RESUMEN
Acute stimulation of M1 or M4 muscarinic cholinergic receptors reduces cocaine abuse-related effects in mice and rats. The combined activation of these receptor subtypes produces synergistic effects on some behavioural endpoints in mice. M1 and M1 + M4 receptor stimulation in a cocaine vs. food choice assay in rats and microdialysis in rats showed delayed and lasting "anticocaine effects". Here, we tested whether these putative lasting neuroplastic changes are sufficient to occlude the reinforcing effects of cocaine at the behavioural level in mice. Mice were pre-treated with the M1 receptor partial agonist VU0364572, M4 receptor positive allosteric modulator VU0152100, or VU0364572 + VU0152100 two weeks prior to acquisition of cocaine intravenous self-administration (IVSA). Male C57BL/6JRj mice received vehicle, VU0364572, VU0152100, or VU0364572 + VU0152100. Female mice were tested with two VU0364572 + VU0152100 dose combinations or vehicle. To attribute potential effects to either reduced rewarding effects or increased aversion to cocaine, we tested VU0364572 alone and VU0364572 + VU0152100 in acquisition of cocaine-conditioned place preference (CPP) in male mice using an unbiased design. The acquisition of cocaine IVSA was drastically reduced and/or slowed in male and female mice receiving VU0364572 + VU0152100, but not either drug alone. Food-maintained operant behaviour was unaffected, indicating that the treatment effects were cocaine-specific. No treatment altered the acquisition of cocaine-CPP, neither in the post-test, nor in a challenge 14 days later. The cocaine IVSA findings confirm unusual long-lasting "anticocaine" effects of muscarinic M1 + M4 receptor stimulation. Thus, in mice, simultaneous stimulation of both receptor subtypes seems to produce potential neuroplastic changes that yield lasting effects.
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Cocaína , Ratones Endogámicos C57BL , Receptor Muscarínico M1 , Receptor Muscarínico M4 , Refuerzo en Psicología , Autoadministración , Animales , Masculino , Cocaína/farmacología , Cocaína/administración & dosificación , Femenino , Receptor Muscarínico M4/metabolismo , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M1/efectos de los fármacos , Ratones , Inhibidores de Captación de Dopamina/farmacología , Inhibidores de Captación de Dopamina/administración & dosificación , Agonistas Muscarínicos/farmacología , Condicionamiento Operante/efectos de los fármacosRESUMEN
The M4 muscarinic acetylcholine receptor (mAChR) is a biological target for neurocognitive disorders. Compound 1 is an ago-PAM for the M4 mAChR. Herein, we report the design, synthesis, and evaluation of novel putative M4 mAChR PAMs based on 1. These analogs were screened and then fully characterized in two functional assays (GoB protein activation and CAMYEL activation) to quantify their allosteric and ago-PAM properties against ACh. A selection of 7 M4 PAMs were assessed for their ability to modulate ACh-mediated ß-arrestin recruitment and revealed 4 distinct clusters of M4 PAM activity: (1) analogs similar to 1 (24d), (2) analogs demonstrating only allosteric agonism (23d), (3) analogs with increased allosteric properties in CAMYEL activation (23b/23f and 24a/24b), and (4) analogs with a biased modulatory effect toward ß-arrestin recruitment (23i). These novel M4 chemical tools disclose discrete molecular determinants, allowing further interrogation of the therapeutic roles of cAMP and ß-arrestin pathways in neurocognitive disorders.
