Structural basis of efficacy-driven ligand selectivity at GPCRs.

A drug's selectivity for target receptors is essential to its therapeutic utility, but achieving selectivity between similar receptors is challenging. The serendipitous discovery of ligands that stimulate target receptors more strongly than closely related receptors, despite binding with similar affinities, suggests a solution. The molecular mechanism of such 'efficacy-driven selectivity' has remained unclear, however, hindering design of such ligands. Here, using atomic-level simulations, we reveal the structural basis for the efficacy-driven selectivity of a long-studied clinical drug candidate, xanomeline, between closely related muscarinic acetylcholine receptors (mAChRs). Xanomeline's binding mode is similar across mAChRs in their inactive states but differs between mAChRs in their active states, with divergent effects on active-state stability. We validate this mechanism experimentally and use it to design ligands with altered efficacy-driven selectivity. Our results suggest strategies for the rational design of ligands that achieve efficacy-driven selectivity for many pharmaceutically important G-protein-coupled receptors.

A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Biochemical signatures of disease severity in multiple sulfatase deficiency.

Sulfatases catalyze essential cellular reactions, including degradation of glycosaminoglycans (GAGs). All sulfatases are post-translationally activated by the formylglycine generating enzyme (FGE) which is deficient in multiple sulfatase deficiency (MSD), a neurodegenerative lysosomal storage disease. Historically, patients were presumed to be deficient of all sulfatase activities; however, a more nuanced relationship is emerging. Each sulfatase may differ in their degree of post-translational modification by FGE, which may influence the phenotypic spectrum of MSD. Here, we evaluate if residual sulfatase activity and accumulating GAG patterns distinguish cases from controls and stratify clinical severity groups in MSD. We quantify sulfatase activities and GAG accumulation using three complementary methods in MSD participants. Sulfatases differed greatly in their tolerance of reduction in FGE-mediated activation. Enzymes that degrade heparan sulfate (HS) demonstrated lower residual activities than those that act on other GAGs. Similarly, HS-derived urinary GAG subspecies preferentially accumulated, distinguished cases from controls, and correlated with disease severity. Accumulation patterns of specific sulfatase substrates in MSD provide fundamental insights into sulfatase regulation and will serve as much-needed biomakers for upcoming clinical trials. This work highlights that biomarker investigation of an ultra-rare disease can simultaneously inform our understanding of fundamental biology and advance clinical trial readiness efforts.

Beta-lactam allergy risk stratification in a maternity population in Australia: Scope for allergy de-labelling.

BACKGROUND: Unconfirmed beta-lactam allergy in pregnant people has been associated with higher morbidity, unnecessary exposure to broad-spectrum antibiotics and prolonged hospitalisation. There are no published data on beta-lactam allergies in pregnant people in Australia. AIMS: The aim was to describe patient-reported beta-lactam allergies and appropriateness for antibiotic allergy de-labelling in a maternity cohort in Australia. METHODS: Maternity patients aged ≥18 years admitted to our institution between March 2021 and June 2021 with a beta-lactam allergy documented in their electronic medical record were interviewed for details of their allergy. The documented allergies were compared to the allergy history obtained from the interview. Severity of the allergy was rated, and appropriateness for allergy de-labelling was assessed using the Victorian Therapeutics Advisory Group beta-lactam antibiotic allergy assessment tool. RESULTS: One hundred and fifty-three beta-lactam allergies (182 reactions) were reported by 145 patients. Penicillin class antibiotics were the most frequently implicated, including unspecified penicillins (95/153, 62%), amoxicillin (19/153, 13%) and amoxicillin-clavulanate (8/153, 5%). Allergy documentation required amending in 52 of 145 patients (36%); 85 of 153 (56%) of the beta-lactam allergies were considered low risk and potentially appropriate for direct oral re-challenge. CONCLUSION: Beta-lactam allergies were inaccurately documented in more than one third of the maternity patients included in our study. As such, education of maternity care providers about the importance of accurate allergy history taking remains an urgent unmet need. Furthermore, allergy assessment and de-labelling during pregnancy should be considered in maternity patients to optimise antibiotic prescribing and to improve maternal and neonatal health outcomes.

Role of Conserved Tyrosine Lid Residues in the Activation of the M2 Muscarinic Acetylcholine Receptor.

