Novel allosteric agonists of M1 muscarinic acetylcholine receptors induce brain region-specific responses that correspond with behavioral effects in animal models

Abstract

M(1) muscarinic acetylcholine receptors (mAChRs) represent a viable target for treatment of multiple disorders of the central nervous system (CNS) including Alzheimer's disease and schizophrenia. The recent discovery of highly selective allosteric agonists of M(1) receptors has provided a major breakthrough in developing a viable approach for the discovery of novel therapeutic agents that target these receptors. Here we describe the characterization of two novel M(1) allosteric agonists, VU0357017 and VU0364572, that display profound differences in their efficacy in activating M(1) coupling to different signaling pathways including Ca(2+) and β-arrestin responses. Interestingly, the ability of these agents to differentially activate coupling of M(1) to specific signaling pathways leads to selective actions on some but not all M(1)-mediated responses in brain circuits. These novel M(1) allosteric agonists induced robust electrophysiological effects in rat hippocampal slices, but showed lower efficacy in striatum and no measureable effects on M(1)-mediated responses in medial prefrontal cortical pyramidal cells in mice. Consistent with these actions, both M(1) agonists enhanced acquisition of hippocampal-dependent cognitive function but did not reverse amphetamine-induced hyperlocomotion in rats. Together, these data reveal that M(1) allosteric agonists can differentially regulate coupling of M(1) to different signaling pathways, and this can dramatically alter the actions of these compounds on specific brain circuits important for learning and memory and psychosis.

Figure 1.

Selectivity profile of VU0357017 among families. A–C, GPCRs. a, Selectivity profile of VU0357017 when tested as an agonist against multiple Family A GPCRs using GPCRProfiler calcium assay; 10 μm VU0357017 was applied to cells expressing various GPCRs. VU0357017 induced a significant response in cells expressing M₁ muscarinic receptors but had little activity at other receptor subtypes. A subsequent addition of a full agonist CRC (ACh) for each receptor allowed measurement of potential PAM or antagonist activity (data not shown). Interestingly, VU0357017 did show potentiation of responses to ACh at D₄ and at β₃ adrenergic receptors. b, Selectivity profile of VU0357017 when tested as an agonist against multiple Family B and C GPCRs. 5-HT, serotonin; M, muscarinic; A, adenosine; Alpha, α-adrenergic; Beta, β-adrenergic; CB, cannabinoid; D, dopamine; H, histamine; P2Y, purinergic; CGRP, calcitonin gene-related peptide; CCK, cholecystokinin; GIP, glucose-dependent insulinotropic peptide; GLP2, glucagon-like peptide receptor; PAC1, pituitary adenylate cyclase-activating polypeptide type I receptor; PTH, parathyroid hormone; VPAC, vasoactive intestinal peptide; CaS, calcium sensing; GABAB, γ amino butyric acid, mGlu, metabotropic glutamate receptor.

Figure 2.

VU0364572 and VU0357017 induce calcium release and ERK phosphorylation but are without effects on β-arrestin recruitment. a, Chemical structures of the two M₁ agonists VU0357017 and VU0364572. b, CRCs of receptor-induced calcium release for CCh (filled circles), VU0364572 (open circles), and VU0357017 (crosses) in CHO-K1 cells stably expressing human M₁ mAChRs. Data are normalized to the CCh maximum response. Data points represent mean ± SEM of four independent experiments performed in duplicate or triplicate. c, CRCs of agonist-induced ERK1/2 phosphorylation (pERK1/2) assessed using the SureFire ERK phosphorylation assay in hM₁ CHO cells. Data are expressed as fold change over basal ERK levels and is normalized to the maximum response elicited by CCh. Data represent the mean ± SEM of 7–8 independent experiments performed in duplicate or triplicate. d, CRCs of agonist-induced β-arrestin recruitment in hM₁ CHO cells using PathHunter detection kit. Data points represent mean ± SEM of three independent experiments performed in duplicate or triplicate and are normalized to % CCh max. e, Saturation isotherms of [³H]-NMS binding to membranes prepared from hM₁ CHO cells. Receptor density values (1479 ± 129 fmol/mg protein) were obtained from three independent experiments. f, Representative saturation isotherms of [³H]-NMS binding to membranes prepared from hM₁ CHO cells used in β-arrestin recruitment assays. Receptor density values (11701.600 ± 1411.21 fmol/mg protein) were obtained from five independent experiments.

