The Activation of Muscarinic Acetylcholine Receptors Protects against Neuroinflammation in a Mouse Model through Attenuating Microglial Inflammation.

Neuroinflammation is a critical factor that contributes to neurological impairment and is closely associated with the onset and progression of neurodegenerative diseases. In the central nervous system (CNS), microglia play a pivotal role in the regulation of inflammation through various signaling pathways. Therefore, mitigating microglial inflammation is considered a promising strategy for restraining neuroinflammation. Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and exhibit clear neuroprotective effects in various disease models. However, whether the activation of mAChRs can harness benefits in neuroinflammation remains largely unexplored. In this study, the anti-inflammatory effects of mAChRs were found in a neuroinflammation mouse model. The expression of various cytokines and chemokines was regulated in the brains and spinal cords after the administration of mAChR agonists. Microglia were the primary target cells through which mAChRs exerted their anti-inflammatory effects. The results showed that the activation of mAChRs decreased the pro-inflammatory phenotypes of microglia, including the expression of inflammatory cytokines, morphological characteristics, and distribution density. Such anti-inflammatory modulation further exerted neuroprotection, which was found to be even more significant by the direct activation of neuronal mAChRs. This study elucidates the dual mechanisms through which mAChRs exert neuroprotective effects in central inflammatory responses, providing evidence for their application in inflammation-related neurological disorders.

Exploration of the mechanism underlying the therapeutic effect of electroacupuncture at chengshan acupoint on post-hemorrhoidectomy anal pain: Insights from the mAChRs/IP3-Ca2+-CaM signaling pathway.

OBJECTIVE: In the context of postoperative anal pain, understanding the intricate mechanisms and effective interventions is paramount. This study investigates the role of Muscarinic Acetylcholine Receptors (mAChRs) and the IP3-Ca2+-CaM signaling pathway in a rat model of postoperative anal pain, exploring the potential analgesic effects of electroacupuncture. METHODS: Comprehensive approaches involving mechanical sensitivity assays, Western blotting, immunohistochemistry, and intracellular calcium concentration measurement were used. RESULTS: The authors found elevated mAChRs expression in the postoperative pain model. Antagonizing mAChRs reduced pain sensitivity and attenuated the IP3-Ca2+-CaM pathway. Remarkably, electroacupuncture treatment further mitigated pain, potentially by suppressing this signaling cascade. INTERPRETATION: These findings reveal a novel connection between mAChRs and the IP3-Ca2+-CaM pathway in postoperative anal pain and suggest electroacupuncture as a promising avenue for pain relief through these mechanisms, offering insights into innovative strategies for postoperative pain management.

Ligand bias at the muscarinic acetylcholine receptor family: Opportunities and challenges.

Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are activated by the endogenous neurotransmitter, acetylcholine (ACh). Disruption of mAChR signalling has been associated with a variety of neurological disorders and non-neurological diseases. Consequently, the development of agonists and antagonists of the mAChRs has been a major avenue in drug discovery. Unfortunately, mAChR ligands are often associated with on-target side effects for two reasons. The first reason is due to the high sequence conservation at the orthosteric ACh binding site among all five receptor subtypes (M1-M5), making on-target subtype selectivity a major challenge. The second reason is due to on-target side effects of mAChR drugs that are associated with the pleiotropic nature of mAChR signalling at the level of a single mAChR subtype. Indeed, there is growing evidence that within the myriad of signalling events produced by mAChR ligands, some will have therapeutic benefits, whilst others may promote cholinergic side effects. This paradigm of drug action, known as ligand bias or biased agonism, is an attractive feature for next-generation mAChR drugs, as it holds the promise of developing drugs devoid of on-target adverse effects. Although relatively simple to detect and even quantify in vitro, ligand bias, as observed in recombinant systems, does not always translate to in vivo systems, which remains a major hurdle in GPCR drug discovery, including the mAChR family. Here we report recent studies that have attempted to detect and quantify ligand bias at the mAChR family, and briefly discuss the challenges associated with biased agonist drug development. This article is part of the Special Issue on "Ligand Bias".

Local anesthetics inhibit muscarinic acetylcholine receptor-mediated calcium responses and the recruitment of ß-arrestin in HSY human parotid cells.

