Thieno[3,2-b]thiophene-2-carboxylic acid derivatives as GPR35 agonists

Article abstract of DOI:10.1016/j.bmcl.2012.04.057

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.

Full text of DOI:10.1016/j.bmcl.2012.04.057

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4148–4152
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thieno[3,2-b]thiophene-2-carboxylic acid derivatives as GPR35 agonists
⇑
Huayun Deng, Jieyu Hu, Haibei Hu, Mingqian He, Ye Fang
Biochemical Technologies, Science and Technology Division, Corning Inc., Corning, NY 14831, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 February 2012
Revised 4 April 2012
Accepted 10 April 2012
Available online 21 April 2012
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 b-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 b-arrestin translocation.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
GPR35
Thieno[3,2-b]thiophene-2-carboxylic acid
Agonists
GPR35 is an orphan G protein-coupled receptor (GPCR) impli-
cated in inflammation, pain, cardiovascular diseases, and metabolic
disorders.1 Several known GPR35 agonists have been reported in
literature; these include clinically used drugs. Cromolyn and dicu-
marol, the two anti-asthma drugs with unknown mechanism of ac-
tion, were recently identified to be GPR35 agonists with moderate
potency.2 Bumetanide and furosemide, two loop diuretics used for
treating cardiovascular disease, were also found to be GPR35 ago-
nists.3 Using label-free phenotypic profiling, we had found that
GPR35 is a target of entacapone,4 a catechol-O-methyl transferase
inhibitor drug for the treatment of Parkinson’s disease, as well as
the anti-nociception niflumic acid.5 We also discovered that cer-
tain abundant natural phytochemicals including myricetin, morin,
and ellagic acid,5 and gallic acid6 are GPR35 agonists. Furthermore,
GPR35 has been found to be up-regulated in several types of in-
flamed cells.1,2,7 Therefore, these findings provide rationales for
proposing activation of GPR35 as a therapeutic target with oppor-
tunity for development in certain diseases including inflammatory
disorders.
Ligand-directed functional selectivity, or biased agonism, de-
scribes the ability of distinct ligands to differentially activate one
of the vectorial pathways mediated through a receptor.8 The biased
agonism is often inferred from the relative potency and efficacy to
modify one signaling molecule/event over another. As a result,
many ligands may give rise to assay readout-dependent potency
and efficacy, suggesting that the efficacy for many ligands is collat-
eral.9 Biased agonism, once thought to be an artifact of recombi-
nant technology, has been now amply demonstrated to be a
natural cellular control mechanism,10,11 and is believed to have
therapeutic impacts.12 Previously, we had discovered several thie-
no[3,2-b]thiophene-2-carboxylic acid (TTAC) derivatives as GPR35
agonists.13 These chemicals were originally designed and synthe-
sized for non-linear optics and organic transistor applications. To
expand pharmacological tools for GPR35, we synthesized a series
of novel TTAC analogs, and characterized their agonist activity
against the GPR35.
TTCA presents several positions for structure–activity relation-
ship (SAR) modifications (Fig. 1). Since we have previously shown
that its carboxylic acid group is critical for its agonist activity at the
GPR35,13 we decided to focus on analogs bearing modifications at
other positions of TTCA.
Compounds 4, 8, 10, 11, 13 and 14 were synthesized from perb-
romothiophene as outlined in Scheme 1. The procedure com-
mences with C-methylation of perbromothiophene using
butyllithium (n-BuLi) and methyl benzesulfonate to produce 1,
the Friedel–Crafts acylation (C-acylation) with AlCl3 and acetyl
chloride (CS2) to produce 2, the ketone-based ring closure reaction
with ethyl 2-mercaptoacetate using 18-crown-6 as the catalytic
agent to produce intermediate 3, and finally hydrolysis to produce
4. Compound 8 was synthesized using lithiation followed by C-
acylation of perbromothiophene, then the ketone-based ring clo-
sure, bromination with N-bromosuccinimide (NBS), and hydrolysis
R2
S
R1
R3
OH
X
O
⇑
Corresponding author. Tel.: +1 607 9747203.
