Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

Abstract

We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M 4 muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligandbinding studies revealed that LY2033298 displayed a preference for the active state of the M 4 mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [ 35 S]GTPgS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3b phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M 4 mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [ 3 H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M 4 mAChR knockout mice, validating the M 4 mAChR as a key target of action of this novel allosteric ligand.

Antipsychotic Drug-Like Effects of the Selective M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator VU0152100

“…Our data with VU0152100 also validate previously reported effects using the structurally distinct M 4 PAM LY2033298 when given in combination with a sub-threshold dose of the non-selective mAChR agonist oxotremorine (Chan et al, 2008;Leach et al, 2010;Suratman et al, 2011). However, LY2033298 does not provide an optimal tool compound for rodent studies in that it has relatively low potency at the rat M 4 mAChR (Chan et al, 2008;Leach et al, 2010) and displays only weak cooperativity with ACh, the endogenous agonist of M 4 (Suratman et al, 2011), but has high cooperativity with the synthetic mAChR agonist oxotremorine.…”

“…However, LY2033298 does not provide an optimal tool compound for rodent studies in that it has relatively low potency at the rat M 4 mAChR (Chan et al, 2008;Leach et al, 2010) and displays only weak cooperativity with ACh, the endogenous agonist of M 4 (Suratman et al, 2011), but has high cooperativity with the synthetic mAChR agonist oxotremorine. As LY2033298 induces robust potentiation of oxotremorine, but not ACh effects on M 4 , it has no behavioral effects when administered alone unless coadministration with oxotremorine (Suratman et al, 2011).…”

“…These studies provided important data supporting the potential for the development of M 4 PAMs for the treatment of psychosis. Conversely, LY2033298, another M 4 PAM, had no effect alone in preclinical models, but did potentiate the effects of a sub-threshold dose of the non-selective mAChR agonist oxotremorine (Chan et al, 2008;Leach et al, 2010, Suratman et al, 2011. The lack of efficacy observed with LY2033298alone when used in vivo was likely due to the lower potency of this M 4 PAM at the rat M 4 mAChR and its low cooperativity with the endogenous agonist ACh at the rodent receptor (Suratman et al, 2011).…”

Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors

“…Experimental data were fitted using the Hill equation and a simplified model of allosterism including the minimum number of parameters (30) because the structure of the experimental data precludes the use of complete expressions of operational models (see details of mathematical modeling in Supplemental Data 2). The usefulness of this procedure in mGlu 5 was evaluated in previous studies (10).…”

“…Final docking and detailed analysis of ligand-receptor interactions was performed in a second molecular homology model of mGlu 4 7TM based on the recent crystal structures of mGlu 1 (16) and mGlu 5 (17). (30). Parameter estimates, SE, and 95% confidence limits produced by global fitting.…”