The development of subtype selective small molecule drugs for the muscarinic acetylcholine receptor (mAChR) family has been challenging. The design of more selective ligands can be improved by understanding the structure and function of key amino acid residues that line ligand binding sites. Here we study the role of three conserved key tyrosine residues [Y1043.33, Y4036.51, and Y4267.39 (Ballesteros and Weinstein numbers in superscript)] at the human M2 mAChR, located at the interface between the orthosteric and allosteric binding sites of the receptor. We specifically focused on the role of the three tyrosine hydroxyl groups in the transition between the inactive and active conformations of the receptor by making phenylalanine point mutants. Single-point mutation at either of the three positions was sufficient to reduce the affinity of agonists by ∼100-fold for the M2 mAChR, whereas the affinity of antagonists remained largely unaffected. In contrast, neither of the mutations affected the efficacy of orthosteric agonists. When mutations were combined into double and triple M2 mAChR mutants, the affinity of antagonists was reduced by more than 100-fold compared with the wild-type M2 receptor. In contrast, the affinity of allosteric modulators, either negative or positive, was retained at all single and multiple mutations, but the degree of allosteric effect exerted on the endogenous ligand acetylcholine was affected at all mutants containing Y4267.39F. These findings will provide insights to consider when designing future mAChR ligands. SIGNIFICANCE STATEMENT: Structural studies demonstrated that three tyrosine residues between the orthosteric and allosteric sites of the M2 muscarinic acetylcholine receptor (mAChR) had different hydrogen bonding networks in the inactive and active conformations. The role of hydroxyl groups of the tyrosine residues on orthosteric and allosteric ligand pharmacology was unknown. We found that hydroxyl groups of the tyrosine residues differentially affected the molecular pharmacology of orthosteric and allosteric ligands. These results provide insights to consider when designing future mAChR ligands.

Positive allosteric modulation of endogenous delta opioid receptor signaling in the enteric nervous system is a potential treatment for gastrointestinal motility disorders.

Allosteric modulators (AMs) are molecules that can fine-tune signaling by G protein-coupled receptors (GPCRs). Although they are a promising therapeutic approach for treating a range of disorders, allosteric modulation of GPCRs in the context of the enteric nervous system (ENS) and digestive dysfunction remains largely unexplored. This study examined allosteric modulation of the delta opioid receptor (DOR) in the ENS and assessed the suitability of DOR AMs for the treatment of irritable bowel syndrome (IBS) symptoms using mouse models. The effects of the positive allosteric modulator (PAM) of DOR, BMS-986187, on neurogenic contractions of the mouse colon and on DOR internalization in enteric neurons were quantified. The ability of BMS-986187 to influence colonic motility was assessed both in vitro and in vivo. BMS-986187 displayed DOR-selective PAM-agonist activity and orthosteric agonist probe dependence in the mouse colon. BMS-986187 augmented the inhibitory effects of DOR agonists on neurogenic contractions and enhanced reflex-evoked DOR internalization in myenteric neurons. BMS-986187 significantly increased DOR endocytosis in myenteric neurons in response to the weakly internalizing agonist ARM390. BMS-986187 reduced the generation of complex motor patterns in the isolated intact colon. BMS-986187 reduced fecal output and diarrhea onset in the novel environment stress and castor oil models of IBS symptoms, respectively. DOR PAMs enhance DOR-mediated signaling in the ENS and have potential benefit for the treatment of dysmotility. This study provides proof of concept to support the use of GPCR AMs for the treatment of gastrointestinal motility disorders.NEW & NOTEWORTHY This study assesses the use of positive allosteric modulation as a pharmacological approach to enhance opioid receptor signaling in the enteric nervous system. We demonstrate that selective modulation of endogenous delta opioid receptor signaling can suppress colonic motility without causing constipation. We propose that allosteric modulation of opioid receptor signaling may be a therapeutic strategy to normalize gastrointestinal motility in conditions such as irritable bowel syndrome.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Three-dimensional quantitative MRI of aerosolized gadolinium-based nanoparticles and contrast agents in isolated ventilated porcine lungs.

