Development of VU6036864: A Triazolopyridine-Based High-Quality Antagonist Tool Compound of the M5 Muscarinic Acetylcholine Receptor.

While the muscarinic acetylcholine receptor mAChR subtype 5 (M5) has been studied over decades, recent findings suggest that more in-depth research is required to elucidate a thorough understanding of its physiological function related to neurological and psychiatric disorders. Our efforts to identify potent, selective, and pharmaceutically favorable next-generation M5 antagonist tool compounds have led to the discovery of a novel triazolopyridine-based series. In particular, VU6036864 (45) showed exquisite potency (human M5 IC50 = 20 nM), good subtype selectivity (>500 fold selectivity against human M1-4), desirable brain exposure (Kp = 0.68, Kp,uu = 0.65), and high oral bioavailability (%F > 100%). VU6036864 (45) and its close analogues will support further studies of M5 as advanced antagonist tool compounds and play an important role in the emerging biology of M5.

Ventral Tegmental Area M5 Muscarinic Receptors Mediate Effort-Choice Responding and Nucleus Accumbens Dopamine in a Sex-Specific Manner .

Optimization of effort-related choices is impaired in depressive disorders. Acetylcholine (ACh) and dopamine (DA) are linked to depressive disorders, and modulation of ACh tone in the ventral tegmental area (VTA) affects mood-related behavioral responses in rats. However, it is unknown if VTA ACh mediates effort-choice behaviors. Using a task of effort-choice, rats can choose to lever press on a fixed-ratio 5 (FR5) schedule for a more-preferred food or consume freely available, less-preferred food. VTA administration of physostigmine (1 µg and 2 µg/side), a cholinesterase inhibitor, reduced FR5 responding for the more-preferred food while leaving consumption of the less-preferred food intact. VTA infusion of the M5 muscarinic receptor negative allosteric modulator VU6000181 (3 µM, 10 µM, 30 µM/side) did not affect lever pressing or chow consumption. However, VU6000181 (30 µM/side) coadministration with physostigmine (2 µg/side) attenuated physostigmine-induced decrease in lever pressing in female and male rats and significantly elevated lever pressing above vehicle baseline levels in male rats. In in vivo voltammetry experiments, VTA infusion of combined physostigmine and VU6000181 did not significantly alter evoked phasic DA release in the nucleus accumbens core (NAc) in female rats. In male rats, combined VTA infusion of physostigmine and VU6000181 increased phasic evoked DA release in the NAc compared with vehicle, physostigmine, or VU6000181 infusion alone. These data indicate a critical role and potential sex differences of VTA M5 receptors in mediating VTA cholinergic effects on effort choice behavior and regulation of DA release. SIGNIFICANCE STATEMENT: Effort-choice impairments are observed in depressive disorders, which are often treatment resistant to currently available thymoleptics. The role of ventral tegmental area (VTA) acetylcholine muscarinic M5 receptors, in a preclinical model of effort-choice behavior, is examined. Using the selective negative allosteric modulator of the M5 receptor VU6000181, we show the role of VTA M5 receptors on effort-choice and regulation of dopamine release in the nucleus accumbens core. This study supports M5 receptors as therapeutic targets for depression.

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1-4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

Muscarinic acetylcholine receptor M5 is involved in spermatogenesis through the modification of cell-cell junctions.

Muscarinic acetylcholine receptor (mAChR) antagonists have been reported to decrease male fertility; however, the roles of mAChRs in spermatogenesis and the underlying mechanisms are not understood yet. During spermatogenesis, extensive remodeling between Sertoli cells and/or germ cells interfaces takes place to accommodate the transport of developing germ cells across the blood-testis barrier (BTB) and adluminal compartment. The cell-cell junctions play a vital role in the spermatogenesis process. This study used ICR male mice and spermatogonial cells (C18-4) and Sertoli cells (TM-4). shRNA of control or M5 gene was injected into 5-week-old ICR mice testes. Ten days post-viral grafting, mice were deeply anesthetized with pentobarbital and the testes were collected. One testicle was fresh frozen for RNA-seq analysis or Western blotting (WB). The second testicle was fixed for immunofluorescence staining (IHF). C18-4 or TM-4 cells were treated with shRNA of control or M5 gene. Then, the cells were collected for RNA-seq analysis, WB, or IHF. Knockdown of mAChR M5 disrupted mouse spermatogenesis and damaged the actin-based cytoskeleton and many types of junction proteins in both Sertoli cells and germ cells. M5 knockdown decreased Phldb2 expression in both germ cells and Sertoli cells which suggested that Phldb2 may be involved in cytoskeleton and cell-cell junction formation to regulate spermatogenesis. Our investigation has elucidated a novel role for mAChR M5 in the regulation of spermatogenesis through the interactions of Phldb2 and cell-cell junctions. M5 may be an attractive future therapeutic target in the treatment of male reproductive disorders.