E-mail address: fangy2@corning.com (Y. Fang).
Figure 1. Structures of TTCA analogs. YE210: X = S, R1 = CH3, R2 = H, R3 = Br.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.057

H. Deng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4148–4152
4149
O
Br
Br
S
S
S
b
c
d
S
S
Br
Br
S
COOC2H5
S
COOH
Br
1 Br
2
3
4
a
Br
S
Br
Br
S
Br
Br
Br
Br
S
Br
S
S
Br
Br
e
f
g
h
O
Br
Br
COOC2H5
S
S
COOC2H5
COOH
Br
i
5
6
7
8
Br
Br
Br
Br
CF3
O
S
S
S
Br
j
k
CF3
CF3
COOH
l
Br
S
S
S
COOC2H5
Br
9
10
11
Br
Br
Br
Br
OCH3
O
S
S
S
S
Br
Br
m
n
o
OH
COOH
OH
COOH
OH
S
S
COOC2H5
Br
11
12
13
14
Scheme 1. Synthesis of TTAC analogs. Reagents and conditions: (a) n-BuLi, THF, 0 °C; methyl benzenesulfonate, THF, rt, 95%; (b) AlCl3, CS2, 0 °C; AcCl, rt, 53%; (c) ethyl 2-
mercaptoacetate, K2CO3, catalytic amount of 18-crown-6, DMF, 70 °C, 55%; (d) LiOH, EtOH/H2O, 40 °C, 31%; (e) AlCl3, CS2, 0 °C; AcCl, 40 °C, 55%; (f) ethyl 2-mercaptoacetate,
K2CO3, catalytic amount of 18-crown-6, DMF, 70 °C, 31%; (g) NBS, DMF, rt, 31%; (h) LiOH, EtOH/H2O, 40 °C, 36%; (i) n-BuLi, THF, À78 °C; 2,2,2-trifluoro-N-methyoxy-N-
methylacetamide, rt, 80%; (j) ethyl 2-mercaptoacetate, sodium bis(trimethylsily)amide, toluene/THF, rt, 50%; (k) LiOH, THF, 70 °C, 90%; (l) n BuLi, THF, À78 °C; ethyl
carbonochloridate, H2O, rt, 50%; (m) ethyl 2-mercaptoacetate, sodium bis(trimethylsily)amide, toluene/THF, rt, 50%; (n) LiOH, THF, 70 °C, 90%; (o) n BuLi, À78 °C, THF; 6 N
HCl, 20%.
in a sequential manner. Compound 11 was synthesized using the
introduction of trifluoroacetyl through C-acylation of perbromothi-
ophene to produced 9, the ketone-base ring closure to produce
intermediate 10, and then hydrolysis to produce 11. Compound
13 was synthesized using the introduction of methoxycarbonyl
through C-acylation to perbromothiophene, the ring closure, and
then hydrolysis. Compound 14 was produced from 13 through
debromination using n-BuLi and 6 N HCl.
Compounds 20 and 24 were synthesized according to Scheme 2.
The synthesis of compound 20 begins with C-acylation of 3,4-
dibromothiophene to produce aldehyde intermediate 15, then
aldehyde oxime formation to produce 16, conversion of oxime to
nitrile 17, coupling followed by condensation to form intermediate
18, diazonium salt formation followed by chlorination to form 19,
and hydrolysis to produce 20. The synthesis of compound 24 was
done using addition of Grignard reagents to aldehyde 15 to pro-
duce 21, oxidization to form ketone 22, the ring closure action to
produce ester 23, and hydrolysis to produce 24.
