19 research outputs found
<sup>13</sup>C NMR Metabolomics: INADEQUATE Network Analysis
The many advantages of <sup>13</sup>C NMR are often overshadowed
by its intrinsically low sensitivity. Given that carbon makes up the
backbone of most biologically relevant molecules, <sup>13</sup>C NMR
offers a straightforward measurement of these compounds. Two-dimensional <sup>13</sup>C–<sup>13</sup>C 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 <sup>1</sup>H 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
<sup>13</sup>C NMR Metabolomics: Applications at Natural Abundance
<sup>13</sup>C 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 <sup>13</sup>C NMR spectra obtained using
a custom <sup>13</sup>C-optimized probe on metabolomic mixtures. A
workflow was developed to use statistical correlations between replicate
1D <sup>13</sup>C and <sup>1</sup>H 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 <sup>13</sup>C–<sup>13</sup>C statistical
correlations from metabolites with as little as 60 nmol of material.
The lower limit of <sup>13</sup>C NMR detection under our experimental
conditions is approximately 40 nmol, slightly lower than the requirement
for statistical analysis. The <sup>13</sup>C and <sup>1</sup>H data
together led to 15 matches in the database compared to just 7 using <sup>1</sup>H alone, and the <sup>13</sup>C correlated peak lists had
far fewer false positives than the <sup>1</sup>H generated lists.
In addition, the <sup>13</sup>C 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 Trp<sup>7</sup> 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 <sup>1</sup>H 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% <sup>13</sup>C, 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% <sup>13</sup>C, 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