Stereocontrolled access to thioisosteres of nucleoside di- and triphosphates.

Nucleoside diphosphates and triphosphates impact nearly every aspect of biochemistry; however, the use of such compounds as tools or medicinal leads for nucleotide-dependent enzymes and receptors is hampered by their rapid in vivo metabolism. Although a successful strategy to address the instability of the monophosphate moiety in oligonucleotide therapeutics has been accomplished by their isosteric replacement with phosphorothioates, no practical methods exist to rapidly and controllably access stereopure di- and triphosphate thioisosteres of both natural and unnatural nucleosides. Here we show how a modular, reagent-based platform can enable the stereocontrolled and scalable synthesis of a library of such molecules. This operationally simple approach provides access to pure stereoisomers of nucleoside α-thiodiphosphates and α-thiotriphosphates, as well as symmetrical or unsymmetrical dinucleoside thiodiphosphates and thiotriphosphates (including RNA cap reagents). We demonstrate that ligand-receptor interactions can be dramatically influenced by P-stereochemistry, showing that such thioisosteric replacements can have profound effects on the potency and stability of lead candidates.

Stereoselective and Divergent Aza-Adenosine and Aza-Guanosine Syntheses from Xylofuranose, the Key Fragments of a STING Cyclic Dinucleotide Agonist.

The stereoselective and divergent synthesis of two aza-nucleosides is reported. Starting from xylofuranose 9, aza-adenosine 2 was prepared in 13 steps and 7% overall yield, and aza-guanosine 3 was prepared in 13 steps and 7.8% overall yield. Compared to the original syntheses, some advantages of these new routes are significant yield improvement, overall step-count reduction, an optimized protecting group strategy, the development of a versatile platform for nitrogenous base incorporation, and the elimination of hazardous reagents (e.g., benzyl isocyanate, Et3N·HF).

Metabolomics as a Truly Translational Tool for Precision Medicine.

Metabolomics is unique among omics technologies in being applicable to metabolism and toxicity studies broadly across organisms (e.g., humans, other mammals, model organisms, and even bacteria) and across biological materials (e.g., blood, urine, saliva, biopsy, and stool), including cultured cells and subcellular fractions. Metabolomics can be used to characterize biologic response patterns in humans as well as to support mechanistic studies in model systems and ex vivo studies. A broad range of resources are available, including publicly accessible data repositories (e.g., Metabolomics Workbench), tools for biostatistics and bioinformatics (e.g., MetaboAnalyst), metabolite identification (e.g., Metlin), and pathway analysis (e.g., Kyoto Encyclopedia of Genes and Genomes). Thus, metabolomics is more than a promise of the future; metabolomics is already available as a translational approach to facilitate precision medicine. This ACT Symposium review will contain an introduction to metabolomics in toxicity studies followed by sections on translational metabolic networks, translational metabolite biomarkers of acetaminophen-induced acute liver injury, translational framework using high-resolution metabolomics for integrated pharmacokinetics and pharmacodynamics, and precision medicine applications: extracting actionable targets from untargeted metabolomics data following one year in space.

DNA-Model-Based Design and Execution of Some Fused Benzodiazepine Hybrid Payloads for Antibody-Drug Conjugate Modality.

A new series with the tetrahydroisoquinoline-fused benzodiazepine (TBD) ring system combined with the surrogates of (1-methyl-1H-pyrrol-3-yl)benzene ("MPB") payloads were designed and executed for conjugation with a monoclonal antibody for anticancer therapeutics. DNA models helped in rationally identifying modifications of the "MPB" binding component and guided structure-activity relationship generation. This hybrid series of payloads exhibited excellent in vitro activity when tested against a panel of various cancer cell lines. One of the payloads was appended with a lysosome-cleavable peptide linker and conjugated with an anti-mesothelin antibody via a site-specific conjugation method mediated by the enzyme bacterial transglutaminase (BTGase). Antibody-drug conjugate (ADC) 50 demonstrated good plasma stability and lysosomal cleavage. A single intravenous dose of ADC 50 (5 or 10 nmol/kg) showed robust efficacy in an N87 gastric cancer xenograft model.

Epigenome-wide meta-analysis of DNA methylation differences in prefrontal cortex implicates the immune processes in Alzheimer's disease.

