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Key Points Molecular-cloning studies have revealed the existence of five molecularly distinct mammalian muscarinic acetylcholine receptors (mAChRs), M 1 –M 5 , which are all members of the superfamily of class I (rhodopsin-like) G-protein-coupled receptors (GPCRs). Each of the five mAChR subtypes exhibits a distinct pattern of distribution and G-protein coupling/signalling profile. The mAChRs regulate many important central and peripheral functions including cognitive, behavioural, sensory, motor and autonomic processes, and the well-known actions of acetylcholine (ACh) on effector tissues innervated by parasympathetic nerves. Interestingly, changes in mAChR function have been implicated in several important pathophysiological disorders including Alzheimer's disease, Parkinson's disease, depression, schizophrenia, urinary incontinence and chronic obstructive pulmonary disease. Primarily owing to the lack of muscarinic ligands that have a high degree of selectivity for the individual mAChR subtypes, it is unclear in many cases which specific mAChR subtypes are involved in mediating the various muscarinic actions of ACh. However, such knowledge is essential for the rational design of novel muscarinic drugs with increased efficacy and reduced side effects. However, recent studies using mutant mouse strains deficient in each of the five mAChR subtypes have led to a wealth of novel information regarding the physiological and potential pathophysiological roles of the individual receptor subtypes. Importantly, such studies have identified specific mAChR-regulated pathways as potentially novel targets for the treatment of various important pathophysiological conditions including Alzheimer's disease, Parkinson's disease, schizophrenia and type 2 diabetes. Consequently, a major challenge remains in the development of small-molecule ligands exhibiting a high degree of selectivity for the individual mAChR subtypes. Such agents will probably include compounds that act on secondary (allosteric) sites that, in contrast to the classical (orthosteric) muscarinic binding site, are less well-conserved among the different mAChR subtypes. Altered muscarinic acetylcholine receptor (mAChR) activity is implicated in many disorders including Alzheimer's disease, schizophrenia, pain, obesity and diabetes, but lack of knowledge of the precise functional roles of the individual mAChR subtypes has hampered drug development. Wess and colleagues review recent insights gained from mAChR -knockout mice that are helping to address this issue and discuss the therapeutic potential of subtype-selective mAChR modulation. Muscarinic acetylcholine receptors (mAChRs), M 1 –M 5 , regulate the activity of numerous fundamental central and peripheral functions. The lack of small-molecule ligands that can block or activate specific mAChR subtypes with high selectivity has remained a major obstacle in defining the roles of the individual receptor subtypes and in the development of novel muscarinic drugs. Recently, phenotypic analysis of mutant mouse strains deficient in each of the five mAChR subtypes has led to a wealth of new information regarding the physiological roles of the individual receptor subtypes. Importantly, these studies have identified specific mAChR-regulated pathways as potentially novel targets for the treatment of various important disorders including Alzheimer's disease, schizophrenia, pain, obesity and diabetes.

Centrally acting muscarinic acetylcholine receptor antagonists like atropine and scopolamine can induce psychosis-like symptoms. Xanomeline, a muscarinic M1/M4 preferring agonist attenuated the effects of amphetamine (animal model for schizophrenia) in the wild-type mice, however, such effects were absent in muscarinic M4 knockout mice. In addition, xanomeline was also found to be effective in attenuating neuropsychiatric symptoms. Thus, muscarinic M4 agents can modulate brain circuitry that is dysregulated in disease states. The effects of SUVN-L3307032 on the allosteric site of the M4 receptor were evaluated using the cell-based reporter gene assay. The pharmacokinetic properties of SUVN-L3307032 were studied both in rodent and non-rodent species. SUVN-L3307032 was assessed for its effects on amphetamine-induced hyperlocomotion in rats at doses of 3, 10, 30, and 60 mg/kg. Preliminary toxicity studies were conducted in rats and dogs to evaluate the safety of SUVN-L3307032. SUVN-L3307032 was found to be a positive allosteric modulator of muscarinic M4 (M4-PAM) receptor. SUVN-L3307032 showed good oral bioavailability in rats and dogs. It also has excellent brain penetration with an adequate free fraction in the brain. SUVN-L3307032 showed dose-dependent receptor occupancy at tested doses of 3, 10, 30, and 60 mg/kg. At the tested doses, SUVN-L3307032 dose-dependently attenuated amphetamine-induced hyperlocomotion. The observation from the amphetamine-induced hyperlocomotion assay correlated well with the occupancy at the allosteric site of muscarinic M4 receptors. Preliminary toxicity studies did not show any concerns for further development. SUVN-L3307032 is being further characterized in animal models for neuropsychiatric symptoms. SUVN-L3307032 could be a promising agent for the management of neuropsychiatric symptoms.