Autoantibodies against M5-muscarinic and beta1-adrenergic receptors in periodontitis patients.

Autoantibodies against muscarinic and beta1-adrenergic receptors are considered a potential cause and/or risk factor for chronic heart failure. Association of periodontitis with such autoantibodies and with impaired heart function has been observed in patients exposed to endemic Chagas' disease, which triggers by itself cardiomyopathy and receptor immunization.Here we studied the association between periodontitis, markers of cardiac injury and receptor autoimmunization in periodontitis patients (n = 147) not exposed to Chagas' disease. The autoantibodies were determined by IgG binding to native intact muscarinic and beta1-adrenergic receptors or to a cyclic peptide mimicking the disease-relevant conformational autoepitope presented by the active beta1-adrenergic receptor. Possible cardiac injury and inflammatory status were judged by serum levels of proBNP/Troponin I and CRP/IL-6, respectively. These parameters were analysed in healthy and periodontally diseased individuals as well as before and after periodontal therapy.Patients with periodontitis had significantly (p < 0.001) higher levels of autoantibodies against M5-muscarinic and beta1-adrenergic receptors, which further increased following periodontal therapy. Receptor autoantibodies were associated with increased inflammatory status but not with increased markers of cardiac injury. Thus, our data indicate that periodontitis triggers systemic inflammation, which is associated with receptor autoimmunization, and, independently thereof, with cardiac injury.

Examining the role of muscarinic M5 receptors in VTA cholinergic modulation of depressive-like and anxiety-related behaviors in rats.

Acetylcholine is implicated in mood disorders including depression and anxiety. Increased cholinergic tone in humans and rodents produces pro-depressive and anxiogenic-like effects. Cholinergic receptors in the ventral tegmental area (VTA) are known to mediate these responses in male rats, as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and the forced swim test (FST). However, these effects have not been examined in females, and the VTA muscarinic receptor subtype(s) mediating the pro-depressive and anxiogenic-like behavioral effects of increased cholinergic tone are unknown. We first examined the behavioral effects of increased VTA cholinergic tone in male and female rats, and then determined whether VTA muscarinic M5 receptors were mediating these effects. VTA infusion of the acetylcholinesterase inhibitor physostigmine (0.5 µg, 1 µg and 2 µg/side) in males and females produced anhedonic-like, anxiogenic, pro-depressive-like responses on the SPT, EPM, and FST. In females, VTA administration of the muscarinic M5 selective negative allosteric modulator VU6000181 (0.68 ng, 2.3 ng, 6.8 ng/side for a 3 µM, 10 µM, 30 µM/side infusion) did not alter SPT, EPM nor FST behavior. However, in males intra-VTA infusion of VU6000181 alone reduced time spent immobile on the FST. Furthermore, co-infusion of VU6000181 with physostigmine, in male and female rats, attenuated the pro-depressive and anxiogenic-like behavioral responses induced by VTA physostigmine alone, in the SPT, EPM, and FST. Together, these data reveal a critical role of VTA M5 receptors in mediating the anhedonic, anxiogenic, and depressive-like behavioral effects of increased cholinergic tone in the VTA.

Simultaneous activation of mGlu2 and muscarinic receptors reverses MK-801-induced cognitive decline in rodents.

The activity of an allosteric agonist of muscarinic M1 receptor, VU0357017, and a positive allosteric modulator (PAM) of M5 receptor, VU0238429, were investigated alone or in combination with the mGlu2 receptor PAM, LY487379 using the following behavioural tests: prepulse inhibition (PPI), novel object recognition (NOR), and spatial delayed alternation (SDA). VU0357017 (10 and 20 mg/kg) and VU0238429 (5 and 10 mg/kg) reversed deficits in PPI while VU0238429 (2.5 and 5 mg/kg) was effective in SDA. The simultaneous administration of subeffective doses of M1 or M5 activators (5, 1, or 0.25 mg/kg) with LY487379 (0.5 mg/kg) induced the same effect as that observed for the active dose of each compound. Selective M1 or M5 receptor blockers antagonized the effect exerted by these combinations, and pharmacokinetic studies confirmed independent transport through the blood-brain barrier. The expression of both receptors (M1 and M5) was established in brain structures involved in cognition (neocortex, hippocampus, and entorhinal cortex) in both the rat and the mouse brains by immunofluorescence staining. Specifically, double neuronal staining of mGlu2-M1 and mGlu2-M5 receptors was observed in many areas of the rat brain, while the number of double-stained mGlu2-M1 receptors was moderate in the mouse brain with no mGlu2-M5 colocalization. Finally, the combined administration of subeffective doses of the compounds did not alter prolactin levels or motor coordination, in contrast to the compounds given alone at the highest dose or in combination with standard neuroleptics.