DNA methylation differences in Alzheimer's disease (AD) have been reported. Here, we conducted a meta-analysis of more than 1000 prefrontal cortex brain samples to prioritize the most consistent methylation differences in multiple cohorts. Using a uniform analysis pipeline, we identified 3751 CpGs and 119 differentially methylated regions (DMRs) significantly associated with Braak stage. Our analysis identified differentially methylated genes such as MAMSTR, AGAP2, and AZU1. The most significant DMR identified is located on the MAMSTR gene, which encodes a cofactor that stimulates MEF2C. Notably, MEF2C cooperates with another transcription factor, PU.1, a central hub in the AD gene network. Our enrichment analysis highlighted the potential roles of the immune system and polycomb repressive complex 2 in pathological AD. These results may help facilitate future mechanistic and biomarker discovery studies in AD.

Multi-omic, Single-Cell, and Biochemical Profiles of Astronauts Guide Pharmacological Strategies for Returning to Gravity.

The National Aeronautics and Space Administration (NASA) Twins Study created an integrative molecular profile of an astronaut during NASA's first 1-year mission on the International Space Station (ISS) and included comparisons to an identical Earth-bound twin. The unique biochemical profiles observed when landing on Earth after such a long mission (e.g., spikes in interleukin-1 [IL-1]/6/10, c-reactive protein [CRP], C-C motif chemokine ligand 2 [CCL2], IL-1 receptor antagonist [IL-1ra], and tumor necrosis factor alpha [TNF-α]) opened new questions about the human body's response to gravity and how to plan for future astronauts, particularly around initiation or resolution of inflammation. Here, single-cell, multi-omic (100-plex epitope profile and gene expression) profiling of peripheral blood mononuclear cells (PBMCs) showed changes to blood cell composition and gene expression post-flight, specifically for monocytes and dendritic cell precursors. These were consistent with flight-induced cytokine and immune system stress, followed by skeletal muscle regeneration in response to gravity. Finally, we examined these profiles relative to 6-month missions in 28 other astronauts and detail potential pharmacological interventions for returning to gravity in future missions.

Serine-Selective Bioconjugation.

This Communication reports the first general method for rapid, chemoselective, and modular functionalization of serine residues in native polypeptides, which uses a reagent platform based on the P(V) oxidation state. This redox-economical approach can be used to append nearly any kind of cargo onto serine, generating a stable, benign, and hydrophilic phosphorothioate linkage. The method tolerates all other known nucleophilic functional groups of naturally occurring proteinogenic amino acids. A variety of applications can be envisaged by this expansion of the toolbox of site-selective bioconjugation methods.

An Enantioselective Total Synthesis of (+)-Duocarmycin SA.

An efficient, concise enantioselective total synthesis of the potent antitumor antibiotic (+)-duocarmycin SA is described. The invented route is based on a disconnection strategy that was devised to facilitate rapid and efficient synthesis of key core compounds to enable preclinical structure-activity relationship investigations. The key tricycle core was constructed with a highly enantioselective indole hydrogenation to set the stereocenter and a subsequent hitherto unexplored vicarious, nucleophilic-substitution/cyclization sequence to effectively forge a final indole ring. Additionally, the development of a stable sulfonamide protecting group capable of mild chemoselective cleavage greatly enhanced sequence yield and throughput. An understanding of key reaction parameters ensured a robust, reproducible sequence easily executable on decagram scales to this highly promising class of compounds.

Metabolomics enables precision medicine: "A White Paper, Community Perspective".

