Potentiation of Muscarinic M3 Receptor Activation through a New Allosteric Site with a Novel Positive Allosteric Modulator ASP8302.

Muscarinic M3 (M3) receptors mediate a wide range of acetylcholine (ACh)-induced functions, including visceral smooth-muscle contraction and glandular secretion. Positive allosteric modulators (PAMs) can avoid various side effects of muscarinic agonists with their spatiotemporal receptor activation control and potentially better subtype selectivity. However, the mechanism of allosteric modulation of M3 receptors is not fully understood, presumably because of the lack of a potent and selective PAM. In this study, we investigated the pharmacological profile of ASP8302, a novel PAM of M3 receptors, and explored the principal site of amino-acid sequences in the human M3 receptor required for the potentiation of receptor activation. In cells expressing human M3 and M5 receptors, ASP8302 shifted the concentration-response curve (CRC) for carbachol to the lower concentrations with no significant effects on other subtypes. In a binding study with M3 receptor-expressing membrane, ASP8302 also shifted the CRC for ACh without affecting the binding of orthosteric agonists. Similar shifts in the CRC of contractions by multiple stimulants were also confirmed in isolated human bladder strips. Mutagenesis analysis indicated no interaction between ASP8302 and previously reported allosteric sites; however, it identified threonine 230 as the amino acid essential for the PAM effect of ASP8302. These results demonstrate that ASP8302 enhances the activation of human M3 receptors by interacting with a single amino acid distinct from the reported allosteric sites. Our findings suggest not only a novel allosteric site of M3 receptors but also the potential application of ASP8302 to diseases caused by insufficient M3 receptor activation. SIGNIFICANCE STATEMENT: The significance of this study is that the novel M3 receptor positive allosteric modulator ASP8302 enhances the activation of human M3 receptor by interacting with a residue distinct from the reported allosteric sites. The finding of Thr230 as a novel amino acid involved in the allosteric modulation of M3 receptors provides significant insight into further research of the mechanism of allosteric modulation of M3 and other muscarinic receptors.

Preparation of a First 18F-Labeled Agonist for M1 Muscarinic Acetylcholine Receptors.

M1 muscarinic acetylcholine receptors (mAChRs) are abundant in postsynaptic nerve terminals of all forebrain regions and have been implicated in the cognitive decline associated with Alzheimer's disease and other CNS pathologies. Consequently, major efforts have been spent in the development of subtype-selective positron emission tomography (PET) tracers for mAChRs resulting in the development of several 11C-labeled probes. However, protocols for the preparation of 18F-labeled mAChR-ligands have not been published so far. Here, we describe a straightforward procedure for the preparation of an 18F-labeled M1 mAChR agonist and its corresponding pinacol boronate radiolabeling precursor and the non-radioactive reference compound. The target compounds were prepared from commercially available aryl fluorides and Boc protected 4-aminopiperidine using a convergent reaction protocol. The radiolabeling precursor was prepared by a modification of the Miyaura reaction and labeled via the alcohol-enhanced Cu-mediated radiofluorination. The developed procedure afforded the radiotracer in a non-decay-corrected radiochemical yield of 17 ± 3% (n = 3) and in excellent radiochemical purity (>99%) on a preparative scale. Taken together, we developed a straightforward protocol for the preparation of an 18F-labeled M1 mAChR agonist that is amenable for automation and thus provides an important step towards the routine production of a 18F-labeled M1 selective PET tracer for experimental and diagnostic applications.

Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Regiospecific Introduction of Halogens on the 2-Aminobiphenyl Subunit Leading to Highly Potent and Selective M3 Muscarinic Acetylcholine Receptor Antagonists and Weak Inverse Agonists.

Muscarinic M3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M3 subtype selectivity over M2, while 3'-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl- and two quinuclidinium-substituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with Ki values from 0.069 to 0.084 nM, as well as high selectivity over the M2 subtype (46- to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.

Discovery of [11C]MK-6884: A Positron Emission Tomography (PET) Imaging Agent for the Study of M4Muscarinic Receptor Positive Allosteric Modulators (PAMs) in Neurodegenerative Diseases.

The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridine-related M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [11C]MK-6884.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo. Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (ß cooperativity). Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

Optical Control of Cardiac Function with a Photoswitchable Muscarinic Agonist.

