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FEBS Letters 585 (2011) 1957–1962

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Tyrphostin analogs are GPR35 ⇑ Huayun Deng 1, Haibei Hu 1, Ye Fang

Biochemical Technologies, Science and Technology Division, Corning Inc., Corning, NY 14831, United States

article info abstract

Article history: GPR35 is an orphan G protein-coupled receptor that is not well-characterized. Here we employ Received 14 April 2011 dynamic mass redistribution (DMR) assays to discover new GPR35 agonists. DMR assays identified Revised 7 May 2011 tyrphostin analogs as GPR35 agonists, which were confirmed with receptor internalization, Tango Accepted 11 May 2011 b-arrestin translocation, and extracellular-signal-regulated kinase phosphorylation assays. These Available online 17 May 2011 agonists provide pharmacological tools to study the biology and function of GPR35. Edited by Christian Griesinger Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Dynamic mass redistribution GPR35 G protein-coupled receptor Optical biosensor Tyrphostin

1. Introduction redistribution (DMR) assay [10], to discover new GPR35 ligands. The DMR assay is enabled by label-free resonant waveguide grat- GPR35 was first identified to be a class A orphan G protein- ing (RWG) biosensor [11], and has attracted much interest in the coupled receptor (GPCR) that contains 309 amino acids [1]. molecular delineation of receptor biology and ligand pharmacol- GPR35b, a splicing variant that contains an N-terminal extension ogy [12–14]. DMR agonism screening in HT-29, a human colon of 31 amino acids, was later discovered in gastric cancer cells in cancer cell line that endogenously expresses GPR35, led to identi- 2004, and shown to be capable of transforming NIH-3T3 cells fication of tyrphostin analogs including entacapone as GPR35 [2]. GPR35 has been found to be expressed in various tissues agonists. including stomach [2], gastrointestinal tissues [3], and mast cells, basophils and eosinophils [4]. Upregulation of GPR35 has also 2. Materials and methods been identified in human mast cells upon challenge with IgE anti- bodies [4], in human macrophages after exposure to benzo(a)pyr- 2.1. Materials and cells ene [5], in failing heart cells [6], and in gastric cancer cells [2].To date, both and 2-acyl lysophosphatidic acids have Tyrphostins and entacapone were obtained from Sigma Chemi- been postulated to be natural agonists for GPR35 [7,8]. Both li- cal Co. (St. Louis, MO, USA). Zaprinast and BioMol 80 kinase inhib- gands elicited cellular responses via GPR35 in engineered cells itor library was purchased from Enzo Life Sciences (Plymouth expressing GPR35. However, it remains controversial whether Meeting, PA). The InhibitorSelect™ 96-well protein kinase inhibi- both agonists are true endogenous agonists, and it is largely un- tor library I (Cat. No. 539744) and II (Cat. No. 539745) were pur- known about the biological functions of GPR35 [9]. Thus, identifi- chased from EMD Chemicals (Gibbstown, NJ, USA). All inhibitors cation of new classes of ligands would be beneficial to elucidate were stocked in dimethyl sulfoxide (DMSO) at 10 mM, and were the biology and pharmacology of GPR35. Here, we applied a path- diluted directly in the assay buffer (1Â Hanks’ balanced salt buffer, way-unbiased, but pathway-sensitive technology, dynamic mass 20 mM Hepes, pH 7.1; HBSS) to the indicated concentrations. EpicÒ 384-well biosensor microplates were obtained from Corning Inc. Abbreviations: DMR, dynamic mass redistribution; GPCR, G protein-coupled (Corning, NY, USA). Mouse monoclonal anti-phosphorylated extra- receptor; RWG, resonant waveguide grating; ERK, extracellular-signal-regulated cellular-signal-regulated kinase 1/2 (anti-pERK1/2) (Cat. #M9682) kinase; CMOT, catechol-O-methyl transferase ⇑ Corresponding author. Fax: +1 607 974 5957. and mouse monoclonal anti-ERK1/2 (#M7431) were obtained from E-mail address: [email protected] (Y. Fang). Sigma. Goat anti-GPR35 antibody (#T14, cytoplasmic) was pur- 1 Equal contributions. chased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.05.026 1958 H. Deng et al. / FEBS Letters 585 (2011) 1957–1962

