¹³C NMR Metabolomics: INADEQUATE Network Analysis

The many advantages of ¹³C NMR are often overshadowed by its intrinsically low sensitivity. Given that carbon makes up the backbone of most biologically relevant molecules, ¹³C NMR offers a straightforward measurement of these compounds. Two-dimensional ¹³C–¹³C correlation experiments like INADEQUATE (incredible natural abundance double quantum transfer experiment) are ideal for the structural elucidation of natural products and have great but untapped potential for metabolomics analysis. We demonstrate a new and semiautomated approach called INETA (INADEQUATE network analysis) for the untargeted analysis of INADEQUATE data sets using an <i>in silico</i> INADEQUATE database. We demonstrate this approach using isotopically labeled <i>Caenorhabditis elegans</i> mixtures

Chemical Detoxification of Small Molecules by <i>Caenorhabditis elegans</i>

<i>Caenorhabditis elegans</i> lives in compost and decaying fruit, eats bacteria and is exposed to pathogenic microbes. We show that <i>C. elegans</i> is able to modify diverse microbial small-molecule toxins via both <i>O-</i> and <i>N-</i>glucosylation as well as unusual 3′-<i>O</i>-phosphorylation of the resulting glucosides. The resulting glucosylated derivatives have significantly reduced toxicity to <i>C. elegans</i>, suggesting that these chemical modifications represent a general mechanism for worms to detoxify their environments

Structure–Activity Relationships of Peptides Incorporating a Bioactive Reverse-Turn Heterocycle at the Melanocortin Receptors: Identification of a 5800-fold Mouse Melanocortin‑3 Receptor (mMC3R) Selective Antagonist/Partial Agonist versus the Mouse Melanocortin‑4 Receptor (mMC4R)

The melanocortin-3 (MC3) and melanocortin-4 (MC4) receptors regulate energy homeostasis, food intake, and associated physiological conditions. The melanocortin-4 receptor (MC4R) has been studied extensively. Less is known about specific physiological roles of the melanocortin-3 receptor (MC3R). A major obstacle to this lack of knowledge is attributed to a limited number of identified MC3R selective ligands. We previously reported a spatial scanning approach of a 10-membered thioether-heterocycle ring incorporated into a chimeric peptide template that identified a lead nM MC4R ligand. Upon the basis of those results, 17 compounds were designed and synthesized that focused upon modification in the pharmacophore domain. Notable results include the identification of a 0.13 nM potent 5800-fold mMC3R selective antagonist/slight partial agonist versus a 760 nM mMC4R full agonist (ligand <b>11</b>). Biophysical experiments (two-dimensional ¹H NMR and computer-assisted molecular modeling) of this ligand resulted in the identification of an inverse γ-turn secondary structure in the ligand pharmacophore domain

¹³C NMR Metabolomics: Applications at Natural Abundance

¹³C NMR has many advantages for a metabolomics study, including a large spectral dispersion, narrow singlets at natural abundance, and a direct measure of the backbone structures of metabolites. However, it has not had widespread use because of its relatively low sensitivity compounded by low natural abundance. Here we demonstrate the utility of high-quality ¹³C NMR spectra obtained using a custom ¹³C-optimized probe on metabolomic mixtures. A workflow was developed to use statistical correlations between replicate 1D ¹³C and ¹H spectra, leading to composite spin systems that can be used to search publicly available databases for compound identification. This was developed using synthetic mixtures and then applied to two biological samples, <i>Drosophila melanogaster</i> extracts and mouse serum. Using the synthetic mixtures we were able to obtain useful ¹³C–¹³C statistical correlations from metabolites with as little as 60 nmol of material. The lower limit of ¹³C NMR detection under our experimental conditions is approximately 40 nmol, slightly lower than the requirement for statistical analysis. The ¹³C and ¹H data together led to 15 matches in the database compared to just 7 using ¹H alone, and the ¹³C correlated peak lists had far fewer false positives than the ¹H generated lists. In addition, the ¹³C 1D data provided improved metabolite identification and separation of biologically distinct groups using multivariate statistical analysis in the <i>D. melanogaster</i> extracts and mouse serum