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Piridinas , Receptor Muscarínico M4 , Regulación Alostérica/efectos de los fármacos , Humanos , Animales , Piridinas/farmacología , Piridinas/síntesis química , Piridinas/química , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/metabolismo , Cricetulus , Trastornos Neurocognitivos/tratamiento farmacológico , Trastornos Neurocognitivos/metabolismo , Células CHO , Relación Estructura-Actividad , Descubrimiento de Drogas , beta-Arrestinas/metabolismo , Pirazoles/farmacología , Pirazoles/química , Pirazoles/síntesis química , Pirazoles/uso terapéuticoRESUMEN
Selective activation of the M4 muscarinic acetylcholine receptor subtype offers a novel strategy for the treatment of psychosis in multiple neurological disorders. Although the development of traditional muscarinic activators has been stymied due to pan-receptor activation, muscarinic receptor subtype selectivity can be achieved through the utilization of a subtype of a unique allosteric site. A major challenge in capitalizing on this allosteric site to date has been achieving a balance of suitable potency and brain penetration. Herein, we describe the design of a brain penetrant series of M4 selective positive allosteric modulators (PAMs), ultimately culminating in the identification of 21 (PF-06852231, now CVL-231/emraclidine), which is under active clinical development as a novel mechanism and approach for the treatment of schizophrenia.
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Encéfalo , Diseño de Fármacos , Receptor Muscarínico M4 , Receptor Muscarínico M4/metabolismo , Receptor Muscarínico M4/agonistas , Regulación Alostérica/efectos de los fármacos , Humanos , Animales , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Relación Estructura-Actividad , Ratas , Cricetulus , Células CHO , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/síntesis química , Agonistas Muscarínicos/química , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismoRESUMEN
Muscarinic neurotransmission is fundamentally involved in supporting several brain functions by modulating flow of information in brain neural circuits including the hippocampus which displays a remarkable functional segregation along its longitudinal axis. However, how muscarinic neuromodulation contributes to the functional segregation along the hippocampus remains unclear. In this study we show that the nonselective muscarinic receptor agonist carbachol similarly suppresses basal synaptic transmission in the dorsal and ventral CA1 hippocampal field, in a concentration-depended manner. Furthermore, using a ten-pulse stimulation train of varying frequency we found that carbachol changes the frequency filtering properties more in ventral than dorsal hippocampus by facilitating synaptic inputs at a wide range of input frequencies in the ventral compared with dorsal hippocampus. Using the M2 receptor antagonist gallamine and the M4 receptor antagonist tropicamide, we found that M2 receptors are involved in controlling basal synaptic transmission and short-term synaptic plasticity (STSP) in the ventral but not the dorsal hippocampus, while M4 receptors participate in modulating basal synaptic transmission and STSP in both segments of the hippocampus. These results were corroborated by the higher protein expression levels of M2 receptors in the ventral compared with dorsal hippocampus. We conclude that muscarinic transmission modulates excitatory synaptic transmission and short-term synaptic plasticity along the entire rat hippocampus by acting through M4 receptors and recruiting M2 receptors only in the ventral hippocampus. Furthermore, M4 receptors appear to exert a permissive role on the actions of M2 receptors on STSP in the ventral hippocampus. This dorsoventral differentiation of muscarinic modulation is expected to have important implications in information processing along the endogenous hippocampal circuitry.
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Hipocampo , Plasticidad Neuronal , Transmisión Sináptica , Animales , Plasticidad Neuronal/fisiología , Plasticidad Neuronal/efectos de los fármacos , Transmisión Sináptica/fisiología , Transmisión Sináptica/efectos de los fármacos , Ratas , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Masculino , Carbacol/farmacología , Receptor Muscarínico M2/metabolismo , Receptores Muscarínicos/metabolismo , Ratas Wistar , Antagonistas Muscarínicos/farmacología , Receptor Muscarínico M4/metabolismo , Agonistas Muscarínicos/farmacología , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: Current pharmacotherapies for Tourette syndrome (TS) are often unsatisfactory and poorly tolerated, underscoring the need for novel treatments. Insufficient striatal acetylcholine has been suggested to contribute to tic ontogeny. Thus, we tested whether activating M1 and/or M4 receptors-the two most abundant muscarinic receptors in the striatum-reduced tic-related behaviours in mouse models of TS. EXPERIMENTAL APPROACH: Studies were conducted using CIN-d and D1CT-7 mice, two TS models characterized by early-life depletion of striatal cholinergic interneurons and cortical neuropotentiation, respectively. First, we tested the effects of systemic and intrastriatal xanomeline, a selective M1/M4 receptor agonist, on tic-like and other TS-related responses. Then, we examined whether xanomeline effects were reduced by either M1 or M4 antagonists or mimicked by the M1/M3 agonist cevimeline or the M4 positive allosteric modulator (PAM) VU0467154. Finally, we measured striatal levels of M1 and M4 receptors and assessed the impact of VU0461754 on the striatal expression of the neural marker activity c-Fos. KEY RESULTS: Systemic and intrastriatal xanomeline reduced TS-related behaviours in CIN-d and D1CT-7 mice. Most effects were blocked by M4, but not M1, receptor antagonists. VU0467154, but not cevimeline, elicited xanomeline-like ameliorative effects in both models. M4, but not M1, receptors were down-regulated in the striatum of CIN-d mice. Additionally, VU0467154 reduced striatal c-Fos levels in these animals. CONCLUSION AND IMPLICATIONS: Activation of striatal M4, but not M1, receptors reduced tic-like manifestations in mouse models, pointing to xanomeline and M4 PAMs as novel putative therapeutic strategies for TS.
