M1 mAChR Research Articles

Restoring Agonist Function at a Chemogenetically Modified M1 Muscarinic Acetylcholine Receptor.

Designer receptors exclusively activated by designer drugs (DREADDs) have been successfully employed to activate signaling pathways associated with specific muscarinic acetylcholine receptor (mAChR) subtypes. The M1 DREADD mAChR displays minimal responsiveness to the endogenous agonist acetylcholine (ACh) but responds to clozapine-N-oxide (CNO), an otherwise pharmacologically inert ligand. We have previously shown that benzyl quinolone carboxylic acid (BQCA), an M1 mAChR positive allosteric modulator (PAM), can rescue ACh responsiveness at these receptors. However, whether this effect is chemotype specific or applies to next-generation M1 PAMs with distinct scaffolds is unknown. Here, we reveal that new M1 PAMs restore ACh function at the M1 DREADD while modulating ACh binding at the M1 wild-type mAChR. Importantly, we demonstrate that the modulation of ACh function by M1 PAMs is translated in vivo using transgenic M1 DREADD mice. Our data provide important insights into mechanisms that define allosteric ligand modulation of agonist affinity vs efficacy and how these effects play out in the regulation of in vivo responses.

Effects mediated by the α7 nicotinic acetylcholine receptor on cell proliferation and migration in rat adipose-derived stem cells.

Adipose-derived stem cells (ASCs) are an attractive source for regenerative medicine as they can be easily isolated, rapidly expandable in culture and show excellent in vitro differentiation potential. Acetylcholine (ACh), one of the main neurotransmitters in central and peripheral nervous systems, plays key roles in the control of several physiological processes also in non-neural tissues. As demonstrated in our previous studies, ACh can contribute to the rat ASCs physiology, negatively modulating ASCs proliferation and migration via M2 muscarinic receptor (mAChR) activation. In the present work we show that rat ASCs also express α7 nicotinic receptors (nAChRs). In particular, we have investigated the effects mediated by the selective activation of α7 nAChRs, which causes a reduction of ASC proliferation without affecting cell survival and morphology, and significantly promotes cell migration via upregulation of the CXCR4 expression. Interestingly, the activation of the α7 nAChR also upregulates the expression of M2 mAChR protein, indicating a cooperation between muscarinic and nicotinic receptors in the inhibition of ASC proliferation.

Development of Novel 4-Arylpyridin-2-one and 6-Arylpyrimidin-4-one Positive Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor.

This study investigated the structure-activity relationships of 4-phenylpyridin-2-one and 6-phenylpyrimidin-4-one M1 muscarinic acetylcholine receptor (M1 mAChRs) positive allosteric modulators (PAMs). The presented series focuses on modifications to the core and top motif of the reported leads, MIPS1650 (1) and MIPS1780 (2). Profiling of our novel analogues showed that these modifications result in more nuanced effects on the allosteric properties compared to our previous compounds with alterations to the biaryl pendant. Further pharmacological characterisation of the selected compounds in radioligand binding, IP1 accumulation and β-arrestin 2 recruitment assays demonstrated that, despite primarily acting as affinity modulators, the PAMs displayed different pharmacological properties across the two cellular assays. The novel PAM 7 f is a potential lead candidate for further development of peripherally restricted M1 PAMs, due to its lower blood-brain-barrier (BBB) permeability and improved exposure in the periphery compared to lead 2.

Bis-Quinolinium Cyclophane Blockers of SK Potassium Channels Are Antagonists of M3 Muscarinic Acetylcholine Receptors.

