Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson's disease at 16q11.2 and MAPT H1 loci.

Parkinson's disease is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies has considerably advanced our understanding of the Parkinson's disease genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial Parkinson's disease, but its relevance to idiopathic Parkinson's disease is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through genome-wide association studies. We identified two new regulators of PINK1-dependent mitophagy initiation, KAT8 and KANSL1, previously shown to modulate lysine acetylation. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic Parkinson's disease. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data do not exclude a possible association between the MAPT gene and Parkinson's disease, they provide strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration.

Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors.

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20-24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.

Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

Human metabolic phenotype diversity and its association with diet and blood pressure.

Metabolic phenotypes are the products of interactions among a variety of factors-dietary, other lifestyle/environmental, gut microbial and genetic. We use a large-scale exploratory analytical approach to investigate metabolic phenotype variation across and within four human populations, based on 1H NMR spectroscopy. Metabolites discriminating across populations are then linked to data for individuals on blood pressure, a major risk factor for coronary heart disease and stroke (leading causes of mortality worldwide). We analyse spectra from two 24-hour urine specimens for each of 4,630 participants from the INTERMAP epidemiological study, involving 17 population samples aged 40-59 in China, Japan, UK and USA. We show that urinary metabolite excretion patterns for East Asian and western population samples, with contrasting diets, diet-related major risk factors, and coronary heart disease/stroke rates, are significantly differentiated (P < 10(-16)), as are Chinese/Japanese metabolic phenotypes, and subgroups with differences in dietary vegetable/animal protein and blood pressure. Among discriminatory metabolites, we quantify four and show association (P < 0.05 to P < 0.0001) of mean 24-hour urinary formate excretion with blood pressure in multiple regression analyses for individuals. Mean 24-hour urinary excretion of alanine (direct) and hippurate (inverse), reflecting diet and gut microbial activities, are also associated with blood pressure of individuals. Metabolic phenotyping applied to high-quality epidemiological data offers the potential to develop an area of aetiopathogenetic knowledge involving discovery of novel biomarkers related to cardiovascular disease risk.

Virtual Screening Directly Identifies New Fragment-Sized Inhibitors of Carboxylesterase Notum with Nanomolar Activity.

Notum is a negative regulator of Wnt signaling acting through the hydrolysis of a palmitoleoylate ester, which is required for Wnt activity. Inhibitors of Notum could be of use in diseases where dysfunctional Notum activity is an underlying cause. A docking-based virtual screen (VS) of a large commercial library was used to shortlist 952 compounds for experimental validation as inhibitors of Notum. The VS was successful with 31 compounds having an IC50 < 500 nM. A critical selection process was then applied with two clusters and two singletons (1-4d) selected for hit validation. Optimization of 4d guided by structural biology identified potent inhibitors of Notum activity that restored Wnt/ß-catenin signaling in cell-based models. The [1,2,4]triazolo[4,3-b]pyradizin-3(2H)-one series 4 represent a new chemical class of Notum inhibitors and the first to be discovered by a VS campaign. These results demonstrate the value of VS with well-designed docking models based on X-ray structures.

Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit.

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.

Structural Analysis and Development of Notum Fragment Screening Hits.

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 µM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 µM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.

TREM2/PLCγ2 signalling in immune cells: function, structural insight, and potential therapeutic modulation.

The central role of the resident innate immune cells of the brain (microglia) in neurodegeneration has become clear over the past few years largely through genome-wide association studies (GWAS), and has rapidly become an active area of research. However, a mechanistic understanding (gene to function) has lagged behind. That is now beginning to change, as exemplified by a number of recent exciting and important reports that provide insight into the function of two key gene products - TREM2 (Triggering Receptor Expressed On Myeloid Cells 2) and PLCγ2 (Phospholipase C gamma2) - in microglia, and their role in neurodegenerative disorders. In this review we explore and discuss these recent advances and the opportunities that they may provide for the development of new therapies.

Changing the HTS Paradigm: AI-Driven Iterative Screening for Hit Finding.

