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Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Cholinergic dysfunction is involved in a range of neurological and psychiatric disorders, including schizophrenia, dementia and Lewy body disease (LBD), leading to widespread use of cholinergic therapies. However, such drugs have focused on increasing the availability of acetylcholine (ACh) generally, with relatively little work done on the muscarinic system and specific muscarinic receptor subtypes. In this review, we provide an overview of the major cholinergic pathways and cholinergic muscarinic receptors in the human brain and evidence for their dysfunction in several neurological and psychiatric disorders. We discuss how the selectivity of cholinergic system dysfunction suggests that targeted cholinergic therapeutics to the muscarinic receptor subtypes will be vital in treating several disorders associated with cognitive dysfunction and behavioural and psychological symptoms.

Funder: Biotechnology and Biological Sciences Research Council, Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.

Highlights • Elabela/Toddler (ELA) is the second endogenous ligand of the apelin receptor identified in zebrafish. • Mature ELA was predicted to be cleaved into [Pyr1]ELA-32, ELA-21 and ELA-11, based on the presence of dibasic amino acid residues. • We have shown that the half-life of [Pyr1]ELA-32 was ∼50 min in human plasma and, Background Elabela/Toddler (ELA) is a novel endogenous ligand of the apelin receptor, whose signalling has emerged as a therapeutic target, for example, in cardiovascular disease and cancer. Shorter forms of ELA-32 have been predicted, including ELA-21 and ELA-11, but metabolism and stability of ELA-32 in humans is poorly understood. We, therefore, developed an LC–MS/MS assay to identify ELA-32 metabolites in human plasma and tissues. Method Human kidney homogenates or plasma were incubated at 37 °C with ELA-32 and aliquots withdrawn over 2−4 h into guanidine hydrochloride. Proteins were precipitated and supernatant solid-phase extracted. Peptides were extracted from coronary artery, brain and kidney by immunoprecipitation or solid-phase extraction following acidification. All samples were reduced and alkylated before analysis on an Orbitrap mass spectrometer in high and nano flow mode. Results The half-life of ELA-32 in plasma and kidney were 47.2 ± 5.7 min and 44.2 ± 3 s, respectively. Using PEAKS Studio and manual data analysis, the most important fragments of ELA-32 with potential biological activity identified were ELA-11, ELA-16, ELA-19 and ELA-20. The corresponding fragments resulting from the loss of C-terminal amino acids were also identified. Endogenous levels of these peptides could not be measured, as ELA peptides are prone to oxidation and poor chromatographic peaks. Conclusions The relatively long ELA plasma half-life observed and identification of a potentially more stable fragment, ELA-16, may suggest that ELA could be a better tool compound and novel template for the development of new drugs acting at the apelin receptor.

Aims HTL0009936 is a selective M-1 muscarinic receptor agonist in development for cognitive dysfunction in Alzheimer's disease. Safety, tolerability and pharmacokinetics and exploratory pharmacodynamic effects of HTL0009936 administered by continuous IV infusion at steady state were investigated in elderly subjects with below average cognitive functioning (BACF).Methods Part A was a four-treatment open label sequential study in healthy elderly investigating 10-83 mg HTL0009936 (IV) and a 24 mg HTL0009936 single oral dose. Part B was a five-treatment randomized, double-blind, placebo and physostigmine controlled cross-over study with IV HTL0009936 in elderly subjects with BACF. Pharmacodynamic assessments were performed using neurocognitive and electrophysiological tests.Results Pharmacokinetics of HTL0009936 showed dose-proportional increases in exposure with a mean half-life of 2.4 hours. HTL0009936 was well-tolerated with transient dose-related adverse events (AEs). Small increases in mean systolic blood pressure of 7.12 mmHg (95% CI [3.99-10.24]) and in diastolic of 5.32 mmHg (95% CI [3.18-7.47]) were noted at the highest dose in part B. Overall, there was suggestive, but no definitive, positive or negative pharmacodynamic effects. Statistically significant effects were observed on P300 with HTL0009936 and adaptive tracking with physostigmine.Conclusions HTL0009936 showed well-characterized pharmacokinetics and single doses were safe and generally well-tolerated in healthy elderly subjects. Due to physostigmine tolerability issues and subject burden, the study design was changed and some pharmacodynamic assessments (neurocognitive) were performed at suboptimal drug exposures. Therefore no clear conclusions can be made on pharmacodynamic effects of HTL0009936, although an effect on P300 is suggestive of central target engagement.