C-acylation of 3,4-dibromothiophene to the form ketone 25, the
ring closure to form the ester 26, Hiyama cross-coupling or Suzki
cross-coupling to produce 28, or 29 to 32, respectively. Compound
34 was produced from ketone 25 through consensation/coupling/
deformylation cascade process, and then hydrolysis. Compounds
36 and 37 were synthesized from the ester 33 through Suzuki
cross-coupling and sequential hydrolysis. Compound 39 was syn-
thesized from the ester 38 through N-methylation and sequential
hydrolysis.
Except for 8, 10, 11, 13 and 14 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 great-
er than 95%.
We employed both label-free dynamic mass redistribution
(DMR)14,15 and Tango b-arrestin translocation assays16 to charac-
terize the pharmacological activity of TTAC analogs at GPR35.
DMR assay is based on whole cell phenotypic responses,14 while
Tango is an endpoint measurement of gene reporter activity linked
to the GPR35 activation-mediated b-arrestin translocation.4,5 The
Compounds 28–32, 34, 36 and 37 were synthesized according
to Scheme 3. The synthesis of compounds 28–32 commences with
S
S
S
S
S
S
S
NH2
Cl
a
b
c
N-OH
d
e
f
Cl
CN
O
Br
Br
Br
Br
Br
Br
Br
S
S
COOC2H5
COOC2H5
S
Br
Br
Br
Br
COOH
15
16
17
18
19
20
g
O
OH
S
S
S
S
h
i
j
Br
Br
Br
Br
S
Br
Br
COOC2H5
S
COOH
21
22
23
24
Scheme 2. Synthesis of TTAC analogs. Reagents and conditions: (a) LDA, THF, N2, À78 °C; DMF, rt, 89.5%; (b) hydroxylamine HCl, NaHCO3, EtOH/H2O, rt, 90.2%; (c) acetic
anhydride, 120 °C, 59.5%; (d) ethyl 2-mercaptoacetate, K2CO3, catalytic amount of 18-crown-6, DMF, 60 °C, 74%; (d) tert-butyl nitrite, CuCl, acetonitrile, 75 °C, 69.8%; (e) LiOH,
EtOH/H2O, 70 °C, 25%; (f) LDA, THF, À78 °C; DMF, À78 °C, 89.5%; (g) bromocyclopropane, magnesium, THF, N2, 0 °C, 21.8%; (h) PCC, CH2Cl2, rt, 83.3%; (i) ethyl 2-
mercaptoacetate, K2CO3, catalytic amount of 18-crown-6, DMF, 60 °C, 56%; (j) LiOH, EtOH/H2O, 70 °C, 23%.

4150
H. Deng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4148–4152
S
S
S
S
S
a
b
c
d
O
Br
R
Br
Br
R
S
S
S
COOC2H5
COOC2H5
S
COOH
Br
Br
25
26
27
28 R = ethynyl
29 R = cyclopropyl
30 R = pyridin-3-yl
e
Br
31 R= thiophen-3-yl
N
H
COOH
f
32 R =1H-pyrazol-4-yl
34
S
Br
S
S
g
N
H
COOC2H5
h
33
R
R
N
N
H
COOC2H5
COOH
H
i
35
36 R = pyridin-3-yl
37 R = cyclopropyl
S
S
j
Br
Br
N
N
COOC2H5
COOH
38
39
Scheme 3. Synthesis of TTAC analogs. Reagents and conditions: (a) AlCl3, CH2Cl2, 0 °C; AcCl, 0 °C, 98%; (b) ethyl 2-mercaptoacetate, K2CO3, catalytic amount of 18-crown-6,
DMF, 80 °C, 65%; (c) ethyltrimethylsilane, cyclopropylboronic acid, pyridin-3-ylboronic acid, thiophrn-3-ylboronic acid, or 1H-pyrazol-4-ylboronic acid for 28–32,
respectively, CuI, Pd(dppf)2Cl3, CH2Cl2, Et3N, N2, 100 °C, 31–74%; (d) LiOH, EtOH/H2O, rt, 12–50%; (e) ethyl 2-isocyanoacetate, CuI, Cs2CO3, N2, 80 °C, 52%; (f) LiOH, EtOH/H2O,
rt, 37%; (g) pyridin-3-ylboronic acid or cyclopropylboronic acid for 36 or 37, respectively; K2CO3, Pd(dppf)2Cl2, dioxane/H2O, N2, 100 °C, 75–80%; (h) LiOH, EtOH/H2O, rt to
40 °C, 24–35%; (i) NaH, THF, 0 °C; CH3I (30), THF, rt, 70%; (j) LiOH, EtOH/H2O, rt, 37%.