Alexa FluorÒ 594 donkey anti-goat IgG (H + L) was purchased from 2.3. Receptor internalization assays Invitrogen (Carlsbad, CA, USA). Human colorectal adenocarcinoma HT-29 was obtained from HT29 cells were plated on an 8-well chamber slide with a American Type Cell Culture (Manassas, VA). The cells were cul- seeding density of 10 000 cells/well and incubated at 37 °C for tured in McCoy’s 5a Medium Modified supplemented with 10% fe- 24 h. Next day, cells were stimulated with a compound or equal tal bovine serum, 4.5 g/l glucose, 2 mM glutamine, and antibiotics amount of DMSO at 37 °C for 1 h. Afterwards, cells were fixed at 37 °C under air/5% CO2. with 4% formaldehyde in 1Â PBS for 15 min, followed by block- Tango™ GPR35-bla U2OS cells were obtained from Invitrogen. ing and permeabilization in a buffer containing 4% goat serum, Tango™ GPR35-bla U2OS cells contain the human GPR35 linked 0.1% bovine serum albumin (BSA), 0.1% Triton X100 in 1Â PBS to a TEV protease site and a Gal4-VP16 transcription factor stably for 2 h. After 5 min wash with PBS, fixed cells were incubated integrated into the Tango™ GPR35-bla U2OS parental cell line. This with the anti-GPR35 (1:500) in 3% BSA/PBS buffer for 24 h, fol- parental cell line stably expresses a b-arrestin/TEV protease fusion lowed by incubation with secondary antibody Alexa FluorÒ 594 protein and the b-lactamase reporter under the control of a donkey anti-goat IgG (H + L) (1:500) in 3% BSA/PBS for 1 h at UAS response element. The cells were cultured according to the room temperature. Cells were finally washed once with PBS protocols recommended by the supplier. Briefly, the cells were and sealed with 1.5 mm thick glass cover-slip. Dried slides were passed using McCoy’s 5A medium (Invitrogen 16600-082) supple- stored at 4 °C until imaging. Confocal imaging was performed mented with 10% dialyzed fetal bovine serum, 0.1 lM NEAA, with Zeiss confocal microscope Axiovert 40. Confocal images 25 lM Hepes (pH 7.3), 1 mM sodium pyruvate, 100 U/ml penicillin, were analyzed using MacBiophotonics Image J software (http:// 100 lg/ml streptomycin, 200 lg/ml zeocin, 50 lg/ml hygromycin, www.macbiophotonics.ca/downloads.htm). and 100 lg/ml geneticin in a humidified 37 °C/5% CO2 incubator. 2.4. ERK MAPK assays 2.2. DMR assays using EpicÒ system The p44/42 MAP kinases were examined using Western blot- All DMR assays were performed using EpicÒ system (Corning ting. Whole cell lysates were collected after the cells were treated Inc.). EpicÒ is a wavelength interrogation reader system tailored with a compound or DMSO for 1 h. Equivalent gel loading was con- for RWG biosensor in microtiter plates. This system consists of a firmed by probing with anti-actin body. The total ERK1/2 and phos- temperature-control unit (26 °C), an optical detection unit, and phorylated ERK1/2 were blotted using respective antibodies. an on-board liquid handling unit with robotics. The detection unit is centered on integrated fiber optics, and enables kinetic measures 2.5. Tango™ b-arrestin assays of cellular responses with a time interval of 15 s. Cells were di- rectly seeded in EpicÒ plates and cultured overnight to form con- The Tango™ GPR35 b-arrestin assay takes advantage of GPR35 fluent monolayer in the cell culture medium. After being washed -induced recruitment of protease tagged beta-arrestin twice, the cells were maintained with HBSS and further incubated molecules to GPR35 that has been modified at the C-terminus inside the system for 1 h. For agonism screen, a 2-min baseline was to include a transcription factor linked by a protease cleavage then established. Immediately after the compound addition using site. As a result of arrestin recruitment, the protease cleaves the the onboard liquid handler, the cellular responses were recorded. transcription factor from the receptor, which then translocates For desensitization assays, cells were initially treated with com- to the nucleus and activates the expression of beta-lactamase. pounds for 1 h, followed by stimulation with zaprinast at a fixed The assay protocol recommended by the supplier was used. dose. The cellular responses were recorded throughout the assays. Briefly, 10 000 cells/well were seeded in 384-well, black-wall,