Dequalinium is used as an antimicrobial compound for oral health and other microbial infections. Derivatives of dequalinium, the bis-quinolinium cyclophanes UCL 1684 and UCL 1848, are high affinity SK potassium channel antagonists. Here we investigated these compounds as M3 muscarinic receptor (mACHR) antagonists. We used the R-CEPIAer endoplasmic reticulum calcium reporter to functionally assay for Gq-coupled receptor signaling, and investigated the bis-quinolinium cyclophanes as antagonists of M3 mACHR activation in transfected CHO cells. Given mACHR roles in airway smooth muscle (ASM) contractility, we also tested the ability of UCL 1684 to relax ASM. We find that these compounds antagonized M3 mACHRs with an IC50 of 0.27 μM for dequalinium chloride, 1.5 μM for UCL 1684 and 1.0 μM for UCL 1848. UCL 1684 also antagonized M1 (IC50 0.12 μM) and M5 (IC50 0.52 μM) mACHR responses. UCL 1684 was determined to be a competitive antagonist at M3 receptors as it increased the EC50 for carbachol without a reduction in the maximum response. The Ki for UCL1684 determined from competition binding experiments was 909 nM. UCL 1684 reduced carbachol-evoked ASM contractions (>90%, IC50 0.43 μM), and calcium mobilization in rodent and human lung ASM cells. We conclude that dequalinium and bis-quinolinium cyclophanes antagonized M3 mACHR activation at sub- to low micromolar concentrations, with UCL 1684 acting as an ASM relaxant. Caution should be taken when using these compounds to block SK potassium channels, as inhibition of mACHRs may be a side-effect if excessive concentrations are used.

Modulation of arousal and sleep/wake architecture by M1 PAM VU0453595 across young and aged rodents and nonhuman primates.

Degeneration of basal forebrain cholinergic circuitry represents an early event in the development of Alzheimer's disease (AD). These alterations in central cholinergic function are associated with disruptions in arousal, sleep/wake architecture, and cognition. Changes in sleep/wake architecture are also present in normal aging and may represent a significant risk factor for AD. M1 muscarinic acetylcholine receptor (mAChR) positive allosteric modulators (PAMs) have been reported to enhance cognition across preclinical species and may also provide beneficial effects for age- and/or neurodegenerative disease-related changes in arousal and sleep. In the present study, electroencephalography was conducted in young animals (mice, rats and nonhuman primates [NHPs]) and in aged mice to examine the effects of the selective M1 PAM VU0453595 in comparison with the acetylcholinesterase inhibitor donepezil, M1/M4 agonist xanomeline (in NHPs), and M1 PAM BQCA (in rats) on sleep/wake architecture and arousal. In young wildtype mice, rats, and NHPs, but not in M1 mAChR KO mice, VU0453595 produced dose-related increases in high frequency gamma power, a correlate of arousal and cognition enhancement, without altering duration of time across all sleep/wake stages. Effects of VU0453595 in NHPs were observed within a dose range that did not induce cholinergic-mediated adverse effects. In contrast, donepezil and xanomeline increased time awake in rodents and engendered dose-limiting adverse effects in NHPs. Finally, VU0453595 attenuated age-related decreases in REM sleep duration in aged wildtype mice. Development of M1 PAMs represents a viable strategy for attenuating age-related and dementia-related pathological disturbances of sleep and arousal.

Striatal and nigral muscarinic type 1 and type 4 receptors modulate levodopa-induced dyskinesia and striato-nigral pathway activation in 6-hydroxydopamine hemilesioned rats

Acetylcholine muscarinic receptors (mAChRs) contribute to both the facilitation and inhibition of levodopa-induced dyskinesia operated by striatal cholinergic interneurons, although the receptor subtypes involved remain elusive. Cholinergic afferents from the midbrain also innervate the substantia nigra reticulata, although the role of nigral mAChRs in levodopa-induced dyskinesia is unknown. Here, we investigate whether striatal and nigral M1 and/or M4 mAChRs modulate dyskinesia and the underlying striato-nigral GABAergic pathway activation in 6-hydroxydopamine hemilesioned rats. Reverse microdialysis allowed to deliver the mAChR antagonists telenzepine (M1 subtype preferring), PD-102807 and tropicamide (M4 subtype preferring), as well as the selective M4 mAChR positive allosteric modulator VU0152100 in striatum or substantia nigra, while levodopa was administered systemically. Dyskinetic movements were monitored along with nigral GABA (and glutamate) and striatal glutamate dialysate levels, taken as neurochemical correlates of striato-nigral pathway and cortico-basal ganglia-thalamo-cortical loop activation. We observed that intrastriatal telenzepine, PD-102807 and tropicamide alleviated dyskinesia and inhibited nigral GABA and striatal glutamate release. This was partially replicated by intrastriatal VU0152100. The M2 subtype preferring antagonist AFDX-116, used to elevate striatal acetylcholine levels, blocked the behavioral and neurochemical effects of PD-102807. Intranigral VU0152100 prevented levodopa-induced dyskinesia and its neurochemical correlates whereas PD-102807 was ineffective. These results suggest that striatal, likely postsynaptic, M1 mAChRs facilitate dyskinesia and striato-nigral pathway activation in vivo. Conversely, striatal M4 mAChRs can both facilitate and inhibit dyskinesia, possibly depending on their localization. Potentiation of striatal and nigral M4 mAChR transmission leads to powerful multilevel inhibition of striato-nigral pathway and attenuation of dyskinesia.