dose responses were performed in two independent measure-
ments, each in duplicate. SAR analysis was carried out based on rel-
ative potency and efficacy of compounds tested.
dose. Furthermore, the potency of CID 2745687 was found to be
similar to block the DMR of these compounds (Table 1). The
dose-dependent inhibition of representative compounds, YE210,
8 and 13, by CID 2745687 were shown in Figure 2d. These results
suggest that these DMR-active TTAC analogs are GPR35 agonists.
Second, we measured the ability of TTAC analogs to cause b-arr-
estin translocation using Tango assay in the engineered U2OS-
GPR35-bla cell line. This cell line stably expresses two fusion pro-
teins: human GPR35 linked to a Gal4-VP16 transcription factor via
a TEV protease site, and b-arrestin/TEV protease fusion protein.
The cell line also stably expresses the b-lactamase reporter gene un-
der the control of a UAS response element. The activation of GPR35
by agonists leads to the recruitment of b-arrestin-TEV protease fu-
sion 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 b-lactamase. Such a b-arrestin translocation is of-
ten specific to the GPR35 activation, given that the test compound is
not fluorescent.5 All Tango assay results were reported after normal-
ized to the maximal response of zaprinast within the same plate.
Results showed that as expected, compound 10 was inactive. All
DMR-active compounds were found to be also active in Tango as-
says. The dose responses were also best fitted with a monophasic
First, we recorded the dose responses of all TTAC analogs in na-
tive HT-29 cells using DMR agonist assays. The real time DMR sig-
nals of representative compounds, 8 and 13, are showed in
Figure 2a and b, respectively. Compound 10 was inactive
(Fig. 2c), consistent with our previous observation that the carbox-
ylic acid group is essential for the agonist activity of TTAC ana-
logs.13 All remaining compounds led to a clear dose-dependent
DMR whose characteristics were similar to YE210 (6-bromo-3-
methylthieno[3,2-b]thiophene-2-carboxylic acid),13 as well as
other known GPR35 agonists including zaprinast, pamoic acid,
and kynurenic acid.4 These compounds were thus termed DMR ac-
tive compounds. The dose responses were best fitted with a mon-
ophasic sigmoidal non-linear regression, leading to a single EC50
for each compound (Fig. 3c, Table 1). 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),13,17 to exam-
ine 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
Figure 2. The dose responses of TTAC analogs. (a,b) Real time dose DMR responses of 8 and 13 in HT-29 cells, respectively. (c) The DMR amplitudes of 8, 10, and 13 as a
function of their doses. (d) The dose-dependent inhibition of compound-induced DMR by CID 2745687. Compound concentration was 250 nM, 2 M and 125 nM for YE210, 8
l
and 13, respectively. The DMR amplitudes 10 min poststimulation in HT-29 cells were calculated for all. The data represents mean s.d. from two independent
measurements, each in duplicate (n = 4).

H. Deng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4148–4152
4151
a
b
YE210
8
13
10
120
100
80
60
40
20
0
120
100
80
60
40
20
0
YE210
31
36
13
-20
-20
-7
-6
-5
-4
-3
-9
-8
-7
-6
-5
-4
Compound, log M
ML-145, log M
Figure 3. The dose-dependent responses of TTAC analogs obtained using Tango assays. (a) The dose responses of four TTAC analogs, YE210, 8, 10 and 13. (b) The CID 2745687
dose-dependent inhibition of the Tango signals induced by 13, 31, 36, and YE210, each at a fixed dose. The compound dose was 10 M, 100 M, 64 M, and 20 M for
l
l
l
l
compound 13, 31, 36, and YE210, respectively. The data represents mean s.d. from two independent measurements, each in duplicate (n = 4).