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Cuerpo Estriado , Modelos Animales de Enfermedad , Agonistas Muscarínicos , Receptor Muscarínico M4 , Síndrome de Tourette , Animales , Síndrome de Tourette/metabolismo , Síndrome de Tourette/tratamiento farmacológico , Receptor Muscarínico M4/metabolismo , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidores , Ratones , Cuerpo Estriado/metabolismo , Cuerpo Estriado/efectos de los fármacos , Masculino , Agonistas Muscarínicos/farmacología , Conducta Animal/efectos de los fármacos , Piridinas/farmacología , Tics/tratamiento farmacológico , Tics/metabolismo , Tiofenos/farmacología , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M1/agonistas , Dioxoles/farmacología , Ratones Endogámicos C57BL , TiadiazolesRESUMEN
Background: Although sporadic Alzheimer's disease (AD) is a neurodegenerative disorder of unknown etiology, familial AD is associated with specific gene mutations. A commonality between these forms of AD is that both display multiple pathogenic events including cholinergic and lipid dysregulation. Objective: We aimed to identify the relevant lipids and the activity of their related receptors in the frontal cortex and correlating them with cognition during the progression of AD. Methods: MALDI-mass spectrometry imaging (MSI) and functional autoradiography was used to evaluate the distribution of phospholipids/sphingolipids and the activity of cannabinoid 1 (CB1), sphingosine 1-phosphate 1 (S1P1), and muscarinic M2/M4 receptors in the frontal cortex (FC) of people that come to autopsy with premortem clinical diagnosis of AD, mild cognitive impairment (MCI), and no cognitive impairment (NCI). Results: MALDI-MSI revealed an increase in myelin-related lipids, such as diacylglycerol (DG) 36:1, DG 38:5, and phosphatidic acid (PA) 40:6 in the white matter (WM) in MCI compared to NCI, and a downregulation of WM phosphatidylinositol (PI) 38:4 and PI 38:5 levels in AD compared to NCI. Elevated levels of phosphatidylcholine (PC) 32:1, PC 34:0, and sphingomyelin 38:1 were observed in discrete lipid accumulations in the FC supragranular layers during disease progression. Muscarinic M2/M4 receptor activation in layers V-VI decreased in AD compared to MCI. CB1 receptor activity was upregulated in layers V-VI, while S1P1 was downregulated within WM in AD relative to NCI. Conclusions: FC WM lipidomic alterations are associated with myelin dyshomeostasis in prodromal AD, suggesting WM lipid maintenance as a potential therapeutic target for dementia.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Humanos , Enfermedad de Alzheimer/patología , Disfunción Cognitiva/patología , Receptor Muscarínico M4 , Lóbulo Frontal/diagnóstico por imagen , Lóbulo Frontal/patología , Colinérgicos , LípidosRESUMEN
Muscarinic acetylcholine receptors (mAChRs) play a significant role in the pathophysiology of schizophrenia. Although activating mAChRs holds potential in addressing the full range of schizophrenia symptoms, clinical application of many non-selective mAChR agonists in cognitive deficits, positive and negative symptoms is hindered by peripheral side effects (gastrointestinal disturbances and cardiovascular effects) and dosage restrictions. Ligands binding to the allosteric sites of mAChRs, particularly the M1 and M4 subtypes, demonstrate activity in improving cognitive function and amelioration of positive and negative symptoms associated with schizophrenia, enhancing our understanding of schizophrenia. The article aims to critically examine current design concepts and clinical advancements in synthesizing and designing small molecules targeting M1/M4, providing theoretical insights and empirical support for future research in this field.