All EC50 or IC50 described in the main text were calculated based on clear bottom assay plates with low fluorescence background the amplitudes of DMR signals at 8 min post-agonist stimulation. (Corning), and cultured in DMEM (Invitrogen, 10569-010) supple- Since all GPR35 agonists led to a sustained positive-DMR (P- mented with 10% dialyzed fetal bovine serum, 0.1 lM NEAA, DMR) signal, the amplitudes at 50 min post-stimulation were also 25 lM Hepes (pH 7.3), 100 U/ml penicillin, and 100 lg/ml strep- used to determine kinetics dependent potency and efficacy of all li- tomycin. After overnight culture, the cells were stimulated with gands (see Table 1). ligands for 5 h in a humidified 37 °C/5% CO2, and then loaded

Table 1

Compound name, and its efficacy relative to the maximal response induced by zaprinast, EC50 at 8 min or 50 min post-stimulation, apparent IC50 to desensitize the cells, all of which were obtained using DMR assays. EC50 obtained using Tango assays were also included.

Compound % zaprinast at EC100 EC50 (lM) EC50 (lM) IC50 (lM) EC50 (lM) (Tango) P-DMR (8 min) P-DMR (50 min) P-DMR (8 min) P-DMR (50 min) Zaprinast 100 100 0.16 ± 0.02 0.05 ± 0.01 0.32 ± 0.04 6.2 ± 0.9 Tyrphostin-1 Inactive Inactive Inactive Inactive Inactive Inactive Tyrphostin-9 11 ± 3 63 ± 1 n.a. 1.0 ± 0.2 Inactive Inactive Tyrphostin-23 94 ± 4 109 ± 5 3.0 ± 0.3 2.0 ± 0.2 4.0 ± 0.5 >60 Tyrphostin-25 107 ± 9 102 ± 8 0.94 ± 0.07 0.70 ± 0.05 0.94 ± 0.07 5.3 ± 1.1 Tyrphostin-46 74 ± 6 83 ± 5 4.6 ± 0.2 1.9 ± 0.2 5.4 ± 0.6 >60 Tyrphostin-47 80 ± 5 101 ± 6 3.9 ± 0.2 2.8 ± 0.4 2.7 ± 0.3 19.2 ± 3.1 Tyrphostin-51 98 ± 4 99 ± 9 0.19 ± 0.02 0.11 ± 0.03 0.21 ± 0.05 7.7 ± 0.9 AG-126 54 ± 13 97 ± 15 11.9 ± 0.4 10.2 ± 0.9 27.1 ± 3.0 Inactive AG-490 Inactive Inactive Inactive Inactive Inactive Inactive AG-494 28 ± 1 95 ± 3 n.a. 12.5 ± 2.1 35 ± 7 Inactive AG-825 55 ± 8 65 ± 13 15.2 ± 3.1 9.5 ± 2.3 16.1 ± 4.1 Inactive AG-1024 40 ± 10 117 ± 11 n.a. 2.4 ± 1.2 >32 Inactive AG-1288 78 ± 2 80 ± 7 0.44 ± 0.04 0.21 ± 0.04 0.70 ± 0.06 25.3 ± 4.7 Entacapone 97 ± 3 140 ± 5 6.3 ± 0.7 31.7 ± 4.1 6.6 ± 0.9 Inactive H. Deng et al. / FEBS Letters 585 (2011) 1957–1962 1959 with the cell permeable LiveBLAzer™ FRET B/G substrate. After 3.9 ± 0.2 nM (n = 4), 1.22 ± 0.10 lM(n = 4), and 234 ± 25 lM the 2 h incubation the coumarin:fluorescein ratio was measured (n = 4) for pamoic acid, zaprinast, and kynurenic acid, respectively using Tecan Safire II microplate reader (Männedorf, Switzerland). (Fig. 1c). Immunostaining with anti-GPR35 showed that GPR35 The FRET B/G substrate contains two fluorophores, coumarin