Compounds and were synthesized according to The synthesis of
Compounds –, , and were synthesized according to . The synthesis of compounds – commences with C-acylation of 3,4-dibromothiophene to the form ketone , the ring closure to form the ester , Hiyama cross-coupling or Suzki cross-coupling to produce , or to , respectively. Compound was produced from ketone through consensation/coupling/deformylation cascade process, and then hydrolysis. Compounds and were synthesized from the ester through Suzuki cross-coupling and sequential hydrolysis. Compound was synthesized from the ester through N-methylation and sequential hydrolysis. Except for , , , and that were synthesized internally, the rest compounds were synthesized by BioDuro Co. (Beijing, China), a CRO company. All internal synthesized compounds have purity greater than 98%, and the rest compounds with purity greater than 95%. We employed both label-free dynamic mass redistribution (DMR), and Tango β-arrestin translocation assays to characterize the pharmacological activity of TTAC analogs at GPR35. DMR assay is based on whole cell phenotypic responses, while Tango is an endpoint measurement of gene reporter activity linked to the GPR35 activation-mediated β-arrestin translocation., The dose responses were performed in two independent measurements, each in duplicate. SAR analysis was carried out based on relative potency and efficacy of compounds tested. First, we recorded the dose responses of all TTAC analogs in native HT-29 cells using DMR agonist assays. The real time DMR signals of representative compounds, and , are showed in a and b, respectively. Compound was inactive (c), consistent with our previous observation that the carboxylic 4-Methylhistamine dihydrochloride group is essential for the agonist activity of TTAC analogs. All remaining compounds led to a clear dose-dependent DMR whose characteristics were similar to YE210 (6-bromo-3-methylthieno[3,2-]thiophene-2-carboxylic acid), as well as other known GPR35 agonists including zaprinast, pamoic acid, and kynurenic acid. These compounds were thus termed DMR active compounds. The dose responses were best fitted with a monophasic sigmoidal non-linear regression, leading to a single EC for each compound (c, ). We further determined the specificity of a compound-induced DMR to GPR35 using DMR antagonist assay. The antagonist assay employed a known GPR35 antagonist, CID 2745687 (also known as SPB05142),, to examine its ability to inhibit a compound-induced DMR. Results showed that CID 2745687 dose-dependently blocked the DMR induced by all DMR-active compounds, when they were assayed at a fixed dose. Furthermore, the potency of CID 2745687 was found to be similar to block the DMR of these compounds (). The dose-dependent inhibition of representative compounds, YE210, and , by CID 2745687 were shown in d. These results suggest that these DMR-active TTAC analogs are GPR35 agonists. Second, we measured the ability of TTAC analogs to cause β-arrestin translocation using Tango assay in the engineered U2OS-GPR35- cell line. This cell line stably expresses two fusion proteins: human GPR35 linked to a Gal4-VP16 transcription factor via a TEV protease site, and β-arrestin/TEV protease fusion protein. The cell line also stably expresses the β-lactamase reporter gene under the control of a UAS response element. The activation of GPR35 by agonists leads to the recruitment of β-arrestin-TEV protease fusion protein to the activated GPR35, leading to cleavage of Gal4-VP16 transcription factor from the receptor by the protease. The transcription factor then translocates to the nucleus and activates the expression of β-lactamase. Such a β-arrestin translocation is often specific to the GPR35 activation, given that the test compound is not fluorescent. All Tango assay results were reported after normalized to the maximal response of zaprinast within the same plate. Results showed that as expected, compound was inactive. All DMR-active compounds were found to be also active in Tango assays. The dose responses were also best fitted with a monophasic sigmoidal non-linear regression, leading to a single EC for each compound (a, ). Further, Tango antagonist assay showed that ML-145 dose-dependently blocked the Tango signals induced by compounds , , or YE210, leading to an IC of 0.085±0.007μM, 1.30±0.10μM, 0.45±0.03μM, and 0.38±0.03μM, respectively (two independent measurements, =4) (b). ML-145 is a potent GPR35 agonist. These assay results further confirmed that all DMR-active compounds were GPR35 agonists.