Crystal structure of the M5 muscarinic acetylcholine receptor.

The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5 mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2 and M5 mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

Acute Negative Allosteric Modulation of M5 Muscarinic Acetylcholine Receptors Inhibits Oxycodone Self-Administration and Cue-Induced Reactivity with No Effect on Antinociception.

Opioid use disorder (OUD) is a debilitating neuropsychiatric condition characterized by compulsive opioid use, dependence, and repeated relapse after periods of abstinence. Given the high risk of developing OUD following prescription opioid use, the continued need for opioid-induced analgesia, and the limitations of current OUD treatments, it is necessary to develop novel, non-opioid-based treatments for OUD and decrease abuse potential of prescription opioids. Recent evidence suggests that negative allosteric modulation (NAM) of the M5 muscarinic acetylcholine receptor (M5 mAChR) may provide an alternative therapeutic approach for the treatment of OUD. Previous studies demonstrated localization of M5 mAChR expression within the mesocorticolimbic reward circuitry and that the selective M5 NAM ML375 attenuates both cocaine and alcohol self-administration in rats. In the present study, the effects of ML375 were evaluated in rats self-administering the µ-opioid agonists oxycodone or remifentanil on a progressive ratio (PR) schedule or on cue reactivity (a rodent model of relapse) in the absence of oxycodone following 72 h of abstinence. ML375 reduced the PR break point for oxycodone and remifentanil self-administration and attenuated cue-elicited responding. Importantly, ML375 did not affect sucrose pellet-maintained responding on a PR schedule or opioid-induced antinociception using the hot-plate and tail-flick assays. We also confirm expression of M5 mAChR mRNA in the ventral tegmental area and show that this is primarily on dopamine (tyrosine hydroxylase mRNA-positive) neurons. Taken together, these findings suggest that selective functional antagonism of the M5 mAChR may represent a novel, non-opioid-based treatment for OUD.

The Muscarinic Acetylcholine Receptor M5: Therapeutic Implications and Allosteric Modulation.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) was the most recent mAChR to be cloned and has since emerged as a potential therapeutic target for a number of indications. Early studies with knockout animals have provided clues to the receptor's role in physiological processes related to Alzheimer's disease, schizophrenia, and addiction, and until recently, useful subtype-selective tools to further probe the pharmacology of M5 have remained elusive. Small-molecule allosteric modulators have since gained traction as a means by which to selectively examine muscarinic pharmacology. This review highlights the discovery and optimization of M5 positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs).

Muscarinic M5 receptors trigger acetylcholine-induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cß (PLCß) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.

Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback.

We have recently identified a cholinergic chemosensory cell in the urethral epithelium, urethral brush cell (UBC), that, upon stimulation with bitter or bacterial substances, initiates a reflex detrusor activation. Here, we elucidated cholinergic mechanisms that modulate UBC responsiveness. We analyzed muscarinic acetylcholine receptor (M1-5 mAChR) expression by using RT-PCR in UBCs, recorded [Ca2+]i responses to a bitter stimulus in isolated UBCs of wild-type and mAChR-deficient mice, and performed cystometry in all involved strains. The bitter response of UBCs was enhanced by global cholinergic and selective M2 inhibition, diminished by positive allosteric modulation of M5, and unaffected by M1, M3, and M4 mAChR inhibitors. This effect was not observed in M2 and M5 mAChR-deficient mice. In cystometry, M5 mAChR-deficient mice demonstrated signs of detrusor overactivity. In conclusion, M2 and M5 mAChRs attenuate the bitter response of UBC via a cholinergic negative autocrine feedback mechanism. Cystometry suggests that dysfunction, particularly of the M5 receptor, may lead to such symptoms as bladder overactivity.-Deckmann, K., Rafiq, A., Erdmann, C., Illig, C., Durschnabel, M., Wess, J., Weidner, W., Bschleipfer, T., Kummer, W. Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback.

Muscarinic M5 receptors modulate ethanol seeking in rats.

