M1 Muscarinic Acetylcholine Receptor Research Articles

Discovery of BAY 2413555, First Selective Positive Allosteric Modulator of the M2 Receptor to Restore Cardiac Autonomic Balance.

Autonomic disbalance, i.e., sympathetic overactivation and parasympathetic withdrawal, is a causal driver of disease progression in heart failure. While sympatholytic drugs are established treatments, no drug therapy restoring vagal control of cardiac function is available. We report here the HTS-based discovery of a novel class of 1,8-naphthyridin-4(1H)-one carboxamides acting as positive allosteric modulators (PAMs) of the M2 muscarinic acetylcholine receptor (M2R). M2R is the main postsynaptic myocyte receptor regulating heart rate, electrical conduction, and contractile strength. Extensive optimization of the screening hit in terms of potency, permeation, metabolic stability, and solubility ultimately resulted in the discovery of the first-in-class clinical candidate BAY 2413555 (27). With an overall technical profile compatible with once-daily oral administration in a phase 1 study, no apparent effects on blood pressure, and a mechanism that largely preserves autonomic regulatory capacity, BAY 2413555 could be the tool to finally study the restoration of autonomic balance.

Investigation of muscarinic acetylcholine receptor M3 activation in atomistic detail: a chemist's viewpoint.

We analyzed the precise ligand:receptor interactions required for activation of the muscarinic acetylcholine receptor M3, a prototypical G protein-coupled receptor and potential diabetes target. Starting from literature-known compounds and docking solutions, ligands were tailored for the modulation of this receptor's activation. Several aspects of the structure-activity relationship of agonists were investigated in atomistic detail, in order to delineate how the receptor can be activated via the orthosteric site. Such exquisitely precise knowledge is instrumental for designing potent and effiacious ligands. We put this strategy into practice and acquired or synthesized and measured a diverse set of 55 ligands ranging from small fragment-like amines coordinating D3.32 to bigger molecules extending towards helices 5 and 6 with diphenyl moieties. In the course of these investigations, we showed that the polarizability of the amine nitrogen and the rigidity and size of the moieties in the space delimited by helices 5 and 6 are the two key elements distinguishing potent and efficacious ligands from those that are not. The resulting data set will be highly useful in drug design and molecular machine learning alike.

The Fatal Mushroom Neurotoxin Muscarine is Released from a Harmless Phosphorylated Precursor upon Cellular Injury

l‐(+)‐Muscarine (1)‐producing mushrooms pose a severe threat to human health as ingestion can result in circulatory collapse or even death. However, their metabolic profile is surprisingly poorly understood, including knowledge of poison release and potentially toxic congeners. In the mycelium of the 1‐producing fool’s funnel mushroom Clitocybe rivulosa, we identified 4’‐phosphomuscarine (2) as the major natural product. Its structure was elucidated by high‐resolution mass spectrometry, nuclear magnetic resonance spectroscopy and by comparison with a synthesized reference. We also detected this previously overlooked phosphorylated compound in the fiber cap mushrooms Pseudosperma spectrale and Inocybe nitidiuscula. Studies on the activation of the muscarinic acetylcholine receptor M3 indicate only weak affinity of 2 to this target. Furthermore, we present biological evidence that muscaridine (3), a quaternary amine congener related to and co‐occurring with 1, does not activate the muscarinic acetylcholine receptor M3 on human embryonic kidney cells. Our work provides important insight into the metabolic profile and the pharmacology of some of the most poisonous mushrooms. As the harmless 2 can liberate the potentially fatal 1 by unspecific enzymatic ester cleavage, these results are highly relevant for emergency medicine to estimate the true toxicity of these mushrooms.