Light-triggered reversible modulation of physiological functions offers the promise of enabling on-demand spatiotemporally controlled therapeutic interventions. Optogenetics has been successfully implemented in the heart, but significant barriers to its use in the clinic remain, such as the need for genetic transfection. Herein, we present a method to modulate cardiac function with light through a photoswitchable compound and without genetic manipulation. The molecule, named PAI, was designed by introduction of a photoswitch into the molecular structure of an M2 mAChR agonist. In vitro assays revealed that PAI enables light-dependent activation of M2 mAChRs. To validate the method, we show that PAI photoisomers display different cardiac effects in a mammalian animal model, and demonstrate reversible, real-time photocontrol of cardiac function in translucent wildtype tadpoles. PAI can also effectively activate M2 receptors using two-photon excitation with near-infrared light, which overcomes the scattering and low penetration of short-wavelength illumination, and offers new opportunities for intravital imaging and control of cardiac function.

Fluorination of Photoswitchable Muscarinic Agonists Tunes Receptor Pharmacology and Photochromic Properties.

Red-shifted azobenzene scaffolds have emerged as useful molecular photoswitches to expand potential applications of photopharmacological tool compounds. As one of them, tetra- ortho-fluoro azobenzene is well compatible for the design of visible-light-responsive systems, providing stable and bidirectional photoconversions and tissue-compatible characteristics. Using the unsubstituted azobenzene core and its tetra- ortho-fluorinated analogue, we have developed a set of uni- and bivalent photoswitchable toolbox derivatives of the highly potent muscarinic acetylcholine receptor agonist iperoxo. We investigated the impact of the substitution pattern on receptor activity and evaluated the different binding modes. Compounds 9b and 15b show excellent photochemical properties and biological activity as fluorination of the azobenzene core alters not only the photochromic behavior but also the pharmacological profile at the muscarinic M1 receptor. These findings demonstrate that incorporation of fluorinated azobenzenes not just may alter photophysical properties but can exhibit a considerably different biological profile that has to be carefully investigated.

TAK-071, a muscarinic M1 receptor positive allosteric modulator, attenuates scopolamine-induced quantitative electroencephalogram power spectral changes in cynomolgus monkeys.

Activation of the muscarinic M1 receptor is a promising approach to improve cognitive deficits associated with cholinergic dysfunction in Alzheimer's disease, dementia with Lewy bodies, and schizophrenia. TAK-071 is an M1-selective positive allosteric modulator that improves cognitive deficits induced by scopolamine, a non-selective muscarinic receptor antagonist, with reduced side effects on gastrointestinal function in rats. In this study, we explored changes in quantitative electroencephalography (qEEG) power bands, with or without scopolamine challenge, as a non-invasive translational biomarker for the effect of TAK-071 in cynomolgus monkeys. Scopolamine has been reported to increase theta and delta power bands and decrease alpha power band in healthy volunteers. In line with the clinical observations, scopolamine (25-100 µg/kg, subcutaneous administration [s.c.]) increased theta and delta power bands in cynomolgus monkeys in a dose-dependent manner, whereas it had the opposite effect on alpha power band. The effects of TAK-071 on scopolamine (25 µg/kg, s.c.)-induced qEEG spectral changes were examined using an acetylcholinesterase inhibitor donepezil and a muscarinic M1/M4 receptor agonist xanomeline as comparative cholinomimetics. TAK-071 (0.3-3 mg/kg, oral administration [p.o.]), donepezil (3 mg/kg, p.o.), and xanomeline (1 mg/kg, s.c.) suppressed the scopolamine-induced increases in alpha, theta, and delta power bands. These results suggest that changes in specific qEEG power bands, in particular theta and delta power bands in the context of scopolamine challenge, could be used as translational biomarkers for the evaluation of TAK-071 in clinical studies.

Discovery of Tricyclic Triazolo- and Imidazopyridine Lactams as M1 Positive Allosteric Modulators.