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Antipsicóticos , Receptor Muscarínico M1 , Esquizofrenia , Antipsicóticos/farmacología , Antipsicóticos/química , Antipsicóticos/uso terapéutico , Estructura Molecular , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inhibidores , Receptor Muscarínico M4/metabolismo , Receptor Muscarínico M4/antagonistas & inhibidores , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismoRESUMEN
Stimulation of the M4 muscarinic acetylcholine receptor reduces striatal hyperdopaminergia, suggesting its potential as a therapeutic target for schizophrenia. Emraclidine (CVL-231) is a novel, highly selective, positive allosteric modulator (PAM) of M4 muscarinic acetylcholine receptors i.e. acts as a modulator that increases the response of these receptors. First, we aimed to further characterize the positron emission tomography (PET) imaging and quantification performance of a recently developed M4 PAM radiotracer, [11C]MK-6884, in non-human primates (NHPs). Second, we applied these results to determine the receptor occupancy of CVL-231 as a function of dose. Using paired baseline-blocking PET scans, we quantified total volume of distribution, binding potential, and receptor occupancy. Both blood-based and reference region-based methods quantified M4 receptor levels across brain regions. The 2-tissue 4-parameter kinetic model best fitted regional [11C]MK-6884-time activity curves. Only the caudate nucleus and putamen displayed statistically significant [11C]MK-6884 uptake and dose-dependent blocking by CVL-231. For binding potential and receptor occupancy quantification, the simplified reference tissue model using the grey cerebellum as a reference region was employed. CVL-231 demonstrated dose-dependent M4 receptor occupancy in the striatum of the NHP brain and shows promise for further development in clinical trials.
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Macaca mulatta , Tomografía de Emisión de Positrones , Receptor Muscarínico M4 , Animales , Tomografía de Emisión de Positrones/métodos , Receptor Muscarínico M4/metabolismo , Regulación Alostérica , Masculino , Radioisótopos de Carbono , Óxidos S-Cíclicos/farmacología , Radiofármacos/farmacocinética , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Cinética , Femenino , Compuestos de Azabiciclo/farmacología , Compuestos de Azabiciclo/farmacocinéticaRESUMEN
Cholinergic signaling plays a crucial role in the regulation of adult hippocampal neurogenesis; however, the mechanisms by which acetylcholine mediates neurogenic effects are not completely understood. Here, we report the expression of muscarinic acetylcholine receptor subtype M4 (M4 mAChR) on a subpopulation of neural precursor cells (NPCs) in the adult mouse hippocampus, and demonstrate that its pharmacological stimulation promotes their proliferation, thereby enhancing the production of new neurons in vivo. Using a targeted ablation approach, we also show that medial septum (MS) and the diagonal band of Broca (DBB) cholinergic neurons support both the survival and morphological maturation of adult-born neurons in the mouse hippocampus. Although the systemic administration of an M4-selective allosteric potentiator fails to fully rescue the MS/DBB cholinergic lesion-induced decrease in hippocampal neurogenesis, it further exacerbates the impairment in the morphological maturation of adult-born neurons. Collectively, these findings reveal stage-specific roles of M4 mAChRs in regulating adult hippocampal neurogenesis, uncoupling their positive role in enhancing the production of new neurons from the M4-induced inhibition of their morphological maturation, at least in the context of cholinergic signaling dysfunction.