From Structure to Clinic: Discovery of A M1 Muscarinic Acetylcholine Receptor Agonist for the Treatment of Memory Loss in Alzheimer's Disease

Current therapies for improving memory in Alzheimer’s disease are focused on correcting for defective cholinergic transmission by increasing acetylcholine levels through inhibition of acetylcholinesterase enzymes. These treatments are however associated with limited clinical efficacy and dose-limiting adverse effects. Here we describe agonist-bound crystal structures of the M1 muscarinic acetylcholine receptor (M1 mAChR) and its application in supporting the rational drug design of a selective M1-orthosteric agonist, HTL0009936. On the basis of pro-cognitive effects across a range of pre-clinical animal models and a favourable safety and toxicology profile HTL0009936 was progressed to first in human trials where the agonist activated central processes corresponding to learning and memory circuitry. Hence, our study not only describes the pharmacological profile of a M1 mAChR agonist with the potential to improve memory in Alzheimer’s disease but also demonstrates how to rationally progress the development of a G protein-coupled receptor ligand from structure to clinical candidate.

Hippocampal knockdown of α2 nicotinic or M1 muscarinic acetylcholine receptors in C57BL/6J male mice impairs cued fear conditioning.

Acetylcholine (ACh) signaling in the hippocampus is important for behaviors related to learning, memory and stress. In this study, we investigated the role of two ACh receptor subtypes previously shown to be involved in fear and anxiety, the M1 mAChR and the α2 nAChR, in mediating the effects of hippocampal ACh on stress-related behaviors. Adeno-associated viral vectors containing short-hairpin RNAs targeting M1 or α2 were infused into the hippocampus of male C57BL/6J mice, and behavior in a number of paradigms related to stress responses and fear learning was evaluated. There were no robust effects of hippocampal M1 mAChR or α2 nAChR knockdown (KD) in the light/dark box, tail suspension, forced swim or novelty-suppressed feeding tests. However, effects on fear learning were observed in both KD groups. Short term learning was intact immediately after training in all groups of mice, but both the M1 and α2 hippocampal knock down resulted in impaired cued fear conditioning 24 h after training. In addition, there was a trend for a deficit in contextual memory the M1 mAChR KD group 24 h after training. These results suggest that α2 nicotinic and M1 muscarinic ACh receptors in the hippocampus contribute to fear learning and could be relevant targets to modify brain circuits involved in stress-induced reactivity to associated cues.

Preparation of a First 18F-Labeled Agonist for M1 Muscarinic Acetylcholine Receptors.

M1 muscarinic acetylcholine receptors (mAChRs) are abundant in postsynaptic nerve terminals of all forebrain regions and have been implicated in the cognitive decline associated with Alzheimer’s disease and other CNS pathologies. Consequently, major efforts have been spent in the development of subtype-selective positron emission tomography (PET) tracers for mAChRs resulting in the development of several 11C-labeled probes. However, protocols for the preparation of 18F-labeled mAChR-ligands have not been published so far. Here, we describe a straightforward procedure for the preparation of an 18F-labeled M1 mAChR agonist and its corresponding pinacol boronate radiolabeling precursor and the non-radioactive reference compound. The target compounds were prepared from commercially available aryl fluorides and Boc protected 4-aminopiperidine using a convergent reaction protocol. The radiolabeling precursor was prepared by a modification of the Miyaura reaction and labeled via the alcohol-enhanced Cu-mediated radiofluorination. The developed procedure afforded the radiotracer in a non-decay-corrected radiochemical yield of 17 ± 3% (n = 3) and in excellent radiochemical purity (>99%) on a preparative scale. Taken together, we developed a straightforward protocol for the preparation of an 18F-labeled M1 mAChR agonist that is amenable for automation and thus provides an important step towards the routine production of a 18F-labeled M1 selective PET tracer for experimental and diagnostic applications.