This Letter describes the chemical optimization of a new series of muscarinic acetylcholine receptor subtype 1 (M1) positive allosteric modulators (PAMs) based on novel tricyclic triazolo- and imidazopyridine lactam cores, devoid of M1 agonism, e.g., no M1 ago-PAM activity, in high expressing recombinant cell lines. While all the new tricyclic congeners afforded excellent rat pharmacokinetic (PK) properties (CLp < 8 mL/min/kg and t1/2 > 5 h), regioisomeric triazolopyridine analogues were uniformly not CNS penetrant ( Kp < 0.05), despite a lack of hydrogen bond donors. However, removal of a single nitrogen atom to afford imidazopyridine derivatives proved to retain the excellent rat PK and provide high CNS penetration ( Kp > 2), despite inclusion of a basic nitrogen. Moreover, 24c was devoid of M1 agonism in high expressing recombinant cell lines and did not induce cholinergic seizures in vivo in mice. Interestingly, all of the new M1 PAMs across the diverse tricyclic heterocyclic cores possessed equivalent CNS MPO scores (>4.5), highlighting the value of both "medicinal chemist's eye" and experimental data, e.g., not sole reliance (or decision bias) on in silico calculated properties, for parameters as complex as CNS penetration.

In Vivo Modulation of Hippocampal Excitability by M4 Muscarinic Acetylcholine Receptor Activator: Implications for Treatment of Alzheimer's Disease and Schizophrenic Patients.

Abnormal hippocampal activity has been linked to impaired cognitive performance in Alzheimer's disease and schizophrenia, leading to a hypothesis that normalization of this activity may be therapeutically beneficial. Our work suggests that one approach for hippocampal normalization may be through activation of the M4 muscarinic acetylcholine receptor. We used a brain penetrant M4 muscarinic acetylcholine receptor selective activator, PT-3763, to show dose-dependent attenuation of field potentials in Schaffer collateral (CA3-CA1) and recurrent associational connections (CA3-CA3) ex vivo in hippocampal slices. In vivo, systemic administration of PT-3763 led to attenuation of glutamate release in CA3 as measured by amperometry and to a dose-dependent decrease in population CA1 pyramidal activity as measured by fiber photometry. This decrease in population activity was also evident with a localized administration of the compound to the recorded site. Finally, PT-3763 reversed scopolamine-induced deficit in Morris water maze. Our results suggest that M4 muscarinic acetylcholine receptor activation may be a suitable therapeutic treatment in diseases associated with hyperactive hippocampal activity.

Muscarinic agonist, (±)-quinuclidin-3-yl-(4-fluorophenethyl)(phenyl)carbamate: High affinity, but low subtype selectivity for human M1 - M5 muscarinic acetylcholine receptors.

Novel quinuclidinyl N-phenylcarbamate analogs were synthesized, and binding affinities at M1-M5 muscarinic acetylcholine receptor (mAChR) subtypes were determined using Chinese hamster ovary (CHO) cell membranes stably expressing one specific subtype of human mAChR. Although not subtype selective, the lead analog (±)-quinuclidin-3-yl-(4-fluorophenethyl)(phenyl)carbamate (3c) exhibited the highest affinity (Ki = 2.0, 13, 2.6, 2.2, 1.8 nM) at each of the M1-M5 mAChRs, respectively. Based on results from the [3H]dopamine release assay using rat striatal slices, 3c acted as an agonist at mAChRs. The effect of 3c was inhibited by the nonselective mAChR antagonist, scopolamine, and 3c augmented release evoked by oxotremorine. A potent analog from the same scaffold, (±)-quinuclidin-3-yl-(4-methoxyphenethyl)(phenyl)-carbamate (3b) exhibited the greatest selectivity (17-fold) at M3 over M2 mAChRs. These analogs could serve as leads for further discovery of novel subtype-selective muscarinic ligands with the goal of providing therapeutics for substance use disorders and chronic obstructive pulmonary disease.

Synthesis and Preliminary Evaluation of 11 C-Labeled VU0467485/AZ13713945 and Its Analogues for Imaging Muscarinic Acetylcholine Receptor Subtype†4.