PURPOSE: The objective of this study is to evaluate the suitability and performance of ultra-short echo time (UTE) sequences for imaging and quantifying the deposition of nebulized MRI contrast agents in human-sized lungs. METHODS: Nebulization of clinically used contrast agent or gadolinium-based nanoparticles were performed using a commercial jet nebulizer in isolated and ventilated porcine lungs connected to a 3D-printed human upper airways replica. MR images of isolated lungs were acquired on a 3T clinical MR scanner using 3D UTE sequences at different flip angles. RESULTS: 3D acquisitions with isotropic millimetric resolution were obtained in less than 4 min. Images exhibit homogeneous and large MR signal enhancement (above 200%) following nebulization of both types of aerosols. Deposition of aerosol down to the level of the bronchi of secondary lobules was visualized. T1 values and the concentration of nanoparticles obtained by MRI were found to correlate with the amount of nebulized gadolinium3+ ions. CONCLUSION: The distribution of aerosolized gadolinium-based contrast agent or nanoparticles can be visualized and quantified using UTE MRI in large animal ventilated lung model on a clinical MRI scanner. This protocol can be used for assessing and quantifying aerosol regional deposition with high spatial resolution (1 mm 3D isotropic) without ionizing radiation and could be applied in the future for diagnostic or therapeutic applications in patients.

Potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acid-sensing ion channel 1a.

Stroke is the second-leading cause of death worldwide, yet there are no drugs available to protect the brain from stroke-induced neuronal injury. Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and a key mediator of acidosis-induced neuronal damage following cerebral ischemia. Genetic ablation and selective pharmacologic inhibition of ASIC1a reduces neuronal death following ischemic stroke in rodents. Here, we demonstrate that Hi1a, a disulfide-rich spider venom peptide, is highly neuroprotective in a focal model of ischemic stroke. Nuclear magnetic resonance structural studies reveal that Hi1a comprises two homologous inhibitor cystine knot domains separated by a short, structurally well-defined linker. In contrast with known ASIC1a inhibitors, Hi1a incompletely inhibits ASIC1a activation in a pH-independent and slowly reversible manner. Whole-cell, macropatch, and single-channel electrophysiological recordings indicate that Hi1a binds to and stabilizes the closed state of the channel, thereby impeding the transition into a conducting state. Intracerebroventricular administration to rats of a single small dose of Hi1a (2 ng/kg) up to 8 h after stroke induction by occlusion of the middle cerebral artery markedly reduced infarct size, and this correlated with improved neurological and motor function, as well as with preservation of neuronal architecture. Thus, Hi1a is a powerful pharmacological tool for probing the role of ASIC1a in acid-mediated neuronal injury and various neurological disorders, and a promising lead for the development of therapeutics to protect the brain from ischemic injury.

Binding to and Inhibition of Insulin-Regulated Aminopeptidase by Macrocyclic Disulfides Enhances Spine Density.

Angiotensin IV (Ang IV) and related peptide analogs, as well as nonpeptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocyclic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N terminus of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09, and of Ang IV in either the extended or γ-turn conformation at the C terminus to human IRAP were predicted by docking and molecular dynamics simulations. The binding free energies calculated with the linear interaction energy method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.

Chronic GLP1 therapy reduces postprandial IL6 in obese humans with prediabetes.

Single-dose glucagon-like peptide 1 (GLP1) therapy increases postprandial plasma IL6 levels in prediabetic, obese humans. GLP1-IL6 interactions underly multiple antidiabetic effects, but these may differ after acute versus chronic therapy. This study examines postprandial effects of GLP1 after chronic therapy. Seven humans (six Black) with prediabetes and obesity completed 6 weeks of exenatide extended release therapy. Then subjects returned for pre- and post-meal measurements of plasma IL6, GLP1, glucagon, and related inflammatory markers. Weight, which was measured before and after therapy, did not change. Plasma IL6 decreased from baseline to postmeal state ( = 0.016), with decreases in free fatty acids (P < 0.001) and increases in insulin (P = 0.002), glucose (P < 0.0001), triglycerides (P = 0.0178), and glucagon (P = 0.018). Baseline GLP1 levels matched 6 weeks of therapy. The fall in postprandial plasma IL6, which contrasts with the increase after acute therapy, highlights the need for more investigation regarding the mechanisms of acute versus chronic GLP1-IL6 signaling.

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa -KO MLD mice transplanted with Arsa -KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

Conversion of Phenolic Acids in Canola Fermentation: Impact on Antimicrobial Activity against Salmonella enterica and Campylobacter jejuni.