and was presented and located primarily at the cell plasma mem- fluorescein, in which excitation of the coumarin results in fluores- brane in unstimulated HT29 cells (Fig. 1d), and got partially inter- cence resonance energy transfer to the fluorescein moiety and nalized by 10 lM pamoic acid (Fig. 1e). Together, these results emission of green fluorescent light. In the absence of beta-lacta- suggest that GPR35 in HT29 is functional, and all three ligands mase expression (i.e., no GPR35 activation), cells generate green are indeed agonists for GPR35. fluorescence. In the presence of beta-lactamase expression upon receptor activation, the substrate is cleaved and the cells generate 3.2. DMR agonism screen blue fluorescence. The coumarin:fluorescein ratio was used as a normalized reporter response. Next, we screened three kinase libraries that include a total of 240 known kinase inhibitors, using DMR agonism assay. Each 3. Results inhibitor was assayed with four replicates. Zaprinast was included as a positive control. Hits were selected based on their maximal 3.1. Characteristics of known GPR35 agonists in HT29 responses within 15 min post-stimulation that are greater than 30% of the zaprinast response. Twenty-five hits were identified To determine the presence of functional GPR35 in HT29, we (Fig. 2). Follow-up structure activity analysis showed that 11 first examined the DMR responses induced by three known out of the 25 hits are tyrphostins, including AG-112, AG-1024, GPR35 agonists including zaprinast [15], pamoic acid [16], and tyrphostin-51, tyrphostin-25, tyrphostin-47, AG-370, AG-1288, kynurenic acid [7]. Results showed that all three ligands gave rise tyrphostin-23, AG-126, tyrphostin-46, and AG-825 (Fig. 3). Fur- to a dose-dependent and saturable response (Fig. 1a); and the ther, AG-494, AG-879, tyrphostin-9, and quercetin also led to

EC50 values were found to be 2.1 ± 0.2 nM (n = 4), 0.16 ± 0.02 lM noticeable DMR. The other five tyrphostins including AG-490, (n = 4), and 152 ± 17 lM(n = 4) for pamoic acid, zaprinast, and AG-1478, AG-1295, AG-1296, and tyrphostin-1 in the libraries kynurenic acid, respectively (Fig. 1b). All three agonists also did not trigger any noticeable DMR. Quercetin was recently iden- dose-dependently desensitized the cells to the repeated stimula- tified to be a GPR35 partial agonist [17]. These results suggest tion with 1 lM zaprinast, leading to an apparent IC50 of that tyrphostins are possible GPR35 agonists.

a b 400 250 Zaprinast kynurenic acid 200 300 Pamoic acid 500µM 150 250µM 1000µM 200 100 125µM 100 µ 50 62.5 M