Discovery and structure-activity relationships study of positive allosteric modulators of the M3 muscarinic acetylcholine receptor

The M3 muscarinic acetylcholine receptor (mAChR) is a member of the family of mAChRs, which are associated with a variety of physiological functions including the contraction of various smooth muscle tissues, stimulation of glandular secretion, and regulation of a range of cholinergic processes in the central nerve system. We report here the discovery and a comprehensive structure­-activity relationships (SARs) study of novel positive allosteric modulators (PAMs) of the M3 mAChR through a high throughput screening (HTS) campaign. Compound 9 exhibited potent in vitro PAM activity towards the M3 mAChR and significant enhancement of muscle contraction in a concentration-dependent manner when applied to isolated smooth muscle strips of rat bladder. Compound 9 also showed excellent subtype selectivity over other subtypes of mAChRs including M1, M2, and M4 mAChRs, and moderate selectivity over the M5 mAChR, indicating that compound 9 is an M3-preferring M3/M5 dual PAM. Moreover, compound 9 displayed acceptable pharmacokinetics profiles after oral dosing to rats. These results suggest that compound 9 may be a promising chemical probe for the M3 mAChR for further investigation of its pharmacological function both in vitro and in vivo.

The Effects of the M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator VU0486846 on Cognitive Performance in Aged Nonhuman Primates

It is estimated that in the United States alone there will be nearly 70 million people over the age of 65 by the year 2030. Yet, there remains a critical unmet need to understand and identify novel therapeutic strategies to address age‐related impairments in arousal, cognitive function, and sleep architecture. Agents such as acetylcholinesterase inhibitors (AChEIs) that enhance overall cholinergic neurotransmission are approved to ameliorate the loss of cognitive function in normal aging and Alzheimer’s disease (AD) populations. However, AChEIs are associated with dose‐limiting side effect due to nonselective activation of peripheral muscarinic acetylcholine receptors (mAChRs). Accumulating evidence indicates that enhancement of central cholinergic neurotransmission through selective activation of the M1 mAChR subtype represents an exciting alternative approach for the treatment of impairments in cognition, arousal and sleep observed in normal aging and AD. Recently we successfully optimized a novel series of M1 positive allosteric modulators (PAMs) as an alternative strategy for the selective activation of M1 receptors, represented by VU0486846. The goal of the present study was to provide the first characterization of the effects of an M1 PAM on cognitive performance in aged nonhuman primates. For these studies, 16 aged (> 20 years old) female and male (N=8/sex) cynomolgus monkeys were trained under two different cognitive tasks using CANTAB touchscreens. Eight monkeys (N=4/sex) were trained under a delayed match‐to‐sample (DMS) task using three delays and 2–3 distractors. The other monkeys were trained under an attention task, the multiple‐choice serial reaction time (MCSRT) task. All monkeys were tested with the following compounds, administered 60‐min before the session: donepezil (0.1–0.56 mg/kg, IM); VU0486846 (1.0–5.6 mg/kg, PO) and d‐amphetamine (0.01–0.056 mg/kg, IM). With no published data on training of aged monkeys in a DMS task, we found that the eight aged monkeys required approximately 1 year of training prior to pharmacologic challenge. The effects of donepezil were variable across monkeys, up to doses that produced adverse effects. In contrast, VU0486846 improved working memory, at doses that did not result in any side effects. As a positive control, d‐amphetamine also improved cognitive performance. In preliminary studies, testing ineffective doses of donepezil + VU0486846 also resulted in improved DMS performance. Regarding MCSRT, effects of both donepezil and VU0486846 were determined on a reaction time task. At doses that improved working memory, VU0486846 did not affect reaction time while in some but not all monkeys, donepezil slowed reaction times. These promising preliminary data will be extended in order to examine sex‐specific and age‐related effects of chronic VU0486846 and the consequences of the M1 PAM and donepezil on sleep architecture in aged nonhuman primates.Support or Funding InformationNIH grant AG054622