Over the past 40 years, the muscarinic acetylcholine receptor family, particularly the M1-receptor and M4-receptor subtypes, have emerged as validated targets for the symptomatic treatment of neurological diseases such as schizophrenia and Alzheimer disease. However, despite considerable effort and investment, no drugs have yet gained clinical approval. This is largely attributable to cholinergic adverse effects that have halted the majority of programmes and resulted in a waning of interest in these G-protein-coupled receptor targets. Recently, this trend has been reversed. Driven by advances in structure-based drug design and an appreciation of the optimal pharmacological properties necessary to deliver clinical efficacy while minimizing adverse effects, a new generation of M1-receptor and M4-receptor orthosteric agonists and positive allosteric modulators are now entering the clinic. These agents offer the prospect of novel therapeutic solutions for ‘hard to treat’ neurological diseases, heralding a new era of muscarinic drug discovery.The M1 and M4 muscarinic acetylcholine receptors represent promising therapeutic targets for Alzheimer disease and schizophrenia. However, the development of agents targeting these receptors has been limited by their adverse cholinergic effects. Here, Tobin discusses how recent advances in the field — including an increased understanding of receptor biology and signalling, as well as the application of structure-based drug design — are enabling a new generation of muscarinic receptor modulators to enter clinical development.

Cholinergic drugs acting at M1/M4 muscarinic receptors hold promise for the treatment of symptoms associated with brain disorders characterized by cognitive impairment, mood disturbances, or psychosis, such as Alzheimer’s disease or schizophrenia. However, the brain-wide functional substrates engaged by muscarinic agonists remain poorly understood. Here we used a combination of pharmacological fMRI (phMRI), resting-state fMRI (rsfMRI), and resting-state quantitative EEG (qEEG) to investigate the effects of a behaviorally active dose of the M1/M4-preferring muscarinic agonist xanomeline on brain functional activity in the rodent brain. We investigated both the effects of xanomeline per se and its modulatory effects on signals elicited by the NMDA-receptor antagonists phencyclidine (PCP) and ketamine. We found that xanomeline induces robust and widespread BOLD signal phMRI amplitude increases and decreased high-frequency qEEG spectral activity. rsfMRI mapping in the mouse revealed that xanomeline robustly decreased neocortical and striatal connectivity but induces focal increases in functional connectivity within the nucleus accumbens and basal forebrain. Notably, xanomeline pre-administration robustly attenuated both the cortico-limbic phMRI response and the fronto-hippocampal hyper-connectivity induced by PCP, enhanced PCP-modulated functional connectivity locally within the nucleus accumbens and basal forebrain, and reversed the gamma and high-frequency qEEG power increases induced by ketamine. Collectively, these results show that xanomeline robustly induces both cholinergic-like neocortical activation and desynchronization of functional networks in the mammalian brain. These effects could serve as a translatable biomarker for future clinical investigations of muscarinic agents, and bear mechanistic relevance for the putative therapeutic effect of these class of compounds in brain disorders.

