M1 mAChR Research Articles

Acetylcholine receptors (muscarinic) in GtoPdb v.2021.3

Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates.

Identification of a Novel Allosteric Site at the M5 Muscarinic Acetylcholine Receptor.

The M5 muscarinic acetylcholine receptor (mAChR) has emerged as an exciting therapeutic target for the treatment of addiction and behavioral disorders. This has been in part due to promising preclinical studies with the M5 mAChR selective negative allosteric modulator (NAM), ML375. The binding site of ML375 remains unknown, however, making it difficult to develop improved M5 mAChR selective modulators. To determine the possible location of the ML375 binding site, we used radioligand binding and functional assays to show that ML375 does not interact with the well-characterized "common" mAChR allosteric site located in the receptor's extracellular vestibule, nor a previously proposed second allosteric site recognized by the modulator, amiodarone. Molecular docking was used to predict potential allosteric sites within the transmembrane (TM) domain of the M5 mAChR. These predicted sites were assessed using M5-M2 mAChR receptor chimeras and further targeted with site-directed mutagenesis, which enabled the identification of a putative binding site for ML375 at the interface of TMs 2-4. Collectively, these results identify a third allosteric site at the M5 mAChR and highlight the ability of allosteric modulators to selectively target highly conserved proteins.

Discovery of VU6028418: A Highly Selective and Orally Bioavailable M4 Muscarinic Acetylcholine Receptor Antagonist.

Herein, we report the SAR leading to the discovery of VU6028418, a potent M4 mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.

Biperiden Challenge Model in Healthy Elderly as Proof-of-Pharmacology Tool: A Randomized, Placebo-Controlled Trial.

Selective M1 muscarinic acetylcholine receptor (mAChR) agonists are being developed as symptomatic treatment for neurodegenerative and neuropsychiatric disorders that lead to cognitive dysfunction. Demonstrating cognition‐enhancing effects in early‐phase clinical development in healthy subjects is difficult. A challenge with the M1 mAChR antagonist biperiden could be used to demonstrate procognitive and pharmacological effects of selective M1 mAChR agonists. The aim of this study was to develop such a model. To this end, 12 healthy elderly subjects participated in a randomized, placebo‐controlled, 3‐way crossover study investigating tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) effects of 2 and 4 mg biperiden. Repeated PD assessments were performed using neurocognitive tasks and electrophysiological measurements. A population PK‐PD model was developed. Four milligrams of biperiden showed significant impairment of sustained attention (−2.1 percentage point in adaptive tracking [95%CI, −3.043 to −1.148], verbal memory (2‐3 fewer words recalled [95%CI, −5.9 to −0.2]) and working memory (up to a 50‐millisecond increase in the n‐back task reaction time [95%CI, 21.854‐77.882]) compared with placebo. The PK data were best fitted by a 2‐compartment model and showed high interoccasion and intersubject variability. Population PK‐PD analysis quantified significant concentration‐effect relationships for the n‐back reaction time, n‐back accuracy, and adaptive tracking. In conclusion, biperiden caused M1 mAChR‐related dose‐ and concentration‐dependent temporary declines in cognitive functioning. Therefore a biperiden pharmacological challenge model can be used for proof‐of‐pharmacology studies and to demonstrate cognition‐enhancing effects of new cholinergic compounds that are being developed.

Acetylcholine receptors (muscarinic) in GtoPdb v.2021.2

Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates. I

M1 and M2 mAChRs activate PDK1 and regulate PKC βI and ε and the exocytotic apparatus at the NMJ.

Neuromuscular junctions (NMJ) regulate cholinergic exocytosis through the M1 and M2 muscarinic acetylcholine autoreceptors (mAChR), involving the crosstalk between receptors and downstream pathways. Protein kinase C (PKC) regulates neurotransmission but how it associates with the mAChRs remains unknown. Here, we investigate whether mAChRs recruit the classical PKCβI and the novel PKCε isoforms and modulate their priming by PDK1, translocation and activity on neurosecretion targets. We show that each M1 and M2 mAChR activates the master kinase PDK1 and promotes a particular priming of the presynaptic PKCβI and ε isoforms. M1 recruits both primed-PKCs to the membrane and promotes Munc18-1, SNAP-25, and MARCKS phosphorylation. In contrast, M2 downregulates PKCε through a PKA-dependent pathway, which inhibits Munc18-1 synthesis and PKC phosphorylation. In summary, our results discover a co-dependent balance between muscarinic autoreceptors which orchestrates the presynaptic PKC and their action on ACh release SNARE-SM mechanism. Altogether, this molecular signaling explains previous functional studies at the NMJ and guide toward potential therapeutic targets.

