Development of [³H]2-Carboxy-4,6-dichloro‑1<i>H</i>‑indole-3-propionic Acid ([³H]PSB-12150): A Useful Tool for Studying GPR17

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1<i>H</i>-indole-3-propionic acid (<b>1</b>) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative <b>8</b> with tritium gas. The radioligand [³H]­PSB-12150 (<b>9</b>) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities

A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides

The marine sponge-derived fungus Stachylidium bicolor 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid N-methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E (1) and F (2). We showed that endolide F (but not E) is a moderate antagonist of the arginine vasopressin V1A receptor, a member of the G protein-coupled receptor superfamily

PGH₁ activates human eosinophils via CRTH2.

<p>Human eosinophils were treated with the indicated concentrations of PGD₂, PGH₁, and PGH₂, respectively, and chemotactic activation was measured in eosinophil shape change assays. Eosinophil shape change is inhibited in the presence of 1 µM of the CRTH2-specific antagonist TM30089. Note: rank order of PG potency matches well with the results obtained in CRTH2-HEK transfectants using DMR assays (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033329#pone-0033329-g001" target="_blank"><b>Figure 1F</b></a>). Results are expressed as the mean ± SEM of 3 experiments conducted in triplicate with a separate donor used in each experiment. Statistical analysis was performed for vehicle vs. TM30089 treated cells and is indicated as (**) for p<0.01 and as (***) for p<0.001.</p

Prostaglandin H₁ (PGH₁) stimulates Ca²⁺ mobilization from intracellular stores in CRTH2 transfectants and primary human eosinophils.

<p><b>A</b>–<b>D</b>: HEK293 cells stably expressing CRTH2 (CRTH2-HEK) were transiently transfected with a chimeric Gαqi5 protein to channel the Gi-sensitive CRTH2 receptor to mobilization of intracellular Ca²⁺. Cells were loaded with a Ca²⁺ fluorophore and CRTH2-specific Ca²⁺ traces were recorded over time upon challenge with the indicated agonists (<b>A</b>: PGD₂, <b>B</b>: PGH₁, <b>C</b>: PGH₂). <b>A</b>–<b>C</b>: representative data (mean + SEM of triplicate determinations. <b>D</b>: Maximum responses of all experiments were normalized to Ca²⁺ flux induced by 1 µM PGD₂ (mean + SEM, n = 3). <b>E</b>: PGH₁ induces Ca²⁺ mobilization in human eosinophils via CRTH2. Intracellular free Ca²⁺ levels were quantified by flow cytometry as described in the methods section. The level of Ca²⁺ mobilization in response to vehicle without agonist was set to 100%. Ca²⁺ mobilization upon addition of PGH₁, PGH₂, and PGD₂ is inhibited in the presence of 1 µM of the CRTH2 specific antagonist TM30089. Data are presented as the mean + SEM from 5 experiments conducted in triplicate, each experiment involving eosinophils from a separate donor.</p

PGH₁ activates human Th2 cells via CRTH2.

<p>Induction of Ca²⁺ mobilization (A–C) and cell migration (D–F) in human Th2 cells in response to the indicated concentrations of PGD₂, PGH₁, and PGH₂, respectively. The level of cell migration in response to medium without agonist was set to 1 fold. Both Ca²⁺ mobilization and cell migration are inhibited in the presence of 1 µM of the CRTH2 specific antagonist TM30089. Pooled data is expressed as the mean ± SEM from 3 experiments conducted in duplicate, each experiment involving Th2 cells from a separate donor. Statistical analysis was performed for vehicle vs. TM30089 treated cells and is indicated as (*) for p<0.05, as (**) for p<0.01 and as (***) for p<0.001.</p

PGH₁ induces eosinophil adhesion to human pulmonary microvascular endothelial cells under flow conditions.

