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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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Dose-dependent responses of GPR35 ligands as measured using Tango β-arrestin translocation gene reporter assays. The coumarin to fluorescein ratio was plotted as a function of ligand doses. Zaprinast was included as a positive control. The data represents mean ± sd from two independent measurements, each in duplicate (n = 4).
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fig9: Dose-dependent responses of GPR35 ligands as measured using Tango β-arrestin translocation gene reporter assays. The coumarin to fluorescein ratio was plotted as a function of ligand doses. Zaprinast was included as a positive control. The data represents mean ± sd from two independent measurements, each in duplicate (n = 4).

Mentions: Last, we examined the ability of these compounds to cause β-arrestin translocation using the Tango GPR35 arrestin assays. This assay uses Tango GPR35-bla U2OS cells to detect GPR35 agonist-induced recruitment of TEV protease tagged β-arrestin molecules to the GPR35-Gal4-VP16 transcription factor fusion protein linked by a TEV protease cleavage site. The β-arrestin recruitment leads to release of the transcription factor from the C-terminus of GPR35, which, in turn, translocates to the nucleus and activates the expression of β-lactamase. The activity of β-lactamase is then assayed with the cell permeable LiveBLAzer fluorescence resonance energy transfer (FRET) B/G substrate. Results showed that zaprinast led to a dose-dependent response in Tango GPR35-bla U2OS cells with a significantly right-shifted potency, EC50 of 4.20 ± 0.25 μM (n = 4) (Figure9; Table 1). Among all the compounds examined, compound 1, 3, 16a, and 16c gave rise to dose-dependent and saturable responses but with distinct potency and efficacy (Figure 9; Table 1). Compounds 7, 9, 10, 11, 12, 13, and 16b also led to detectable responses (Figure9 and Table 1). However, 2, 4, 5, 6, 8, 15a, 16d, and 16e all at a dose up to 128 μM were inactive in this assay. These results suggest that 16a is a full agonist in the arrestin translocation assay, while 1, 3, 7, 9, 10, 11, 12, 13, 16b, and 16c are partial agonists. However, considering the common right-shifted potency of GPR35 agonists obtained using the Tango assay, we can not rule out the possibility that the other compounds may also be active but with much lower potency. Collectively, we demonstrated that the two chemical series compounds are GPR35 agonists.


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)

Dose-dependent responses of GPR35 ligands as measured using Tango β-arrestin translocation gene reporter assays. The coumarin to fluorescein ratio was plotted as a function of ligand doses. Zaprinast was included as a positive control. The data represents mean ± sd from two independent measurements, each in duplicate (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: Dose-dependent responses of GPR35 ligands as measured using Tango β-arrestin translocation gene reporter assays. The coumarin to fluorescein ratio was plotted as a function of ligand doses. Zaprinast was included as a positive control. The data represents mean ± sd from two independent measurements, each in duplicate (n = 4).
Mentions: Last, we examined the ability of these compounds to cause β-arrestin translocation using the Tango GPR35 arrestin assays. This assay uses Tango GPR35-bla U2OS cells to detect GPR35 agonist-induced recruitment of TEV protease tagged β-arrestin molecules to the GPR35-Gal4-VP16 transcription factor fusion protein linked by a TEV protease cleavage site. The β-arrestin recruitment leads to release of the transcription factor from the C-terminus of GPR35, which, in turn, translocates to the nucleus and activates the expression of β-lactamase. The activity of β-lactamase is then assayed with the cell permeable LiveBLAzer fluorescence resonance energy transfer (FRET) B/G substrate. Results showed that zaprinast led to a dose-dependent response in Tango GPR35-bla U2OS cells with a significantly right-shifted potency, EC50 of 4.20 ± 0.25 μM (n = 4) (Figure9; Table 1). Among all the compounds examined, compound 1, 3, 16a, and 16c gave rise to dose-dependent and saturable responses but with distinct potency and efficacy (Figure 9; Table 1). Compounds 7, 9, 10, 11, 12, 13, and 16b also led to detectable responses (Figure9 and Table 1). However, 2, 4, 5, 6, 8, 15a, 16d, and 16e all at a dose up to 128 μM were inactive in this assay. These results suggest that 16a is a full agonist in the arrestin translocation assay, while 1, 3, 7, 9, 10, 11, 12, 13, 16b, and 16c are partial agonists. However, considering the common right-shifted potency of GPR35 agonists obtained using the Tango assay, we can not rule out the possibility that the other compounds may also be active but with much lower potency. Collectively, we demonstrated that the two chemical series compounds are GPR35 agonists.

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