The Fatal Mushroom Neurotoxin Muscarine is Released from a Harmless Phosphorylated Precursor upon Cellular Injury.

l-(+)-Muscarine (1)-producing mushrooms pose a severe threat to human health as ingestion can result in circulatory collapse or even death. However, their metabolic profile is surprisingly poorly understood, including knowledge of poison release and potentially toxic congeners. In the mycelium of the 1-producing fool's funnel mushroom Clitocybe rivulosa, we identified 4'-phosphomuscarine (2) as the major natural product. Its structure was elucidated by high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and by comparison with a synthesized reference. We also detected this previously overlooked phosphorylated compound in the fiber cap mushrooms Pseudosperma spectrale and Inocybe nitidiuscula. Studies on the activation of the muscarinic acetylcholine receptor M3 indicate only weak affinity of 2 to this target. Furthermore, we present biological evidence that muscaridine (3), a quaternary amine congener related to and co-occurring with 1, does not activate the muscarinic acetylcholine receptor M3 on human embryonic kidney cells. Our work provides important insight into the metabolic profile and the pharmacology of some of the most poisonous mushrooms. As the harmless 2 can liberate the potentially fatal 1 by unspecific enzymatic ester cleavage, these results are highly relevant for emergency medicine to estimate the true toxicity of these mushrooms.

Effects of Different Photoperiods on Growth Performance, Glucose Metabolism, Acetylcholine, and Its Relative Acetylcholine Receptor Modulation in Broiler Chickens.

Photoperiods are crucial environmental factors in the growth and health of modern intensive broiler chicken production. To date, the effects of different photoperiods on glucose metabolism, acetylcholine (ACh), and its relative acetylcholine receptor modulation in broilers remain elusive. Herein, we aimed to identify the effects of different photoperiods on regulating glucose metabolism, ACh, nicotinic acetylcholine receptor alpha 4 (α4 nAChR) mRNA, and M3 muscarinic acetylcholine receptor (M3 mAChR) modulation in broilers. A total of 216 healthy 5-day-old Arbor Acres (AA) male broilers was randomly assigned to 12L:12D, 18L:6D, and 24L:0D photoperiods for 4 weeks. The results show that, compared with the 12L:12D photoperiod, the 18L:6D and 24L:0D photoperiods significantly increase the average daily gain (ADG) and average daily feed intake (ADFI) of broilers (p < 0.05). However, the feed efficiency (FE) of broilers significantly decreased in the 18L:6D and 24L:0D photoperiods (p < 0.05). Moreover, compared with the 12L:12D photoperiod, the ACh concentrations and α4 nAChR mRNA expression levels in the hypothalamus and medulla oblongata of broilers significantly increased (p < 0.05); M3 mAChR mRNA expression levels in cecum significantly reduced in the 18L:6D photoperiod and the 24L:0D photoperiod (p < 0.05). Compared with the 12L:12D photoperiod, the serum glucose (GLU), serum insulin (INS), serum triglyceride (TG) levels, and homeostasis model assessment of insulin resistance (HOMA-IR) of broilers significantly enhanced in the 18L:6D and 24L:0D photoperiods (p < 0.05). Our results indicate that extending the photoperiod can promote the growth rate, ACh expression, and α4 nAChR mRNA expression of broilers while reducing the feed efficiency, inhibiting M3 mAChR mRNA expression, and inducing glucose metabolism disorders in broilers.

Long-Term Neurotoxic Effects and Alzheimer's Disease Risk of Early EHDPP Exposure in Zebrafish: Insights from Molecular Mechanisms to Adult Pathology.

2-Ethylhexyl diphenyl phosphate (EHDPP), ubiquitously monitored in environmental media, is highly bioaccumulative and may pose long-term risks, even after short-term exposure. In this investigation, larval zebrafish were exposed to 0.05, 0.5, and 5.0 μg/L EHDPP from 4 to 120 h postfertilization (hpf) to examine the long-term neurotoxicity effects of early exposure. Exposure to 5.0 μg/L EHDPP yielded hyperactive locomotor behavior, which was characterized by increased swimming speed, larger turning angles, and heightened sensitivity to light-dark stimulation. The predicted targets of EHDPP (top 100 potential macromolecules) were primarily associated with brain diseases like Alzheimer's disease (AD). Comparisons of differentially expressed genes (DEGs) from AD patients (GSE48350) and RNA-seq data from EHDPP-exposed zebrafish confirmed consistently abnormal regulatory pathways. EHDPP's interaction with M1 and M5 muscarinic acetylcholine receptors likely disrupted calcium homeostasis, leading to mitochondrial dysfunction and neurotransmitter imbalance as well as abnormal locomotor behavior. Especially, 5.0 μg/L EHDPP exposure during early development (4-120 hpf) triggered early- and midstage AD-like symptoms in adulthood (180 dpf), characterized by cognitive confusion, aggression, blood-brain barrier disruption, and mitochondrial damage in brains. These findings provide deep insights into the long-term neurotoxicity effects and Alzheimer's disease risks of early EHDPP exposure at extremely low dosages.