Despite the cost to both individual and society, alcohol use disorders (AUDs) remain a major health risk within society, and both relapse and heavy drinking are still poorly controlled with current medications. Here we demonstrate for the first time that a centrally active and selective negative allosteric modulator for the rat M5 muscarinic acetylcholine receptor (mAChR), ML375, decreases ethanol self-administration and attenuates cue-induced reinstatement of ethanol seeking in ethanol-preferring (iP) rats. Importantly, ML375 did not affect sucrose self-administration or general locomotor activity indicative of a selective effect on ethanol seeking. Based on the expression profile of M5 mAChRs in the brain and the distinct roles different aspects of the dorsal striatum have on long-term and short-term ethanol use, we studied whether intra-striatal microinjection of ML375 modulated ethanol intake in rats. We show in iP rats with an extensive history of ethanol intake that intra-dorsolateral (DL), but not intra-dorsomedial, striatal injections of ML375 reduced ethanol self-administration to a similar extent as the nicotinic acetylcholine receptor ligand varenicline, which has preclinical and clinical efficacy in reducing the reinforcing effects of ethanol. These data implicate the DL striatum as a locus for the effects of cholinergic-acting drugs on ethanol seeking in rats with a history of long-term ethanol use. Accordingly, we demonstrate in rats that selectively targeting the M5 mAChR can modulate both voluntary ethanol intake and cue-induced ethanol seeking and thereby provide direct evidence that the M5 mAChR is a potential novel target for pharmacotherapies aimed at treating AUDs.

Selective inhibition of M5 muscarinic acetylcholine receptors attenuates cocaine self-administration in rats.

Cocaine use disorder (CUD) remains a debilitating health problem in the United States for which there are no Food and Drug Administration-approved treatment options. Accumulating anatomical and electrophysiological evidence indicates that the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5 ) plays a critical role in the regulation of the mesolimbic dopaminergic reward circuitry, a major site of action for cocaine and other psychostimulants. In addition, M5 knockout mice exhibit reduced cocaine self-administration behaviors with no differences in sugar pellet-maintained responding relative to wild-type mice. These findings suggest that selective inhibition of M5 mAChR may provide a novel pharmacological approach for targeting CUD. Recently, we reported the synthesis and characterization of ML375, a selective negative allosteric modulator (NAM) for the rat and human M5 mAChR with optimized pharmacokinetic properties for systemic dosing in rodents. In the present study, male Sprague-Dawley rats were trained to self-administer intravenous cocaine (0.1-0.75 mg/kg/infusion) under a 10-response fixed ratio or a progressive ratio schedule of reinforcement. Under both schedules of reinforcement, ML375 produced dose-related reductions in cocaine self-administration. ML375 also modestly reduced sugar pellet-maintained responding on the 10-response, fixed ratio schedule but had no effect under a progressive ratio schedule of reinforcement. Further, ML375 did not affect general motor output as assessed by a rotarod test. Collectively, these results provide the first demonstration that selective inhibition of M5 using the M5 NAM ML375 can attenuate both the reinforcing effects and the relative strength of cocaine and suggest that M5 NAMs may represent a promising, novel treatment approach for CUD.

Gßγ subunit activation promotes dopamine efflux through the dopamine transporter.

The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants-like cocaine and amphetamine-and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gßγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gßγ induces the release of DA through DAT. Specifically, a Gßγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gßγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gßγ. A TAT peptide containing the Gßγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gßγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gßγ-dependent mechanism. Collectively, our data show that Gßγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.

Promoter IV-BDNF deficiency disturbs cholinergic gene expression of CHRNA5, CHRM2, and CHRM5: effects of drug and environmental treatments.

Brain-derived neurotrophic factor (BDNF) promotes maturation of cholinergic neurons. However, how activity-dependent BDNF expression affects specific cholinergic gene expression remains unclear. This study addressed this question by determining mRNA levels of 22 acetylcholine receptor subunits, the choline transporter (CHT), and the choline acetyltransferase (ChAT) in mice deficient in activity-dependent BDNF via promoter IV (KIV) and control wild-type mice. Quantitative RT-PCR revealed significant reductions in nicotinic acetylcholine receptor alpha 5 (CHRNA5) in the frontal cortex and hippocampus and M5 muscarinic acetylcholine receptor (CHRM5) in the hippocampus, but significant increases in M2 muscarinic acetylcholine receptor (CHRM2) in the frontal cortex of KIV mice compared to wild-type mice. Three-week treatments with fluoxetine, phenelzine, duloxetine, imipramine, or an enriched environment treatment (EET) did not affect the altered expression of these genes except that EET increased CHRNA5 levels only in KIV frontal cortex. EET also increased levels of CHRNA7, CHT, and ChAT, again only in the KIV frontal cortex. The imipramine treatment was most prominent among the four antidepressants; it up-regulated hippocampal CHRM2 and frontal cortex CHRM5 in both genotypes, and frontal cortex CHRNA7 only in KIV mice. To the best of our knowledge, this is the first evidence that BDNF deficiency disturbs expression of CHRNA5, CHRM2, and CHRM5. Our results suggest that promoter IV-BDNF deficiency - which occurs under chronic stress - causes cholinergic dysfunctions via these receptors. EET is effective on CHRNA5, while its compensatory induction of other cholinergic genes or drugs targeting CHRNA5, CHRM2, and CHRM5 may become an alternative strategy to reverse these BDNF-linked cholinergic dysfunctions.

