M1 Muscarinic Acetylcholine Receptor Research Articles

The Anti-Inflammatory Effects of Fermented Herbal Roots of Asparagus cochinchinensis in an Ovalbumin-Induced Asthma Model.

Introduction: Roots of Asparagus cochinchinensis, which have pharmacologically active ingredients, have received great attention because they show good therapeutic effects for various inflammatory diseases without specific toxicity. This study investigated the anti-asthmatic effects of a butanol extract of Asparagus cochinchinensis roots that had been fermented with Weissella cibaria (BAW) and its possible underlying cholinergic regulation. Methods: Alterations of the anti-asthmatic markers and the molecular response factors were measured in an ovalbumin (OVA)-induced asthma model after treatment with BAW. Results: Treatment with BAW decreased the intracellular reactive oxygen species (ROS) production in lipopolysaccharides (LPS) activated RAW264.7 cells. The results of the animal experiments revealed lower infiltration of inflammatory cells and bronchial thickness, and a significant reduction in the number of macrophages and eosinophils, concentration of OVA-specific IgE, and expression of Th2 cytokines in the OVA + BAW treated group. In addition, a significant recovery of goblet cell hyperplasia, MMP-9 expression, and the VEGF signaling pathway was observed upon airway remodeling in the OVA + BAW treated group. Furthermore, these responses of BAW were linked to recovery of acetylcholine esterase (AChE) activity and muscarinic acetylcholine receptor (mAChR) M3 downstream signaling pathway in epithelial cells, smooth muscle cells, and afferent sensory nerves of OVA + BAW-treated mice. Conclusion: Overall, these findings are the first to provide evidence that the therapeutic effects of BAW can prevent airway inflammation and remodeling through the recovery of cholinergic regulation in structural cells and inflammatory cells of the chronic asthma model.

In Vivo Modulation of Hippocampal Excitability by M4 Muscarinic Acetylcholine Receptor Activator: Implications for Treatment of Alzheimer's Disease and Schizophrenic Patients.

Abnormal hippocampal activity has been linked to impaired cognitive performance in Alzheimer's disease and schizophrenia, leading to a hypothesis that normalization of this activity may be therapeutically beneficial. Our work suggests that one approach for hippocampal normalization may be through activation of the M4 muscarinic acetylcholine receptor. We used a brain penetrant M4 muscarinic acetylcholine receptor selective activator, PT-3763, to show dose-dependent attenuation of field potentials in Schaffer collateral (CA3-CA1) and recurrent associational connections (CA3-CA3) ex vivo in hippocampal slices. In vivo, systemic administration of PT-3763 led to attenuation of glutamate release in CA3 as measured by amperometry and to a dose-dependent decrease in population CA1 pyramidal activity as measured by fiber photometry. This decrease in population activity was also evident with a localized administration of the compound to the recorded site. Finally, PT-3763 reversed scopolamine-induced deficit in Morris water maze. Our results suggest that M4 muscarinic acetylcholine receptor activation may be a suitable therapeutic treatment in diseases associated with hyperactive hippocampal activity.

The Effect of Autoantibody against M2-Muscarinic Acetylcholine Receptor in Heart Failure Patients on Digoxin Treatment