OBJECTIVES: Local anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and ß-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca2+ responses and formation of receptor-ß-arrestin complexes in the HSY human parotid cell line. METHODS: Ca2+ responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and ß-arrestin were examined using a ß-arrestin GPCR assay kit. RESULTS: Lidocaine reduced mAChR-mediated Ca2+ responses but did not change the intracellular Ca2+ concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca2+ responses, with EC50 values of 48.0 and 20.4 µM, respectively, for 50 µM carbachol-stimulated Ca2+ responses. In the absence of extracellular Ca2+, the pretreatment of cells with QX314 reduced carbachol-induced Ca2+ release, indicating that QX314 reduced Ca2+ release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca2+ entry as they did not alter the thapsigargin-induced Ca2+ response. QX314 and procaine reduced the carbachol-mediated recruitment of ß-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice. CONCLUSION: Local anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca2+ release from intracellular stores and the recruitment of ß-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.

M2 receptors are required for spatiotemporal sequence learning in mouse primary visual cortex.

Acetylcholine is a neurotransmitter that plays a variety of roles in the central nervous system. It was previously shown that blocking muscarinic receptors with a nonselective antagonist prevents a form of experience-dependent plasticity termed "spatiotemporal sequence learning" in the mouse primary visual cortex (V1). Muscarinic signaling is a complex process involving the combined activities of five different G protein-coupled receptors, M1-M5, all of which are expressed in the murine brain but differ from each other functionally and in anatomical localization. Here we present electrophysiological evidence that M2, but not M1, receptors are required for spatiotemporal sequence learning in mouse V1. We show in male mice that M2 is highly expressed in the neuropil in V1, especially in thalamorecipient layer 4, and colocalizes with the soma in a subset of somatostatin-expressing neurons in deep layers. We also show that expression of M2 receptors is higher in the monocular region of V1 than it is in the binocular region but that the amount of experience-dependent sequence potentiation is similar in both regions and that blocking muscarinic signaling after visual stimulation does not prevent plasticity. This work establishes a new functional role for M2-type receptors in processing temporal information and demonstrates that monocular circuits are modified by experience in a manner similar to binocular circuits.NEW & NOTEWORTHY Muscarinic acetylcholine receptors are required for multiple forms of plasticity in the brain and support perceptual functions, but the precise role of the five subtypes (M1-M5) are unclear. Here we show that the M2 receptor is specifically required to encode experience-dependent representations of spatiotemporal relationships in both monocular and binocular regions of mouse V1. This work identifies a novel functional role for M2 receptors in coding temporal information into cortical circuits.

M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells.

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

Design, synthesis and preclinical evaluation of muscarine receptor antagonists via a scaffold-hopping approach.

Our research group recently identified a rearrangement product of pirenzepine as starting point for a comprehensive rational drug design approach towards orthosteric muscarinic acetylcholine receptor ligands. Chemical reduction and bioscaffold hop lead to the development of sixteen promising compounds featuring either a benzimidazole or carbamate moiety, all exhibiting comparable pharmacophoric characteristics. The synthesized compounds were characterized by NMR, HR-MS, and RP-HPLC techniques. Subsequent evaluation encompassed binding affinity assessment on CHO-hM1-5 cells, mode of action determination, and analysis of physico-chemical parameters. The CNS MPO score indicated favorable drug-like attributes and potential CNS activity for the antagonistic ligands. The most promising compounds displayed Ki-values within a desirable low nanomolar range, and their structural features allow for potential carbon-11 radiolabeling. Our optimization efforts resulted in compounds with a remarkable 138-fold increase in binding affinity compared to the previously mentioned rearrangement product towards human M5, suggesting their prospective utility in positron emission tomography applications.

Long-Term-But Not Short-Term-Plasticity at the Mossy Fiber-CA3 Pyramidal Cell Synapse in Hippocampus Is Altered in M1/M3 Muscarinic Acetylcholine Receptor Double Knockout Mice.

Muscarinic acetylcholine receptors are well-known for their crucial involvement in hippocampus-dependent learning and memory, but the exact roles of the various receptor subtypes (M1-M5) are still not fully understood. Here, we studied how M1 and M3 receptors affect plasticity at the mossy fiber (MF)-CA3 pyramidal cell synapse. In hippocampal slices from M1/M3 receptor double knockout (M1/M3-dKO) mice, the signature short-term plasticity of the MF-CA3 synapse was not significantly affected. However, the rather unique NMDA receptor-independent and presynaptic form of long-term potentiation (LTP) of this synapse was much larger in M1/M3-deficient slices compared to wild-type slices in both field potential and whole-cell recordings. Consistent with its presynaptic origin, induction of MF-LTP strongly enhanced the excitatory drive onto single CA3 pyramidal cells, with the effect being more pronounced in M1/M3-dKO cells. In an earlier study, we found that the deletion of M2 receptors in mice disinhibits MF-LTP in a similar fashion, suggesting that endogenous acetylcholine employs both M1/M3 and M2 receptors to constrain MF-LTP. Importantly, such synergism was not observed for MF long-term depression (LTD). Low-frequency stimulation, which reliably induced LTD of MF synapses in control slices, failed to do so in M1/M3-dKO slices and gave rise to LTP instead. In striking contrast, loss of M2 receptors augmented LTD when compared to control slices. Taken together, our data demonstrate convergence of M1/M3 and M2 receptors on MF-LTP, but functional divergence on MF-LTD, with the net effect resulting in a well-balanced bidirectional plasticity of the MF-CA3 pyramidal cell synapse.

