Novel M₂‐selective, G_i‐biased agonists of muscarinic acetylcholine receptors

Abstract

Background and Purpose: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. Experimental Approach: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M 2 receptors was modelled in silico. Key Results: Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,-6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2Hpyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M 2 muscarinic receptors and displaying bias towards the G i signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. Conclusions and Implications: The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M 2 and M 4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

Fusion with Promiscuous Gα16 Subunit Reveals Signaling Bias at Muscarinic Receptors

“…Comparison of parameters of functional response of selected agonists at Gα 16 _fused and Gα 16 cotransfected wt receptors suggest better coupling of fused Gα subunit. The better coupling of Gα 16 in fusion proteins was demonstrated by a decrease in the value of equilibrium dissociation constant (K A ) of agonists at Gα 16_ fused receptors ( Table 2 vs. our previous data Randakova et al [ 19 ], Table 3 ), except iperoxo at M 5 _Gα 16 ( Table 3 , discussed below). The elimination of interaction with other competitive Gα subunits as well as fusion alone could lead to better coupling of fused Gα subunits.…”

“…Our data demonstrate that oxotremorine stimulates accumulation of IPx at M 2 _Gα 16 more efficiently in comparison with co-transfected system M 2 +Gα 16 , where the competition of endogenous G i/o and G q/11 occurs and more efficiently than at wt M 2 via endogenous G q/11 ( Table 3 ). In our previous study of Randáková et al [ 19 ], oxotremorine displayed lower RA i to stimulate the accumulation of IP X in the co-expressed system M 2 + Gα 16 than in the presented study. This discrepancy can be explained by different levels of expression of Gα 16 in co-expressed systems and points to the advantage of using fusion proteins with 1:1 stoichiometry for easier spotting of agonist bias.…”

“…At the rest of the Gα 16 _fused receptors, the operational efficacies of oxotremorine and carbachol were the same. In contrast, oxotremorine stimulated accumulation of IP X at M 2 + Gα 16 and M 2 wt with efficacy comparable ( Table 3 ) or lower [ 19 ] to carbachol. Operational efficacies τ of functional responses of carbachol and oxotremorine at M 2 _Gα 16 and M 2 + Gα 16 ( Figure 4 ) are summarized in Table 3 .…”

“…Structurally different agonists vary in interactions in the orthosteric binding site of the muscarinic receptor [ 55 ]. The portfolio of used agonists included reference balanced full agonist carbachol, classic muscarinic agonists arecoline, furmethide, pilocarpine, oxotremorine, super-agonist iperoxo [ 32 , 33 ], bitopic agonists xanomeline [ 56 ], and McN-A343 [ 57 ], and Gi/o-biased agonists JR6 and JR7 [ 19 ] ( Supplementary Materials Figure S2 ).…”

“…Selective targeting on the G i/o versus G q/11 mediated pathway by biased agonist could be a way to achieve selectivity to even or odd muscarinic subtypes [ 19 ]. Moreover, agonists biased to individual isoforms of G-proteins could lead to tissue-specific activation of mAChRs, due to the predominant expression of some G-proteins in specific tissues (e.g., G o in the central nervous system or G 16 in hematopoietic cells) [ 10 , 20 ].…”