Background: Autoantibody against M₂-muscarinic acetylcholine receptor (anti-M₂AChR) has a biological effect similar to a vagus agonist. Digoxin has a function of vagus nervous system stimulation. We hypothesized that anti-M₂AChR is highly correlated with digoxin in patients with chronic heart failure (CHF). Methods: Synthetic M₂AChR peptides served as the target antigen in an ELISA were used to screen the sera of 80 CHF patients, who were separated into a negative (–) or positive (+) anti-M₂AChR group according to their anti-M₂AChR reactivity. Echocardiography and serum digoxin concentration (SDC) were performed at baseline and after 1 year of digoxin in combination with the standard treatment regime. The end-point events were compared over 1 year of follow-up. Results: Seventy-two CHF patients completed the final data analysis, including 32 (+)anti-M₂AChR and 40 (–)anti-M₂AChR patients. The resting heart rate of the positive group was higher than that of the negative group at baseline (p < 0.05; 89.0 ± 1.6 vs. 83.8 ± 1.1 bpm). Both groups showed improvement in the left ventricular end-diastolic and end-systolic dimensions and ejection fraction with digoxin in combination with the standard treatment regime for 1 year (all p < 0.01). However, the 32 patients with (–)anti-M₂AChR had greater improvements than the 40 patients with (+)anti-M₂AChR, and this was accompanied by a marked decrease of rehospitalization (all p < 0.01) but not of cardiovascular mortality after 1 year. The SDC of patients with (–)anti-M₂AChR was significantly lower than that of patients with (+)anti-M₂AChR (p < 0.05; 0.63 ± 0.05 vs.1.16 ± 0.06 ng/mL) and had a positive correlation with anti-M₂AChR (r = 0.81, p < 0.001). Conclusion: These results suggested that anti-M₂AChR could be a useful biomarker of vagus nerve overactivation and is associated with a poor response to digoxin treatment in CHF patients.

Antihypertensive effects of orally administered eggplant (Solanum melongena) rich in acetylcholine on spontaneously hypertensive rats.

Our previous results (Nakamura et al., 2013, 2016) indicated that acetylcholine (ACh) in orally administered foods exerts antihypertensive effects. Eggplants (Solanum melongena) contain abundant ACh (Horiuchi et al., 2003), and their food functionality was discovered, using spontaneously hypertensive rats, by measuring blood pressure after oral administration of a suspension of lyophilized eggplant powder. We found that lyophilized eggplant powder induced significantly lowered acute and chronic blood pressure levels at very low doses of 0.0650 mg/kg body weight (b.w.) and 0.821 mg/(kg b.w.·day), respectively. Chronic administration suppressed adrenaline and noradrenaline excretion in the urine, and aorta assays showed that eggplant acted on the M3 muscarinic ACh receptor (M3 mAChR). ACh was conclusively shown to function as the main component of eggplant contributing to antihypertensive activity by suppressing sympathetic nervous activity via M3 mAChR. This report reveals a new food functionality of eggplant and its potential as a novel antihypertensive food.

A0543 Autoantibody against M2 Muscarinic Acetylcholine Receptor and Clinical Outcomes of Heart Failure in response to Digoxin

A0543 Autoantibody against M2 Muscarinic Acetylcholine Receptor and Clinical Outcomes of Heart Failure in response to Digoxin

Utility of an “Allosteric Site-Impaired” M2 Muscarinic Acetylcholine Receptor as a Novel Construct for Validating Mechanisms of Action of Synthetic and Putative Endogenous Allosteric Modulators

Muscarinic acetylcholine receptors (mAChRs) are exemplar models for understanding G protein-coupled receptor (GPCR) allostery, possessing a "common" allosteric site in an extracellular vestibule (ECV) for synthetic modulators including gallamine, strychnine, and brucine. In addition, there is intriguing evidence of endogenous peptides/proteins that may target this region at the M2 mAChR. A common feature of synthetic and endogenous M2 mAChR negative allosteric modulators (NAMs) is their cationic nature. Using a structure-based approach, we previously designed a mutant M2 mAChR (N410K+T423K) to specifically abrogate binding of ECV cationic modulators (Dror et al., 2013). Herein, we used this "allosteric site-impaired" receptor to investigate allosteric interactions of synthetic modulators as well as basic peptides (poly-l-arginine, endogenously produced protamine, and major basic protein). Using [3H]N-methylscopolamine equilibrium and kinetic binding and functional assays of guanosine 5'-O-[γ-thio]triphosphate [35S] binding and extracellular signal-regulated kinases 1 and 2 phosphorylation, we found modest effects of the mutations on potencies of orthosteric antagonists and an increase in the affinity of the cognate agonist, acetylcholine, likely reflecting the effect of the mutations on the access/egress of these ligands into the orthosteric pocket. More importantly, we noted a significant abrogation in affinity for all synthetic or peptidic modulators at the mutant mAChR, validating their allosteric nature. Collectively, these findings provide evidence for a hitherto-unappreciated role of endogenous cationic peptides interacting allosterically at the M2 mAChR and identify the allosteric site-impaired GPCR as a tool for validating NAM activity as well as a potential candidate for future chemogenetic strategies to understand the physiology of endogenous allosteric substances.