Decoding the mechanisms of allostery.

A complex interplay between structure, conformational dynamics and pharmacology defines distant regulation of G protein-coupled receptors.

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics.

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.

Novel Xanomeline-Containing Bitopic Ligands of Muscarinic Acetylcholine Receptors: Design, Synthesis and FRET Investigation.

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M1, M2, M4, and M5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M1/M4-preferring orthosteric agonist Xanomeline 10 and the M1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M1 mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M1 and M4 mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M1 subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.

A Rab10-ACAP1-Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling.

Membrane trafficking processes regulate the G protein-coupled receptor activity. The muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, but the cellular machineries that control the trafficking of these receptors remain largely elusive. Here, we revealed the role of the small GTPase Rab10 as a negative regulator for the post-activation trafficking of M4 mAChR and the underlying mechanism. We show that constitutively active Rab10 arrests the receptor within Rab5-positive early endosomes and significantly hinders the resensitization of M4-mediated Ca2+ signaling. Mechanistically, M4 binds to Rab10-GTP, which requires the motif 386RKKRQMAA393 (R386-A393) within the third intracellular loop. Moreover, Rab10-GTP inactivates Arf6 by recruiting the Arf6 GTPase-activating protein, ACAP1. Strikingly, deletion of the motif R386-A393 causes M4 to bypass the control by Rab10 and switch to the Rab4-facilitated fast recycling pathway, thus reusing the receptor. Therefore, Rab10 couples the cargo sorting and membrane trafficking regulation through cycle between GTP-bound and GDP-bound state. Our findings suggest a model that Rab10 binds to the M4 like a molecular brake and controls the receptor's transport through endosomes, thus modulating the signaling, and this regulation is specific among the mAChR subtypes.

Muscarinic Acetylcholine Receptors Modulate HCN Channel Properties in Vestibular Ganglion Neurons.

Efferent modulation of vestibular afferent excitability is linked to muscarinic signaling cascades that close low-voltage-gated potassium channels (i.e., KCNQ). Here, we show that muscarinic signaling cascades also depolarize the activation range of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We compared the voltage activation range and kinetics of HCN channels and induced firing patterns before and after administering the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M) in dissociated vestibular ganglion neurons (VGNs) from rats of either sex using perforated whole-cell patch-clamp methods. Oxo-M depolarized HCN channels' half-activation voltage (V 1/2) and sped up the rate of activation near resting potential twofold. HCN channels in large-diameter and/or transient firing VGN (putative cell bodies of irregular firing neuron from central epithelial zones) had relatively depolarized V 1/2 in control solution and were less sensitive to mAChR activation than those found in small-diameter VGN with sustained firing patterns (putatively belonging to regular firing afferents). The impact of mAChR on HCN channels is not a direct consequence of closing KCNQ channels since pretreating the cells with Linopirdine, a KCNQ channel blocker, did not prevent HCN channel depolarization by Oxo-M. Efferent signaling promoted ion channel configurations that were favorable to highly regular spiking in some VGN, but not others. This is consistent with previous observations that low-voltage gated potassium currents in VGN are conducted by mAChR agonist-sensitive and -insensitive channels. Connecting efferent signaling to HCN channels is significant because of the channel's impact on spike-timing regularity and nonchemical transmission between Type I hair cells and vestibular afferents.SIGNIFICANCE STATEMENT Vestibular afferents express a diverse complement of ion channels. In vitro studies identified low-voltage activated potassium channels and hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Moreover, a network of acetylcholine-releasing efferent neurons controls afferent excitability by closing a subgroup of low-voltage activated potassium channels on the afferent neuron. This work shows that these efferent signaling cascades also enhance the activation of HCN channels by depolarizing their voltage activation range. The size of this effect varies depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system exquisite control over afferent neuron activity.