Canola meal (CM) is commonly used in poultry feeds. CM has a high protein content but also contains high levels of antimicrobial phenolic acids. Lactic acid bacteria can alter CM phenolic composition during fermentation and influence its antimicrobial activity against pathogens. Fermented CM was analyzed for phenolic composition using tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). Sinapic acid and derivatives were the major phenolic acids in CM. Growth of lactobacilli in CM was attenuated when compared to cereal substrates. Glucosides and esters of sinapic acid were extensively hydrolyzed during fermentation with Lactiplantibacillus plantarum and Furfurilactobacillus milii. Lp. plantarum transformed hydroxycinnamic acids to dihydro, 4-vinyl, and 4-ethyl derivatives, Ff. milii reduced hydroxycinnamic acids to dihydroderivatives, but Limosilactobacillus reuteri did not convert hydroxycinnamic acids. The minimum inhibitory concentration of phenolic extracts was assessed with lactobacilli, Salmonella, and Campylobacter jejuni as indicator strains. Fermentation of CM with Lp. plantarum or Ff. milii increased the antimicrobial activity of phenolic extracts against Salmonella enterica and Campylobacter jejuni. Fermentation with Lm. reuteri TMW1.656 but not fermentation with Lm. reuteri TMW1.656ΔrtcN increased the antimicrobial activity of extracts owing to the production of reutericyclin. This study demonstrates that fermentation of CM with lactobacilli converts hydroxycinammic esters and may increase the antimicrobial activity of phenolic compounds in CM against pathogens.

Inhibition of insulin-regulated aminopeptidase confers neuroprotection in a conscious model of ischemic stroke.

Stroke is a leading cause of mortality and morbidity with a paucity of effective pharmacological treatments. We have previously identified insulin-regulated aminopeptidase (IRAP) as a potential target for the development of a new class of drugs for the treatment of stroke, as global deletion of this gene in mice significantly protected against ischemic damage. In the current study, we demonstrate that small molecular weight IRAP inhibitors reduce infarct volume and improve neurological outcome in a hypertensive animal model of ischemic stroke. The effects of two structurally distinct IRAP inhibitors (HFI419 or SJM164) were investigated in a model of stroke where the middle cerebral artery was transiently occluded with endothelin-1 in the conscious spontaneously hypertensive rat. IRAP inhibitor was administered into the lateral ventricle at 2 or 6 h after stroke, with subsequent doses delivered at 24, 48 and 70 h post-stroke. Functional outcomes were assessed prior to drug treatment, and on day 1 and 3 post-stroke. Histological analyses and neuroinflammatory cytokine profiling were conducted at 72 and 24 h post-stroke respectively. IRAP inhibitor treatment following stroke significantly reduced infarct volume and improved neurological and motor deficits. These protective effects were maintained even when the therapeutic window was extended to 6 h. Examination of the cellular architecture at 72 h post-stroke demonstrated that IRAP expression was upregulated in CD11b positive cells and activated astrocytes. Furthermore, IRAP inhibitor treatment significantly increased gene expression for interleukin 6 and C-C motif chemokine ligand 2 in the ischemic core. This study provides proof-of-principle that selective inhibition of IRAP activity with two structurally distinct IRAP inhibitors reduces infarct volume and improves functional outcome even when the first dose is administered 6 h post-stroke. This is the first direct evidence that IRAP inhibitors are a class of drug with potential use in the treatment of ischemic stroke.

Cohort profile: The SmartSleep Study, Denmark, combining evidence from survey, clinical and tracking data.

PURPOSE: The SmartSleep Study is established to comprehensively assess the impact of night-time smartphone use on sleep patterns and health. An innovative combination of large-scale repeated survey information, high-resolution sensor-driven smartphone data, in-depth clinical examination and registry linkage allows for detailed investigations into multisystem physiological dysregulation and long-term health consequences associated with night-time smartphone use and sleep impairment. PARTICIPANTS: The SmartSleep Study consists of three interconnected data samples, which combined include 30 673 individuals with information on smartphone use, sleep and health. Subsamples of the study population also include high-resolution tracking data (n=5927) collected via a customised app and deep clinical phenotypical data (n=245). A total of 7208 participants are followed in nationwide health registries with full data coverage and long-term follow-up. FINDINGS TO DATE: We highlight previous findings on the relation between smartphone use and sleep in the SmartSleep Study, and we evaluate the interventional potential of the citizen science approach used in one of the data samples. We also present new results from an analysis in which we use 803 000 data points from the high-resolution tracking data to identify clusters of temporal trajectories of night-time smartphone use that characterise distinct use patterns. Based on these objective tracking data, we characterise four clusters of night-time smartphone use. FUTURE PLANS: The unprecedented size and coverage of the SmartSleep Study allow for a comprehensive documentation of smartphone activity during the entire sleep span. The study has been expanded by linkage to nationwide registers, which allow for further investigations into the long-term health and social consequences of night-time smartphone use. We also plan new rounds of data collection in the coming years.