Activation of the mGlu1 metabotropic glutamate receptor has antipsychotic-like effects and is required for efficacy of M4 muscarinic receptor allosteric modulators

Recent clinical and preclinical studies suggest that selective activators of the M4 muscarinic acetylcholine receptor have potential as a novel treatment for schizophrenia. M4 activation inhibits striatal dopamine release by mobilizing endocannabinoids, providing a mechanism for local effects on dopamine signaling in the striatum but not in extrastriatal areas. G protein-coupled receptors (GPCRs) typically induce endocannabinoid release through activation of Gαq/11-type G proteins whereas M4 transduction occurs through Gαi/o-type G proteins. We now report that the ability of M4 to inhibit dopamine release and induce antipsychotic-like effects in animal models is dependent on co-activation of the Gαq/11-coupled mGlu1 subtype of metabotropic glutamate (mGlu) receptor. This is especially interesting in light of recent findings that multiple loss of function single nucleotide polymorphisms (SNPs) in the human gene encoding mGlu1 (GRM1) are associated with schizophrenia, and points to GRM1/mGlu1 as a gene within the “druggable genome” that could be targeted for the treatment of schizophrenia. Herein, we report that potentiation of mGlu1 signaling following thalamo-striatal stimulation is sufficient to inhibit striatal dopamine release, and that a novel mGlu1 positive allosteric modulator (PAM) exerts robust antipsychotic-like effects through an endocannabinoid-dependent mechanism. However, unlike M4, mGlu1 does not directly inhibit dopamine D1 receptor signaling and does not reduce motivational responding. Taken together, these findings highlight a novel mechanism of cross talk between mGlu1 and M4 and demonstrate that highly selective mGlu1 PAMs may provide a novel strategy for the treatment of positive symptoms associated with schizophrenia.

Correction: The combination of Cassia obtusifolia L. and Foeniculum vulgare M. exhibits a laxative effect on loperamide-induced constipation of rats.

[This corrects the article DOI: 10.1371/journal.pone.0195624.].

Most calbindin-immunoreactive neurons, but few calretinin-immunoreactive neurons, express the m1 acetylcholine receptor in the middle temporal visual area of the macaque monkey.

IntroductionRelease of the neuromodulator acetylcholine into cortical circuits supports cognition, although its precise role and mechanisms of action are not well understood. Little is known about functional differences in cholinergic modulatory effects across cortical model systems, but anatomical evidence suggests that such differences likely exist because, for example, the expression of cholinergic receptors differs profoundly both within and between species.MethodsIn the primary visual cortex (V1) of macaque monkeys, cholinergic receptors are strongly expressed by inhibitory interneurons. Using dual‐immunofluorescence confocal microscopy, we examine m1 muscarinic acetylcholine receptor expression by two subclasses of inhibitory interneurons—identified by their expression of the calcium‐binding proteins calbindin and calretinin—in the middle temporal extrastriate area (MT) of the macaque.Results and ConclusionsWe find that the majority of calbindin‐immunoreactive neurons (55%) and only few calretinin‐immunoreactive neurons (10%) express the m1 acetylcholine receptor. These results differ from the pattern observed in V1 of the same species, lending further support to the notion that cholinergic modulation in the cortex is tuned such that different cortical compartments will respond to acetylcholine release in different ways.