Discovery of a novel class of heteroaryl-pyrrolidinones as positive allosteric modulators of the muscarinic acetylcholine receptor M1

This Letter describes the synthesis and optimization of a series of heteroaryl-pyrrolidinone positive allosteric modulators (PAMs) of the muscarinic acetylcholine receptor M1 (mAChR M1). Through the continued optimization of M1 PAM tool compound VU0453595, with a focus on replacement of the 6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one with a wide variety of alternative 4,5-dihydropyrrolo-fused heteroaromatics, the generation of M1 PAMs with structurally novel chemotypes is disclosed. Two compounds from these subseries, 8b (VU6005610) and 20a (VU6005852), show robust selectivity for the M1 mAChR, and no M1 agonism. Both compounds have favorable preliminary PK profiles in vitro;8b additionally demonstrates high brain exposure in a rodent IV cassette model.

Metformin Improves Autonomic Nervous System Imbalance and Metabolic Dysfunction in Monosodium L-Glutamate-Treated Rats.

Metformin is an antidiabetic drug used for the treatment of diabetes and metabolic diseases. Imbalance in the autonomic nervous system (ANS) is associated with metabolic diseases. This study aimed to test whether metformin could improve ANS function in obese rats. Obesity was induced by neonatal treatment with monosodium L-glutamate (MSG). During 21–100 days of age, MSG-rats were treated with metformin 250 mg/kg body weight/day or saline solution. Rats were euthanized to evaluate biometric and biochemical parameters. ANS electrical activity was recorded and analyzed. Metformin normalized the hypervagal response in MSG-rats. Glucose-stimulated insulin secretion in isolated pancreatic islets increased in MSG-rats, while the cholinergic response decreased. Metformin treatment normalized the cholinergic response, which involved mostly the M3 muscarinic acetylcholine receptor (M3 mAChR) in pancreatic beta-cells. Protein expression of M3 mAChRs increased in MSG-obesity rats, while metformin treatment decreased the protein expression by 25%. In conclusion, chronic metformin treatment was effective in normalizing ANS activity and alleviating obesity in MSG-rats.

5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated activation of Gαq/11 in postmortem dorsolateral prefrontal cortex of opiate addicts.

Chronic exposure to opiates causes the development of tolerance and physical dependence as well as persistent brain neuroplasticity. Despite a wealth of postmortem human studies for opiate addicts, little direct information regarding the functional status of serotonergic and cholinergic receptor-mediated signaling pathways in the human brain of opiate addicts is yet available. Functional activation of Gαq/11 proteins coupled to 5-HT2A and M1 type muscarinic acetylcholine receptor (mAChR) was assessed by using the method named [35S]GTPγS binding/immunoprecipitation in frontal cortical membrane preparations from postmortem human brains obtained from opiate addicts and matched controls. Concentration-response curves for 5-HT and carbachol in individual subjects were analyzed according to a nonlinear regression model, which generated the values of maximum percent increase (%Emax), negative logarithm of the half-maximal effect (pEC50) and slope factor. As for 5-HT2A receptor-mediated Gαq/11 activation, the %Emax values were reduced significantly and the pEC50 values were decreased significantly in opiate addicts as compared to the control group. Regarding carbachol-induced Gαq/11 activation, no significant difference in %Emax or pEC50 values was detected between the both groups, whereas the slope factor was increased significantly in opiate addicts as compared to the control group. Our data demonstrate that the signaling pathways mediated by Gαq/11 proteins coupled with 5-HT2A receptors and M1 mAChRs in prefrontal cortex are functionally altered in opiate addicts in comparison with control subjects. These alterations may underpin some aspects of addictive behavior to opiate as well as neuropsychological consequences or comorbid mental disorders associated with opioid use.

Pharmaceutical Assessment Suggests Locomotion Hyperactivity in Zebrafish Triggered by Arecoline Might Be Associated with Multiple Muscarinic Acetylcholine Receptors Activation.