Photo-BQCA: Positive Allosteric Modulators Enabling Optical Control of the M1 Receptor.

The field of G protein-coupled receptor (GPCR) research has greatly benefited from the spatiotemporal resolution provided by light controllable, i.e., photoswitchable ligands. Most of the developed tools have targeted the Rhodopsin-like family (Class A), the largest family of GPCRs. However, to date, all such Class A photoswitchable ligands were designed to act at the orthosteric binding site of these receptors. Herein, we report the development of the first photoswitchable allosteric modulators of Class A GPCRs, designed to target the M1 muscarinic acetylcholine receptor. The presented benzyl quinolone carboxylic acid (BQCA) derivatives, Photo-BQCisA and Photo-BQCtrAns, exhibit complementary photopharmacological behavior and allow reversible control of the receptor using light as an external stimulus. This makes them valuable tools to further investigate M1 receptor signaling and a proof of concept for photoswitchable allosteric modulators at Class A receptors.

M1 muscarinic receptor activation reverses age-related memory updating impairment in mice

Previously consolidated memories can become temporarily labile upon reactivation.Reactivation-based memory updating is chiefly studied in young subjects, so we aimed to assess this process across the lifespan. To do this, we developed a behavioural paradigm wherein a reactivated object memory is updated with contextual information; 3-month-old and 6-month-old male C57BL/6 mice displayed object memory updating, but 12-month-old mice did not. We found that M1 muscarinic acetylcholine receptor signaling during reactivation was necessary for object memory updating in the young mice. Next, we targeted this mechanism in an attempt to facilitate object memory updating in aging mice. Remarkably, systemic pharmacological M1 receptor activation reversed the age-related deficit. Quantification of cholinergic system markers within perirhinal cortex revealed subtle cellular changes that may contribute to differential performance across age groups. These findings suggest that natural cholinergic change across the lifespan contributes to inflexible memory in the aging brain.

Discovery of VU6016235: A Highly Selective, Orally Bioavailable, and Structurally Distinct Tricyclic M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator (PAM).

Herein, we report structure-activity relationship (SAR) studies to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace a 5-amino-2,4-dimethylthieno[2,3-d]pyrimidine-6-carboxamide core, which led to the discovery of two novel tricyclic cores. While both tricyclic cores displayed low nanomolar potency against both human and rat M4 and were highly brain-penetrant, the 2,4-dimethylpyrido[4',3':4,5]thieno[2,3-d]pyrimidine tricycle core provided lead compound, VU6016235, with an overall superior pharmacological and drug metabolism and pharmacokinetics (DMPK) profile, as well as efficacy in a preclinical antipsychotic animal model.

Positive allosteric modulation of a GPCR ternary complex.

The activation of a G protein-coupled receptor (GPCR) leads to the formation of a ternary complex between agonist, receptor, and G protein that is characterized by high-affinity binding. Allosteric modulators bind to a distinct binding site from the orthosteric agonist and can modulate both the affinity and the efficacy of orthosteric agonists. The influence allosteric modulators have on the high-affinity active state of the GPCR-G protein ternary complex is unknown due to limitations on attempting to characterize this interaction in recombinant whole cell or membrane-based assays. Here, we use the purified M2 muscarinic acetylcholine receptor reconstituted into nanodiscs to show that, once the agonist-bound high-affinity state is promoted by the G protein, positive allosteric modulators stabilize the ternary complex that, in the presence of nucleotides, leads to an enhanced initial rate of signaling. Our results enhance our understanding of how allosteric modulators influence orthosteric ligand signaling and will aid the design of allosteric therapeutics.

Protective role of M3 muscarinic acetylcholine receptor in indomethacin-induced small intestinal injury.