Synthesis, Biophysical, and Pharmacological Evaluation of the Melanocortin Agonist AST3-88: Modifications of Peptide Backbone at Trp 7 Position Lead to a Potent, Selective, and Stable Ligand of the Melanocortin 4 Receptor (MC4R)

The melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors are expressed in the brain and are implicated in the regulation of food intake and energy homeostasis. The endogenous agonist ligands for these receptors (α-, β-, γ-MSH and ACTH) are linear peptides with limited receptor subtype selectivity and metabolic stability, thus minimizing their use as probes to characterize the overlapping pharmacological and physiological functions of the melanocortin receptor subtypes. In the present study, an engineered template, in which the peptide backbone was modified by a heterocyclic reverse turn mimetic at the Trp⁷ residue, was synthesized using solid phase peptide synthesis and characterized by a β-galactosidase cAMP based reporter gene assay. The functional assay identified a ∼5 nM mouse MC4R agonist (AST3-88) with more than 50-fold selectivity over the mMC3R. Biophysical studies (2D ¹H NMR spectroscopy and molecular dynamics) of AST3-88 identified a type VIII β-turn secondary structure spanning the pharmacophore domain stabilized by the intramolecular interactions between the side chains of the His and Trp residues. Enzymatic studies of AST3-88 revealed enhanced stability of AST3-88 over the α-MSH endogenous peptide in rat serum. Upon central administration of AST3-88 into rats, a decreased food intake response was observed. This is the first study to probe the in vivo physiological activity of this engineered peptide-heterocycle template. These findings advance the present knowledge of pharmacophore design for potent, selective, and metabolically stable melanocortin ligands

Isotopic Ratio Outlier Analysis Global Metabolomics of Caenorhabditis elegans

We demonstrate the global metabolic analysis of Caenorhabditis elegans stress responses using a mass-spectrometry-based technique called isotopic ratio outlier analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95 and 5% ¹³C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis. This labeling strategy provides several advantages over conventional approaches: (1) compounds arising from biosynthesis are easily distinguished from artifacts, (2) errors from sample extraction and preparation are minimized because the control and experiment are combined into a single sample, (3) measurement of both the molecular weight and the exact number of carbon atoms in each molecule provides extremely accurate molecular formulas, and (4) relative concentrations of all metabolites are easily determined. A heat-shock perturbation was conducted on C. elegans to demonstrate this approach. We identified many compounds that significantly changed upon heat shock, including several from the purine metabolism pathway. The metabolomic response information by IROA may be interpreted in the context of a wealth of genetic and proteomic information available for C. elegans. Furthermore, the IROA protocol can be applied to any organism that can be isotopically labeled, making it a powerful new tool in a global metabolomics pipeline

Chill coma recovery time for cold-hardy and susceptible flies

Chill coma recovery times assayed on the day metabolomics samples were collected for the experiments

Isotopic Ratio Outlier Analysis Global Metabolomics of Caenorhabditis elegans

We demonstrate the global metabolic analysis of Caenorhabditis elegans stress responses using a mass-spectrometry-based technique called isotopic ratio outlier analysis (IROA). In an IROA protocol, control and experimental samples are isotopically labeled with 95 and 5% ¹³C, and the two sample populations are mixed together for uniform extraction, sample preparation, and LC-MS analysis. This labeling strategy provides several advantages over conventional approaches: (1) compounds arising from biosynthesis are easily distinguished from artifacts, (2) errors from sample extraction and preparation are minimized because the control and experiment are combined into a single sample, (3) measurement of both the molecular weight and the exact number of carbon atoms in each molecule provides extremely accurate molecular formulas, and (4) relative concentrations of all metabolites are easily determined. A heat-shock perturbation was conducted on C. elegans to demonstrate this approach. We identified many compounds that significantly changed upon heat shock, including several from the purine metabolism pathway. The metabolomic response information by IROA may be interpreted in the context of a wealth of genetic and proteomic information available for C. elegans. Furthermore, the IROA protocol can be applied to any organism that can be isotopically labeled, making it a powerful new tool in a global metabolomics pipeline