Funder: Sosei Heptares, BACKGROUND: The cholinergic system and M1 receptor remain an important target for symptomatic treatment of cognitive dysfunction. The selective M1 receptor partial agonist HTL0018318 is under development for the symptomatic treatment of Dementia's including Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). We investigated the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of multiple doses of HTL0018318 in healthy younger adults and elderly subjects. METHODS: This randomised, double blind, placebo-controlled study was performed, investigating oral doses of 15-35 mg/day HTL0018318 or placebo in 7 cohorts of healthy younger adult (n = 36; 3 cohorts) and elderly (n = 50; 4 cohorts) subjects. Safety, tolerability and pharmacokinetic measurements were performed. Pharmacodynamics were assessed using a battery of neurocognitive tasks and electrophysiological biomarkers of synaptic and cognitive functions. RESULTS: HTL0018318 was generally well-tolerated in multiple doses up to 35 mg/day and were associated with mild or moderate cholinergic adverse events. There were modest increases in blood pressure and pulse rate when compared to placebo-treated subjects, with tendency for the blood pressure increase to attenuate with repeated dosing. There were no clinically significant observations or changes in blood and urine laboratory measures of safety or abnormalities in the ECGs and 24-h Holter assessments. HTL0018318 plasma exposure was dose-proportional over the range 15-35 mg. Maximum plasma concentrations were achieved after 1-2 h. The apparent terminal half-life of HTL0018318 was 16.1 h (± 4.61) in younger adult subjects and 14.3 h (± 2.78) in elderly subjects at steady state. HTL0018318 over the 10 days of treatment had significant effects on tests of short-term (working) memory (n-back) and learning (Milner maze) with moderate to large effect sizes. CONCLUSION: Multiple doses of HTL0018138 showed well-characterised pharmacokinetics and were safe and generally well-tolerated in the dose range studied. Pro-cognitive effects on short-term memory and learning were demonstrated across the dose range. These data provide encouraging data in support of the development of HTL0018138 for cognitive dysfunction in AD and DLB. TRIAL REGISTRATION: Netherlands Trial Register identifier NTR5781 . Registered on 22 March 2016.

Aims HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease (AD) and other dementias. We investigated safety, tolerability, pharmacokinetics (PK) and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. Methods This randomized, double-blind, placebo-controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1-35 mg HTL0018318. PD assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. CSF concentrations of HTL0018318 and food effects on PK of HTL0018318 were investigated in an open label and partial cross-over design in 14 healthy subjects. Results Pharmacokinetics of HTL0018318 were well-characterized showing dose proportional increases in exposure from 1-35 mg. Single doses of HTL0018318 were associated with mild dose-related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mm Hg in younger adults (95% CI [4.2-16.3], 35 mg dose) and up to 11.9 mm Hg in elderly subjects (95% CI [4.9-18.9], 15 mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. Conclusions HTL0018318 showed well-characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for AD and other dementias. Netherlands Trial Register identifier NTR5648.

Current therapies for improving memory in Alzheimer’s disease are focused on correcting for defective cholinergic transmission by increasing acetylcholine levels through inhibition of acetylcholinesterase enzymes. These treatments are however associated with limited clinical efficacy and dose-limiting adverse effects. Here we describe agonist-bound crystal structures of the M1 muscarinic acetylcholine receptor (M1 mAChR) and its application in supporting the rational drug design of a selective M1-orthosteric agonist, HTL0009936. On the basis of pro-cognitive effects across a range of pre-clinical animal models and a favourable safety and toxicology profile HTL0009936 was progressed to first in human trials where the agonist activated central processes corresponding to learning and memory circuitry. Hence, our study not only describes the pharmacological profile of a M1 mAChR agonist with the potential to improve memory in Alzheimer’s disease but also demonstrates how to rationally progress the development of a G protein-coupled receptor ligand from structure to clinical candidate.

The development of G protein-biased agonists for the μ-opioid receptor (MOR) offers a clear drug discovery rationale for improved analgesia and reduced side-effects of opiate pharmacotherapy. However, our understanding of the molecular mechanisms governing ligand bias is limited, which hinders our ability to rationally design biased compounds. We have investigated the role of MOR binding site residues W320 and Y328 in controlling bias, by receptor mutagenesis. The pharmacology of a panel of ligands in a cAMP and a β-arrestin2 assay were compared between the wildtype and mutated receptors, with bias factors calculated by operational analysis using ΔΔlog(τ/KA) values. [3H]diprenorphine competition binding was used to estimate affinity changes. Introducing the mutations W320A and Y328F caused changes in pathway bias, with different patterns of change between ligands. For example, DAMGO increased relative β-arrestin2 activity at the W320A mutant, whilst its β-arrestin2 response was completely lost at Y328F. In contrast, endomorphin-1 gained activity with Y328F but lost activity at W320A, in both pathways. For endomorphin-2 there was a directional shift from cAMP bias at the wildtype towards more β-arrestin2 bias at W320A. We also observe clear uncoupling between mutation-driven changes in function and binding affinity. These findings suggest that the mutations influenced the balance of pathway activation in a ligand-specific manner, thus identifying residues in the MOR binding pocket that govern ligand bias. This increases our understanding of how ligand/receptor binding interactions can be translated into agonist-specific pathway activation.