Muscarinic Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

In recent years, we have studied by immunohistochemistry, intracellular recording, and western blotting the role of the muscarinic acetylcholine receptors (mAChRs; M1, M2, and M4 subtypes) in the mammalian neuromuscular junction (NMJ) during development and in the adult. Here, we evaluate our published data to emphasize the mAChRs' relevance in developmental synaptic elimination and their crosstalk with other metabotropic receptors, downstream kinases, and voltage-gated calcium channels (VGCCs). The presence of mAChRs in the presynaptic membrane of motor nerve terminals allows an autocrine mechanism in which the secreted acetylcholine influences the cell itself in feedback. mAChR subtypes are coupled to different downstream pathways, so their feedback can move in a broad range between positive and negative. Moreover, mAChRs allow direct activity-dependent interaction through ACh release between the multiple competing axons during development. Additional regulation from pre- and postsynaptic sites (including neurotrophic retrograde control), the agonistic and antagonistic contributions of adenosine receptors (AR; A1 and A2A), and the tropomyosin-related kinase B receptor (TrkB) cooperate with mAChRs in the axonal competitive interactions which lead to supernumerary synapse elimination that achieves the optimized monoinnervation of musculoskeletal cells. The metabotropic receptor-driven balance between downstream PKA and PKC activities, coupled to developmentally regulated VGCC, explains much of how nerve terminals with different activities finally progress to their withdrawal or strengthening.

Opportunities and challenges for the development of M1 muscarinic receptor positive allosteric modulators in the treatment for neurocognitive deficits.

Targeting allosteric sites of M1 muscarinic acetylcholine receptors (M1 receptors) is a promising strategy to treat neurocognitive disorders, such as Alzheimer's disease and schizophrenia. Indeed, the last two decades have seen an impressive body of work focussing on the design and development of positive allosteric modulators (PAMs) for the M1 receptor. This has led to the identification of a structurally diverse range of highly selective M1 PAMs. In preclinical models, M1 PAMs have shown rescue of cognitive deficits and improvement of endpoints predictive of symptom domains of schizophrenia. Yet, to date only a few M1 PAMs have reached early-stage clinical trials, with many of them failing to progress further due to on-target mediated cholinergic adverse effects that have plagued the development of this class of ligand. This review covers the recent preclinical and clinical studies in the field of M1 receptor drug discovery for the treatment of Alzheimer's disease and schizophrenia, with a specific focus on M1 PAM, highlighting both the undoubted potential but also key challenges for the successful translation of M1 PAMs from bench-side to bedside.

Cognitive Impairment, Sleep Disturbance, and Depression in Women with Silicone Breast Implants: Association with Autoantibodies against Autonomic Nervous System Receptors.

BACKGROUND: Silicone breast implants (SBIs) has been shown to be associated with an increased risk of autoimmune diseases. In the current study, we aimed to explore the potential association between circulating autoantibodies against the autonomic nervous system and cognitive impairment, memory deficit, and depressive symptoms reported by women with SBIs. METHODS: ELISA assays were used to quantify anti-adrenergic receptors (α1, α2, ß1, ß2), anti-muscarinic receptors (M1-M5), anti-endothelin receptor type A, and anti-angiotensin II type 1 receptor titers in the sera of 93 symptomatic female subjects with SBIs and 36 age-matched healthy female controls. RESULTS: A significant difference was detected in the level of autoantibodies against the autonomic nervous system receptors in women with SBIs who reported memory impairment, cognitive impairment, and sleep disturbance as compared with both women with SBIs who did not complain of these symptoms or with healthy individuals without SBIs. CONCLUSIONS: Clinical symptoms such as depression, cognitive impairment, and sleep disturbances were found to be associated with dysregulation of the levels of circulating autoantibodies targeting the autonomous nervous system receptors in women with SBIs. These autoantibodies may have diagnostic significance in diseases associated with breast implants.

Activation of M3-AChR and IP3/Ca2+/PKC signaling pathways by pilocarpine increases glycine-induced currents in ventral horn neurons of the spinal cord.

Our study aimed to determine the effects of pilocarpine and the mechanisms involving muscarinic acetylcholine receptors (mAChRs) on glycine receptors (GlyRs) in neurons of the spinal cord ventral horn. An enzymatic digestion combined with acute mechanical separation was applied to isolate neurons from the spinal cord ventral horn. Patch-clamp recording was then used to investigate the outcomes of pilocarpine. Our results indicate that pilocarpine increased the glycine currents in a concentration-dependent manner, which was blocked by the M3-AChR selective antagonists 4-DAMP and J104129. Pilocarpine also enhanced the glycine currents in nominally Ca2+-free extracellular solution. Conversely, the enhancement of glycine currents by pilocarpine disappeared when intracellular Ca2+ was chelated by BAPTA. Heparin and Xe-C, which are IP3 receptor antagonists, also totally abolished the pilocarpine effect. Furthermore, Bis-IV, a PKC inhibitor, eliminated the pilocarpine effect. Additionally, PMA, a PKC activator, mimicked the pilocarpine effect. These results indicate that pilocarpine may increase the glycine currents by activating the M3-AChRs and IP3/Ca2+/PKC pathways.