Figure 3.

VU0364572 and VU0357017 induce responses in a cell line with variable receptor expression. a, Saturation isotherms of [³H] NMS binding to membranes prepared from hM₁ TREx CHO cells treated with varying concentrations of tet. Membranes were prepared 24 h after treatment with tet and specific binding values increased following treatment indicating increased receptor density (in fmol/mg protein; 0 ng/ml tet = 268.5 ± 88.0, 10 ng/ml tet = 188.33 ± 37.0, 25 ng/ml tet = 666.9 ± 130, 50 ng/ml tet = 2277.2 ± 619.6, 1 μg/ml tet = 4435.6 ± 1431.1, n = 3). b–d, CRCs of calcium release for CCh, VU0364572, and VU0357017 in hM₁ TREx CHO cells that were treated overnight with 1 μg/ml (closed circles), 50 ng/ml (closed triangles), 25 ng/ml (closed squares), 10 ng/ml (crosses), or 0 ng/ml tet (open circles). Data points represent mean ± SEM of three independent experiments performed in duplicate or triplicate. Data are normalized to % ionomycin (1 μm). e–g, M₁-induced ERK phosphorylation measured in TREx CHO cells treated across a range of tet concentrations. An increase in CCh's maximal response was present in cells treated with 1 μg/ml tet. There was little effect on the EC₅₀ values. Induction of M₁ expression with 50 ng/ml or 1 μg/ml tet had a small effect on the maximal response to VU0364572, but had little effect on the maximal response to VU0357017. Data points represent the mean ± SEM of two or three independent experiments performed in duplicate or triplicate. Data are expressed as fold over basal ERK response.

Figure 4.

CCh and M₁ compound VU0364572 induce β-arrestin recruitment in TREx CHO cells. a, Preagonist and postagonist confocal scans of hM₁ TREx CHO cells expressing β-arrestin2-YFP. Treatment of cells with CCh (100 μm) induces β-arrestin recruitment (black arrows, bottom) in cells that were exposed overnight to tet (50 ng/ml). Treatment of cells with VU0364572 (100 μm) induces β-arrestin2 recruitment (black arrows, bottom) in cells that were exposed overnight to tet (1 μg/ml). VU0357017 (100 μm) did not induce β-arrestin2 recruitment in cells treated overnight with 50 ng/ml or 1 μg/ml tet. b, Quantification of the effects of each agonist on the number of puncta. A one-way ANOVA revealed that puncta in CCh-treated cells (50 ng/ml tet) differed significantly when compared with VU0357017 (1 μg/ml tet) and VU0364572 (1 μg/ml tet)-treated cells (F_(3,26) = 4.82, *p < 0.001). Neither CCh nor M₁ agonist induced arrestin recruitment in cells that were not treated with tet (data not shown).

Figure 5.

M₁ agonists VU0364572 and VU0357017 significantly enhance threshold theta-burst LTP and VU0364572 induces LTD at the Schaffer collateral-CA1 synapse of rodent hippocampal slices. Insets for each figure are representative fEPSP traces measured at baseline (black) or 50 (LTD) or 55 (LTP) minutes after compound washout (gray). Scale bars: x-axis, 2 ms; y-axis, 0.6 mV. a, The standard TBS protocol (TBS saturation) induces significant LTP (n = 9), whereas the threshold TBS protocol induces only a slight potentiation of fEPSP slope (n = 8) at the SC-CA1 synapse. b, Bath application of 500 nm VU0364572 (n = 5) or VU0357017 (n = 5 of 9 experiments) for 10 min before threshold TBS induced a significant potentiation of fEPSP slope. c, Significant differences (*p < 0.01) were observed in the mean percentage potentiation induced by threshold TBS compared with TBS saturation or TBS threshold plus compound. d, Addition of 50 μm (n = 5) CCh for 10 min induced LTD of fEPSP slope, whereas addition of 30 μm CCh (n = 4) had no effect. e, Bath application of 30 μm VU0364572 (n = 6) for 10 min induced LTD, whereas addition of 30 μm VU0357017 (n = 6) had no effect on LTD. f, Mean percentage maximal depression induced by each compound. VU0364572 induced a significant depression in fEPSP slope compared with 30 μm CCh (*p < 0.01).