Iterative screening is a process in which screening is done in batches, with each batch filled by using machine learning to select the most promising compounds from the library based on the previous results. We believe iterative screening is poised to enhance the screening process by improving hit finding while at the same time reducing the number of compounds screened. In addition, we see this process as a key enabler of next-generation high-throughput screening (HTS), which uses more complex assays that better describe the biology but demand more resource per screened compound. To demonstrate the utility of these methods, we retrospectively analyze HTS data from PubChem with a focus on machine learning-based screening strategies that can be readily implemented in practice. Our results show that over a variety of HTS experimental paradigms, an iterative screening setup that screens a total of 35% of the screening collection over as few as three iterations has a median return rate of approximately 70% of the active compounds. Increasing the portion of the library screened to 50% yields median returns of approximately 80% of actives. Using six iterations increases these return rates to 78% and 90%, respectively. The best results were achieved with machine learning models that can be run on a standard desktop. By demonstrating that the utility of iterative screening holds true even with a small number of iterations, and without requiring significant computational resources, we provide a roadmap for the practical implementation of these techniques in hit finding.

Structural Insights into Notum Covalent Inhibition.

The carboxylesterase Notum hydrolyzes a palmitoleate moiety from Wingless/Integrated(Wnt) ligands and deactivates Wnt signaling. Notum inhibitors can restore Wnt signaling which may be of therapeutic benefit for pathologies such as osteoporosis and Alzheimer's disease. We report the identification of a novel class of covalent Notum inhibitors, 4-(indolin-1-yl)-4-oxobutanoate esters. High-resolution crystal structures of the Notum inhibitor complexes reveal a common covalent adduct formed between the nucleophile serine-232 and hydrolyzed butyric esters. The covalent interaction in solution was confirmed by mass spectrometry analysis. Inhibitory potencies vary depending on the warheads used. Mechanistically, the resulting acyl-enzyme intermediate carbonyl atom is positioned at an unfavorable angle for the approach of the active site water, which, combined with strong hydrophobic interactions with the enzyme pocket residues, hinders the intermediate from being further processed and results in covalent inhibition. These insights into Notum catalytic inhibition may guide development of more potent Notum inhibitors.

Enhanced insulin signalling ameliorates C9orf72 hexanucleotide repeat expansion toxicity in Drosophila.

G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/IGF signalling is reduced in fly models of C9orf72 repeat expansion using RNA sequencing of adult brain. We further demonstrate that activation of insulin/IGF signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/IGF signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/IGF signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.

Integrated cytokine and metabolic analysis of pathological responses to parasite exposure in rodents.

Parasitic infections cause a myriad of responses in their mammalian hosts, on immune as well as on metabolic level. A multiplex panel of cytokines and metabolites derived from four parasite-rodent models, namely, Plasmodium berghei-mouse, Trypanosoma brucei brucei-mouse, Schistosoma mansoni-mouse, and Fasciola hepatica-rat were statistically coanalyzed. (1)H NMR spectroscopy and multivariate statistical analysis were used to characterize the urine and plasma metabolite profiles in infected and noninfected animals. Each parasite generated a unique metabolic signature in the host. Plasma cytokine concentrations were obtained using the 'Meso Scale Discovery' multi cytokine assay platform. Multivariate data integration methods were subsequently used to elucidate the component of the metabolic signature which is associated with inflammation and to determine specific metabolic correlates with parasite-induced changes in plasma cytokine levels. For example, the relative levels of acetyl glycoproteins extracted from the plasma metabolite profile in the P. berghei-infected mice were statistically correlated with IFN-gamma, whereas the same cytokine was anticorrelated with glucose levels. Both the metabolic and the cytokine data showed a similar spatial distribution in principal component analysis scores plots constructed for the combined murine data, with samples from all infected animals clustering according to the parasite species and whereby the protozoan infections (P. berghei and T. b. brucei) grouped separately from the helminth infection (S. mansoni). For S. mansoni, the main infection-responsive cytokines were IL-4 and IL-5, which covaried with lactate, choline, and d-3-hydroxybutyrate. This study demonstrates that the inherently differential immune response to single- and multicellular parasites not only manifests in the cytokine expression, but also consequently imprints on the metabolic signature, and calls for in-depth analysis to further explore direct links between immune features and biochemical pathways.

Metabolome-wide association study identifies multiple biomarkers that discriminate north and south Chinese populations at differing risks of cardiovascular disease: INTERMAP study.