Arecoline is one of the nicotinic acid-based alkaloids, which is found in the betel nut. In addition to its function as a muscarinic agonist, arecoline exhibits several adverse effects, such as inducing growth retardation and causing developmental defects in animal embryos, including zebrafish, chicken, and mice. In this study, we aimed to study the potential adverse effects of waterborne arecoline exposure on zebrafish larvae locomotor activity and investigate the possible mechanism of the arecoline effects in zebrafish behavior. The zebrafish behavior analysis, together with molecular docking and the antagonist co-exposure experiment using muscarinic acetylcholine receptor antagonists were conducted. Zebrafish larvae aged 96 h post-fertilization (hpf) were exposed to different concentrations (0.001, 0.01, 0.1, and 1 ppm) of arecoline for 30 min and 24 h, respectively, to find out the effect of arecoline in different time exposures. Locomotor activities were measured and quantified at 120 hpf. The results showed that arecoline caused zebrafish larvae locomotor hyperactivities, even at a very low concentration. For the mechanistic study, we conducted a structure-based molecular docking simulation and antagonist co-exposure experiment to explore the potential interactions between arecoline and eight subtypes, namely, M1a, M2a, M2b, M3a, M3b, M4a, M5a, and M5b, of zebrafish endogenous muscarinic acetylcholine receptors (mAChRs). Arecoline was predicted to show a strong binding affinity to most of the subtypes. We also discovered that the locomotion hyperactivity phenotypes triggered by arecoline could be rescued by co-incubating it with M1 to M4 mAChR antagonists. Taken together, by a pharmacological approach, we demonstrated that arecoline functions as a highly potent hyperactivity-stimulating compound in zebrafish that is mediated by multiple muscarinic acetylcholine receptors.

Design, Synthesis, and Structure-Activity Relationships Study of N-Pyrimidyl/Pyridyl-2-thiazolamine Analogues as Novel Positive Allosteric Modulators of M3 Muscarinic Acetylcholine Receptor.

The M3 muscarinic acetylcholine receptor (mAChR) plays an essential pharmacological role in mediating a broad range of actions of acetylcholine (ACh) released throughout the periphery and central nerve system (CNS). Nevertheless, its agonistic functions remain unclear due to the lack of available subtype-selective agonists or positive allosteric modulators (PAMs). In the course of our extended structure-activity relationships (SARs) study on 2-acylaminothiazole derivative 1, a previously reported PAM of the M3 mAChR, we successfully identified N-pyrimidyl/pyridyl-2-thiazolamine analogues as new scaffolds. The SARs study was rationalized using conformational analyses based on intramolecular interactions. A comprehensive study of a series of analogues described in this paper suggests that a unique sulfur-nitrogen nonbonding interaction in the N-pyrimidyl/pyridyl-2-thiazolamine moiety enable conformations that are essential for activity. Further, a SARs study around the N-pyrimidyl/pyridyl-2-thiazolamine core culminated in the discovery of compound 3g, which showed potent in vitro PAM activity for the M3 mAChR with excellent subtype selectivity. Compound 3g also showed a distinct pharmacological effect on isolated smooth muscle tissue from rat bladder and favorable pharmacokinetics profiles, suggesting its potential as a chemical tool for probing the M3 mAChR in further research.

Calcium-Dependent Regulation of the Neuronal Glycine Transporter GlyT2 by M2 Muscarinic Acetylcholine Receptors.

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission and plays a key role in regulating nociceptive signal progression. The cholinergic system acting through muscarinic acetylcholine receptors (mAChRs) also mediates important regulations of nociceptive transmission being the M2 subtype the most abundantly expressed in the spinal cord. Here we studied the effect of M2 mAChRs stimulation on GlyT2 function co-expressed in a heterologous system with negligible levels of muscarinic receptor activity. We found GlyT2 is down-regulated by carbachol in a calcium-dependent manner. Different components involved in cell calcium homeostasis were analysed to establish a role in the mechanism of GlyT2 inhibition. GlyT2 down-regulation by carbachol was increased by thapsigargin and reduced by internal store depletion, although calcium release from endoplasmic reticulum or mitochondria had a minor role on GlyT2 inhibition. Our results are consistent with a GlyT2 sensitivity to intracellular calcium mobilized by M2 mAChRs in the subcortical area of the plasma membrane. A crucial role of the plasma membrane sodium calcium exchanger NCX is proposed.

