/11-dependent signaling. Furthermore, iper-7-naph and iper-8-naph showed no significant differences in both Gq/11- and Gsassays. Our data indicate that it might be possible to gain subtype selectivity by exploiting the allosteric moiety or the length of the linker chain. In particular, the bitopic derivative iper-6-naph was shown to be a partial agonist at the M2 mAChR, but was not able to activate the M1 mAChR [2]. Taken together, these data demonstrate 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 is possible to control signaling of the M1 mAChR by allosteric constraint of the conformational flexibility. 1. Antony, J. et al.: FASEB J. 2009, 23:442-450. 2. Bock, A. et al.: Nat. Commun. 2012, 3:1044 doi: 10.1038/ncomms2028. 3. Gregory, K.J.; Sexton P.M.; Christopoulos A.: Curr. Neuropharmacol. 2007, 5:157-167.