Acetylcholine Muscarinic M4 Receptors as a Therapeutic Target for Alcohol Use Disorder: Converging Evidence From Humans and Rodents

BackgroundAlcohol use disorder (AUD) is a major socioeconomic burden on society, and current pharmacotherapeutic treatment options are inadequate. Aberrant alcohol use and seeking alters frontostriatal function. MethodsWe performed genome-wide RNA sequencing and subsequent quantitative polymerase chain reaction and receptor binding validation in the caudate–putamen of human AUD samples to identify potential therapeutic targets. We then back-translated our top candidate targets into a rodent model of long-term alcohol consumption to assess concordance of molecular adaptations in the rat striatum. Finally, we adopted rat behavioral models of alcohol intake and seeking to validate a potential therapeutic target. ResultsWe found that G protein–coupled receptors were the top canonical pathway differentially regulated in individuals with AUD. The M4 muscarinic acetylcholine receptor (mAChR) was downregulated at the gene and protein levels in the putamen, but not in the caudate, of AUD samples. We found concordant downregulation of the M4 mAChR, specifically on dopamine D1 receptor–expressing medium spiny neurons in the rat dorsolateral striatum. Systemic administration of the selective M4 mAChR positive allosteric modulator, VU0467154, reduced home cage and operant alcohol self-administration, motivation to obtain alcohol, and cue-induced reinstatement of alcohol seeking in rats. Local microinjections of VU0467154 in the rat dorsolateral striatum reduced alcohol self-administration and cue-induced reinstatement of alcohol seeking. ConclusionsCollectively, these results identify the M4 mAChR as a potential therapeutic target for the treatment of AUD and the D1 receptor–positive medium spiny neurons in the dorsolateral striatum as a key site mediating the actions of M4 mAChR in relation to alcohol consumption and seeking.

Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs.

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.

Tacrine-xanomeline and tacrine-iperoxo hybrid ligands: Synthesis and biological evaluation at acetylcholinesterase and M1 muscarinic acetylcholine receptors.

We synthesized a set of new hybrid derivatives (7-C8, 7-C10, 7-C12 and 8-C8, 8-C10, 8-C12), in which a polymethylene spacer chain of variable length connected the pharmacophoric moiety of xanomeline, an M1/M4-preferring orthosteric muscarinic agonist, with that of tacrine, a well-known acetylcholinesterase (AChE) inhibitor able to allosterically modulate muscarinic acetylcholine receptors (mAChRs). When tested in vitro in a colorimetric assay for their ability to inhibit AChE, the new compounds showed higher or similar potency compared to that of tacrine. Docking analyses were performed on the most potent inhibitors in the series (8-C8, 8-C10, 8-C12) to rationalize their experimental inhibitory power against AChE. Next, we evaluated the signaling cascade at M1 mAChRs by exploring the interaction of Gαq-PLC-β3 proteins through split luciferase assays and the myo-Inositol 1 phosphate (IP1) accumulation in cells. The results were compared with those obtained on the known derivatives 6-C7 and 6-C10, two quite potent AChE inhibitors in which tacrine is linked to iperoxo, an exceptionally potent muscarinic orthosteric activator. Interestingly, we found that 6-C7 and 6-C10 behaved as partial agonists of the M1 mAChR, at variance with hybrids 7-Cn and 8-Cn containing xanomeline as the orthosteric molecular fragment, which were all unable to activate the receptor subtype response.

Allosteric modulation of muscarinic receptors in alcohol and substance use disorders.