Rates of heart disease and stroke vary markedly between north and south China. A (1)H NMR spectroscopy-based metabolome-wide association approach was used to identify urinary metabolites that discriminate between southern and northern Chinese population samples, to investigate population biomarkers that might relate to the difference in cardiovascular disease risk. NMR spectra were acquired from two 24-h urine specimens per person for 523 northern and 244 southern Chinese participants in the INTERMAP Study of macro/micronutrients and blood pressure. Discriminating metabolites were identified using orthogonal partial least squares discriminant analysis and assessed for statistical significance with conservative family wise error rate < 0.01 to minimize false positive findings. Urinary metabolites significantly (P < 1.2 × 10(-16) to 2.9 × 10(-69)) higher in northern than southern Chinese populations included dimethylglycine, alanine, lactate, branched-chain amino acids (isoleucine, leucine, valine), N-acetyls of glycoprotein fragments (including uromodulin), N-acetyl neuraminic acid, pentanoic/heptanoic acid, and methylguanidine; metabolites significantly (P < 1.1 × 10(-12) to 2 × 10(-127)) higher in the south were gut microbial cometabolites (hippurate, 4-cresyl sulfate, phenylacetylglutamine, 2-hydroxyisobutyrate), succinate, creatine, scyllo-inositol, prolinebetaine, and trans-aconitate. These findings indicate the importance of environmental influences (e.g., diet), endogenous metabolism, and mammalian-gut microbial cometabolism, which may help explain north-south China differences in cardiovascular disease risk.

5-Phenyl-1,3,4-oxadiazol-2(3H)-ones Are Potent Inhibitors of Notum Carboxylesterase Activity Identified by the Optimization of a Crystallographic Fragment Screening Hit.

Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment screen was performed, which identified fragment hit 1,2,3-triazole 7 as an attractive starting point for a structure-based drug design hit-to-lead program. Optimization of 7 identified oxadiazol-2-one 23dd as a preferred example with properties consistent with drug-like chemical space. Screening 23dd in a cell-based TCF/LEF reporter gene assay restored the activation of Wnt signaling in the presence of Notum. Mouse pharmacokinetic studies with oral administration of 23dd demonstrated good plasma exposure and partial blood-brain barrier penetration. Significant progress was made in developing fragment hit 7 into lead 23dd (>600-fold increase in activity), making it suitable as a new chemical tool for exploring the role of Notum-mediated regulation of Wnt signaling.

Screening of a Custom-Designed Acid Fragment Library Identifies 1-Phenylpyrroles and 1-Phenylpyrrolidines as Inhibitors of Notum Carboxylesterase Activity.

The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions. Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were identified as hits for Notum inhibition, and 14 of these fragments were shown to bind in the palmitoleate pocket of Notum. Optimization of 1-phenylpyrrole 20, guided by structure-based drug design, identified 20z as the most potent compound from this series. Similarly, the optimization of 1-phenylpyrrolidine 8 gave acid 26. This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules possessing favorable in vitro ADME profiles.

Biomarkers in nutritional epidemiology: applications, needs and new horizons.

Modern epidemiology suggests a potential interactive association between diet, lifestyle, genetics and the risk of many chronic diseases. As such, many epidemiologic studies attempt to consider assessment of dietary intake alongside genetic measures and other variables of interest. However, given the multi-factorial complexities of dietary exposures, all dietary intake assessment methods are associated with measurement errors which affect dietary estimates and may obscure disease risk associations. For this reason, dietary biomarkers measured in biological specimens are being increasingly used as additional or substitute estimates of dietary intake and nutrient status. Genetic variation may influence dietary intake and nutrient metabolism and may affect the utility of a dietary biomarker to properly reflect dietary exposures. Although there are many functional dietary biomarkers that, if utilized appropriately, can be very informative, a better understanding of the interactions between diet and genes as potentially determining factors in the validity, application and interpretation of dietary biomarkers is necessary. It is the aim of this review to highlight how some important biomarkers are being applied in nutrition epidemiology and to address some associated questions and limitations. This review also emphasizes the need to identify new dietary biomarkers and highlights the emerging field of nutritional metabonomics as an analytical method to assess metabolic profiles as measures of dietary exposures and indicators of dietary patterns, dietary changes or effectiveness of dietary interventions. The review will also touch upon new statistical methodologies for the combination of dietary questionnaire and biomarker data for disease risk assessment. It is clear that dietary biomarkers require much further research in order to be better applied and interpreted. Future priorities should be to integrate high quality dietary intake information, measurements of dietary biomarkers, metabolic profiles of specific dietary patterns, genetics and novel statistical methodology in order to provide important new insights into gene-diet-lifestyle-disease risk associations.

