Bitopic binding mode of an M1 muscarinic acetylcholine receptor agonist associated with adverse clinical trial outcomes

The realisation of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (M1 mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702, described previously as a potent M1 receptor allosteric agonist, which showed pro-cognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side-effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702 together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. We conclude that these properties, whilst imparting beneficial effects on learning and memory, are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data supports the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses

Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus

Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans

Cannabinoid receptors in GtoPdb v.2023.1

Cannabinoid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Cannabinoid Receptors [119]) are activated by endogenous ligands that include N-arachidonoylethanolamine (anandamide), N-homo-γ-linolenoylethanolamine, N-docosatetra-7,10,13,16-enoylethanolamine and 2-arachidonoylglycerol. Potency determinations of endogenous agonists at these receptors are complicated by the possibility of differential susceptibility of endogenous ligands to enzymatic conversion [5].There are currently three licenced cannabinoid medicines each of which contains a compound that can activate CB1 and CB2 receptors [111]. Two of these medicines were developed to suppress nausea and vomiting produced by chemotherapy. These are nabilone (Cesamet®), a synthetic CB1/CB2 receptor agonist, and synthetic Δ9-tetrahydrocannabinol (Marinol®; dronabinol), which can also be used as an appetite stimulant. The third medicine, Sativex®, contains mainly Δ9-tetrahydrocannabinol and cannabidiol, both extracted from cannabis, and is used to treat multiple sclerosis and cancer pain

Acetylcholine receptors (muscarinic) (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Muscarinic acetylcholine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [45]) are GPCRs of the Class A, rhodopsin-like family where the endogenous agonist is acetylcholine. In addition to the agents listed in the table, AC-42, its structural analogues AC-260584 and 77-LH-28-1, N-desmethylclozapine, TBPB and LuAE51090 have been described as functionally selective agonists of the M1 receptor subtype via binding in a mode distinct from that utilized by non-selective agonists [243, 242, 253, 155, 154, 181, 137, 11, 230]. There are two pharmacologically characterised allosteric sites on muscarinic receptors, one defined by it binding gallamine, strychnine and brucine, and the other defined by the binding of KT 5720, WIN 62,577, WIN 51,708 and staurosporine [161, 162]

Acetylcholine receptors (muscarinic) in GtoPdb v.2023.1

Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [53]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates. Of note, it has been observed that mAChRs dimerise reversibly [134] and that dimerisation/oligomerisation can be affected by ligands [183, 196]

Acetylcholine receptors (muscarinic) in GtoPdb v.2021.3

Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates

Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi

ABSTRACT: BACKGROUND: Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans. RESULTS: 97% of the 22.5 megabase genome sequences of A. benhamiae and T. verrucosum are unambiguously alignable and collinear. To unravel dermatophyte-specific virulence-associated traits, we compared sets of potentially pathogenicity-associated proteins, such as secreted proteases and enzymes involved in secondary metabolite production, with those of closely related onygenales (Coccidioides species) and the mould Aspergillus fumigatus. The comparisons revealed expansion of several gene families in dermatophytes and disclosed the peculiarities of the dermatophyte secondary metabolite gene sets. Secretion of proteases and other hydrolytic enzymes by A. benhamiae was proven experimentally by a global secretome analysis during keratin degradation. Molecular insights into the interaction of A. benhamiae with human keratinocytes were obtained for the first time by global transcriptome profiling. Given that A. benhamiae is able to undergo mating, a detailed comparison of the genomes further unraveled the genetic basis of sexual reproduction in this species. CONCLUSIONS: Our results enlighten the genetic basis of fundamental and putatively virulence-related traits of dermatophytes, advancing future research on these medically important pathogens

Improved annotation with <i>de novo</i> transcriptome assembly in four social amoeba species

Background: Annotation of gene models and transcripts is a fundamental step in genome sequencing projects. Often this is performed with automated prediction pipelines, which can miss complex and atypical genes or transcripts. RNA sequencing (RNA-seq) data can aid the annotation with empirical data. Here we present de novo transcriptome assemblies generated from RNA-seq data in four Dictyostelid species: D. discoideum, P. pallidum, D. fasciculatum and D. lacteum. The assemblies were incorporated with existing gene models to determine corrections and improvement on a whole-genome scale. This is the first time this has been performed in these eukaryotic species. Results: An initial de novo transcriptome assembly was generated by Trinity for each species and then refined with Program to Assemble Spliced Alignments (PASA). The completeness and quality were assessed with the Benchmarking Universal Single-Copy Orthologs (BUSCO) and Transrate tools at each stage of the assemblies. The final datasets of 11,315-12,849 transcripts contained 5,610-7,712 updates and corrections to >50% of existing gene models including changes to hundreds or thousands of protein products. Putative novel genes are also identified and alternative splice isoforms were observed for the first time in P. pallidum, D. lacteum and D. fasciculatum. Conclusions: In taking a whole transcriptome approach to genome annotation with empirical data we have been able to enrich the annotations of four existing genome sequencing projects. In doing so we have identified updates to the majority of the gene annotations across all four species under study and found putative novel genes and transcripts which could be worthy for follow-up. The new transcriptome data we present here will be a valuable resource for genome curators in the Dictyostelia and we propose this effective methodology for use in other genome annotation projects

Cannabinoid Receptor 2 Signaling Does Not Modulate Atherogenesis in Mice

BACKGROUND:Strong evidence supports a protective role of the cannabinoid receptor 2 (CB(2)) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB(2) receptor in Murine atherogenesis. METHODS AND FINDINGS:Low density lipoprotein receptor-deficient (LDLR(-/-)) mice subjected to intraperitoneal injections of the selective CB(2) receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB(2) activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB(2) (-/-)/LDLR(-/-) mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB(2) (+/+)/LDLR(-/-) controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-elicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB(2) receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro. CONCLUSION:Our study demonstrates that both activation and deletion of the CB(2) receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB(2) in other inflammatory processes. However, in the context of atherosclerosis, CB(2) does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque