Potentiation of Muscarinic M₃Receptor Activation through a New Allosteric Site with a Novel Positive Allosteric Modulator ASP8302

Abstract

Muscarinic M 3 (M 3 ) receptors mediate a wide range of acetylcholine (ACh)-induced functions, including visceral smooth muscle contraction and glandular secretion. Positive allosteric modulators (PAMs) can avoid various side effects of muscarinic agonists with their spatiotemporal receptor activation control and potentially better subtype selectivity. However, the mechanism of allosteric modulation of M 3 receptors is not fully understood, presumably due to the lack of a potent and selective PAM. In this study, we investigated the pharmacological profile of ASP8302, a novel PAM of M 3 receptors, and explored the principal site of amino acid sequences in the human M 3 receptor required for the potentiation of receptor activation. In cells expressing human M 3 and M 5 receptors, ASP8302 shifted the concentration-response curve (CRC) for carbachol to the lower concentrations with no significant effects on other subtypes. In a binding study with M 3 receptor-expressing membrane, ASP8302 also shifted the CRC for ACh without affecting the binding of orthosteric agonists. Similar shifts in the CRC of contractions by multiple stimulants were also confirmed in isolated human bladder strips. Mutagenesis analysis indicated no interaction between ASP8302 and previously reported allosteric sites; however, identified threonine 230 as the amino acid essential for the PAM effect of ASP8302. These results demonstrate that ASP8302 enhances the activation of human M 3 receptors by interacting with a single amino acid distinct from the reported allosteric sites. Our findings suggest not only a novel allosteric site of M 3 receptors but also the potential application of ASP8302 to diseases caused by insufficient M 3 receptor activation.

Muscarinic M3 positive allosteric modulator ASP8302 enhances bladder contraction and improves voiding dysfunction in rats

“…These results show that the M 3 PAM ASP8302 enhances cholinergic contraction of bladder smooth muscle in rats, consistent with findings in human tissues. 18 While M 5 receptors expressed in the central nervous system (CNS) are implicated in the modulation of dopamine transmission and behavioral changes, [23][24][25] their peripheral functions are largely unknown. We speculate that ASP8302 likely has a low risk of causing CNS-related side effects through M 5 PAM activity because it has low brain penetration, and did not produce any abnormal changes in spontaneous behaviors at the highest dose tested in rats (data not shown).…”

“…Intracellular Ca 2+ mobilization was measured using a fluorometric imaging plate reader as described in our previous study. 18 PAM activity of ASP8302 was represented by a fold-shift in the CCh-concentration-response curve (CRC).…”

“…The effect of ASP8302 (final 1 μmol/L) on CCh-or ACh-induced concentration-dependent contraction of isolated bladder strips was determined as described in our previous study. 18…”

“…39 In our study, the distigmineinduced increase in TIP was suppressed by the M 2 antagonist methoctramine (Figure 5). With its higher M 3 receptor selectivity achieved by targeting the allosteric site, 18 ASP8302 is expected to offer a lower risk of side effects than currently available cholinergic drugs. Further characterization of the potential benefits of stimulation-dependent activation of M 3 receptors will enable clearer differentiation of ASP8302 from existing cholinomimetics.…”

“…12,13 Recent progress in the discovery and characterization of PAMs of M 3 receptors [14][15][16][17] includes findings from our previous study, which elucidated the in vitro pharmacological profile of the novel PAM of human M 3 receptor ASP8302. 18 However, no study has investigated the in vivo modulation of bladder function by a PAM of M 3 receptors.…”