Alcohol and substance use disorders are chronic relapsing disorders, characterized by a compulsion to take drugs and loss of control in limiting intake. The impact of alcohol and substance abuse on morbidity and mortality is very high worldwide, and treatment options are inadequate at a population level. There is a need for the discovery and development of novel pharmacotherapies to treat alcohol and substance use disorders. The recent development of subtype selective muscarinic acetylcholine receptor (mAChR) allosteric modulators, is rapidly increasing our understanding of this system and revealing great promise for disease treatment. In this chapter, we will present and integrate the current literature regarding the role of mAChRs in drug and alcohol abuse with emerging evidence using novel allosteric modulators targeting specific mAChR subtypes. We will focus on the potential of M1, M4 and M5 mAChRs in the treatment of alcohol, psychostimulant and opioid dependence, using evidence from preclinical studies to predict their utility in treating different stages of the addiction cycle. Overall, preclinical evidence suggests allosteric modulation of mAChRs may provide promise as potential treatment options for alcohol and substance abuse in reducing consumption, facilitating abstinence and preventing relapse.

M1 muscarinic acetylcholine receptors: A therapeutic strategy for symptomatic and disease-modifying effects in Alzheimer's disease?

The M1 muscarinic acetylcholine receptor (mAChR) plays a crucial role in learning and memory processes and has long been identified as a promising therapeutic target for the improvement of cognitive decline in Alzheimer's disease (AD). As such, clinical trials with xanomeline, a mAChR orthosteric agonist, showed an improvement in cognitive and behavioral symptoms associated with AD. Despite this, the clinical utility of xanomeline was hampered by a lack of M1 receptor selectivity and adverse cholinergic responses attributed to activation of peripheral M2 and M3 mAChRs. More recently, efforts have focused on developing more selective M1 compounds via targeting the less-conserved allosteric binding pockets. As such, positive allosteric modulators (PAMs) have emerged as an exciting strategy to achieve exquisite selectivity for the M1 mAChR in order to deliver improvements in cognitive function in AD. Furthermore, over recent years it has become increasingly apparent that M1 therapeutics may also offer disease-modifying effects in AD, due to the modulation of pathogenic amyloid processing. This article will review the progress made in the development of M1 selective ligands for the treatment of cognitive decline in AD, and will discuss the current evidence that selective targeting of the M1 mAChR could also have the potential to modify AD progression.

Functional coupling of M1 muscarinic acetylcholine receptor to Gαq/11 in dorsolateral prefrontal cortex from patients with psychiatric disorders: a postmortem study.

Accumulating studies have implicated intracellular signaling through muscarinic acetylcholine receptors (mAChRs) in psychiatric illness. In the present study, carbamylcholine chloride (carbachol)-induced Gαi/o and Gαq/11 activation was identified in postmortem human prefrontal cortical membranes. The following two sample cohorts were used: subjects [1], consisting of 40 controls without neuropsychiatric disorders, and subjects [2], consisting of 20 with bipolar disorder (BP), 20 major depressive disorder (MDD), 20 schizophrenia, and 20 controls, strictly sex- and age-matched. Carbachol-stimulated [35S]GTPγS binding to human brain membranes was assessed by the two methods, i.e., conventional method using filtration techniques (Gαi/o activation coupled to M2/M4 mAChRs) applied to subjects [1], and [35S]GTPγS binding/immuno precipitation assay (Gαq/11 activation coupled to M1 mAChR) applied to subjects [1] and [2]. The concentration eliciting the half-maximal effect (EC50), maximum percent increase (%Emax), and slope factor were obtained from concentration-response curve of carbachol-induced Gαi/o and Gαq/11 activation. The pEC50 values of both carbachol-induced Gαi/o and Gαq/11 activations in subjects [1] were significantly correlated, though its implications or underlying molecular processes are unclear. The results of M1 mAChR-mediated Gαq/11 activation in subjects [2] indicated no significant disorder-specific alterations. However, the distribution patterns of the pEC50 values showed unequal variances among the groups. There was a significant inverse correlation between the %Emax values and the pEC50 values in subjects with schizophrenia, but not in those with BP or MDD, or controls. These data support the notion that schizophrenia patients consist of biologically heterogeneous subgroups with respect to M1 mAChR-mediated signaling pathways.

