Frontal Cortex Lipid Alterations During the Onset of Alzheimer's Disease.

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.

M1/M4 receptors as potential therapeutic treatments for schizophrenia: A comprehensive study.

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.

Stimulation of the muscarinic receptor M4 regulates neural precursor cell proliferation and promotes adult hippocampal neurogenesis.

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.

[Muscarinic M1 and/or M4 receptor agonists as potential novel treatments for psychoses]. / Muscarine 1- en/of 4-receptoragonisten als potentieel nieuwe behandeling voor psychosen.

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.

Muscarinic M1 and M4 receptor agonists for schizophrenia: promising candidates for the therapeutic arsenal.

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.

Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic Receptor.

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.

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.

CHRM4/AKT/MYCN upregulates interferon alpha-17 in the tumor microenvironment to promote neuroendocrine differentiation of prostate cancer.

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.

The muscarinic M4 acetylcholine receptor exacerbates symptoms of movement disorders.

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.

Cross-diagnostic determinants of cognitive functioning: the muscarinic cholinergic receptor as a model system.

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.

M1/M4-Preferring Muscarinic Cholinergic Receptor Agonist Xanomeline Reverses Wake and Arousal Deficits in Nonpathologically Aged Mice.

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.

Inactivation of the cholinergic M4 receptor results in a disinhibited endophenotype predicting alcohol use.

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.

Biased Profile of Xanomeline at the Recombinant Human M4 Muscarinic Acetylcholine Receptor.

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.

Cobratoxin Alleviates Cancer-Induced Bone Pain in Rats via Inhibiting CaMKII Signaling Pathway after Acting on M4 Muscarinic Cholinergic Receptors.

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.

Effects of acute and repeated administration of the selective M4 PAM VU0152099 on cocaine versus food choice in male rats.

Ligands that stimulate muscarinic acetylcholine receptors 1 and 4 (M1 , M4 ) have shown promising effects as putative pharmacotherapy for cocaine use disorder in rodent assays. We have previously shown reductions in cocaine effects with acute M4 stimulation, as well as long-lasting, delayed reductions in cocaine taking and cocaine seeking with combined M1 /M4 receptor stimulation or with M1 stimulation alone. M4 stimulation opposes dopaminergic signalling acutely, but direct dopamine receptor antagonists have proved unhelpful in managing cocaine use disorder because they lose efficacy with long-term administration. It is therefore critical to determine whether M4 approaches themselves can remain effective with repeated or chronic dosing. We assessed the effects of repeated administration of the M4 positive allosteric modulator (PAM) VU0152099 in rats trained to choose between intravenous cocaine and a liquid food reinforcer to obtain quantitative measurement of whether M4 stimulation could produce delayed and lasting reduction in cocaine taking. VU0152099 produced progressively augmenting suppression of cocaine choice and cocaine intake, but produced neither rebound nor lasting effects after treatment ended. To compare and contrast effects of M1 versus M4 stimulation, we tested whether the M4 PAM VU0152100 suppressed cocaine self-administration in mice lacking CalDAG-GEFI signalling factor, required for M1 -mediated suppression of cocaine self-administration. CalDAG-GEFI ablation had no effect on M4 -mediated suppression of cocaine self-administration. These findings support the potential usefulness of M4 PAMs as pharmacotherapy to manage cocaine use disorder, alone or in combination with M1 -selective ligands, and show that M1 and M4 stimulation modulate cocaine-taking behaviour by distinct mechanisms.

Muscarinic Receptors Expression in the Peripheral Blood Cells Differentiate Dementia with Lewy Bodies from Alzheimer's Disease.

BACKGROUND: Central nervous system disruption of cholinergic (ACh) signaling, which plays a major role in cognitive processes, is well documented in dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). The expression of muscarinic ACh receptors type 1 and 4 (CHRM1 and CHRM4) has been reported to be altered in the brain of DLB patients. OBJECTIVE: We aim to assess the peripheral gene expression of CHRM1 and 4 in DLB as a possible marker as compared to AD and healthy control (HC) subjects. METHODS: Peripheral blood mononuclear cells were collected from 21 DLB, 13 AD, and 8 HC matched subjects. RT-PCR was performed to estimate gene expression of CHRM1 and CHRM4. RESULTS: Peripheral CHRM1 expression was higher and CHRM4 was lower in DLB and AD compared to HC, whereas both CHRM1 and CHRM4 levels were higher in AD compared to DLB patients. Receiver operating characteristics curves, with logistic regression analysis, showed that combining peripheral CHRM1 and CHRM4 levels, DLB and AD subjects were classified with an accuracy of 76.0%. CONCLUSION: Alterations of peripheral CHRM1 and CHRM4 was found in both AD and DLB patients as compared to HC. CHRM1 and CHRM4 gene expression resulted to be lower in DLB patients compared to AD. In the future, peripheral CHRM expression could be studied as a possible marker of neurodegenerative conditions associated with cholinergic deficit and a possible marker of response to acetylcholinesterase inhibitors.

Synthesis and characterization of chiral 6-azaspiro[2.5]octanes as potent and selective antagonists of the M4 muscarinic acetylcholine receptor.

In this manuscript, we report a series of chiral 6-azaspiro[2.5]octanes and related spirocycles as highly potent and selective antagonists of the muscarinic acetylcholine receptor subtype 4 (mAChR4). Chiral separation and subsequent X-ray crystallographic analysis of early generation analogs revealed the R enantiomer to possess excellent human and rat M4 potency, and further structure-activity relationship (SAR) studies on this chiral scaffold led to the discovery of VU6015241 (compound 19). Compound 19 is characterized by high M4 potency and selectivity across multiple species, excellent aqueous solubility, and moderate brain exposure in rodents after intraperitoneal administration.

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes - Part 2.

This Letter details our efforts to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace the 3-amino-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core which lead to the discovery of two novel tricyclic cores: a 7,9-dimethylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine core and 2,4-dimethylthieno[2,3-b:5,4-c']dipyridine core. Both tricyclic cores displayed low nanomolar potency against the human M4 receptor.

Monoamine Oxidase Inhibition by Major Tanshinones from Salvia miltiorrhiza and Selective Muscarinic Acetylcholine M4 Receptor Antagonism by Tanshinone I.

Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson's disease (PD). Lipophilic tanshinones are major phytoconstituents in the dried roots of Salvia miltiorrhiza that have demonstrated neuroprotective effects against dopaminergic neurotoxins and the inhibition of MAO-A. Since MAO-B inhibition is considered an effective therapeutic strategy for PD, we tested the inhibitory activities of three abundant tanshinone congeners against recombinant human MAO (hMAO) isoenzymes through in vitro experiments. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone, with an IC50 less than 10 µM. They also suppressed hMAO-B activity, with an IC50 below 25 µM. Although tanshinones are known to inhibit hMAO-A, their enzyme inhibition mechanism and binding sites have yet to be investigated. Enzyme kinetics and molecular docking studies have revealed the mode of inhibition and interactions of tanshinones during enzyme inhibition. Proteochemometric modeling predicted mAChRs as possible pharmacological targets of 1, and in vitro functional assays confirmed the selective M4 antagonist nature of 1 (56.1% ± 2.40% inhibition of control agonist response at 100 µM). These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.

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.