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ABSTRACT: Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-protein-coupled receptor agonist library revealed that a subset of receptors, including dopamine D(1) and D(4) receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.
Journal of Biomolecular Screening 08/2012; 17(9):1180-91. · 2.05 Impact Factor
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ABSTRACT: The optimization of a series of thieno[3,2-b]thiophene-2-carboxylic acid derivatives for agonist activity against the GPR35 is reported. Compounds were optimized to achieve β-arrestin-biased agonism for developing probe molecules that may be useful for elucidating the biology and physiology of GPR35. Compound 13 was identified to the most potent GPR35 agonist, and compounds 30 and 36 exhibited the highest efficacy to cause β-arrestin translocation.
Bioorganic & medicinal chemistry letters 04/2012; 22(12):4148-52. · 2.65 Impact Factor
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ABSTRACT: 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.Keywords: GPR35; natural phenols; ellagic acid; biased agonism
01/2012;
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ABSTRACT: Fluorescent and luminescent probes are essential to both in vitro molecular assays and in vivo imaging techniques, and have been extensively used to measure biological function. However, little is known about the biological activity, thus potential interferences with the assay results, of these probe molecules. Here we show that D-luciferin, one of the most widely used bioluminescence substrates, is a partial agonist for G protein-coupled receptor-35 (GPR35). Label-free phenotypic profiling using dynamic mass redistribution (DMR) assays showed that D-luciferin led to a DMR signal in native HT-29 cells, whose characteristics are similar to those induced by known GPR35 agonists including zaprinast and pamoic acid. DMR assays further showed that D-luciferin is a partial agonist competitive to several known GPR35 agonists and antagonists. D-luciferin was found to cause the phosphorylation of ERK that was suppressed by known GPR35 antagonists, and also result in β-arrestin translocation signal but with low efficacy. These results not only suggest that D-luciferin is a partial agonist of GPR35, but also will evoke careful interpretation of biological data obtained using molecular and in vivo imaging assays when these probe molecules are used.
PLoS ONE 01/2012; 7(4):e34934. · 4.09 Impact Factor
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ABSTRACT: Both kynurenic acid and 2-acyl lysophosphatidic acid have been postulated to be the endogenous agonists of GPR35. However, controversy remains whether alternative endogenous agonists exist. The molecular targets accounted for many nongenomic actions of thyroid hormones are mostly unknown. Here we report the agonist activity of multiple tyrosine metabolites at the GPR35. Tyrosine metabolism intermediates that contain carboxylic acid and/or catechol functional groups were first selected. Whole cell dynamic mass redistribution (DMR) assays enabled by label-free optical biosensor were then used to characterize their agonist activity in native HT-29. Molecular assays including β-arrestin translocation, ERK phosphorylation and receptor internalization confirmed that GPR35 functions as a receptor for 5,6-dihydroxyindole-2-carboxylic acid, 3,3',5'-triiodothyronine, 3,3',5-triiodothyronine, gentisate, rosmarinate, and 3-nitrotyrosine. These results suggest that multiple tyrosine metabolites are alternative endogenous ligands of GPR35, and GPR35 may represent a druggable target for treating certain diseases associated with abnormality of tyrosine metabolism.
Scientific Reports 01/2012; 2:373.
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ABSTRACT: 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(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives, as GPR35 agonists. Of these, 2-(3-cyano-5-(3,4-dichlorophenyl)-4,5-dimethylfuran-2(5H)-ylidene)malononitrile (YE120) and 6-bromo-3-methylthieno[3,2-b]thiophene-2-carboxylic acid (YE210) were found to be the two most potent GPR35 agonists with an EC(50) 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.
Journal of Medicinal Chemistry 09/2011; 54(20):7385-96. · 4.80 Impact Factor
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ABSTRACT: GPR35 is an orphan G protein-coupled receptor that is not well-characterized. Here we employ dynamic mass redistribution (DMR) assays to discover new GPR35 agonists. DMR assays identified tyrphostin analogs as GPR35 agonists, which were confirmed with receptor internalization, Tango β-arrestin translocation, and extracellular-signal-regulated kinase phosphorylation assays. These agonists provide pharmacological tools to study the biology and function of GPR35.
FEBS letters 06/2011; 585(12):1957-62. · 3.54 Impact Factor