Introduction Fluorescent ligands are a promising tool in the study of GPCR pharmacology as they are safe and more cost effective than conventional probes such as radioligands. In this study, we investigated the use of a novel fluorescent ligand, apelin-488, to successfully show qualitative and quantitative apelin receptor internalisation, which may help further our understanding of apelin receptor pharmacology. The apelin receptor, a class A GPCR that unusually binds two endogenous peptide ligands, apelin and elabela/toddler, is expressed and functional in the cardiovascular system. Mutations in the human apelin receptor have been identified in rare diseases and our aim is to identify tool compounds to explore the potential functional consequence of these mutations. Methods CHO-K1 cells stably expressing the wild-type apelin receptor were treated with apelin-488, fixed, and stained with Hoechst 33342 and a wheat germ agglutinin-AlexaFluor-594 conjugate as nuclear and membrane markers respectively. Triple fluorescence confocal images were generated using Opera PhenixTM High Content Screening System and data were quantified using Harmony High Content Imaging and Analysis Software. Fluorescent ligand intensity at the membrane or in the cytoplasm was determined, providing cellular localisation for the apelin receptor following ligand binding. Activity of the fluorescent ligand at the apelin receptor in comparison to the endogenous ligand, [Pyr1]apelin-13 or CMF-019, a G protein biased small molecule apelin receptor ligand, was determined using a dynamic mass redistribution assay and radioligand binding assays. Results The fluorescent ligand, apelin-488 binds specifically to the apelin receptor in stably transfected CHO-K1 cells. High content screening revealed that in these cells, the apelin receptor is internalised over time and was still present at the cell membrane at an early time point, 30 secs (fig 1A). However, by 60 mins, apelin-488 membrane localisation was reduced while internal localisation had increased, suggesting substantial apelin receptor internalisation had occurred. (fig 1B). Untransfected CHO-K1 cells showed no apelin-488 signal, supporting specific binding of the fluorescent ligand to the apelin receptor. A dynamic mass redistribution assay demonstrated that apelin-488 is functional at the apelin receptor, at a similar potency as the endogenous ligand [Pyr1]apelin-13 and the G protein biased small molecule apelin receptor ligand CMF-019 (Fig 2). Conclusion Apelin-488 shows specific binding to the apelin receptor while retaining activity in a dynamic mass redistribution and binding assays. This work provides evidence for apelin-488 as a novel fluorescent tool for assessing internalisation of the apelin receptor following receptor binding. A new method for studying apelin receptor internalisation is particularly interesting due to the implication of apelin receptor desensitisation in pulmonary arterial hypertension, mediated by arrestin and internalisation pathways. Additionally, apelin-488 may provide insight into binding and receptor trafficking in mutated apelin receptors previously identified in patients with rare cardiovascular diseases. As such, apelin-488 and other fluorescent ligands may help streamline development of drugs that could be beneficial in clinical settings. Conflict of interest none