Long-acting muscarinic antagonists and small airways in asthma: Which link?

Involvement of small airways, those of <2mm in internal diameter, is present in all stages of asthma and contributes substantially to its pathophysiologic expression. Therefore, small airways are a potential target to achieve optimal asthma control. Airway tone, which is increased in asthma, is mainly controlled by the vagus nerve that releases acetylcholine (ACh) and activates muscarinic ACh receptors (mAChRs) post-synaptically on airway smooth muscle (ASM). In small airways, M3 mAChRs are expressed, but there is no vagal innervation. Non-neuronal ACh released from the epithelial cells that may express choline acetyltransferase in response to inflammatory stimuli, as well as from other structural cells in the airways, including fibroblasts and mast cells, can activate mAChRs. By antagonizing M3 mAChR, the contraction of the ASM is prevented and, potentially, local inflammation can be reduced and the progression of remodeling may be averted. In fact, ACh also contributes to inflammation and remodeling of the airways and regulates the growth of ASM. Several experimental studies have demonstrated the potential benefit derived from the use of mAChR antagonists, mainly long-acting mAChR antagonists (LAMAs), on small airways in asthma. However, there are several confounding factors that may cause a wrong estimation of the relationship between LAMAs and small airways in asthma. Further studies are needed to differentiate broncholytic and anti-inflammatory effects of LAMAs and to better understand the interaction between LAMAs and corticosteroids, also in the context of a triple therapy that includes a β2 -AR agonist, at different levels of the bronchial tree.

McN-A-343, a muscarinic agonist, reduces inflammation and oxidative stress in an experimental model of ulcerative colitis

AimThe aim of the present study was to investigate the anti-inflammatory response mediated of the M1 muscarinic acetylcholine receptor (mAChR) during experimental colitis. Material and methodsAfter the induction of 6% acetic acid colitis, mice were treated with McN-A-343 0.5, 1.0, and 1.5 mg/kg or dexamethasone (DEXA, 2.0 mg/kg) or pirenzepine (PIR, 10 mg/kg; M1 mAChR antagonist). Colonic inflammation was assessed by macroscopic and microscopic lesion scores, colonic wet weight, myeloperoxidase (MPO) activity, interleukin-1 beta (IL1-β) levels and tumor necrosis factor alpha (TNF-α), glutathione (GSH), malondialdehyde (MDA) and nitrate and nitrite (NO3/NO2), mRNA expression of IKKα, nuclear factor kappa beta (NF-kB) and cyclooxygenase-2 (COX-2), as well protein expression of NF-kB and COX-2. ResultsTreatment with McN-A-343 at a concentration of 1.5 mg/kg showed a significant reduction in intestinal damage as well as a decrease in wet weight, MPO activity, pro-inflammatory cytokine concentration, markers of oxidative stress and expression of inflammatory mediators. The action of the M1 agonist by the administration of pirenzepine, which promoted the blocking of the mAChR M1-mediated anti-inflammatory response, has also been proven. ConclusionThe results suggest that peripheral colonic M1 mAChR is involved in reversing the pro-inflammatory effect of experimentally induced colitis, which may represent a promising therapeutic alternative for patients with ulcerative colitis.

Huperzine A lowers intraocular pressure via the M3 mAChR and provides retinal neuroprotection via the M1 mAChR: a promising agent for the treatment of glaucoma.