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Células-Madre Neurales , Receptor Muscarínico M4 , Ratones , Animales , Receptor Muscarínico M4/metabolismo , Células-Madre Neurales/metabolismo , Hipocampo/metabolismo , Neurogénesis/genética , Colinérgicos/metabolismo , Colinérgicos/farmacología , Proliferación CelularRESUMEN
INTRODUCTION: Successful phase 3 trials of KarXT in people with schizophrenia herald a new era of treating the disorder with drugs that do not target the dopamine D2 receptor. The active component of KarXT is xanomeline, a muscarinic (CHRM) M1 and M4 agonist, making muscarinic receptors a viable target for treating schizophrenia. AREAS COVERED: This review covers the process of taking drugs that activate the muscarinic M1 and M4 receptors from conceptualization to the clinic and details the mechanisms by which activating the CHRM1 and 4 can affect the broad spectrum of symptoms experienced by people with schizophrenia. EXPERT OPINION: Schizophrenia is a syndrome which means drugs that activate muscarinic M1 and M4 receptors, as was the case for antipsychotic drugs acting on the dopamine D2 receptor, will not give optimal outcomes in everyone within the syndrome. Thus, it would be ideal to identify people who are responsive to drugs activating the CHRM1 and 4. Given knowledge of the actions of these receptors, it is possible treatment non-response could be restricted to sub-groups within the syndrome who have deficits in cortical CHRM1 or those with one of the cognitive endophenotypes that may be identifiable by changes in the blood transcriptome.
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Antipsicóticos , Esquizofrenia , Humanos , Esquizofrenia/tratamiento farmacológico , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/genética , Receptor Muscarínico M4/uso terapéutico , Antipsicóticos/farmacología , Antipsicóticos/uso terapéutico , Receptores de Dopamina D2/uso terapéutico , Receptor Muscarínico M1RESUMEN
BACKGROUND: Research suggests that cholinergic muscarinic 1 (M1) and/or muscarinic 4 (M4) receptors may be involved in the pathophysiology of psychotic disorders. Agonistic modulation of these receptors can offer new treatment options. AIM: To provide an overview of current research on the role of cholinergic M1 and M4 receptors in the development and treatment of psychoses, with special attention to the development of new drugs such as xanomeline and emraclidine. METHOD: To obtain an overview, we searched for English-language studies published in PubMed, Embase, and PsycInfo up until June 1, 2023. We examined the role and effects of M1 and/or M4 agonists in schizophrenia. Additionally, we consulted clinical trial registers. RESULTS: Our search strategy resulted in nine published articles on five clinical studies. These studies revealed that reduced presence of M1 receptors, primarily in the frontal cortex, and M4 receptors, primarily in the basal ganglia, are associated with psychoses. M1 and M4 receptors modulate dopaminergic activity in the ventral tegmentum and striatum through various pathways. Several M1 and/or M4 agonists, partial agonists, and positive allosteric modulators (PAMs) have been developed. Drugs exhibiting agonistic activity on M1 and/or M4 receptors, such as xanomeline-trospium (phase 2 and 3 studies) and emraclidine (phase 1b studies), have shown positive effects on cognitive and potentially negative symptoms in patients with schizophrenia. CONCLUSION: M1 and/or M4 receptor agonists show potential as new treatment strategies for individuals with psychotic disorders. Although initial studies with xanomeline-trospium and emraclidine have shown positive results, further research is needed to assess their long-term efficacy, safety, and tolerability before these new medications can be evaluated.
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Trastornos Psicóticos , Receptor Muscarínico M1 , Humanos , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Trastornos Psicóticos/tratamiento farmacológico , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/metabolismoRESUMEN
M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.
