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Discovery of 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as G protein-coupled receptor 35 (GPR35) agonists.

Deng H, Hu H, He M, Hu J, Niu W, Ferrie AM, Fang Y - J. Med. Chem. (2011)

Bottom Line: 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.The present study provides novel chemical series as a starting point for further investigations of GPR35 biology and pharmacology.

View Article: PubMed Central - PubMed

Affiliation: Biochemical Technologies, Science and Technology Division, Corning Inc., Corning, New York 14831, United States.

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.

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Compound 15a was unable to activate GPR35. (a) Structures of 15a and 16a. (b) The net-zero DMR induced by 15a, in comparison with that by 16a. The data represents mean ± sd from four replicates (n = 4). (c–e) Representative fluorescence images of HT-29 under different conditions: unstimulated (c), stimulated with 1 μM 16a (d), or stimulated with 10 μM compound 15a (e). The images were obtained after compound treatment for 1 h, permeabilized, and stained with anti-GPR35, followed by fluorescent secondary antibody. Red: GPR35 stains. Blue: nuclei stains with DAPI.
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fig8: Compound 15a was unable to activate GPR35. (a) Structures of 15a and 16a. (b) The net-zero DMR induced by 15a, in comparison with that by 16a. The data represents mean ± sd from four replicates (n = 4). (c–e) Representative fluorescence images of HT-29 under different conditions: unstimulated (c), stimulated with 1 μM 16a (d), or stimulated with 10 μM compound 15a (e). The images were obtained after compound treatment for 1 h, permeabilized, and stained with anti-GPR35, followed by fluorescent secondary antibody. Red: GPR35 stains. Blue: nuclei stains with DAPI.

Mentions: Next, we examined the ability of representative compounds to cause receptor internalization, a hallmark of the activation of most GPCRs. We specifically compared compound 16a with its analogue, compound 15a (Figure 8a). Unlike 16a, 15a was found to be inactive in DMR assays (Figure 8b). To study agonist-induced internalization of endogenous GPR35, the cells were first exposed to an agonist at a given dose for 1 h at 37 °C. After fixation and permeabilization, the cells were incubated with an anti-GPR35 antibody (T-14, intracellular domain), followed by staining with a fluorescently labeled secondary antibody. Fluorescent confocal imaging showed that GPR35 in the unstimulated cells was primarily located at the cell surface (Figure 8c), and stimulation with 16a caused significant internalization of GPR35 (Figure 8d). As expected, 15a was inactive in the receptor internalization assay (Figure 8e).


Discovery of 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as G protein-coupled receptor 35 (GPR35) agonists.

Deng H, Hu H, He M, Hu J, Niu W, Ferrie AM, Fang Y - J. Med. Chem. (2011)

Compound 15a was unable to activate GPR35. (a) Structures of 15a and 16a. (b) The net-zero DMR induced by 15a, in comparison with that by 16a. The data represents mean ± sd from four replicates (n = 4). (c–e) Representative fluorescence images of HT-29 under different conditions: unstimulated (c), stimulated with 1 μM 16a (d), or stimulated with 10 μM compound 15a (e). The images were obtained after compound treatment for 1 h, permeabilized, and stained with anti-GPR35, followed by fluorescent secondary antibody. Red: GPR35 stains. Blue: nuclei stains with DAPI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198121&req=5

fig8: Compound 15a was unable to activate GPR35. (a) Structures of 15a and 16a. (b) The net-zero DMR induced by 15a, in comparison with that by 16a. The data represents mean ± sd from four replicates (n = 4). (c–e) Representative fluorescence images of HT-29 under different conditions: unstimulated (c), stimulated with 1 μM 16a (d), or stimulated with 10 μM compound 15a (e). The images were obtained after compound treatment for 1 h, permeabilized, and stained with anti-GPR35, followed by fluorescent secondary antibody. Red: GPR35 stains. Blue: nuclei stains with DAPI.
Mentions: Next, we examined the ability of representative compounds to cause receptor internalization, a hallmark of the activation of most GPCRs. We specifically compared compound 16a with its analogue, compound 15a (Figure 8a). Unlike 16a, 15a was found to be inactive in DMR assays (Figure 8b). To study agonist-induced internalization of endogenous GPR35, the cells were first exposed to an agonist at a given dose for 1 h at 37 °C. After fixation and permeabilization, the cells were incubated with an anti-GPR35 antibody (T-14, intracellular domain), followed by staining with a fluorescently labeled secondary antibody. Fluorescent confocal imaging showed that GPR35 in the unstimulated cells was primarily located at the cell surface (Figure 8c), and stimulation with 16a caused significant internalization of GPR35 (Figure 8d). As expected, 15a was inactive in the receptor internalization assay (Figure 8e).

Bottom Line: 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.The present study provides novel chemical series as a starting point for further investigations of GPR35 biology and pharmacology.

View Article: PubMed Central - PubMed

Affiliation: Biochemical Technologies, Science and Technology Division, Corning Inc., Corning, New York 14831, United States.

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.

Show MeSH
Related in: MedlinePlus