<p>Eosinophils were pre-incubated with vehicle (<b>A</b>–<b>C</b>) or 10 µM CRTH2-specific antagonist TM30089 (<b>D</b>–<b>F</b>) for 10 min at room temperature followed by treatment with vehicle (<b>A</b>, <b>D</b>), 1 µM PGH₁ (<b>B</b>, <b>E</b>) or 30 nM PGD₂ (<b>C</b>, <b>F</b>) for 10 min at 37°C. Eosinophils were then superfused over human pulmonary microvascular endothelial cells grown on VenaEC biochips (Cellix, Dublin) for 5 min at 37°C. Representative images were taken 5 min after start of the superfusion (<b>A</b>–<b>F</b>). <b>G</b>: averaged data from <b>A</b>–<b>F</b>, quantified by computerized image analysis. Data are shown as mean + SEM of 4 experiments. *P<0.05 PGD₂ versus TM30089+PGD₂ and PGH₁ versus TM30089+PGH₁.</p

Prostaglandin H₁ (PGH₁) fully activates CRTH2 in living CRTH2-HEK cell transfectants.

<p><b>A</b>–<b>E</b>, abilities of PGH₁, selected prostaglandins, and the PGH₁ precursor dihomo-γ-linolenic acid (DGLA) to stimulate CRTH2 signaling using dynamic mass redistribution (DMR) technology. Cells were challenged with increasing concentrations of the indicated ligands and DMR was recorded as a measure of receptor activity (representative optical traces). <b>F</b>, transformation of optical traces (<b>A</b>–<b>E</b>) into concentration effect curves. Molar log EC₅₀ values were </p><p>PGH₁: −6.37±0.12; PGH₂: −7.09±0.08; PGD₁: −6.92±0.16; PGD₂: −7.95±0.09</p> (mean values ± SEM, n = 3).<p></p

Applying Molecular Networking for the Detection of Natural Sources and Analogues of the Selective Gq Protein Inhibitor FR900359

The cyclic depsipeptide FR900359 (FR), isolated from the traditional Chinese medicine plant <i>Ardisia crenata</i>, is a potent Gq protein inhibitor and thus a valuable tool to study Gq-mediated signaling of G protein-coupled receptors. Two new FR analogues (<b>3</b> and <b>4</b>) were isolated from <i>A. crenata</i> together with the known analogues <b>1</b> and <b>2</b>. The structures of compounds <b>3</b> and <b>4</b> were established by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS² analysis. The latter approach led to the annotation of further FR analogues <b>5</b>–<b>9</b>. Comparative bioactivity tests of compounds <b>1</b>–<b>4</b> along with the parent molecule FR showed high-affinity binding to Gq proteins in the low nanomolar range (IC₅₀ = 2.3–16.8 nM) for all analogues as well as equipotent inhibition of Gq signaling, which gives important SAR insights into this valuable natural product. Additionally, FR was detected from leaves of five other <i>Ardisia</i> species, among them the non-nodulated leaves of <i>Ardisia lucida</i>, implying a much broader distribution of FR than originally anticipated

Dualsteric Muscarinic Antagonists–Orthosteric Binding Pose Controls Allosteric Subtype Selectivity

Bivalent ligands of G protein-coupled receptors have been shown to simultaneously either bind to two adjacent receptors or to bridge different parts of one receptor protein. Recently, we found that bivalent agonists of muscarinic receptors can simultaneously occupy both the orthosteric transmitter binding site and the allosteric vestibule of the receptor protein. Such dualsteric agonists display a certain extent of subtype selectivity, generate pathway-specific signaling, and in addition may allow for designed partial agonism. Here, we want to extend the concept to bivalent antagonism. Using the phthal- and naphthalimide moieties, which bind to the allosteric, extracellular site, and atropine or scopolamine as orthosteric building blocks, both connected by a hexamethonium linker, we were able to prove a bitopic binding mode of antagonist hybrids for the first time. This is demonstrated by structure–activity relationships, site-directed mutagenesis, molecular docking studies, and molecular dynamics simulations. Findings revealed that a difference in spatial orientation of the orthosteric tropane moiety translates into a divergent M₂/M₅ subtype selectivity of the corresponding bitopic hybrids

Free Fatty Acid Receptor 1 (FFA1/GPR40) Agonists: Mesylpropoxy Appendage Lowers Lipophilicity and Improves ADME Properties

FFA1 (GPR40) is a new target for treatment of type 2 diabetes. We recently identified the potent FFA1 agonist TUG-469 (<b>5</b>). Inspired by the structurally related TAK-875, we explored the effects of a mesylpropoxy appendage on <b>5</b>. The appendage significantly lowers lipophilicity and improves metabolic stability while preserving potency, resulting in discovery of the potent FFA1 agonist <b>13</b>