Crystal structure of the M5 muscarinic acetylcholine receptor

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2 and M5 mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

PKC and Ras are Involved in M1 Muscarinic Receptor-Mediated Modulation of AMPA Receptor GluA1 Subunit.

M1 muscarinic acetylcholine receptors (M1 mAChRs) have long been an attractive target for the treatment of Alzheimer's disease (AD), the most common cause of dementia in the elderly. M1 mAChR agonists show desirably preclinical activities; however, most have not gone further into late clinical trials due to ineffectiveness or side effects. Thus, to understand the signaling pathways involved in M1 mAChR-mediated memory improvement may be important for design of biased agonists with on-target therapeutic effects. M1 mAChRs are classically coupled to Gαq or ectopically to Gαs to activate multiple kinases such as protein kinase C (PKC), Ras and protein kinase A (PKA). Our previous studies have found that M1 mAChRs could improve learning and memory through modulating AMPA receptor GluA1 subunit via PKA-PI3K-Akt signaling. Here, we further investigated whether PKC and Ras were involved in M1 mAChR-mediated modulation of GluA1. We demonstrated the role of PKC and Ras in the signaling pathway, as both PKC inhibitors Ro-31-8425 or Gö6983 and Ras inhibitor salirasib abolished the membrane insertion of GluA1 and enhancement of its phosphorylation at Ser845 induced by M1 mAChRs in the primary cultured neurons and hippocampus in vivo. We further showed that PKC and Ras modulated PKA-PI3K-Akt signaling since the increases of PKA, Akt and mTOR activities by M1 mAChR activation were blocked by PKC and Ras inhibitors. These data demonstrated the detailed mechanism underlying M1 mAChR-mediated modulation of GluA1 through Gαq/11 coupling, broadening the knowledge of the downstream signaling after M1 mAChR-Gαq/11 coupling.

Antinociceptive Effect of Spirocyclopiperazinium Salt Compound DXL-A-24 and the Underlying Mechanism.

The antinociceptive effects of spirocyclopiperazinium salt compound DXL-A-24 on neuropathic pain and chemical-stimulated pain were investigated in this study. After the administration of DXL-A-24, the paw withdrawal latency (PWL) and mechanical withdrawal threshold (MWT) were increased in rats suffering from neuropathic pain (chronic constriction injury, CCI) on days 1, 3, 5, 7 and 14 after surgery, and pain responses were inhibited in mice stimulated with chemicals (formalin or acetic acid). In the analysis of antinociceptive targets, the effect of DXL-A-24 was blocked by a peripheral nicotinic acetylcholine receptor (nAChR) antagonist (hexamethonium, Hex) or α7 nAChR antagonist (methyllycaconitine, MLA) in the formalin test. Meanwhile, the effect of DXL-A-24 was also blocked by a peripheral muscarinic acetylcholine receptor (mAChR) antagonist (atropine methylnitrate, Amn) or M4 mAChR antagonist (tropicamide, TRO). The antinociceptive signalling pathway was explored using molecular biology methods in ipsilateral dorsal root ganglions (DRGs) of CCI rats after the administration of DXL-A-24 for 7days. Western blot analyses showed that the increased levels of phosphorylation of calcium/calmodulin-dependent protein kinaseII alpha (CaMKIIα) and cAMP response element-binding protein (CREB) were eliminated, and the qRT-PCR assay showed that the increase in the expression of Tumor necrosis factor alpha (TNF-α) mRNA was reduced. Meanwhile, immunofluorescence staining revealed that the increase in calcitonin gene related peptide (CGRP) expression was inhibited by the administration of DXL-A-24, and the effect was blocked by MLA or TRO. In conclusion, DXL-A-24 exerts significant antinociceptive effects on neuropathic pain and chemical-stimulated pain. The antinociceptive effect of DXL-A-24 is probably attributed to the activation of peripheral α7 nAChR and M4 mAChR, the subsequent inhibition of the CaMKIIα/CREB signalling pathway, and finally the inhibition of TNF-α and CGRP expression.