Synthesis, Biological Evaluation, and Docking Studies of Antagonistic Hydroxylated Arecaidine Esters Targeting mAChRs.

The muscarinic acetylcholine receptor family is a highly sought-after target in drug and molecular imaging discovery efforts aimed at neurological disorders. Hampered by the structural similarity of the five subtypes' orthosteric binding pockets, these efforts largely failed to deliver subtype-selective ligands. Building on our recent successes with arecaidine-derived ligands targeting M1, herein we report the synthesis of a related series of 11 hydroxylated arecaidine esters. Their physicochemical property profiles, expressed in terms of their computationally calculated CNS MPO scores and HPLC-logD values, point towards blood-brain barrier permeability. By means of a competitive radioligand binding assay, the binding affinity values towards each of the individual human mAChR subtypes hM1-hM5 were determined. The most promising compound of this series 17b was shown to have a binding constant towards hM1 in the single-digit nanomolar region (5.5 nM). Similar to our previously reported arecaidine-derived esters, the entire series was shown to act as hM1R antagonists in a calcium flux assay. Overall, this study greatly expanded our understanding of this recurring scaffolds' structure-activity relationship and will guide the development towards highly selective mAChRs ligands.

Design, Synthesis, and Biological Evaluation of 4,4'-Difluorobenzhydrol Carbamates as Selective M1 Antagonists.

Due to their important role in mediating a broad range of physiological functions, muscarinic acetylcholine receptors (mAChRs) have been a promising target for therapeutic and diagnostic applications alike; however, the list of truly subtype-selective ligands is scarce. Within this work, we have identified a series of twelve 4,4'-difluorobenzhydrol carbamates through a rigorous docking campaign leveraging commercially available amine databases. After synthesis, these compounds have been evaluated for their physico-chemical property profiles, including characteristics such as HPLC-logD, tPSA, logBB, and logPS. For all the synthesized carbamates, these characteristics indicate the potential for BBB permeation. In competitive radioligand binding experiments using Chinese hamster ovary cell membranes expressing the individual human mAChR subtype hM1-hM5, the most promising compound 2 displayed a high binding affinitiy towards hM1R (1.2 nM) while exhibiting modest-to-excellent selectivity versus the hM2-5R (4-189-fold). All 12 compounds were shown to act in an antagonistic fashion towards hM1R using a dose-dependent calcium mobilization assay. The structural eligibility for radiolabeling and their pharmacological and physico-chemical property profiles render compounds 2, 5, and 7 promising candidates for future position emission tomography (PET) tracer development.

Association of autoantibodies to muscarinic acetylcholine receptors with gastrointestinal symptoms and disease severity in patients with postural orthostatic tachycardia syndrome.

Previous studies have reported a relationship between postural orthostatic tachycardia syndrome (POTS) and positivity for serum autoantibodies against G-protein-coupled receptors (GPCRs). However, the role of these autoantibodies in POTS is unclear. The present retrospective study analyzed the autoimmune etiology of POTS in 24 patients using a head-up tilt test to assess for any correlation between the clinical features of POTS and serum levels of autoantibodies against diverse GPCRs. In total, ten assessment items, including autonomic function tests, were analyzed. Of these, persistent, gastrointestinal symptoms and disease severity showed a significant association with the serum level of anti-muscarinic acetylcholine receptor (mAChRs) antibodies (gastrointestinal symptoms, M1, M2, M5; disease severity, M1, M3, M4, M5) [P <0.05]), while no significant association was found between the clinical features and autoantibodies against adrenergic receptors (α1, α2, ß1, ß2), angiotensin receptor 1, or endothelin receptor A. The patients were further divided into two groups based on the presence or absence of persistent gastrointestinal symptoms and then were characterized by the ten assessment items and neuropsychological tests, including the Wechsler Adult Intelligence Scale score and Self-Rating Depression Scale score. The results demonstrated a clear difference between the two groups in terms of disease severity, age at onset (older or younger than 20 years), and processing speed index (P <0.05), which were highly consistent with the association between these clinical features and the levels of serum anti-mAChR antibodies, particularly the anti-M5 receptor antibody. These findings suggested that anti-mAChR antibodies may play an important role in a subgroup of POTS patients with persistent gastrointestinal symptoms.