Continued optimization of the M5 NAM ML375: Discovery of VU6008667, an M5 NAM with high CNS penetration and a desired short half-life in rat for addiction studies.

This letter describes the continued optimization of M5 NAM ML375 (VU0483253). While a valuable in vivo tool compound, ML375has an excessively long elimination half-life in rat (t1/2=80h), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement). Thus, we required an M5 NAM of comparable potency to ML375, but with a rat t1/2 of less than 4h. Steep SAR plagued this chemotype, and here we detail aniline replacements that offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a single methyl group to the 9b-phenyl ring acted as a metabolic shunt, providing (S)-11 (VU6008667), an equipotent M5 NAM, with high CNS penetration, excellent selectivity versus M1-4 and the desired short half-life (t1/2=2.3h) in rat.

Ligand-based virtual screen for the discovery of novel M5 inhibitor chemotypes.

This Letter describes a ligand-based virtual screening campaign utilizing SAR data around the M5 NAMs, ML375 and VU6000181. Both QSAR and shape scores were employed to virtually screen a 98,000-member compound library. Neither approach alone proved productive, but a consensus score of the two models identified a novel scaffold which proved to be a modestly selective, but weak inhibitor (VU0549108) of the M5 mAChR (M5 IC50=6.2µM, M1-4 IC50s>10µM) based on an unusual 8-((1,3,5-trimethyl-1H-pyrazol-4-yl)sulfonyl)-1-oxa-4-thia-8-azaspiro[4,5]decane scaffold. [(3)H]-NMS binding studies showed that VU0549108 interacts with the orthosteric site (Ki of 2.7µM), but it is not clear if this is negative cooperativity or orthosteric binding. Interestingly, analogs synthesized around VU0549108 proved weak, and SAR was very steep. However, this campaign validated the approach and warranted further expansion to identify additional novel chemotypes.

Molecular Mechanisms of Action of M5 Muscarinic Acetylcholine Receptor Allosteric Modulators.

Recently, the first subtype-selective allosteric modulators of the M5 muscarinic acetylcholine receptor (mAChR) have been described, but their molecular mechanisms of action remain unknown. Using radioligand-binding and functional assays of inositol phosphate (IP) accumulation and Ca(2+) mobilization in a recombinant cell line stably expressing the human M5 mAChR, we investigated the effects of the positive allosteric modulator (PAM), ML380, and negative allosteric modulator, ML375. In functional assays, ML380 caused robust enhancements in the potency of the full agonists, acetylcholine (ACh), carbachol, and oxotremorine-M, while significantly increasing the maximal response to the partial agonist, pilocarpine. ML380 also demonstrated direct allosteric agonist activity. In contrast, ML375 displayed negative cooperativity with each of the agonists in a manner that varied with the pathway investigated and progressively reduced the maximal pilocarpine response. Radioligand-binding affinity cooperativity estimates were consistent with values derived from functional assays in some instances but not others, suggesting additional allosteric effects on orthosteric ligand efficacy. For ML375 this was confirmed in IP assays performed after reduction of receptor reserve by the alkylating agent, phenoxybenzamine, as it reduced the maximal ACh response. In contrast, ML380 enhanced only ACh potency after receptor alkylation, with no effect on maximal response, consistent with studies of the M1 mAChR with the prototypical PAM, BQZ12. Interaction studies between ML380 and ML375 also indicated that they most likely used an overlapping allosteric site. Our findings indicate that novel small-molecule modulators of the M5 mAChR display mixed mechanisms of action compared with previously characterized modulators of other mAChRs.

Muscarinic acetylcholine receptor binding affinities of pethidine analogs.

A series of pethidine analogs were synthesized and their affinities for the [(3)H]N-methyl-scopolamine (NMS) binding site on muscarinic acetylcholine receptors (mAChRs) were determined using M1, M3 or M5 human mAChRs expressed by Chinese hamster ovary (CHO) cell membranes. Compound 6b showed the highest binding affinities at M1, M3 and M5 mAChRs (Ki=0.67, 0.37, and 0.38 µM, respectively).