Muscarinic acetylcholine receptors (mAChRs) have five distinct subunits (M1 -M5 ) and are involved in the action of the neurotransmitter acetylcholine in the central and peripheral nervous system. Attributed to the promising clinical efficacy of xanomeline, an M1 /M4 -preferring agonist, in patients of schizophrenia and Alzheimer's disease, M1 - or M4 -selective mAChR modulators have been developed that target the topographically distinct allosteric sites. Herein we report the synthesis and preliminary evaluation of 11 C-labeled positron emission tomography (PET) ligands based on a validated M4 R positive allosteric modulator VU0467485 (AZ13713945) to facilitate drug discovery. [11 C]VU0467485 and two other ligands were prepared in high radiochemical yields (>30 %, decay-corrected) with high radiochemical purity (>99 %) and high molar activity (>74 GBq µmol-1 ). In vitro autoradiography studies indicated that these three ligands possess moderate-to-high in vitro specific binding to M4 R. Nevertheless, further physiochemical property optimization is necessary to overcome the challenges associated with limited brain permeability.

Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor.

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 µM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms.

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.

Structure-Based Design and Discovery of New M2 Receptor Agonists.

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

Discovery of the first low-shift positive allosteric modulators for the muscarinic M1 receptor.

Positive modulation of the muscarinic M1-receptor has for a long time attracted scientists and drug developers for the potential treatment of Alzheimer's disease or Schizophrenia. The precognitive potential of M1 activation has however not been clinically demonstrated as a result of side effects associated both with agonists and positive allosteric modulators (PAM's) of the M1-receptor. To avoid excessive activation of the M1-receptor we have designed a new screening format and developed the first low-shift positive allosteric modulators for the M1 receptor. Low-shift PAM's offer the potential of "use-dependent" attenuation of transmitter-signaling while avoiding pseudo-agonistic behavior in vivo as a common limitation of the so far described high-shift PAM's. With these novel M1-PAM's, the M1 receptor is potentially the first GPCR for which both, high- and low shift PAM's have become available.

Structural determinants at the M2 muscarinic receptor modulate the RGS4-GIRK response to pilocarpine by impairment of the receptor voltage sensitivity.

Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K+ channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors.

Broad analgesic activity of a novel, selective M1 agonist.

Although the muscarinic receptor family has long been a source of potentially compelling targets for small molecule drug discovery, it was difficult to achieve agonist selectivity within the family. A new class of M1 muscarinic agonists has emerged, and these compounds have been characterized as agonists that activate the receptor at an allosteric site. Members of this class of M1 agonists have been shown to be selective across the muscarinic receptors. However, upon introduction of a novel pharmacologic mechanism, it is prudent to ensure that no new off-target activities have arisen, particularly within the context of in vivo experiments. Reported here, is the in vitro and in vivo characterization of a novel M1 agonist tool compound, PPBI, and demonstrations that the primary biological effects of PPBI are mediated through M1. PPBI reverses d-amphetamine locomotor activity, but fails to do so in transgenic mice that do not express M1. PPBI also reverses a natural deficit in a rat cognition model at a level of exposure which also activates cortical circuitry. Most notably, PPBI is analgesic in a variety of rat and mouse models and the analgesic effect of PPBI is reversed by an M1-preferring antagonist and an M1-selective toxin. Finally, the pharmacokinetic/pharmacodynamic measures of PPBI are compared across multiple endpoints which highlights that activity in models of psychosis and pain require higher exposures than that required in the cognition model.

Classics in Chemical Neuroscience: Xanomeline.

Xanomeline (1) is an orthosteric muscarinic acetylcholine receptor (mAChR) agonist, often referred to as M1/M4-preferring, that received widespread attention for its clinical efficacy in schizophrenia and Alzheimer's disease (AD) patients. Despite the compound's promising initial clinical results, dose-limiting side effects limited further clinical development. While xanomeline, and related orthosteric muscarinic agonists, have yet to receive approval from the FDA for the treatment of these CNS disorders, interest in the compound's unique M1/M4-preferring mechanism of action is ongoing in the field of chemical neuroscience. Specifically, the promising cognitive and behavioral effects of xanomeline in both schizophrenia and AD have spurred a renewed interest in the development of safer muscarinic ligands with improved subtype selectivity for either M1 or M4. This Review will address xanomeline's overall importance in the field of neuroscience, with a specific focus on its chemical structure and synthesis, pharmacology, drug metabolism and pharmacokinetics (DMPK), and adverse effects.