Structure-activity relationship of pyrazol-4-yl-pyridine derivatives and identification of a radiofluorinated probe for imaging the muscarinic acetylcholine receptor M4.

There is an accumulating body of evidence implicating the muscarinic acetylcholine receptor 4 (M4) in schizophrenia and dementia with Lewy bodies, however, a clinically validated M4 positron emission tomography (PET) radioligand is currently lacking. As such, the aim of this study was to develop a suitable M4 PET ligand that allows the non-invasive visualization of M4 in the brain. Structure-activity relationship studies of pyrazol-4-yl-pyridine derivates led to the discovery of target compound 12 - a subtype-selective positive allosteric modulator (PAM). The radiofluorinated analogue, [18F]12, was synthesized in 28 ± 10% radiochemical yield, >37 GBq/µmol and an excellent radiochemical purity >99%. Initial in vitro autoradiograms on rodent brain sections were performed in the absence of carbachol and showed moderate specificity as well as a low selectivity of [18F]12 for the M4-rich striatum. However, in the presence of carbachol, a significant increase in tracer binding was observed in the rat striatum, which was reduced by >60% under blocking conditions, thus indicating that orthosteric ligand interaction is required for efficient binding of [18F]12 to the allosteric site. Remarkably, however, the presence of carbachol was not required for high specific binding in the non-human primate (NHP) and human striatum, and did not further improve the specificity and selectivity of [18F]12 in higher species. These results pointed towards significant species-differences and paved the way for a preliminary PET study in NHP, where peak brain uptake of [18F]12 was found in the putamen and temporal cortex. In conclusion, we report on the identification and preclinical development of the first radiofluorinated M4 PET radioligand with promising attributes. The availability of a clinically validated M4 PET radioligand harbors potential to facilitate drug development and provide a useful diagnostic tool for non-invasive imaging.

Xanomeline displays concomitant orthosteric and allosteric binding modes at the M4 mAChR.

The M4 muscarinic acetylcholine receptor (M4 mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M4 mAChR in complex with the heterotrimeric Gi1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M4 mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M4 mAChR. These findings provide a basis for further understanding xanomeline's complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics.

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.

Predicting stress and depressive symptoms using high-resolution smartphone data and sleep behavior in Danish adults.

STUDY OBJECTIVES: The early detection of mental disorders is crucial. Patterns of smartphone behavior have been suggested to predict mental disorders. The aim of this study was to develop and compare prediction models using a novel combination of smartphone and sleep behavior to predict early indicators of mental health problems, specifically high perceived stress and depressive symptoms. METHODS: The data material included two separate population samples nested within the SmartSleep Study. Prediction models were trained using information from 4522 Danish adults and tested in an independent test set comprising of 1885 adults. The prediction models utilized comprehensive information on subjective smartphone behavior, objective night-time smartphone behavior, and self-reported sleep behavior. Receiver operating characteristics area-under-the-curve (ROC AUC) values obtained in the test set were recorded as the performance metrics for each prediction model. RESULTS: Neither subjective nor objective smartphone behavior was found to add additional predictive information compared to basic sociodemographic factors when forecasting perceived stress or depressive symptoms. Instead, the best performance for predicting poor mental health was found in the sleep prediction model (AUC = 0.75, 95% CI: 0.72-0.78) for perceived stress and (AUC = 0.83, 95%CI: 0.80-0.85) for depressive symptoms, which included self-reported information on sleep quantity, sleep quality and the use of sleep medication. CONCLUSIONS: Sleep behavior is an important predictor when forecasting mental health symptoms and it outperforms novel approaches using objective and subjective smartphone behavior.