Hippocampal place cell dysfunction and the effects of muscarinic M1 receptor agonism in a rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease that disproportionately impacts memory and the hippocampus. However, it is unclear how AD pathology influences the activity of surviving neurons in the hippocampus to contribute to the memory symptoms in AD. One well-understood connection between spatial memory and neuronal activity in healthy brains is the activity of place cells, neurons in the hippocampus that fire preferentially in a specific location of a given environment (the place field of the place cell). In the present study, place cells were recorded from the hippocampus in a recently-developed rat model of AD (Tg-F344 AD) at an age (12-20 months) at which the AD rats showed marked spatial memory deficits. Place cells in the CA2 and CA3 pyramidal regions of the hippocampus in AD rats showed sharply reduced spatial fidelity relative to wild-type (WT) rats. In contrast, spiking activity of place cells recorded in region CA1 in AD rats showed good spatial fidelity that was similar to CA1 place cells in WT rats. Oral administration of the M1 muscarinic acetylcholine receptor agonist VU0364572 impacted place cell firing rates in CA1 and CA2/3 hippocampal regions, but did not improve the spatial fidelity of CA2/3 hippocampal place cells in AD rats. The results indicated that, to the extent the spatial memory impairment in AD rats was attributable to hippocampal dysfunction, the memory impairment was more attributable to dysfunction in hippocampal regions CA2 and CA3 rather than CA1.

Effects of Cortisol-Induced Acetylcholine Accumulation on Tissue Damage and Regeneration in Steatotic Livers in the Context of Partial Hepatectomy Under Vascular Occlusion

Introduction Steatosis is a known risk factor in hepatic surgery and it is associated with postoperative morbidity and mortality. Acetylcholine (ACh) is the major neurotransmitter of vagus nerve and it is released as a response to stressful events like trauma or infection. Stress can also activate the HPA axis that drives the production of cortisol. Both cortisol and ACh have been proved beneficial in experimental models of sepsis, liver transplantation or myocardial ischemia, among other pathologies. In this study, we will study the relationship between cortisol and ACh and their effects on regeneration and injury in seatotic livers submitted to partial hepatectomy (PH) under vascular occlusion, a common technique used in hepatic surgery to avoid bleeding. Materials & Methods We determined hepatic levels of ACh and its receptors in obese Zucker rats that underwent partial hepatectomy under vascular occlusion. The ACTH-cortisol pathway was pharmacologically modulated and the effects on hepatic injury and regeneration, as well as its underlying mechanisms of action, were evaluated. Results There is a significant ACh accumulation driven by the administration of cortisol in steatotic livers submitted to PH under vascular occlusion, that consequently leads to an overexpression of a7 nicotinic and M3 muscarinic ACh receptors. However, administration of ACh prior to surgery increases injury and impairs regeneration via M3 mACh receptor. Interestingly, M3 mAChR antagonist, but not a7 nAChR, alone or in combination with cortisol, did confer eprotection against liver damage caused by the surgical procedure and enhanced regeneration. Moreover, administration of ACh reduced PI3K/Akt hepatic expression and consequently reduced levels of markers of oxidative stress, neutrophil accumulation and edema. In consonance with the results presented above, treatment with an M3 mAChR antagonist, but not a7 nAChR, alone or in combination with cortisol, reduced the hepatic levels of these harmful markers as a result of an overexpression of PI3K and pAKT in these steatotic livers. Thus, this indicates a lesser role of a7 nAChR. Conclusions The reported benefits of cortisol in PH cannot be extrapolated in situations of PH under vascular occlusion in steatotic livers. Cortisol is responsible of the accumulation of acetylcholine in these livers submitted to partial hepatectomy with vascular occlusion. ACh exerts its action through the M3 ACh receptor, as it exacerbates liver damage and oxidative stress and impairs regeneration. Strategies aimed to block cortisol activity in order to block ACh accumulation and/or blockade of M3 mAChR are probably worthwhile to protect steatotic livers and reduce the incidence of postoperative complications following PH under vascular occlusion.

Characterization of methanthelinium binding and function at human M1-M5 muscarinic acetylcholine receptors.