INTRODUCTION BACKGROUND TO METABOLOMICS: Metabolomics is the comprehensive study of the metabolome, the repertoire of biochemicals (or small molecules) present in cells, tissues, and body fluids. The study of metabolism at the global or "-omics" level is a rapidly growing field that has the potential to have a profound impact upon medical practice. At the center of metabolomics, is the concept that a person's metabolic state provides a close representation of that individual's overall health status. This metabolic state reflects what has been encoded by the genome, and modified by diet, environmental factors, and the gut microbiome. The metabolic profile provides a quantifiable readout of biochemical state from normal physiology to diverse pathophysiologies in a manner that is often not obvious from gene expression analyses. Today, clinicians capture only a very small part of the information contained in the metabolome, as they routinely measure only a narrow set of blood chemistry analytes to assess health and disease states. Examples include measuring glucose to monitor diabetes, measuring cholesterol and high density lipoprotein/low density lipoprotein ratio to assess cardiovascular health, BUN and creatinine for renal disorders, and measuring a panel of metabolites to diagnose potential inborn errors of metabolism in neonates. OBJECTIVES OF WHITE PAPER­EXPECTED TREATMENT OUTCOMES AND METABOLOMICS ENABLING TOOL FOR PRECISION MEDICINE: We anticipate that the narrow range of chemical analyses in current use by the medical community today will be replaced in the future by analyses that reveal a far more comprehensive metabolic signature. This signature is expected to describe global biochemical aberrations that reflect patterns of variance in states of wellness, more accurately describe specific diseases and their progression, and greatly aid in differential diagnosis. Such future metabolic signatures will: (1) provide predictive, prognostic, diagnostic, and surrogate markers of diverse disease states; (2) inform on underlying molecular mechanisms of diseases; (3) allow for sub-classification of diseases, and stratification of patients based on metabolic pathways impacted; (4) reveal biomarkers for drug response phenotypes, providing an effective means to predict variation in a subject's response to treatment (pharmacometabolomics); (5) define a metabotype for each specific genotype, offering a functional read-out for genetic variants: (6) provide a means to monitor response and recurrence of diseases, such as cancers: (7) describe the molecular landscape in human performance applications and extreme environments. Importantly, sophisticated metabolomic analytical platforms and informatics tools have recently been developed that make it possible to measure thousands of metabolites in blood, other body fluids, and tissues. Such tools also enable more robust analysis of response to treatment. New insights have been gained about mechanisms of diseases, including neuropsychiatric disorders, cardiovascular disease, cancers, diabetes and a range of pathologies. A series of ground breaking studies supported by National Institute of Health (NIH) through the Pharmacometabolomics Research Network and its partnership with the Pharmacogenomics Research Network illustrate how a patient's metabotype at baseline, prior to treatment, during treatment, and post-treatment, can inform about treatment outcomes and variations in responsiveness to drugs (e.g., statins, antidepressants, antihypertensives and antiplatelet therapies). These studies along with several others also exemplify how metabolomics data can complement and inform genetic data in defining ethnic, sex, and gender basis for variation in responses to treatment, which illustrates how pharmacometabolomics and pharmacogenomics are complementary and powerful tools for precision medicine. CONCLUSIONS KEY SCIENTIFIC CONCEPTS AND RECOMMENDATIONS FOR PRECISION MEDICINE: Our metabolomics community believes that inclusion of metabolomics data in precision medicine initiatives is timely and will provide an extremely valuable layer of data that compliments and informs other data obtained by these important initiatives. Our Metabolomics Society, through its "Precision Medicine and Pharmacometabolomics Task Group", with input from our metabolomics community at large, has developed this White Paper where we discuss the value and approaches for including metabolomics data in large precision medicine initiatives. This White Paper offers recommendations for the selection of state of-the-art metabolomics platforms and approaches that offer the widest biochemical coverage, considers critical sample collection and preservation, as well as standardization of measurements, among other important topics. We anticipate that our metabolomics community will have representation in large precision medicine initiatives to provide input with regard to sample acquisition/preservation, selection of optimal omics technologies, and key issues regarding data collection, interpretation, and dissemination. We strongly recommend the collection and biobanking of samples for precision medicine initiatives that will take into consideration needs for large-scale metabolic phenotyping studies.

Soil microbial communities respond differently to three chemically defined polyphenols.

High molecular weight polyphenols (e.g. tannins) that enter the soil may affect microbial populations, by serving as substrates for microbial respiration or by selecting for certain microbes. In this study we examined how three phenolic compounds that represent some environmentally widespread tannins or their constituent functional groups were respired by soil microorganisms and how the compounds affected the abundance and diversity of soil bacteria and archaea, including ammonia oxidizers. An acidic, silt loam soil from a pine forest was incubated for two weeks with the monomeric phenol methyl gallate, the small polyphenol epigallocatechin gallate, or the large polyphenol oenothein B. Respiration of the polyphenols during the incubation was measured using the Microresp™ system. After incubation, metabolic diversity was determined by community level physiological profiling (CLPP), and genetic diversity was determined using denaturing gradient gel electrophoresis (DGGE) analysis on DNA extracted from the soil samples. Total microbial populations and ammonia-oxidizing populations were measured using real time quantitative polymerase chain reaction (qPCR). Methyl gallate was respired more efficiently than the higher molecular weight tannins but not as efficiently as glucose. Methyl gallate and epigallocatechin gallate selected for genetically or physiologically unique populations compared to glucose. None of the polyphenols supported microbial growth, and none of the polyphenols affected ammonia-oxidizing bacterial populations or ammonia-oxidizing archaea. Additional studies using both a wider range of polyphenols and a wider range of soils and environments are needed to elucidate the role of polyphenols in determining soil microbiological diversity.

Metal mobilization in soil by two structurally defined polyphenols.