EP4 prostanoid receptor (EP4R) contributes to the intestinal epithelial Cl- secretion, and inhibition of prostaglandin E (PGE) production by non-steroidal anti-inflammatory drugs (NSAIDs) plays a central role in NSAID-induced enteropathy. Although M3 muscarinic acetylcholine receptor (M3R) also contributes to the intestinal epithelial Cl- secretion, it remains unclear whether M3R is involved in NSAID-induced enteropathy due to a lack of selective agents. The present study explored how M3R is involved in the regulation of the intestinal epithelial Cl- secretion and its pathophysiological role in NSAID-induced enteropathy. Using the novel highly-selective M3 positive allosteric modulator PAM-369 that we recently developed, we evaluated the role of M3R in the intestinal epithelial secretion ex vivo by measuring the short circuit current (Isc) of intestinal epithelium with a Ussing chamber system and examined whether or not M3R protects against small intestinal injury in indomethacin-treated mice. Both the PGE1 derivative misoprostol and carbachol similarly increased the Isc in a concentration-dependent manner. The Isc increases were abolished either by receptor antagonists (an EP4R antagonist and a M3R antagonist, respectively) or by removal of extracellular Cl-. PAM-369 enhanced the carbachol-induced Isc by potentiating M3R, which could contribute to enhanced intestinal epithelial secretion. Treatment with PAM-369 ameliorated small intestinal injury in indomethacin-treated mice. Importantly, the M3R expression was significantly up-regulated, and PAM-369 potentiation of M3R was augmented in indomethacin-treated mice compared to untreated mice. These findings show that M3R plays a role in maintaining the intestinal epithelial secretion, which could contribute to protection against indomethacin-induced small intestinal injury. M3R is a promising target for treating or preventing NSAID-induced enteropathy. KEY MESSAGES: PAM-369, the M3 positive allosteric modulator, was used to potentiate M3R. PAM-369 enhanced carbachol-induced Isc in mouse ileum. PAM-369 ameliorated small intestinal injury in indomethacin-treated mice. M3R is a promising target for treating or preventing NSAID-induced enteropathy.

Reactivation-dependent transfer of fear memory between contexts requires M1 muscarinic receptor stimulation in dorsal hippocampus of male rats.

Memory updating is essential for integrating new information into existing representations. However, this process could become maladaptive in conditions like post-traumatic stress disorder (PTSD), when fear memories generalize to neutral contexts. Previously, we have shown that contextual fear memory malleability in rats requires activation of M1 muscarinic acetylcholine receptors in the dorsal hippocampus. Here, we investigated the involvement of this mechanism in the transfer of contextual fear memories to other contexts using a novel fear memory updating paradigm. Following brief reexposure to a previously fear conditioned context, male rats (n = 8-10/group) were placed into a neutral context to evaluate the transfer of fear memory. We also infused the selective M1 receptor antagonist pirenzepine into the dorsal hippocampus before memory reactivation to try to block this effect. Results support the hypothesis that fear memory can be updated with novel contextual information, but only if rats are reexposed to the originally trained context relatively recently before the neutral context; evidence for transfer was not seen if the fear memory reactivation was omitted or if it occurred 6 h before neutral context exposure. The transferred fear persisted for 4 weeks, and the effect was blocked by M1 antagonism. These findings strongly suggest that fear transfer requires reactivation and destabilization of the original fear memory. The novel preclinical model introduced here, and its implication of muscarinic receptors in this process, could therefore inform therapeutic strategies for PTSD and similar conditions.

Neuroprotective effect of long-term resistance physical exercise against memory damage elicited by a lipopolysaccharide-induced neuroinflammation model in malerats.