BackgroundGlaucoma is a neurodegenerative disease that shares similar pathological mechanisms with Alzheimer’s disease (AD). Drug treatments for glaucoma increasingly rely upon both lowering of intraocular pressure (IOP) and optic nerve protection, as lowering of IOP alone has been unsatisfactory. Huperzine A (HupA) is an acetylcholinesterase inhibitor (AChEI) used for AD. This study investigated the potential of HupA as a treatment for glaucoma.MethodsThe ability of HupA to lower IOP via causing pupil constriction was assessed using New Zealand rabbits. The retinal neuroprotective effects of HupA were assessed in vivo using rat retinas subjected to ischemia-reperfusion (I/R) and in vitro using primary retinal neurons (PRNs) suffering from oxygen-glucose deprivation (OGD).ResultsHupA caused pupil constriction in a dose-time dependent manner which was reversed by the nonselective muscarinic acetylcholine receptor (mAChR) antagonist atropine and the selective M3 mAChR antagonist 4-DAMP. However, HupA had no effect on isolated iris muscle tension and calcium flow indicating an indirect M3 mAChR mediated effect. HupA exerted a neuroprotective effect against I/R and OGD to attenuate the retinal pathological lesion, improve retinal neuronal cell viability, reverse oxidative stress injury by increasing GSH levels and SOD activity, and decreasing MDA content and reduce the retinal neuronal apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 expression with no effect on the calcium flow tests. The effects were abolished by atropine and the selective M1 mAChR antagonist pirenzepine in OGD-induced PRNs suggesting an indirect M1 mAChR-mediated effect via inhibiting AChE activity to increase endogenous ACh level. Furthermore, HupA increased phosphorylated AKT level and decreased the levels of phosphorylated JNK, P38 MAPK and ERK via M1 mAChR antagonists indicating an involvement of activating the M1 mAChR and the downstream AKT/MAPK signaling pathway in the protective effects of HupA.ConclusionsHupA could significantly decrease IOP via activating M3 mAChR indirectly and produce retinal neuroprotective effect through M1 mAChR/AKT/MAPK by increasing endogenous ACh level. These investigations demonstrated that HupA was an effective drug in glaucoma treatment and the clinical application of HupA and other AChEIs for glaucoma patients should be further investigated.

Fine Tuning Muscarinic Acetylcholine Receptor Signaling Through Allostery and Bias.

The M1 and M4 muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, in particular for Alzheimer’s disease and schizophrenia. Due to high sequence homology, selective targeting of any of the M1-M5 mAChRs through the endogenous ligand binding site has been notoriously difficult to achieve. With the discovery of highly subtype selective mAChR positive allosteric modulators in the new millennium, selectivity through targeting an allosteric binding site has opened new avenues for drug discovery programs. However, some hurdles remain to be overcome for these promising new drug candidates to progress into the clinic. One challenge is the potential for on-target side effects, such as for the M1 mAChR where over-activation of the receptor by orthosteric or allosteric ligands can be detrimental. Therefore, in addition to receptor subtype selectivity, a drug candidate may need to exhibit a biased signaling profile to avoid such on-target adverse effects. Indeed, recent studies in mice suggest that allosteric modulators for the M1 mAChR that bias signaling toward specific pathways may be therapeutically important. This review brings together details on the signaling pathways activated by the M1 and M4 mAChRs, evidence of biased agonism at these receptors, and highlights pathways that may be important for developing new subtype selective allosteric ligands to achieve therapeutic benefit.

Rab43 GTPase directs postsynaptic trafficking and neuron-specific sorting of G protein–coupled receptors

G protein–coupled receptors (GPCRs) are important modulators of synaptic functions. A fundamental but poorly addressed question in neurobiology is how targeted GPCR trafficking is achieved. Rab GTPases are the master regulators of vesicle-mediated membrane trafficking, but their functions in the synaptic presentation of newly synthesized GPCRs are virtually unknown. Here, we investigate the role of Rab43, via dominant-negative inhibition and CRISPR–Cas9–mediated KO, in the export trafficking of α2-adrenergic receptor (α2-AR) and muscarinic acetylcholine receptor (mAChR) in primary neurons and cells. We demonstrate that Rab43 differentially regulates the overall surface expression of endogenous α2-AR and mAChR, as well as their signaling, in primary neurons. In parallel, Rab43 exerts distinct effects on the dendritic and postsynaptic transport of specific α2B-AR and M3 mAChR subtypes. More interestingly, the selective actions of Rab43 toward α2B-AR and M3 mAChR are neuronal cell specific and dictated by direct interaction. These data reveal novel, neuron-specific functions for Rab43 in the dendritic and postsynaptic targeting and sorting of GPCRs and imply multiple forward delivery routes for different GPCRs in neurons. Overall, this study provides important insights into regulatory mechanisms of GPCR anterograde traffic to the functional destination in neurons.

