8 research outputs found
The DMR arising from co-stimulation with two agonists.
<p>(a) The dose-dependent responses of pamoic acid in the absence and presence of D-luciferin at three different fixed doses; the cell seeding density was 25000 cells per well; and (b) the dose-dependent response of D-luciferin in the absence and presence of pamoic acid at three fixed doses, the cell seeding density was 32000 cells per well. The ligand P-DMR amplitudes were used to calculate all dose responses. All data represent mean ± s.d. from 2 independent measurements, each in duplicate (n = 4).</p
D-Luciferin caused β-arrestin translocation via GPR35.
<p>(a) The dose-dependent responses of three ligands as measured using Tango™ β-arrestin translocation gene reporter assays. (b) The dose-dependent responses of zaprinast in the presence of D-luciferin at different fixed doses. (c) The dose-dependent responses of D-luciferin in the presence of 10 µM zaprinast. All data were normalized to the zaprinast maximal response. The data represents mean ± s.d. from two independent measurements, each in duplicate (n = 4).</p
D-Luciferin triggered internalization of endogenous GPR35 in HT-29 cells.
<p>Representative confocal fluorescence images of HT-29 under different conditions: (<b>a</b>) treated with the assay vehicle containing 0.1% DMSO; (<b>b</b>) treated with 32 µM D-luciferin. The images were obtained after compound treatment for 1 hr, permeabilized, stained with anti-GPR35, followed by fluorescent secondary antibody. Red: GPR35 stains. Representative images obtained from 2 independent measurements were used.</p
D-Luciferin triggered ERK phosphorylation via GPR35.
<p>(a) Western blot of phosphorylated ERK1/2 (pERK1/2) after treated with 32 µM D-luciferin for different times; (b) Western blot of phosphorylated ERK 30 min after treated with three different known GPR35 agonists, YE210 (1 µM), zaprinast (1 µM) and pamoic acid (1 µM); (c) Western blot of phosphorylated ERK 5 min after treated with 32 µM in the presence of SPB05142 at different doses; (d) Western blot of phosphorylated ERK 5 min after treated with 32 µM in the presence of ML145 at different doses. The phosphorylated ERK and total ERK were blotted, and actin was used as control. Representative images obtained from 2 independent measurements were used.</p
The desensitization of HT-29 to 250 nM zaprinast after 1 hr pretreatment with D-luciferin at different doses.
<p>Only the zaprinast-induced DMR signals were shown: (a) the real time kinetic response of zaprinast; and (b) the zaprinast P-DMR amplitudes as a function of D-luciferin concentration. All data represent mean ± s.d. from 2 independent measurements, each in duplicate (n = 4).</p
DMR characteristics of ligands in HT-29 cells.
<p>(a) The dose-dependent kinetic DMR signals induced by D-luciferin; (b) the dose-dependent kinetic DMR signals induced by pamoic acid; and (c) the P-DMR amplitudes of three ligands as a function of ligand dose. All data represent mean ± s.d. from 2 independent measurements, each in duplicate (n = 4), except for zaprinast (n = 8).</p
Discovery of Natural Phenols as G Protein-Coupled Receptor-35 (GPR35) Agonists
We report the discovery and characterization of natural
phenols
as G protein-coupled receptor-35 (GPR35) agonists. Pharmacological
characterization using label-free dynamic mass redistribution and
Tango β-arrestin translocation assays revealed that GPR35-active
natural phenols are divergent in their biased agonism
Discovery of 2-(4-Methylfuran-2(5<i>H</i>)-ylidene)malononitrile and Thieno[3,2-<i>b</i>]thiophene-2-carboxylic Acid Derivatives as G Protein-Coupled Receptor 35 (GPR35) Agonists
Screening with dynamic mass redistribution (DMR) assays in a native cell line HT-29 led to identification of two novel series of chemical compounds, 2-(4-methylfuran-2(5<i>H</i>)-ylidene)malononitrile and thieno[3,2-<i>b</i>]thiophene-2-carboxylic acid derivatives, as GPR35 agonists. Of these, 2-(3-cyano-5-(3,4-dichlorophenyl)-4,5-dimethylfuran-2(5<i>H</i>)-ylidene)malononitrile (YE120) and 6-bromo-3-methylthieno[3,2-<i>b</i>]thiophene-2-carboxylic acid (YE210) were found to be the two most potent GPR35 agonists with an EC<sub>50</sub> of 32.5 ± 1.7 nM and 63.7 ± 4.1 nM, respectively. Both agonists exhibited better potency than that of zaprinast, a known GPR35 agonist. DMR antagonist assays, knockdown of GPR35 with interference RNA, receptor internalization assays, and Tango β-arrestin translocation assays confirmed that the agonist activity of these ligands is specific to GPR35. The present study provides novel chemical series as a starting point for further investigations of GPR35 biology and pharmacology