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Acetilcolina , Receptores Muscarínicos , Ratas , Humanos , Ratones , Animales , Acetilcolina/metabolismo , Receptores Muscarínicos/metabolismo , Receptor Muscarínico M4/metabolismo , Atropina , Ligandos , Colinérgicos , Antagonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/metabolismo , Receptor Muscarínico M2/metabolismo , Ensayo de Unión Radioligante , Receptor Muscarínico M1/metabolismoRESUMEN
Current treatment options for prostate cancer focus on targeting androgen receptor (AR) signaling. Inhibiting effects of AR may activate neuroendocrine differentiation and lineage plasticity pathways, thereby promoting the development of neuroendocrine prostate cancer (NEPC). Understanding the regulatory mechanisms of AR has important clinical implications for this most aggressive type of prostate cancer. Here, we demonstrated the tumor-suppressive role of the AR and found that activated AR could directly bind to the regulatory sequence of muscarinic acetylcholine receptor 4 (CHRM4) and downregulate its expression. CHRM4 was highly expressed in prostate cancer cells after androgen-deprivation therapy (ADT). CHRM4 overexpression may drive neuroendocrine differentiation of prostate cancer cells and is associated with immunosuppressive cytokine responses in the tumor microenvironment (TME) of prostate cancer. Mechanistically, CHRM4-driven AKT/MYCN signaling upregulated the interferon alpha 17 (IFNA17) cytokine in the prostate cancer TME after ADT. IFNA17 mediates a feedback mechanism in the TME by activating the CHRM4/AKT/MYCN signaling-driven immune checkpoint pathway and neuroendocrine differentiation of prostate cancer cells. We explored the therapeutic efficacy of targeting CHRM4 as a potential treatment for NEPC and evaluated IFNA17 secretion in the TME as a possible predictive prognostic biomarker for NEPC.
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Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Proteínas Proto-Oncogénicas c-akt , Antagonistas de Andrógenos/uso terapéutico , Interferón-alfa/uso terapéutico , Microambiente Tumoral , Línea Celular Tumoral , Diferenciación Celular , Receptores Androgénicos/metabolismo , Receptor Muscarínico M4/uso terapéuticoRESUMEN
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.
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Receptor Muscarínico M4 , Receptores Muscarínicos , Humanos , Acetilcolina/metabolismo , Regulación Alostérica , Sitio Alostérico , Microscopía por Crioelectrón , Ligandos , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/metabolismoRESUMEN
Barbeau's seesaw hypothesis of dopamine-acetylcholine balance has predominated movement disorders literature for years. Both the simplicity of the explanation and the matching efficacy of anticholinergic treatment in movement disorders seem to support this hypothesis. However, evidence from translational and clinical studies in movement disorders indicates that many features of this simple balance are lost, broken, or absent from movement disorders models or in imaging studies of patients with these disorders. This review reappraises the dopamine-acetylcholine balance hypothesis in light of recent evidence and describes how the Gαi/o coupled muscarinic M4 receptor acts in opposition to dopamine signaling in the basal ganglia. We highlight how M4 signaling can ameliorate or exacerbate movement disorders symptoms and physiological correlates of these symptoms in specific disease states. Furthermore, we propose future directions for investigation of this mechanisms to fully understand the potential efficacy of M4 targeting therapeutics in movement disorders. Overall, initial evidence suggest that M4 is a promising pharmaceutical target to ameliorate motor symptoms of hypo- and hyper-dopaminergic disorders.
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Acetilcolina , Trastornos del Movimiento , Humanos , Receptor Muscarínico M4 , Dopamina , ColinérgicosRESUMEN
Cognitive impairment is a predictor of disability across different neuropsychiatric conditions, and cognitive abilities are also strongly related to educational attainment and indices of life success in the general population. Previous attempts at drug development for cognitive enhancement have commonly attempted to remedy defects in transmitters systems putatively associated with the conditions of interest such as the glutamate system in schizophrenia. Recent studies of the genomics of cognitive performance have suggested influences that are common in the general population and in different neuropsychiatric conditions. Thus, it seems possible that transmitter systems that are implicated for cognition across neuropsychiatric conditions and the general population would be a viable treatment target. We review the scientific data on cognition and the muscarinic cholinergic receptor system (M1 and M4) across different diagnoses, in aging, and in the general population. We suggest that there is evidence suggesting potential beneficial impacts of stimulation of critical muscarinic receptors for the enhancement of cognition in a broad manner, as well as the treatment of psychotic symptoms. Recent developments make stimulation of the M1 receptor more tolerable, and we identify the potential benefits of M1 and M4 receptor stimulation as a trans-diagnostic treatment model.