An adenoviral vector encoded with the GPx-1 gene attenuates memory impairments induced by β-amyloid (1-42) in GPx-1 KO mice via activation of M1 mAChR-mediated signalling

In the present study, we examined whether glutathione peroxidase-1 (GPx-1), a major H2O2 scavenger in the brain, affects memory deficits induced by Aβ (1-42) in mice. Treatment with 400 pmol/5 μl Aβ (1-42) (i.c.v.) resulted in a reduction of GPx-1 expression in wild-type (WT) mice. An Aβ (1-42)-induced reduction in acetylcholine (ACh) level was observed in the hippocampus. Treatment with Aβ (1-42) consistently resulted in reduced expression and activity of choline acetyltransferase (ChAT) and in an increase in expression and activity of acetylcholinesterase (AChE). Upon examining each of the muscarinic acetylcholine receptors (mAChRs) and nicotinic AChRs, we noted that Aβ (1-42) treatment selectively reduced the levels of M1 mAChR. In addition, Aβ (1-42) induced a significant reduction in phospho-cAMP response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) expression. The cholinergic impairments induced by Aβ (1-42) were more pronounced in GPx-1 knockout mice than in WT mice. Importantly, an adenoviral vector encoded with the GPx-1 gene (Ad-GPx-1) significantly rescued Aβ (1-42)-induced cholinergic impairments in GPx-1 knockout mice. In addition, M1 mAChR antagonist dicyclomine significantly counteracted Ad-GPx-1-mediated increases in p-CREB and BDNF expression, as well as memory-enhancing effects in GPx-1 knockout mice, thus indicating that M1 mAChR might be a critical mediator for the rescue effects of Ad-GPx-1. Combined, our results suggest that GPx-1 gene protected against Aβ (1-42)-induced memory impairments via activation of M1 mAChR-dependent CREB/BDNF signalling.

Muscarinic Acetylcholine Receptors Modulate Interleukin-6 Production and Immunoglobulin Class Switching in Daudi Cells.

B cells express muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs, respectively). Following immunization with ovalbumin, serum immunoglobulin G (IgG) and interleukin (IL)-6 levels were lower in M1 and M5 mAChR double-deficient mice and higher in α7 nAChR-deficient mice than in wild-type mice. This suggests mAChRs participate in the cytokine production involved in B cell differentiation into plasma cells, which induces immunoglobulin class switching from IgM to IgG. However, because these results were obtained with conventional knockout mice, in which all cells in the body were affected, the specific roles of these receptors expressed in B cells remains unclear. In the present study, Daudi B lymphoblast cells were used to investigate the specific roles of mAChRs and nAChR in B cells. Stimulating Daudi cells using Pansorbin cells (heat-killed, formalin-fixed Staphylococcus aureus coated with protein A) upregulated expression of M1-M4 mAChRs and the α4 nAChR subunit. Under these conditions, mAChRs, but not nAChRs, mediated immunoglobulin class switching to IgG. This effect was blocked by scopolamine, a non-selective mAChR antagonist, and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), a Gq/11-coupled M1, M3, M5 antagonist. In addition, IL-6 secretion was further enhanced following mAChR activation. Thus, Gq/11-coupled mAChRs expressed in B cells thus appear to contribute to IL-6 production and B cell maturation into IgG-producing plasma cells.

Synthesis, Biological, and Computational Evaluation of Antagonistic, Chiral Hydrobenzoin Esters of Arecaidine Targeting mAChR M1

Muscarinic acetylcholine receptors (mAChRs) are a pivotal constituent of the central and peripheral nervous system. Yet, therapeutic and diagnostic applications thereof are hampered by the lack of subtype selective ligands. Within this work, we synthesized and chemically characterized three different stereoisomers of hydrobenzoin esters of arecaidine by NMR, HR-MS, chiral chromatography, and HPLC-logP. All compounds are structurally eligible for carbon-11 labeling and show appropriate stability in Dulbecco’s phosphate-buffered saline (DPBS) and F12 cell culture medium. A competitive radioligand binding assay on Chinese hamster ovary cell membranes comprising the human mAChR subtypes M1-M5 showed the highest orthosteric binding affinity for subtype M1 and a strong influence of stereochemistry on binding affinity, which corresponds to in silico molecular docking experiments. Ki values toward M1 were determined as 99 ± 19 nM, 800 ± 200 nM, and 380 ± 90 nM for the (R,R)-, (S,S)-, and racemic (R,S)-stereoisomer, respectively, highlighting the importance of stereochemical variations in mAChR ligand development. All three stereoisomers were shown to act as antagonists toward mAChR M1 using a Fluo-4 calcium efflux assay. With respect to future positron emission tomography (PET) tracer development, the (R,R)-isomer appears especially promising as a lead structure due to its highest subtype selectivity and lowest Ki value.