Table 1
Compounds, their EC50 and efficacy (that is, the maximal responses) as measured using DMR and Tango assays, and the IC50 of CID 2745687 to block the DMR of each compound at
a fixed dose*
Compound
EC50
(lM)
IC50
(
l
M)
Efficacy
Tango (% zaprinast)
antagonist**
DMR
152 17
0.16 0.02
0.061 0.004
1.29 0.11
0.70 0.05
Inactive
Tango
>500
6.2 0.9
8.30 0.6
22.2 1.8
36.5 3.8
DMR (pm)
Kynurenic acid
Zaprinast
1 (YE210)
4
8
4.41 0.47
10.5 0.3
8.06 0.54
9.80 0.71
9.61 0.79
202
269
290
268
232
>60
100
113
52
52
10
Inactive
11
13
14
20
24
28
29
30
31
32
34
36
37
39
0.51 0.04
0.016 0.002
9.78 0.81
1.03 0.09
2.63 0.18
1.48 0.09
0.95 0.07
0.24 0.02
0.26 0.02
0.37 0.02
11.8 0.9
31.1 2.5
2.46 0.19
340 29
39.2 2.7
504 41
54.8 4.2
52.4 5.1
11.8 0.9
17.6 1.2
52.5 4.7
113 12
26.9 1.9
119 21
87.5 7.2
4.18 0.43
4.09 0.31
4.77 0.37
7.82 0.56
13.6 1.2
8.20 0.72
8.92 0.65
10.0 0.9
8.70 0.53
1.97 0.12
9.33 0.77
10.2 1.0
8.45 0.62
13.5 1.1
230
228
159
262
246
257
262
223
232
180
222
210
228
290
52
96
64
68
46
102
101
118
107
102
24
120
35
77
0.63 0.04
8.37 0.67
5.30 0.42
*
The data represents mean s.d. from two independent measurements (n = 4).
The antagonist IC50 was obtained against a specific compound at a fixed dose: 1 (0.5
**
l
l
M), 4 (8
M), 39 (32 l
l
M), 8 (2
M).
l
M), 11 (2
lM), 13 (0.125
l
M), 14 (32
lM), 20 (4 lM), 24 (16 lM),
28 (4
lM), 29 (2
l
M), 30 (2
lM), 31 (2
l
M), 32 (2
lM), 34 (32
lM), 36 (2
lM), 37 (32
sigmoidal non-linear regression, leading to a single EC50 for each
compound (Fig. 3a, Table 1). Further, Tango antagonist assay
showed that ML-145 dose-dependently blocked the Tango signals
induced by compounds 13, 31, 36 or YE210, leading to an IC50 of
than those to result in DMR in the native HT-29 cells. The third
group includes compounds such as 34 and 37 which exhibited
higher efficacy to trigger DMR than those to induce Tango signal.
Compounds 30 and 36 exhibited the highest efficacy to cause b-
arrestin translocation.
0.085 0.007
lM, 1.30 0.10 lM, 0.45 0.03 lM, and 0.38
0.03 M, respectively (two independent measurements, n = 4)
l
Comparing their relative potency obtained using the two dis-
tinct assays showed that Tango assays generally gave rise to a
right-shifted potency, compared to DMR assay results (Fig. 4b).
Using YE210 as the reference, we found that most TTAC analogs
led to a less right-shifted potency obtained using Tango assay.
Compound 4 displayed a moderate potency but the least right-
shifted potency, compared to YE210. Compound 13 was the most
potent GPR35 agonist among all TTAC analogs tested.
(Fig. 3b). ML-145 is a potent GPR35 agonist.18 These assay results fur-
ther confirmed that all DMR-active compounds were GPR35 agonists.
