Dualsteric compounds to induce signaling pathway selectivity in CHO-M1 cells

Muscarinic acetylcholine receptors (mAChRs) belong to the large superfamily of G protein-coupled receptors (GPCRs). Stimulation of GPCRs leads to a conformational change in the receptor protein allowing the receptor to transduce extracellular stimuli onto intracellular adaptor proteins which then further propagate the signal inside the cell [1]. GPCR-induced signaling can be the result of the interplay between rather complex molecular events as several GPCRs can activate multiple different adaptor proteins, for instance different classes of heterotrimeric G proteins. Additionally, GPCRs can be targeted by compounds not only via the binding site for the endogenous messenger (orthosteric binding site) but also via distinct druggable “allosteric” sites [2]. In the present work, we investigated the influence of spatial restriction of the allosteric vestibule located in the extracellular loops of the receptor protein on muscarinic M1 acetylcholine receptor (M1 mAChR) -mediated signaling pathways. To this end, we employed several dualsteric compounds which are able to simultaneously occupy the orthosteric and an allosteric binding site of mAChRs, thereby constraining flexibility of the extracellular vestibule which constitutes the common allosteric binding site [3]. At the muscarinic M2 receptor such spatial restriction of the allosteric area of the receptor protein has been demonstrated to allow selective activation of particular signaling pathways [2]. Here, we want to transfer this principle to the muscarinic M1 subtype, which preferentially signals into Gq/11-mediated pathways, but can also promiscuously stimulate Gs and Gi proteins [4]. We investigated M1 receptor-mediated signaling induced by dualsteric compounds consisting of highly affine and efficacious agonist iperoxo [5] as an orthosteric building block linked to an allosteric phthalimide (phth) moiety or a bulkier naphthalimide (naph) residue through alkyl chains of different lengths (6, 7 ,8 or 10 carbon atoms). Gq/11- and Gs-dependent signaling pathways were analyzed in HTRF-based IP1 and cAMP accumulation assays, respectively. We found that, in general, Gs protein activation in CHO-M1 cells was highly sensitive to the restriction of spatial flexibility of the extracellular receptor area, because application of the bulky allosteric naph residue compromised M1 receptor-mediated cAMP production to a greater extent than IP1 accumulation. Moreover, compounds with a rather short linker length (i.e. C6) displayed weaker potency and efficacy for both Gq/11- and Gs-mediated signaling than dualsteric ligands with elongated linker chains (i.e. C8). In particular, iper-6-phth was a partial agonist for the Gq/11 pathway, but totally lost affinity for Gs-mediated signaling, whereas iper-7-phth was a strong partial agonist for both pathways under investigation. For compounds carrying the rather voluminous naph moiety as allosteric residue an even stronger impact of a short linker length was observed: iper-6-naph acted as a neutral antagonist on both pathways investigated up to a concentration of 10 µM, although it was able to bind to the M1 mAChRs with an affinity in the low micromolar range. Interestingly, previous studies demonstrated that this ligand is a partial agonist at M2 and M3 subtypes of mAChRs [2 and unpublished data]. Dualsteric compounds with 7, 8 or 10 carbon atoms as linker chains were all strong partial agonists for Gq and moderate partial agonists for Gs protein activation. We conclude that a C6 linker connecting the allosteric and the orthosteric moiety compromises receptor flexibility to the greatest extent and thus has the strongest impact on M1-mediated signaling. Taken together, we present iper-6-naph as the first dualsteric compound with functional subtype selectivity at muscarinic acetylcholine receptors, as it is a rather strong partial agonist at M2 [2] and M3 receptors, but clearly acts as a neutral antagonist at M1. Furthermore, our findings show that it is possible to control M1-receptor mediated signaling by restriction of the conformational flexibility in the allosteric area of the receptor protein. 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 3. Antony, J. et al.: FASEB J. 2009, 23:442-450 4. Thomas, R. et al.: J. Pharmacol. Exp. Ther. 2008, 327(2):365-74 5. Schrage, R. et al.: BJP 2012, doi: 10.1111/bph.12003