31µM Response (pm) Response (pm) µ 0 0 16 M

-50 -100 0 1020304050 -10 -9 -8 -7 -6 -5 -4 -3 Time (min) Compound, log M

cde400 Zaprinast kynurenic acid 300 Pamoic acid

200

100 Response (pm) 0

-100 -10 -9 -8 -7 -6 -5 -4 -3 Compound, log M

Fig. 1. Characterization of three known GPR35 agonists including zaprinast, pamoic acid and kynurenic acid. (a) The dose DMR responses of kynurenic acid in HT29. (b) The DMR amplitudes of three agonists as a function of their doses. (c) The DMR amplitudes of 1 lM zaprinast as a function of doses of three agonists. Here the cells were pre- stimulated with distinct agonists at different doses for 1 h. The amplitudes at 8 min post-stimulation were used to calculate their potency for (b) and (c). (d) The confocal image of unstimulated HT29 cells. (e) The confocal image of HT29 cells 1 h after stimulated with 10 lM pamoic acid. The images were obtained after the cells were permeabilized and stained with anti-GPR35, followed staining with the fluorescent secondary antibody. 1960 H. Deng et al. / FEBS Letters 585 (2011) 1957–1962

400 (CMOT) inhibitor for the treatment of Parkinson’s disease [18]. Zaprinast Thus, we characterized entacapone using DMR assays. Results showed that entacapone led to a dose-dependent DMR with an

EC50 of 6.3 ± 0.7 lM(n = 4), whose DMR characteristics is similar 200 to other GPR35 agonists including zaprinast and tyrphostin-51 (Fig. 4b). Similar to tyrphostin-51, entacapone also dose-depen- DMSO dently desensitized the cells responding to the repeated stimula- 0 tion with 1 lM zaprinast, leading to an apparent IC50 of

Response (pm) 31.7 ± 4.1 lM(n =4)(Fig. 4c). These results suggest that entaca- pone may also be a GPR35 agonist.

-200 3.4. GPR35 internalization, ERK phosphorylation and arrestin -40 0 40 80 120 160 200 240 translocation induced by tyrphostins Compound

Fig. 2. The DMR responses as a function of compounds. Each compound was Since tyrphostin-51 is the most potent ligand among tyrphos- assayed at four replicates. The DMR amplitudes of all compounds at 8 min post- tins in DMR assays, we examined its ability to cause receptor inter- simulation were plotted. nalization. Results showed that stimulation of HT29 with 10 lM tyrphostin-51 led to significant internalization of GPR35 (Fig. 5a). 3.3. Dose-dependent DMR signals of tyrphostins Since ERK phosphorylation is a hallmark of the activation and sig- naling of many GPCRs including GPR35 [16], we examined the ability The three kinase libraries consist of 19 tyrphostin analogs, 14 of of both tyrphostin-51 and entacapone to result in ERK phosphoryla- which led to detectable DMR when being screened at 10 lM. Thus, tion. Results showed that stimulation of HT-29 cells with both ago- we were focused on characterization of tyrphostins. We first exam- nists led to robust phosphorylation of ERK (Fig. 5b). As a control, the ined 13 tyrphostins for their dose-dependent responses in HT29 known GPR35 agonist zaprinast also led to robust ERK phosphoryla- cells using DMR assays. Results showed that except for tyrphostin- tion, while tyrphostin-1 was inactive as expected (Fig. 5b). 1 and AG-490, all tyrphostins tested gave rise to dose-dependent re- Finally, we examined the ability of tyrphostins to cause arrestin sponses (Fig. 4a). Further, these tyrphostins differ greatly in potency translocation using the Tango™ GPR35 arrestin assays. Results and efficacy (Table 1). Tyrphostin-51, tyrphostin-23 and tyrphostin- showed that zaprinast led to a dose dependent response in Tango™

25 acted as full agonists, while others were partial agonists. GPR35-bla U2OS cells with an EC50 of 6.2 ± 0.9 lM(n =4)(Fig. 6a; Searching the DrugBank database (http://www.drugbank.ca/) Table 1). Similarly, tyrphostin-51 also led to a comparable response, identified entacapone that is a tyrphostin analogy drug (Fig. 3). leading to an EC50 of 7.7 ± 0.9 lM(n =4)(Fig. 6b), suggesting that Entacapone is a selective, reversible catechol-O-methyl transferase similar to zaprinast tyrphostin-51 acted as a full agonist for GPR35

NH NH2 2 CN CN CN CN CN CN CN CN CN CN O HO HO N H Tyrphostin 1 Tyrphostin 9 AG-112 AG-370