Resistance exercise training (RET) is considered an excellent tool for preventing diseases with an inflammatory background. Its neuroprotective, antioxidant, and anti-inflammatory properties are responsible for positively modulating cholinergic and oxidative systems, promoting neurogenesis, and improving memory. However, the mechanisms behind these actions are largely unknown. In order to investigate the pathways related to these effects of exercise, we conducted a 12-week long-term exercise training protocol and used lipopolysaccharide (LPS) to induce damage to the cortex and hippocampus of male Wistar rats. The cholinergic system, oxidative stress, and histochemical parameters were analyzed in the cerebral cortex and hippocampus, and memory tests were also performed. It was observed that LPS: (1) caused memory loss in the novel object recognition (NOR) test; (2) increased the activity of acetylcholinesterase (AChE) and Iba1 protein density; (3) reduced the protein density of brain-derived neurotrophic factor (BDNF) and muscarinic acetylcholine receptor M1 (CHRM1); (4) elevated the levels of lipid peroxidation (TBARS) and reactive species (RS); and (5) caused inflammatory damage to the dentate gyrus. RET, on the other hand, was able to prevent all alterations induced by LPS, as well as increase per se the protein density of the alpha-7 nicotinic acetylcholine receptor (nAChRα7) and Nestin, and the levels of protein thiols (T-SH). Overall, our study elucidates some mechanisms that support resistance physical exercise as a valuable approach against LPS-induced neuroinflammation and memory loss.

Targeting the muscarinic M1 receptor with a selective, brain-penetrant antagonist to promote remyelination in multiple sclerosis

Multiple sclerosis (MS) is a chronic and debilitating neurological disease that results in inflammatory demyelination. While endogenous remyelination helps to recover function, this restorative process tends to become less efficient over time. Currently, intense efforts aimed at the mechanisms that promote remyelination are being considered promising therapeutic approaches. The M1 muscarinic acetylcholine receptor (M1R) was previously identified as a negative regulator of oligodendrocyte differentiation and myelination. Here, we validate M1R as a target for remyelination by characterizing expression in human and rodent oligodendroglial cells (including those in human MS tissue) using a highly selective M1R probe. As a breakthrough to conventional methodology, we conjugated a fluorophore to a highly M1R selective peptide (MT7) which targets the M1R in the subnanomolar range. This allows for exceptional detection of M1R protein expression in the human CNS. More importantly, we introduce PIPE-307, a brain-penetrant, small-molecule antagonist with favorable drug-like properties that selectively targets M1R. We evaluate PIPE-307 in a series of in vitro and in vivo studies to characterize potency and selectivity for M1R over M2-5R and confirm the sufficiency of blocking this receptor to promote differentiation and remyelination. Further, PIPE-307 displays significant efficacy in the mouse experimental autoimmune encephalomyelitis model of MS as evaluated by quantifying disability, histology, electron microscopy, and visual evoked potentials. Together, these findings support targeting M1R for remyelination and support further development of PIPE-307 for clinical studies.

Discovery of 2-Methyl-5-(1H-pyrazol-4-yl)pyridines and Related Heterocycles as Promising M4 mAChR Positive Allosteric Modulators for the Treatment of Neurocognitive Disorders.

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 β-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 β-arrestin recruitment (23i). These novel M4 chemical tools disclose discrete molecular determinants, allowing further interrogation of the therapeutic roles of cAMP and β-arrestin pathways in neurocognitive disorders.

Assessing the interplay between off-target promiscuity, cytotoxicity, and tolerability in rodents to improve the safety profile of novel anti-malarial plasmepsin X inhibitors.

Within drug development, high off-target promiscuity as well as potent cytotoxicity, are associated with a high attrition rate. We investigated the safety profile of novel plasmepsin X (PMX) inhibitors for the treatment of malaria. In our screening cascade, a total of 249 PMX compounds were profiled in a panel of in vitro secondary pharmacology assays containing 44 targets (SafetyScreen44 panel) and in a cytotoxicity assay in HepG2 cells using ATP as an endpoint. Six of the lead compounds were subsequently tested in a 7-d rat toxicology study, and/or in a cardiovascular study in guinea pigs. Overall, compounds with high cytotoxicity in HepG2 cells correlated with high promiscuity (off-target hit rate >20%) in the SafetyScreen44 panel and were associated with poor tolerability in vivo (decedents, morbidity, adverse clinical signs, or severe cardiovascular effects). Some side effects observed in rats or guinea pigs could putatively be linked with hits in the secondary pharmacological profiling, such as the M1 or M2 muscarinic acetylcholine receptor, opioid µ and/or κ receptors or hERG/CaV1.2/Na+ channels, which were common to >50% the compounds tested in vivo. In summary, compounds showing high cytotoxicity and high promiscuity are likely to be poorly tolerated in vivo. However, such associations do not necessarily imply a causal relationship. Identifying the targets that cause these undesirable effects is key for early safety risk assessment. A tiered approach, based on a set of in vitro assays, helps selecting the compounds with highest likelihood of success to proceed to in vivo toxicology studies.