Third, we performed correlation analysis between the results
obtained using DMR agonist assay in HT-29 and Tango assay in
the engineered U2OS cells. First, we compared their relative effi-
cacy, the maximal responses after normalized to the maximal re-
sponses of zaprinast obtained using both DMR and Tango assays.
Results showed that TTAC analogs displayed assay readout-depen-
dent efficacy (Fig. 4a). These compounds can be classified to three
categories using zaprinast as the reference compound. The first
group includes compounds 11, 14, 28 and 29 that gave rise to com-
parable efficacy between the two assay readouts. The second group
includes compounds such as 30, 32 and 36 that exhibited higher
efficacy to cause b-arrestin translocation in the engineered cells
Finally, we examined the ability of compound 30 to cause the
internalization of endogenous GPR35 in HT-29 cells using a previ-
ously reported protocol.13 Results showed that similar to zaprinast
at 10 lM, compound 30 at 12.5 lM caused significant internaliza-
tion of GPR35 in the native cell lines (Fig. 5). Together with DMR
and Tango assays, this result further confirmed that compound 30
is a potent GPR35 agonist.

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H. Deng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4148–4152
a
b
140
120
100
80
1000
36
30
28
29
100
10
32
14
Zaprinast
11
YE210
13
4
Zaprinast
60
1
40
37
34
0.1
20
0
0.01
0
20 40 60 80 100 120 140
% Zaprinast (DMR)
0.01 0.1
1
10
µ
100 1000
EC50 (DMR, M)
Figure 4. Correlation analyses between DMR and Tango assays. (a) The maximal responses of all agonists after normalized to the maximal response of zaprinast within the
same plate. (b) The correlation plot between EC50 values of all agonists obtained using DMR and Tango assays. The data represents mean s.d. from two independent
measurements, each in duplicate (n = 4).
Figure 5. Compound 30 triggered GPR35 internalization in HT-29 cells. Representative confocal fluorescence images of HT-29 under different conditions: (a) treated with the
assay vehicle containing 0.1% DMSO; (b) treated with 12.5 lM 30; (c) treated with 10 lM zaprinast. The images were obtained after compound treatment for 1hr,
permeabilized, stained with anti-GPR35, followed by fluorescent secondary antibody. Red: GPR35 stains; Blue: nuclei stains with DAPI. Representative images obtained from
2 independent measurements were used.
13. Deng, H.; Hu, H.; He, M.; Hu, J.; Niu, W.; Ferrie, A. M.; Fang, Y. J. Med. Chem.
In summary, we synthesized a series of TTAC analogs and found
that only compound 13 exhibited higher potency than YE210.
2011, 54, 7385.
14. Fang, Y.; Ferrie, A. M.; Fontaine, N. H.; Mauro, J.; Balakrishnan, J. Biophys. J.
However, a couple of analogs including 30 and 36 displays higher
efficacy than zaprinast to cause b-arrestin translocation. Further,
correlation analysis suggests that many ligands displayed biased
agonism. Future exploration of SAR may lead to the discovery of
TTAC analogs with further improved potency, and more impor-
tantly, compounds having b-arrestin biased agonism for GPR35.
Considering the importance of b-arrestin in regulating a diverse ar-
ray of cell signaling pathway downstream GPCRs, such a class of
GPR35 agonists may open possibility to further elucidate the biol-
ogy and physiology of GPR35.
2006, 91, 1925.