O NH2 CN CN C H HO HO CN 2 5 HO O N N CN CN 2 CN CN CN C2H5 HO HO HO HO OH Tyrphostin 25 Tyrphostin 23 OH OH Entacapone Tyrphostin 51

O o CONH2 CONH2 HO HO CN HO N N CN H H CN CN HO HO HO HO Tyrphostin 47 OH Tyrphostin 46 AG-494 AG-490

CN CN CN N CONH Br HO 2 CN CN CN S CN S HO HO O2N HO OH AG-126 O NO2 AG-1024 AG-1288 AG-825

Fig. 3. Structures of a series of tyrphostins tested. All ligands, but tyrphostin-1 and AG-490, exhibit agonism activity on GPR35 in HT29 cells. H. Deng et al. / FEBS Letters 585 (2011) 1957–1962 1961

a 300 b c 300 300 32µM 2000nM 250 16µM 200 1000nM 200 200 500nM 8µM 250nM 150 100 125nM 100 100 4µM 63nM 31nM

Response (pm) 50 µ Response (pm) 2 M

0 Response (pm) 0 16nM µ 1 M Entacapone 0 0.5µM Tyrphostin 51 -100 -50 -100 01020304050 01020304050 -9 -8 -7 -6 -5 -4 Time (min) Time (min) Compound, log M

Fig. 4. Characteristics of the DMR signals induced by tyrphostin-51 and entacapone. (a) The dose DMR responses of tyrphostin-51; (b) the dose DMR responses of entacapone; and (c) the DMR amplitudes (8 min post-stimulation) of both tyrphostin-51 and entacapone as a function of ligand doses.

ab Zaprinast Tyrphostin-1 Entacapone DMSO Tyrphostin 51 pERK1/2

ERK2

µ 10 m Actin

Fig. 5. Receptor internalization induced by 10 lM tyrphostin-51 (a) and ERK phosphorylation induced by 10 lM tyrphostin-51, 32 lM entacapone, 32 lM tyrphostin-1 or 1 lM zaprinast (b). The confocal image was obtained after the cells were stimulated with tyrphostin-51 for 1 h, permeabilized and stained with anti-GPR35, followed staining with the fluorescent secondary antibody.

a 15 Zaprinast b 15 Zaprinast Tryphostin-25 Tyrphostin-51 12 Tryphostin-47 12 Tyrphostin-46 AG-1288 Tyrphostin-23 Entacapone Tryphostin-1 9 9