Introduction The apelin receptor is a class A GPCR that binds two endogenous peptide ligands, apelin and elabela/toddler, to regulate the cardiovascular system. Apelin peptide levels are downregulated in pulmonary arterial hypertension (PAH) but the receptor remains present and clinically targetable. The NIHR BioResource BRIDGE study, a prospective component of the Genomics England 100,000 Genomes Project, has performed case-control genomic analysis of ∼7423 patients with rare cardiovascular diseases including PAH, and has identified a number of mutations in the apelin receptor in this cohort. Of these, 11 were selected for further assessment using high content imaging, following previous saturation binding studies that showed mutational effects on receptor expression and ligand binding. Methods The Opera PhenixTM High Content Screening System was used to generate high-throughput triple fluorescence confocal images in fixed CHO-K1 cells transiently transfected with eGFP-tagged wild-type or mutant apelin receptor. Cells were stained with wheat germ agglutinin-AlexaFluor-594 and Hoechst 33342 as membrane and nuclear markers respectively. Harmony High Content Imaging and Analysis Software was used to empirically quantify fluorescence intensities of the eGFP-tagged apelin receptor in the cytoplasm and membrane of transfected cells. Results Images showed high expression of wild-type receptor with some degree of co-localisation with the membrane stain (fig 1A; fig 2). Mutation 2 showed visibly lower total protein and reduced membrane expression (fig 1B; fig 2). However, mutations 6 and 9 (Fig 1C and 1E respectively) showed similar expression and no significant difference in membrane co-localisation compared to wild-type (fig 2). Mutation 8 showed a trend for reduced membrane co-localisation (fig 1D) compared to wild-type. Note that membrane co-localisation differences closely matched differences in overall eGFP signal. Future work will aim to use machine learning to accurately isolate cell populations that show definite apelin receptor-eGFP signal at the membrane before re-running analysis. Conclusion We report the successful use of high content imaging to quantify differences in expression and membrane co-localisation of the apelin receptor when naturally occurring mutations identified in the NIHR BioResource BRIDGE study are introduced into the protein. When considered with receptor affinities and densities in previous saturation binding data, this work suggests that certain mutations may exhibit an overall impact on receptor expression (i.e. mutation 2), whilst others may directly alter ligand binding without significantly influencing protein levels (i.e. mutant 8). This work may provide great insight on the effects of real human apelin receptor mutations that may be contributing to disease phenotypes. Conflict of interest none

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

Residence time describes the how long a ligand is bound to its target, and is attracting interest in drug discovery as a potential means of improving clinical efficacy by increasing target coverage. This concept, as originally applied to antagonists, is more complicated for G-protein-coupled receptor (GPCR) agonists because of the transiency of receptor responses (via desensitization and internalization). However, in some cases sustained GPCR agonist responses have been observed, with evidence consistent with a role for slow binding kinetics. We propose a model to explain our understanding of how residence time and rebinding might influence sustained signaling by internalized receptors. We also highlight the anticipated benefit for drug discovery of fully understanding and exploiting these phenomena to target desirable receptor response profiles selectively.

G protein-coupled receptor 119 (GPR119) is involved in regulating metabolic homoeostasis, with GPR119 agonists targeted for the treatment of type-2 diabetes and obesity. Using the endogenous agonist oleoylethanolamide and a number of small molecule synthetic agonists we have investigated the temporal dynamics of receptor signalling. Using both a dynamic luminescence biosensor-based assay and an endpoint cAMP accumulation assay we show that agonist-driven desensitization is not a major regulatory mechanism for GPR119 despite robust activation responses, regardless of the agonist used. Temporal analysis of the cAMP responses demonstrated sustained signalling resistant to washout for some, but not all of the agonists tested. Further analysis indicated that the sustained effects of one synthetic agonist AR-231,453 were consistent with a role for slow dissociation kinetics. In contrast, the sustained responses to MBX-2982 and AZ1 appeared to involve membrane deposition. We also detect wash-resistant responses to AR-231,453 at the level of physiologically relevant responses in an endogenous expression system (GLP-1 secretion in GLUTag cells). In conclusion, our findings indicate that in a recombinant expression system GPR119 activation is sustained, with little evidence of pronounced receptor desensitization, and for some ligands persistent agonist responses continue despite removal of excess agonist. This provides novel understanding of the temporal responses profiles of potential drug candidates targetting GPR119, and highlights the importance of carefully examining the the mechanisms through which GPCRs generate sustained responses.

Ghrelin plays a major physiological role in the control of food intake, and inverse agonists of the ghrelin receptor (GHS-R1a) are widely considered to offer utility as antiobesity agents by lowering the set-point for hunger between meals. We identified an acylurea series of ghrelin modulators from high throughput screening and optimized binding affinity through structure-activity relationship studies. Furthermore, we identified specific substructural changes, which switched partial agonist activity to inverse agonist activity, and optimized physicochemical and DMPK properties to afford the non-CNS penetrant inverse agonist 22 (AZ-GHS-22) and the CNS penetrant inverse agonist 38 (AZ-GHS-38). Free feeding efficacy experiments showed that CNS exposure was necessary to obtain reduced food intake in mice, and it was demonstrated using GHS-R1a null and wild-type mice that this effect operates through a mechanism involving GHS-R1a.

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Galpha(i). Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Galpha(i) signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Galpha(i) signalling mediated by GPR43 in SCFA-stimulated leptin secretion.

Nature 546, 254–258 (2017); doi:10.1038/nature22800 In the Supplementary Information of this Article, on pages 3, 8 and 11, we refer to “UK Patent Application No. 1522431.4”, which pertains to the discovery of the truncated peptide analogues we used1, and specifically to the use of the modified N terminus or ‘Cap’.