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Trastornos Psicóticos , Receptor Muscarínico M4 , Humanos , Cognición/fisiología , Trastornos Psicóticos/diagnóstico , Receptor Muscarínico M1 , ColinérgicosRESUMEN
Degeneration of the cholinergic basal forebrain is implicated in the development of cognitive deficits and sleep/wake architecture disturbances in mild cognitive impairment (MCI) and Alzheimer's disease (AD). Indirect-acting muscarinic cholinergic receptor agonists, such as acetylcholinesterase inhibitors (AChEIs), remain the only FDA-approved treatments for the cognitive impairments observed in AD that target the cholinergic system. Novel direct-acting muscarinic cholinergic receptor agonists also improve cognitive performance in young and aged preclinical species and are currently under clinical development for AD. However, little is known about the effects of direct-acting muscarinic cholinergic receptor agonists on disruptions of sleep/wake architecture and arousal observed in nonpathologically aged rodents, nonhuman primates, and clinical populations. The purpose of the present study was to provide the first assessment of the effects of the direct-acting M1/M4-preferring muscarinic cholinergic receptor agonist xanomeline on sleep/wake architecture and arousal in young and nonpathologically aged mice, in comparison with the AChEI donepezil, when dosed in either the active or inactive phase of the circadian cycle. Xanomeline produced a robust reversal of both wake fragmentation and disruptions in arousal when dosed in the active phase of nonpathologically aged mice. In contrast, donepezil had no effect on either age-related wake fragmentation or arousal deficits when dosed during the active phase. When dosed in the inactive phase, both xanomeline and donepezil produced increases in wake and arousal and decreases in nonrapid eye movement sleep quality and quantity in nonpathologically aged mice. Collectively, these novel findings suggest that direct-acting muscarinic cholinergic agonists such as xanomeline may provide enhanced wakefulness and arousal in nonpathological aging, MCI, and AD patient populations.
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Nivel de Alerta , Agonistas Muscarínicos , Trastornos Neurocognitivos , Receptor Muscarínico M1 , Receptor Muscarínico M4 , Sueño , Animales , Ratones , Acetilcolinesterasa/metabolismo , Nivel de Alerta/efectos de los fármacos , Nivel de Alerta/fisiología , Colinérgicos/farmacología , Colinérgicos/uso terapéutico , Donepezilo/farmacología , Donepezilo/uso terapéutico , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/metabolismo , Tiadiazoles/farmacología , Tiadiazoles/uso terapéutico , Vigilia/efectos de los fármacos , Vigilia/fisiología , Sueño/efectos de los fármacos , Sueño/fisiología , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Trastornos Neurocognitivos/tratamiento farmacológico , Trastornos Neurocognitivos/metabolismoRESUMEN
The muscarinic cholinergic M4 receptor subtype (M4 mAChR) is densely expressed in brain areas known to be involved in the reinforcing effects of drugs of abuse and we were the first to show that mice lacking M4 mAChRs exhibit elevated operant responding for alcohol and reduced capacity to extinguish this alcohol-seeking behaviour. Here we explore possible underlying determinants of this phenotype. We subjected M4 mAChR knockout mice and their littermate wildtype controls to tests of spontaneous activity, learning and memory, novelty seeking, as well as anxiety and examined the relationship of a newly discovered "disinhibited" endophenotype of these mice with voluntary alcohol consumption and relapse. We found a positive correlation between "disinhibited" behaviour on the plus maze and alcohol preference as well as relapse to alcohol drinking after a period of abstinence. Taken together, these data point to M4 mAChRs as a potential target for improved treatment strategies for alcohol use disorder. This receptor should be further investigated for its involvement in modulating behavioural inhibition in relation to loss of control over consumption of alcohol.
Asunto(s)
Endofenotipos , Receptor Muscarínico M4 , Consumo de Bebidas Alcohólicas/genética , Animales , Etanol/farmacología , Ratones , Ratones Noqueados , Agonistas Muscarínicos/farmacología , RecurrenciaRESUMEN
Many Food and Drug Administration (FDA)-approved drugs are structural analogues of the endogenous (natural) ligands of G protein-coupled receptors (GPCRs). However, it is becoming appreciated that chemically distinct ligands can bind to GPCRs in conformations that lead to different cellular signaling events, a phenomenon termed biased agonism. Despite this, the rigorous experimentation and analysis required to identify biased agonism are often not undertaken in most clinical candidates and go unrealized. Recently, xanomeline, a muscarinic acetylcholine receptor (mAChR) agonist, has entered phase III clinical trials for the treatment of schizophrenia. If successful, xanomeline will be the first novel FDA-approved antipsychotic drug in almost 50 years. Intriguingly, xanomeline's potential for biased agonism at the mAChRs and, in particular, the M4 mAChR, the most promising receptor target for schizophrenia, has not been assessed. Here, we quantify the biased agonism profile of xanomeline and three other mAChR agonists in Chinese hamster ovary cells recombinantly expressing the M4 mAChR. Agonist activity was examined across nine distinct signaling readouts, including the activation of five different G protein subtypes, ERK1/2 phosphorylation, ß-arrestin recruitment, calcium mobilization, and cAMP regulation. Relative to acetylcholine (ACh), xanomeline was biased away from ERK1/2 phosphorylation and calcium mobilization compared to Gαi2 protein activation. These findings likely have important implications for our understanding of the therapeutic action of xanomeline and call for further investigation into the in vivo consequences of biased agonism in drugs targeting the M4 mAChR for the treatment of schizophrenia.
Asunto(s)
Calcio , Tiadiazoles , Acetilcolina/metabolismo , Acetilcolina/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Ligandos , Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/uso terapéutico , Piridinas , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M4/agonistas , Receptores Acoplados a Proteínas G , Receptores Muscarínicos , Tiadiazoles/químicaRESUMEN
Cancer-induced bone pain (CIBP) is a common pain in clinics, which can reduce the quality of life and increase the mortality of patients, but the treatment of CIBP is limited. This study was designed to investigate the analgesic effect of α-cobratoxin on CIBP and further to explore the molecular target and potential signal pathway. As shown by the mechanical allodynia test in a CIBP rat model, administration of α-cobratoxin produced significant analgesia in a dose-dependent manner, and the analgesic effects were blocked by pretreatment with an intrathecal injection of M4 mAChR-siRNA or intraperitoneal injection of tropicamide, an antagonist of M4 muscarinic cholinergic receptor. Whole-cell patch-clamp recording showed that α-cobratoxin can decrease the spontaneous firing and spontaneous excitatory postsynaptic currents of SDH neurons in CIBP rats. In primary lumber SDH neurons, intracellular calcium measurement revealed that α-cobratoxin decreased intracellular calcium concentration, and immunofluorescence demonstrated that M4 muscarinic cholinergic receptor and CaMKII/CREB were co-expressed. In the CIBP model and primary SDH neurons, Western blot showed that the levels of p-CaMKII and p-CREB were increased by α-cobratoxin and the effect of α-cobratoxin was antagonized by M4 mAChR-siRNA. The quantitative polymerase chain reaction (qPCR) results showed that α-cobratoxin downregulated the expression of proinflammatory cytokines through M4 muscarinic cholinergic receptor in SDH. These results suggest that α-cobratoxin may activate M4 muscarinic cholinergic receptor, triggering the inhibition of SDH neuronal excitability via CaMKII signaling pathway, thereby resulting in antagonistic effects in the CIBP rat model.