6 6 Response ratio Response (pm) 3 3

0 0 -9 -8 -7 -6 -5 -4 -3 -9 -8 -7 -6 -5 -4 -3 Compound, log M Compound, log M

Fig. 6. Arrestin translocation induced beta-lactamase expression upon the activation of GPR35 by tyrphostins in Tango™ GPR35-bla U2OS cells. The coumarin to fluorescein ratio was plotted as a function of ligand doses. Zaprinast was included as a positive control, while tyrphostin-1 as a negative control. to cause arrestin translocation. Interestingly, several other tyrphos- 4. Discussion tins including tyrphostin-23, tyrphostin-25, tyrphostin-46, tyrpho- stin-47 and AG-1288 acted as partial agonists (Fig. 6), while other GPR35 is a poorly-characterized orphan GPCR with a few known tyrphostins tested were inactive. It is worthy noting that the Tango agonists. To identify GPR35 ligands, we first showed that GPR35 is arrestin assay generally led to a right shift in potency. Nonetheless, present and located primarily at the cell surface of native HT29 cells these results suggest that tyrphostins including entacapone are (Fig. 1d). DMR assays further showed that endogenous GPR35 in GPR35 agonists. HT29 is functional, whose activation by three known GPR35 agonists 1962 H. Deng et al. / FEBS Letters 585 (2011) 1957–1962 leads to robust DMR signals (Fig. 1a and b). Screening three kinase [2] Okumura, S., Baba, H., Kumada, T., Nanmoku, K., Nakajima, H., Nakane, Y., inhibitor libraries using DMR agonism assay led to identification of Hioki, K. and Ikenaka, K. (2004) Cloning of a G-protein-coupled receptor that shows an activity to transform NIH3T3 cells and is expressed in gastric cancer 14 tyrphostin analogs that acted as GPR35 agonists. Among all tyr- cells. Cancer Sci. 95, 131–135. phostins examined, tyrphostin-51 was found to be the most potent [3] Imielinski, M. et al. (2009) Common variants at five new loci associated with early-onset inflammatory bowel disease. Nat. Genet. 41, 1335–1340. ligand with an EC50 of 190 nM (Fig. 4a), which is comparable to the [4] Yang, Y., Lu, J.Y.L., Wu, X., Summer, S., Whoriskey, J., Saris, C. and Reagan, J.D. potency of zaprinast. Tyrpostin-51 resulted in significant internali- (2010) G-protein-coupled receptor 35 is a target of the asthma drugs cromolyn zation of GPR35, the degree of which is comparable to that induced disodium and nedocromil sodium. Pharmacology 86, 1–5. by pamoic acid (Fig. 5a). Further, tyrophstin-51 also led to robust [5] Sparfel, L., Pinel-Marie, M.-L., Boize, M., Koscielny, S., Desmots, S., Pery, A. and Fardel, O. (2010) Transcriptional signature of human macrophages exposed to ERK phosphorylation (Fig. 5b) and b-arrestin translocation the environmental contaminant benzo(a)pyrene. Toxicol. Sci. 114, 247–259. (Fig. 6b). Tyrphostins were originally designed and developed for [6] Min, K.D., Asakura, M., Liao, Y., Nakamaru, K., Okazaki, H., Takahashi, T., inhibiting tyrosine kinases [19]. Like all kinase inhibitors tyrphos- Fujimoto, K., Ito, S., Takahashi, A., Asanuma, H., Yamazaki, S., Minamino, T., Sanada, S., Seguchi, O., Nakano, A., Ando, Y., Otsuka, T., Furukawa, H., Isomura, tins often interact with multiple targets. Thus, the identification of T., Takashima, S., Mochizuki, N. and Kitakaze, M. (2010) Identification of tyrphostins as potent GPR35 agonists adds new target class to these related to heart failure using global profiling of human failing kinase inhibitors. myocardium. Biochem. Biophys. Res. Commun. 393, 55–60. One of the most notable findings of this study is that entacapone [7] Wang, J., Simonavicius, N., Wu, X., Swaminath, G., Reagan, J., Tian, H. and Ling, L. (2006) Kynurenic acid as a ligand for orphan G protein-coupled receptor was identified to be a GPR35 agonist with a moderate potency. GPR35. J. Biol. Chem. 281, 22021–22028. Chemical similarity searching of the Drug Bank database allowed [8] Oka, S., Ota, R., Shima, M., Yamashita, A. and Sugiura, T. (2010) GPR35 is a us to identify entacapone as a tyrphostin analog drug. Follow-up novel receptor. Biochem. Biophys. Res. Commun. 395, 232–237. DMR and receptor signaling assays showed that (1) entacapone re- [9] Milligan, G. (2011) Orthologue selectivity and ligand bias: translating the sulted in a DMR similar to other GPR35 agonists; (2) entacapone pharmacology of GPR35. Trends Pharmacol. Sci. 32, 317–325. caused cells desensitized upon the repeated stimulation with zapri- [10] Fang, Y. (2010) Label-free receptor assays. Drug Discov. Today Technol. 7, e5– e11. nast; and (3) entacapone behaved similarly to tyrphostin-51 in [11] Fang, Y., Ferrie, A.M., Fontaine, N.H., Mauro, J. and Balakrishnan, J. (2006) receptor signaling assays. Entacapone was apparently inactive in Resonant waveguide grating biosensor for living cell sensing. Biophys. 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