15. For DMR assays, human colorectal adenocarcinoma HT-29 cells (American
Type Cell Culture, Manassas, VA, USA) were seeded at a density of 32,000 cells
per well in EpicÒ 384well cell culture compatible microplates (Corning Inc.,
Corning, NY, USA), and cultured in McCoy’s 5a Medium Modified
supplemented with 10% fetal bovine serum, 4.5 g/liter glucose, 2 mM
glutamine, and antibiotics at 37 °C under air/5% CO2. After overnight culture,
the confluent cells were washed twice and maintained in the assay buffer (1Â
HBSS). After incubated inside EpicÒ system (Corning Inc.) for 1 h, a 2-min
baseline was established, compounds were then added using on-board liquid
handler and cellular responses were then monitored in real time. For
antagonist assays, the GPR35 antagonists used were pre-incubated for
10 min, and then the agonists examined were then added, and the agonist-
induced responses were then monitored. All DMR signals were background
corrected.
References and notes
16. For Tango assays, Tango™ GPR35-bla U2OS cells (Invitrogen) were seeded at a
density of 10,000 cells per well in 384-well, black-wall, clear bottom assay
plates with low fluorescence background (Corning), and cultured in Dulbecco’s
modified eagle medium (Invitrogen) supplemented with 10% dialyzed fetal
1. MacKenzie, A. E.; Lappin, J. E.; Taylor, D. L.; Nicklin, S. A.; Milligan, G. Front.
Endocrin. 2011, 2, e68.
2. Yang, Y.; Lu, J. Y. L.; Wu, X.; Summer, S.; Whoriskey, J.; Saris, C.; Reagan, J. D.
Pharmacology 2010, 86, 1.
3. Yang, Y.; Fu, A.; Wu, X.; Reagan, J. D. Pharmacology 2012, 89, 13.
4. Deng, H.; Hu, H.; Fang, Y. FEBS Lett. 2011, 585, 1957.
5. Deng, H.; Hu, H.; Ling, S.; Ferrie, A. M.; Fang, Y. ACS Med. Chem. Lett. 2012, 3,
165.
6. Deng, H.; Fang, Y. Pharmacology 2012, 89, 211.
7. Wang, J.; Simonavicius, N.; Wu, X.; Swaminath, G.; Reagan, J.; Tian, H.; Ling, L. J.
Biol. Chem. 2006, 281, 22021.
8. Galandrin, S.; Oligny-Longpre, G.; Bouvier, M. Trends Pharmacol. Sci. 2007, 8,
423.
9. Kenakin, T. Nat. Rev. Drug Disc. 2005, 4, 919.
10. Fang, Y.; Ferrie, A. M. FEBS Lett. 2008, 582, 558.
11. Fang, Y. Expert Opin. Drug Discov. 2011, 6, 1285.
12. Mailmana, R. B. Trends Pharmacol. Sci. 2007, 8, 390.
bovine serum, 0.1
ml penicillin, and 100
l
M non-essential amino acids, 25
lM Hepes (pH 7.3), 100 U/
lg/ml streptomycin. After overnight culture, the cells
were stimulated with ligands for 5 h in a humidified 37 °C/5% CO2, and then
loaded with the cell permeable LiveBLAzer™ FRET B/G substrate. After the 2 h
incubation the coumarin to fluorescein ratio was measured using Tecan Safire
II microplate reader (Männedorf, Switzerland). The coumarin to fluorescein
ratio was calculated, and all results obtained were then normalized to the
zaprinast maximal responses using an intra-plate referencing protocol. The
maximal response of zaprinast was set to be 100%.
17. Zhao, P.; Sharir, H.; Kapur, A.; Cowan, A.; Geller, E. B.; Adler, M. W.; Seltzman,
H. H.; Reggio, P. H.; Heynen-Genel, S.; Sauer, M.; Chung, T. D. Y.; Bai, Y.; Chen,
W.; Caron, M. G.; Barak, L. S.; Abood, M. E. Mol. Pharmacol. 2010, 78, 560.
18. Heynen-Genel, S.; Dahl, R.; Shi, S.; Sauer, M.; Hariharan, S.; Sergienko, E.; Dad,
S.; Chung, T.D.Y.; Stonich, D.; Su, Y.; Caron, M.; Zhao, P.; Abood, M.E.; Barak, L.S.
Probe Reports from the Molecular Libraries, Program 2010, NBK5070.