Cholinergic treatments with an emphasis on M1 muscarinic acetylcholine receptor (mAChR) agonists as potential modulating agents are a new approach in Alzheimer’s disease (AD) therapy. In previous research, we designed and characterized novel thiazolidine-2,4-dione (TZD)-derived compounds that possess anti-AD properties and enhance the expression of mAChRM1 in rats. This study evaluated a novel orthosteric agonist of mAChRM1 from related pathways that has shown promising anti-Alzheimer’s disease activity. PC12 cells were exposed to various concentrations of TZ4M before they were exposed to scopolamine (3 µM). Immunocytochemistry and western blot analyses revealed that TZ4M increased the expression of mAChRM1 in differentiated cells induced by scopolamine-treated PC12 cells. The results showed that TZ4M (3 and 5 µM) markedly upregulated PKC and ChAT protein expression, and the cells were significantly protected against increased ROS levels followed by neuronal cell loss, as evidenced by the MTT assay. TUNEL staining indicated that TZ4M impeded the shaping of apoptotic bodies. Analysis of the amino acid sequences of the ligand–protein binding site indicated that TZ4M is bound to the orthosteric site (acetylcholine site). This study revealed that TZ4M, a derivative of TZD, effectively protects against scopolamine-induced damage. TZ4M, a novel mACRM1 orthosteric agonist, is promising for treating AD.

Habitual chewing of \"betel nut\" preparations constitutes the fourth most common human self-administration of a psychoactive substance after alcohol, caffeine, and nicotine. The primary active ingredient in these preparations is arecoline, which comes from the areca nut, the key component of all such preparations. Arecoline is known to be a relatively non-selective muscarinic partial agonist, accounting for many of the overt peripheral and central nervous system effects, but not likely to account for the addictive properties of the drug. We report that arecoline has activity on select nicotinic acetylcholine receptor (nAChR) subtypes, including the two classes of nAChR most related to the addictive properties of nicotine: receptors containing α4 and β2 subunits and those which also contain α6 and β3 subunits. Arecoline is a partial agonist with about 6-10% efficacy for the α4* and α6* receptors expressed in Xenopus oocytes. Additionally, arecoline is a silent agonist of α7 nAChR; while it does not activate α7 receptors when applied alone, it produces substantial activation when co-applied with the positive allosteric modulator PNU-120696. Some α7 silent agonists are effective inhibitors of inflammation, which might account for anti-inflammatory effects of arecoline. Arecoline's activity on nAChR associated with addiction may account for the habitual use of areca nut preparations in spite of the well-documented risk to personal health associated with oral diseases and cancer. The common link between betel and tobacco suggests that partial agonist therapies with cytisine or the related compound varenicline may also be used to aid betel cessation attempts.

Stimulating muscarinic M /M receptors can blunt reinforcing and other effects of cocaine. A hallmark of addiction is continued drug seeking/craving after abstinence and relapse. We tested whether stimulating M and/or M receptors could facilitate extinction of cocaine seeking, and whether this was mediated via memory consolidation. Experimentally naïve C57BL/6J mice were allowed to acquire self-administration of intravenous cocaine (1 mg/kg/infusion) under a fixed-ratio 1 schedule of reinforcement. Then, saline was substituted for cocaine until responding extinguished to ≤30% of cocaine-reinforced responding. Immediately after each extinction session, mice received saline, the M /M receptor-preferring agonist xanomeline, the M receptor-selective allosteric agonist VU0357017, the M receptor-selective positive allosteric modulator VU0152100, or VU0357017 + VU0152100. In additional experiments, xanomeline was administered delayed after the session or in the home cage before extinction training began. In the latter group, reinstatement of responding by a 10-mg/kg cocaine injection was also tested. Stimulating M  + M receptors significantly expedited extinction from 17.2 sessions to 8.3 using xanomeline or 7.8 using VU0357017 + VU0152100. VU0357017 alone and VU0152100 alone did not significantly modify rates of extinction (12.6 and 14.6 sessions). The effect of xanomeline was fully preserved when administered delayed after or unpaired from extinction sessions (7.5 and 6.4 sessions). Xanomeline-treated mice showed no cocaine-induced reinstatement. These findings show that M /M receptor stimulation can decrease cocaine seeking in mice. The effect lasted beyond treatment duration and was not dependent upon extinction learning. This suggests that M /M receptor stimulation modulated or reversed some neurochemical effects of cocaine exposure.

