Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia

Abstract

Current antipsychotics provide symptomatic relief for patients suffering from schizophrenia and related psychoses; however, their effectiveness is variable and many patients discontinue treatment due to side effects. Although the etiology of schizophrenia is still unclear, a leading hypothesis implicates an imbalanced dopaminergic system. Muscarinic acetylcholine (ACh) receptors regulate dopamine levels in key areas of the brain involved in psychosis, with the M(4) subtype emerging as a key regulator of dopaminergic hyperactivity. Unfortunately, no selective small molecule tools exist to provide pharmacological validation of this hypothesis. Here, we describe the discovery of a small molecule modulator, LY2033298, that is highly selective for human M(4) receptors by virtue of targeting an allosteric site on this receptor. Pharmacological assays confirmed the selectivity of LY2033298 for the M(4) receptor and revealed the highest degree of positive allosteric enhancement of ACh potency thus far identified. Radioligand binding assays also show this compound to directly potentiate agonist binding while having minimal effects on antagonist binding. Mutational analysis identified a key amino acid (D(432)) in the third extracellular loop of the human M(4) receptor to be critical for selectivity and agonist potentiation by LY2033298. Importantly, LY2033298 was active in animal models predictive of clinical antipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent.

Fig. 1.

LY2033298 enhances ACh responses selectively at hM₄. The effect and selectivity of LY2033298 on ACh were tested by FLIPR (A) and [³⁵S]GTPγS (B) binding, using recombinant cell lines (AV12 G_α15 hM₂, or hM₄ and CHO hM₁, hM₃, or hM₅) and CHO cell membranes of hM₁–M₅, respectively. Responses in the presence of LY2033298 were normalized to the control maximal ACh response (100%) and basal response (B; 0%) for each receptor. In both assays, no significant allosteric effect was observed in hM₁, hM₃, or hM₅ cell lines. A very modest allosteric effect on hM₂ receptors was observed and a small agonist effect was detected at hM₄. (A Inset) Illustrated is the paradigm of functional screening for the concentration-dependent effect of LY2033298 on a submaximal dose of ACh (3 nM), which was obtained by plotting the data points along the dotted line.

Fig. 2.

LY2033298 allosterically increases agonist binding to M₄ receptors. (A) The specific binding of [³H]Oxo-M to CHO hM4 cell membranes and rat striatal membranes was potentiated by LY2033298 in a dose-dependent manner. (B) The specific binding of [³H]NMS to hM4 receptors in the presence of different concentrations of unlabeled ACh and LY2033298 was measured in the presence of 0.2 mM GTP. The incubation time was 2 h to allow equilibrium to be reached. The low slope factor of the ACh inhibition curve (0.67) was unchanged in the presence of increasing concentrations of LY2033298. The presence of LY2033298 increased ACh potency up to 40-fold. At high concentrations, LY2033298 appears to weakly and negatively modulate [³H]NMS binding at hM₄ receptors. The data are from one experiment repeated three times with quantitatively similar results.

Fig. 3.

Sequential mutations in the o3 loop of muscarinic receptors reveal a critical residue (D⁴³²) for LY2033298-mediated potentiation. Receptor mutants were transiently expressed in AV12 G_α15 cells. The effect of LY2033298 on ACh stimulated calcium mobilization was tested upon coapplication of LY2033298 (1 nM to 1 μM) with the EC₂₅ concentration of ACh for each receptor. The results were normalized to the potentiation mediated by LY2033298 on hM₄ receptors, where 0% and 100% corresponded to zero and maximum potentiation mediated by LY2033298 on hM₄ receptors (see Fig. S4). (A) Mutants hM4-Δ6.1 and hM4-Δ7.1 were significantly less responsive to LY2033298 (*) when compared with hM₄ receptors. The data points represents mean ± S.E.M. of more than eight experiments. (B) The enhancement of ACh function by LY2033298 for the humanized rM₄ was not significantly different from rM₄ receptors, whereas ratinized hM₄ was significantly different from hM₄ but not from rM₄ receptors. The receptor selectivity of LY2033298-mediated potentiation between rM₄ and rM₂ receptors was sustained. Data shown are mean ± SEM (n ≥ 3).

Fig. 4.

LY2033298-mediated potentiation is effective in rat CAR and PPI psychosis models. (A) LY2033298 reduced conditioned avoidance responding (CAR) in trained male HSD rats in a dose-dependent manner in the presence of an inactive dose of oxotremorine (*, P < 0.05 versus vehicle). (B) Apomorphine-induced suppression of the acoustic startle response was reversed by LY2033298 in a dose-dependent manner in the presence of a subeffective dose of Oxo. Data are mean percentage prepulse inhibition (PPI) values (V, vehicle; Apo, apomorphine; Oxo, oxotremorine; LY, 2033298) (#, P < 0.05 versus V/V/Apo; *, P < 0.05 versus V/V/V).