Transferring Fungi to a Deuterium-Enriched Medium Results in Assorted, Conditional Changes in Secondary Metabolite Production

Deuterium is one of the few stable isotopes that have the capacity to significantly alter a compound’s chemical and biological properties. The addition of a single neutron to a protium atom results in the near doubling of its mass, which gives rise to deuterium’s characteristic isotope effects. Since the incorporation of deuterium into organic substrates is known to alter enzyme/protein–substrate interactions, we tested the extent to which deuterium enrichment would modify fungal secondary metabolite production. Several fungal cultures were tested, and in all cases their secondary metabolomes were marked by changes in natural product production. Workup of one <i>Aspergillus</i> sp. grown under deuterium-enrichment conditions resulted in the production of several secondary metabolites not previously detected from the fungus. Bioassay testing revealed that in comparison to the inactive crude fungal extract derived from growing the fungus under non-deuterium-enriched conditions, an extract derived from the same isolate cultured in a deuterium-enriched medium inhibited methicillin-resistant <i>Staphylococcus aureus</i>. Using an assortment of NMR and mass spectrometry experiments, we were able to identify the bacterial inhibitor as an isotope-labeled version of pigmentosin A (<b>6</b>). Five additional isotopically labeled metabolites were also obtained from the fungus including brevianamide F (<b>1</b>), stephacidin A (<b>2</b>), notoamide D (<b>3</b>), notoamide L (<b>4</b>), and notoamide C (<b>5</b>). Given the assorted changes observed in the secondary metabolite profiles of this and other fungi grown in deuterium-enriched environments, as well as the fact that <b>1</b> and <b>3</b>–<b>6</b> had not been previously observed from the <i>Aspergillus</i> sp. isolate used in this study, we propose that deuterium enrichment might offer an effective method for further expanding a fungus’s chemical diversity potential

A Modular Library of Small Molecule Signals Regulates Social Behaviors in <em>Caenorhabditis elegans</em>

<div><p>The nematode <em>C. elegans</em> is an important model for the study of social behaviors. Recent investigations have shown that a family of small molecule signals, the ascarosides, controls population density sensing and mating behavior. However, despite extensive studies of <em>C. elegans</em> aggregation behaviors, no intraspecific signals promoting attraction or aggregation of wild-type hermaphrodites have been identified. Using comparative metabolomics, we show that the known ascarosides are accompanied by a series of derivatives featuring a tryptophan-derived indole moiety. Behavioral assays demonstrate that these indole ascarosides serve as potent intraspecific attraction and aggregation signals for hermaphrodites, in contrast to ascarosides lacking the indole group, which are repulsive. Hermaphrodite attraction to indole ascarosides depends on the ASK amphid sensory neurons. Downstream of the ASK sensory neuron, the interneuron AIA is required for mediating attraction to indole ascarosides instead of the RMG interneurons, which previous studies have shown to integrate attraction and aggregation signals from ASK and other sensory neurons. The role of the RMG interneuron in mediating aggregation and attraction is thought to depend on the neuropeptide Y-like receptor NPR-1, because solitary and social <em>C. elegans</em> strains are distinguished by different <em>npr-1</em> variants. We show that indole ascarosides promote attraction and aggregation in both solitary and social <em>C. elegans</em> strains. The identification of indole ascarosides as aggregation signals reveals unexpected complexity of social signaling in <em>C. elegans</em>, which appears to be based on a modular library of ascarosides integrating building blocks derived from lipid β-oxidation and amino-acid metabolism. Variation of modules results in strongly altered signaling content, as addition of a tryptophan-derived indole unit to repellent ascarosides produces strongly attractive indole ascarosides. Our findings show that the library of ascarosides represents a highly developed chemical language integrating different neurophysiological pathways to mediate social communication in <em>C. elegans</em>.</p> </div