Preclinical evaluation of [18F]SYN1 and [18F]SYN2, novel radiotracers for PET myocardial perfusion imaging

BackgroundPositron emission tomography (PET) is now an established diagnostic method for myocardial perfusion imaging (MPI) in coronary artery disease, which is the main cause of death globally. The available tracers show several limitations, therefore, the 18F-labelled tracer is in high demand nowadays. The preclinical studies on normal Wistar rats aimed to characterise two potential, novel radiotracers, [18F]SYN1 and [18F]SYN2, to evaluate which is a better candidate for PET MPI cardiotracer.ResultsThe dynamic microPET images showed rapid myocardial uptake for both tracers. However, the uptake was higher and also stable for [18F]SYN2, with an average standardized uptake value of 3.8. The biodistribution studies confirmed that [18F]SYN2 uptake in the cardiac muscle was high and stable (3.02%ID/g at 15 min and 2.79%ID/g at 6 h) compared to [18F]SYN1 (1.84%ID/g at 15 min and 0.32%ID/g at 6 h). The critical organs determined in dosimetry studies were the small intestine and the kidneys. The estimated effective dose for humans was 0.00714 mSv/MBq for [18F]SYN1 and 0.0109 mSv/MBq for [18F]SYN2. The tested dose level of 2 mg/kg was considered to be the No Observed Adverse Effect Level (NOAEL) for both candidates. The better results were achieved for [18F]SYN2, therefore, further preclinical studies were conducted only for this tracer. Radioligand binding assays showed significant responses in 3 from 68 assays: muscarinic acetylcholine M1 and M2 receptors and potassium channel hERG. The compound was mostly metabolised via an oxidative N-dealkylation, while the fluor substituent was not separated from the molecule.Conclusion[18F]SYN2 showed a favourable pharmacodynamic and pharmacokinetic profile, which enabled a clear visualization of the heart in microPET. The compound was well-tolerated in studies in normal rats with moderate radiation exposure. The results encourage further exploration of [18F]SYN2 in clinical studies.

Co-stimulation of muscarinic M1 and M4 acetylcholine receptors prevents later cocaine reinforcement in male and female mice, but not place-conditioning

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.

Design and Synthesis of Clinical Candidate PF-06852231 (CVL-231): A Brain Penetrant, Selective, Positive Allosteric Modulator of the M4 Muscarinic Acetylcholine Receptor.

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.

Predicting the potential risks posed by antidepressants as emerging contaminants in fish based on network pharmacological analysis

This study conducted a network pharmacology-based analysis to simultaneously discern a broad spectrum of potential environmental risks and health effects of antidepressants, a common class of pharmaceutical emerging contaminants (PECs) possessing a complex pharmacological profile, and in silico predict the adverse phenotypes potentially occurring in fish associated with exposure to antidepressants and their mixtures under realistic exposure scenarios. Results showed that 24 of the included 39 antidepressants had been detected worldwide in water environment across 50 countries. Using the environmentally realistic exposure scenario for China as an example, the predicted blood concentrations of antidepressant residues that were generated based on the Fish Plasma Model ranged from 37.89 (Alprazolam) to 16,772.05 (Sertraline) ng/L in exposed fish. Hazard-based bioactivity network without regard to concentration data was composed of 148 potential targets and 701 antidepressant-target interactions. After filtering each antidepressant-target interaction node using the predicted drug concentrations in the blood of fish under realistic exposure scenarios in China, an environmental risk-based network was refined and showed that 11 targets, including muscarinic acetylcholine receptor M1, alpha-2B adrenergic receptor, serotonin 2 A receptor, etc. might be modulated by antidepressants at concentrations equal to or below the environmental exposure levels and their mixtures in fish. Environmentally relevant concentrations of antidepressants in water samples from China might perturb the behavior, stress response, phototaxis, and development in exposed fish.