Despite great advances in our understanding of the pathobiology of colorectal cancer and the genetic and environmental factors that mitigate its onset and progression, a paucity of effective treatments persists. The five-year survival for advanced, stage IV disease remains substantially less than 20%. This review examines a relatively untapped reservoir of potential therapies to target muscarinic receptor expression, activation, and signaling in colorectal cancer. Most colorectal cancers overexpress M3 muscarinic receptors (M3R), and both in vitro and in vivo studies have shown that activating these receptors stimulates cellular programs that result in colon cancer growth, survival, and spread. In vivo studies using mouse models of intestinal neoplasia have shown that using either genetic or pharmacological approaches to block M3R expression and activation, respectively, attenuates the development and progression of colon cancer. Moreover, both in vitro and in vivo studies have shown that blocking the activity of matrix metalloproteinases (MMPs) that are induced selectively by M3R activation, i.e., MMP1 and MMP7, also impedes colon cancer growth and progression. Nonetheless, the widespread expression of muscarinic receptors and MMPs and their importance for many cellular functions raises important concerns about off-target effects and the safety of employing similar strategies in humans. As we highlight in this review, highly selective approaches can overcome these obstacles and permit clinicians to exploit the reliance of colon cancer cells on muscarinic receptors and their downstream signal transduction pathways for therapeutic purposes.

Pilocarpine is a selective M 1 /M 3 agonist of muscarinic acetylcholine receptor subtypes. Muscarinic acetylcholine receptors are G protein-coupled receptors. These receptors are different drug targets. The aim of the present work was to investigate the effect of pilocarpine on the expression of M 3 muscarinic acetylcholine receptor, the AChE activity, IL-8 release response, and proliferation in K562 cells, via muscarinic receptor activation. Human chronic myeloid leukemic cell cultures were incubated with drugs. Proliferation assays were performed by BrdU assay. Expression of M 3 muscarinic acetylcholine receptor and apoptosis proteins such as bcl, bax, cyt C, and caspases was assessed with the semiquantitative Western blotting method. Pilocarpine inhibits chronic myeloid cell proliferation and M 3 muscarinic acetylcholine receptor protein expression. Pilocarpine increases caspase-8 and -9 expression levels, upregulating the proapoptotic protein Bax and downregulating the expression levels of the antiapoptotic protein Bcl-2. The apoptotic activity of pilocarpine is associated with an increase in AChE activity. M 3 muscarinic acetylcholine receptors can activate multiple signal transduction systems and mediate inhibitory effects on chronic myeloid K562 cell proliferation depending on the presence of 1% FBS conditions. This apoptotic effect of pilocarpine may be due to the concentration of pilocarpine and the increase in AChE level. Our results suggest that inhibition of cell proliferation by inducing apoptosis of pilocarpine in K562 cells may be one of the targets. M 3 selective agonist may have therapeutic potential in chronic myeloid leukemia. Graphical Abstract

IL-17 is associated with asthma, and T H H17 cells are found in the airways of individuals with asthma. Dean Sheppard and his colleagues now report that IL-17A (but not IL-17F) directly enhances contractile responses in airway smooth muscle cells. Mice lacking T H 17 cells in the lungs exhibit reduced airway hyper-responsiveness in response to allergen challenge. Emerging evidence suggests that the T helper 17 (T H 17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate T H 17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T H 17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T H 17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)–IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)–ras homolog gene family, member A (RhoA)–Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T H 17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.