Polyphenols including tannins comprise a large percentage of plant detritus such as leaf litter, and affect soil processes including metal dynamics. We tested the effects of tannins on soil metal mobilization by determining the binding stoichiometries of two model polyphenols to Al(III) and Fe(III) using micelle-mediated separation and inductively coupled plasma optical emission spectroscopy (ICP-OES). By fitting the data to the Langmuir model we found the higher molecular weight polyphenol (oenothein B) was able to bind more metal than the smaller polyphenol (epigallocatechin gallate, EGCg). For example, oenothein B bound 9.43 mol Fe mol(-1), while EGCg bound 4.41 mol of Fe mol(-1). Using the parameters from the binding model, we applied the Langmuir model for competitive binding to predict binding for mixtures of Al(III) and Fe(III). Using the parameters from the single metal experiments and information about polyphenol sorption to soils we built a model to predict metal mobilization from soils amended with polyphenols. We tested the model with three natural soils and found that it predicted mobilization of Fe and Al with r(2)=0.92 and r(2)=0.88, respectively. The amount of metal that was mobilized was directly proportional to the maximum amount of metal bound to the polyphenol. The secondary parameter in each model was the amount of weak organically chelated Fe or Al that was in the soil. This study provides the first compound-specific information about how natural polyphenols interact with metals in the environment. We propose a model that is applicable to developing phytochelation agents for metal detoxification, and we discuss how tannins may play a role in metal mobilization from soils.

Qualitative variation in proanthocyanidin composition of Populus species and hybrids: genetics is the key.

The literature on proanthocyanidins (tannins) in ecological systems is dominated by quantitative studies. Despite evidence that the qualitative characteristics (subunit type, polymer chain length) of these complex polyphenolics are important determinants of biological activity, little is known about genetic and environmental controls on the type of proanthocyanidins produced by plants. We tested the hypothesis that genetics, season, developmental stage, and environment determine proanthocyanidin qualitative characteristics by using four Populus "cross types" (narrowleaf [P. angustifolia], Fremont [P. fremontii], F1 hybrids, and backcrosses to narrowleaf). We used thiolysis and HPLC analysis to characterize the proanthocyanidins, and found that genetics strongly control composition. The narrowleaf plants accumulate mixed procyanidin/prodelphinidins with average composition epicatechin(11)-epigallocatechin(8)-catechin(2)-catechin((terminal)). Backcross genotypes produce mixed procyanidin/prodelphinidins similar to narrowleaf, while Fremont makes procyanidin dimers, and the F1 plants contain procyanidin heptamers. Less striking effects were noted for genotype × environment, while season and developmental zone had little effect on proanthocyanidin composition or chain length. We discuss the metabolic and ecological consequences of differences in condensed tannin qualitative traits.

Calf flow reserve with H(2)(15)O PET as a quantifiable index of lower extremity flow.

UNLABELLED: Objective measures of recruitable blood flow are of importance in angiogenesis trials. We validated a new PET-derived flow reserve (FR) measurement in healthy subjects and subjects with peripheral artery disease (PAD). METHODS: Five healthy volunteers and 5 subjects with PAD underwent cannulation of the femoral artery and vein. Basal and maximal flow (100 micro g/kg/min of adenosine infused intraarterially) in the lower extremity was determined using thermodilution (TD) techniques. Subjects then underwent plethysmography (PL) followed by PET measurements of blood flow at the calf level. For the PET studies, a transmission scan followed by injection of 1.85 GBq (50 mCi) H(2)(15)O and dynamic scanning for 5 min were acquired in five 1-min frames. Regions of interest were drawn on successive PET image slices, and radioactivity was quantified from the first-minute scan after injection. FR for each of the 3 modalities was expressed as the ratio of adenosine to basal flow. RESULTS: PET-derived FR correlated strongly with TD (r = 0.82; P = 0.004) but not with PL (r = 0.17; P = 0.85). The mean average difference in FR between healthy volunteers and PAD subjects was 13.0 with PET and 4.5 with TD. The intra- and intersubject variability for PET expressed as the coefficient of variation was 10.5% and 29.0% for healthy subjects and 7.0% and 52.9% in PAD, respectively. CONCLUSION: As expected, FR was significantly lower in PAD subjects compared with healthy subjects as assessed with TD and PET but not with PL. PET-derived FR appears to be reproducible and generates sharper and higher indices of recruitable flow in healthy subjects and PAD. These findings have implications for the use of PET-derived FR as a sensitive index of recruitable flow in angiogenesis trials.