Firstly, it was determined whether methanthelinium bromide (MB) binds to human M1-M5 (hM1-hM5) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [3H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100μM of MB ranging from none in hM3 to 4.6-fold in hM2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [3H]NMS (0.2 and 2.0nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logKI (± S.E.) hM3 = 8.71 ± 0.15, hM1 = 8.68 ± 0.14, hM5 = 8.58 ± 0.07, hM2 = 8.27 ± 0.07 to hM4 = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logKB: hM1 = 9.53 ± 0.05, hM4 = 9.33 ± 0.05, hM5 = 8.80 ± 0.05, hM2 = 8,79± 0.06, to hM3 = 8.43 ± 0.04. In conclusion, MB, below 1μM, binds competitively and non-selectively (except for the difference between hM3 vs. hM4) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM1 and hM4.

A Positive Allosteric Modulator Enhances the Ability of Beta Cell M3 Muscarinic Receptors to Simulate Insulin Secretion In Vitro and In Vivo

Several studies have shown that activation of M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic beta-cells leads to striking increases in glucose-stimulated insulin release. As a result, we hypothesized that agents that can promote signaling through beta-cell M3Rs might become useful in the treatment of type 2 diabetes. The M3R is a prototypic class A GPCR that is selectively coupled to G proteins of the Gq family. At present, selective M3R agonists that bind to the orthosteric ACh binding site are not available. However, the lab of Craig Lindsley (Bridges et al., 2010) recently described a compound (VU0119498) that binds to an allosteric site on the M3R, thus enhancing ACh-induced signaling through M3Rs. In this study, we tested the ability of this positive allosteric modulator (PAM) to stimulate insulin release in vitro and in vivo. The use of PAMs has the great advantage that it respects the spatio-temporal control of receptor activation, i.e., the PAM is only active upon receptor occupation by an endogenous orthosteric agonist. Studies with cultured MIN6 cells showed that VU0119498 enhanced ACh-induced insulin secretion in an M3R-dependent fashion. In vivo studies demonstrated that PAM treatment of WT mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These VU0119498 effects were mediated by beta-cell M3Rs since they were absent in mutant mice selectively lacking M3Rs in beta-cells. In future studies, we will use mouse models of diabetes to examine whether this M3R-PAM is endowed with antidiabetic activity. This approach may lead to the development of a novel generation of antidiabetic drugs. Disclosure L. Zhu: None. J. Wess: None.

Acetylcholine Receptor Stimulation for Cognitive Enhancement: Better the Devil You Know?

Drug treatments to improve memory focus on enhancing acetylcholine. However, Vijayraghavan and colleagues (2018) show that direct stimulation of the M1 muscarinic acetylcholine receptor adversely affected neuronal activity in prefrontal cortex related to working memory for behavioral rules.

Probing the binding site of novel selective positive allosteric modulators at the M1 muscarinic acetylcholine receptor

Subtype-selective allosteric modulation of the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) is an attractive approach for the treatment of numerous disorders, including cognitive deficits. The discovery of benzyl quinolone carboxylic acid, BQCA, a selective M1 mAChR positive allosteric modulator (PAM), spurred the subsequent development of newer generation M1 PAMs representing diverse chemical scaffolds, different pharmacodynamic properties and, in some instances, improved pharmacokinetics. Key exemplar molecules from such efforts include PF-06767832 (N-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-4-(4-(thiazol-4-yl)benzyl)pyridine-2-carboxamide), VU6004256 (4,6-difluoro-N-(1S,2S)-2-hydroxycyclohexyl-1-((6-(1-methyl-1H-pyrazol-4-yl)pyridine-3-yl)methyl)-1H-indole-3-carboxamide) and MIPS1780 (3-(2-hydroxycyclohexyl)-6-(2-((4-(1-methyl-1H-pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3H)-one). Given these diverse scaffolds and pharmacodynamics, the current study combined pharmacological analysis and site-directed mutagenesis to explore the potential binding site and function of newer M1 mAChR PAMs relative to BQCA. Interestingly, the mechanism of action of the novel PAMs was consistent with a common model of allostery, as previously described for BQCA. Key residues involved in the activity of BQCA, including Y179 in the second extracellular loop (ECL) and W4007.35 in transmembrane domain (TM) 7, were critical for the activity of all PAMs tested. Overall, our data indicate that structurally distinct PAMs share a similar binding site with BQCA, specifically, an extracellular allosteric site defined by residues in TM2, TM7 and ECL2. These findings provide valuable insights into the structural basis underlying modulator binding, cooperativity and signaling at the M1 mAChR, which is essential for the rational design of PAMs with tailored pharmacological properties.

Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer’s disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that “pure” positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.

Assessment of the Molecular Mechanisms of Action of Novel 4-Phenylpyridine-2-One and 6-Phenylpyrimidin-4-One Allosteric Modulators at the M1 Muscarinic Acetylcholine Receptors

Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism but whose molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, benzyl quinolone carboxylic acid (BQCA), in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signal-regulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and β-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated.

Activation of M2 muscarinic acetylcholine receptors by a hybrid agonist enhances cytotoxic effects in GB7 glioblastoma cancer stem cells

In previous studies, we found that the orthosteric muscarinic agonist arecaidine propargyl ester (APE) (100 μM) induced a decreased cell proliferation and severe apoptosis in glioblastoma cancer stem cells (GSCs). In this report, we have investigated the effects mediated by hybrid (orthosteric/allosteric) muscarinic agonists P-6-Iper and N-8-Iper on GSCs survival. At variance with APE, the agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper action appear to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. Moreover, analysis of the expression of phosphorylated histone H2AX (γ-H2AX) indicated that the treatment with N-8-Iper produced a decreased cell survival by induction of DNA damage. The ability of N-8-Iper to produce a cytotoxic effect and apoptosis at low doses indicates that this muscarinic agonist is a suitable probe in a putative therapeutic intervention on glioblastoma through M2 receptor activation.

Discovery and Optimization of Potent and CNS Penetrant M5-Preferring Positive Allosteric Modulators Derived from a Novel, Chiral N-(Indanyl)piperidine Amide Scaffold.

The pharmacology of the M5 muscarinic acetylcholine receptor (mAChR) is the least understood of the five mAChR subtypes due to a historic lack of selective small molecule tools. To address this shortcoming, we have continued the optimization effort around the prototypical M5 positive allosteric modulator (PAM) ML380 and have discovered and optimized a new series of M5 PAMs based on a chiral N-(indanyl)piperidine amide core with robust SAR, human and rat M5 PAM EC50 values <100 nM and rat brain/plasma Kp values of ∼0.40. Interestingly, unlike M1 and M4 PAMs with unprecedented mAChR subtype selectivity, this series of M5 PAMs displayed varying degrees of PAM activity at the other two natively Gq-coupled mAChRs, M1 and M3, yet were inactive at M2 and M4.

Ligand-Triggered Structural Changes in the M2 Muscarinic Acetylcholine Receptor.

The muscarinic M2 acetylcholine receptor, one of the few G-protein coupled receptors that has not only been crystallized in both active and inactive conformations but also in the presence of a positive allosteric modulator, is an interesting system to study the molecular mechanisms of GPCR activation and ligand allosterism. Here, we have employed molecular dynamics (MD) simulations (adding to 14 μs in total) to study conformational changes triggered by the inverse agonist R-(-)-3-quinuclidinyl-benzilate (QNB) in the structure of the active M2 receptor (PBD ID 4MQS ) after replacement of the agonist iperoxo by the inverse agonist QNB. This permitted us to identify the sequence of events in the deactivation mechanism of the M2 acetylcholine receptor, which results first in the rearrangement of the transmission switch, the subsequent opening of the extracellular portion of the receptor and finally, the closure of the intracellular part. We also evaluate the effect of the positive allosteric modulator LY2119620 when bound simultaneously with the orthosteric agonist iperoxo and find that it restricts the conformation of Trp4227.35 in a position that modulates the orientation of the Tyr4267.39 at the orthosteric-binding pocket.