Figure 6.

M₁-selective agonists VU0364572 and VU0357017 induce small changes in action potential spiking frequency in MSNs. a, The membrane potential response to a current step before and after application of CCh (10 μm) in MSNs of rats. CCh (n = 7) induces a robust increase in evoked action potential firing. b, Response to current injection following application of VU0364572 (n = 5). c, Response to current injection following application of VU0357017 (n = 5). d, Bar graph showing the change in number of spikes/pulse following addition of test compounds. *p < 0.0002 for VU0364572; *p ≤ 0.001 for VU0357017. For comparison of VU0364572 and VU0357017, *p ≥ 0.05. See Results for details.

Figure 7.

VU0357017 and VU0364572 are devoid of agonist activity in mouse mPFC. a, Sample traces from single neurons and changes in probability plots of the IEI from representative cells following treatment with CCh showing that CCh induces increases in sEPSCs (n = 15). b, c Sample traces and probability plots of IEI in representative cells treated with VU0357017 (n = 7) or VU0364572 (n = 4). d, Bar graphs depicting mean changes in sEPSC frequency. All changes in frequency represent the mean ± SEM and are compared with baseline controls. ^#p < 0.0001, **p < 0.0001, for VU037017 and **p = 0.0002 for VU0364572. See Results for details.

Figure 8.

M₁ agonists VU0357017 and VU0364572 enhance performance in MWM and CFC in rats. a, Swim distance across the 5 d of testing in the water maze. Data are collapsed across four daily trials and across groups of VU0364572 or vehicle-treated animals. b, Effects of treatment with VU0364572 on spatial memory on days 4 (*p < 0.05) and 5 (*p < 0.001) of testing in MWM assays. c, Effects of treatment with VU0364572 on memory retention between day 4 Trial 4 and day 5 Trial 1 (n = 8) ⁺p < 0.05 versus day 4 for vehicle-treated animals only. *p ≤ 0.02 versus vehicle-treated animals for day 5 (n = 8). d, Effects of treatment with VU0364572 on platform crossings during the first 30 s of the probe trial. *p < 0.05. e, Swim distance across the 5 d of testing in the water maze. Data are collapsed across four daily trials and across groups of VU0357017 or vehicle-treated animals. f, Effects of treatment with VU0357017 on spatial memory on days 4 and 5 of testing (n = 8) (p > 0.445). g, Effects of treatment with VU357017 on spatial memory retention between day 4 Trial 4 and day 5 Trial 1 (n = 8) (p > 0.755). h, Effects of treatment with VU0357017 on platform crossings during the first 30 s of the probe trial (p > 0.981). i, Effects of treatment of VU0364572 on acquisition of contextual fear in rats (n = 4–6, each group) (*p < 0.05). j, Effects of VU0357017 treatment on acquisition of contextual fear in rats (n = 4–6, each group) (*p < 0.05).

Figure 9.

VU0357017 and VU0364572 do not reverse amphetamine-induced hyperlocomotion in rats. a, An amphetamine dose–response curve showing that 1 mg/kg amphetamine induces robust hyperlocomotion. b, VU0357017 fails to reverse hyperlocomotion induced by amphetamine treatment in rats suggesting that M₁ agonism by this compound does not have an antipsychotic-like profile. VU0357017 or vehicle was injected intraperitoneally 30 min before amphetamine injection (4 mg/kg, s.c.). c, A broad dose range of VU0364572 also fails to reverse this response.