Metabolic profiling and population screening of analgesic usage in nuclear magnetic resonance spectroscopy-based large-scale epidemiologic studies.

The application of a (1)H nuclear magnetic resonance (NMR) spectroscopy-based screening method for determining the use of two widely available analgesics (acetaminophen and ibuprofen) in epidemiologic studies has been investigated. We used samples and data from the cross-sectional INTERMAP Study involving participants from Japan (n = 1145), China (n = 839), U.K. (n = 501), and the U.S. (n = 2195). An orthogonal projection to latent structures discriminant analysis (OPLS-DA) algorithm with an incorporated Monte Carlo resampling function was applied to the NMR data set to determine which spectra contained analgesic metabolites. OPLS-DA preprocessing parameters (normalization, bin width, scaling, and input parameters) were assessed systematically to identify an optimal acetaminophen prediction model. Subsets of INTERMAP spectra were examined to verify and validate the presence/absence of acetaminophen/ibuprofen based on known chemical shift and coupling patterns. The optimized and validated acetaminophen model correctly predicted 98.2%, and the ibuprofen model correctly predicted 99.0% of the urine specimens containing these drug metabolites. The acetaminophen and ibuprofen models were subsequently used to predict the presence/absence of these drug metabolites for the remaining INTERMAP specimens. The acetaminophen model identified 415 out of 8436 spectra as containing acetaminophen metabolite signals while the ibuprofen model identified 245 out of 8604 spectra as containing ibuprofen metabolite signals from the global data set after excluding samples used to construct the prediction models. The NMR-based metabolic screening strategy provides a new objective approach for evaluation of self-reported medication data and is extendable to other aspects of population xenometabolome profiling.

Discovery of 2-phenoxyacetamides as inhibitors of the Wnt-depalmitoleating enzyme NOTUM from an X-ray fragment screen.

NOTUM is a carboxylesterase that has been shown to act by mediating the O-depalmitoleoylation of Wnt proteins resulting in suppression of Wnt signaling. Here, we describe the development of NOTUM inhibitors that restore Wnt signaling for use in in vitro disease models where NOTUM over activity is an underlying cause. A crystallographic fragment screen with NOTUM identified 2-phenoxyacetamide 3 as binding in the palmitoleate pocket with modest inhibition activity (IC50 33 µM). Optimization of hit 3 by SAR studies guided by SBDD identified indazole 38 (IC50 0.032 µM) and isoquinoline 45 (IC50 0.085 µM) as potent inhibitors of NOTUM. The binding of 45 to NOTUM was rationalized through an X-ray co-crystal structure determination which showed a flipped binding orientation compared to 3. However, it was not possible to combine NOTUM inhibition activity with metabolic stability as the majority of the compounds tested were rapidly metabolized in an NADPH-independent manner.

Publisher Correction: Molecular and functional variation in iPSC-derived sensory neurons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular and functional variation in iPSC-derived sensory neurons.

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools for modeling biological processes, particularly in cell types that are difficult to obtain from living donors. Here we present a map of regulatory variants in iPSC-derived neurons, based on 123 differentiations of iPSCs to a sensory neuronal fate. Gene expression was more variable across cultures than in primary dorsal root ganglion, particularly for genes related to nervous system development. Using single-cell RNA-sequencing, we found that the number of neuronal versus contaminating cells was influenced by iPSC culture conditions before differentiation. Despite high differentiation-induced variability, our allele-specific method detected thousands of quantitative trait loci (QTLs) that influenced gene expression, chromatin accessibility, and RNA splicing. On the basis of these detected QTLs, we estimate that recall-by-genotype studies that use iPSC-derived cells will require cells from at least 20-80 individuals to detect the effects of regulatory variants with moderately large effect sizes.