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

Article abstract of DOI:10.1021/jm200999f

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 EC₅₀ 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.

Full text of DOI:10.1021/jm200999f

ARTICLE
pubs.acs.org/jmc
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
Huayun Deng, Haibei Hu, Mingqian He, Jieyu Hu, Weijun Niu, Ann M. Ferrie, and Ye Fang*
Biochemical Technologies, Science and Technology Division, Corning Inc., Corning, New York 14831, United States
S Supporting Information
b
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 EC50 of
3
2.5 ( 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.
’
INTRODUCTION
assays could offer an unprecedented advantage to discover novel
19
ligands for receptors, particularly orphan receptors. We set
out to test such a hypothesis by screening an internally made
chemical library against endogenous GPR35 in a native cell line,
HT-29. The library consists of 660 intermediates synthesized in
house, and these intermediates were originally designed for
G protein-coupled receptor 35 (GPR35) is an orphan G
protein-coupled receptor (GPCR) and is believed to play a role
in hypertension, coronary artery disease, asthma, pain, early
onset inflammatory bowel disease, and cancers. Both kynurenic
acid and 2-acyl lysophosphatidic acid have been postulated
to be natural agonists for GPR35. However, there are only a
handful compounds that have been reported in literature to exhibit
either agonist or antagonist activity on GPR35. These compounds
include zaprinast, pamoic acid, NPPB (5-nitro-2-(3-phenyl-
propylamino)-benzoate), cromolyn and dicumarol, luteolin,
1
2
3
4,5
6
7
8
9
20
applications in nonlinear optics and organic field-effect tran-
21
sistors. The chemical library was chosen because (1) these
chemicals were not designed for pharmacological applications so
their pharmacological activities were unknown, and (2) some
chemicals contain malononitrile group, similar to tyrphostins.
Certain tyrphostin analogues have been demonstrated to be
1
0
4
1
1
3
12
quercetin, and niflumic acid, all of which are GPR35 agonists. A
couple of GPR35 antagonists including compound 17 (CID-
22
GPR35 agonists in our laboratory. Using DMR assays, we had
discovered two novel chemical series, 2-(4-methylfuran-2(5H)-
ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic
acid derivatives, as GPR35 agonists.
2
745687, methyl-5-[(tert-butylcarbamothioylhydrazinylidene)-
methyl]-1-(2,4-difluorophenyl)-pyrazole-4-carboxylate) have also
4
been reported. Thus, identification of novel GPR35 ligands would be
beneficial to further elucidate the biology and therapeutic potentials
of GPR35.
’
CHEMISTRY
-(4-Methylfuran-2(5H)-ylidene)malononitrile compounds
Conventional pharmacological assays often measure single
signaling molecules, one at a time, downstream receptor activa-
tion and signaling. Thus, these assays often encounter difficul-
ties to identify ligands for poorly characterized receptors such as
GPR35, whose signaling pathways and biology are largely
unknown. Recently, dynamic mass redistribution (DMR) assays
have been emerging as a powerful tool to delineate receptor
2
were synthesized via the reaction of α-ketols with 2 equiv of
malononitrile under basic conditions. This reaction starts with a
Knoevenagel condensation, followed by the hydroxyl group
attacking one of the nitriles to form an iminolactone intermedi-
ate, which reacts again with malononitrile to result in the final
products. The detailed synthesis of compounds 1 (YE120) and
13ꢀ17
biology and drug pharmacology in native cells.
DMR assays
20
are enabled by a label-free optical biosensor, which transforms a
cellular response into an integrated and kinetic response, termed
2
ꢀ8 have been reported previously. Followed the same
1
8
DMR. Because of its pathway-unbiased yet pathway-sensitive
nature in measurement, it has been hypothesized that DMR
Received: July 26, 2011
Published: September 27, 2011
r 2011 American Chemical Society
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Scheme 1
procedure, compound 9 was prepared from 3-hydroxy-3-methyl-
butan-2-one.
Thieno[3,2-b]thiophene-2-carboxylic acid derivatives were
synthesized using an aldehyde or ketone-based ring closure
21
reaction, followed by hydrolysis. Specifically, compounds 10,
1, 12, and 13 were synthesized through hydrolysis of the
1
products from a ring closure reaction of ethyl mercaptoacetate
with 3-bromothiophene-2-carbaldehyde, 1-(3-bromothiophen-
2
-yl)heptan-1-one, 3-bromo-4-hexylthiophene-2-carbaldehyde,
or 3-bromo-6-methylthieno[3,2-b]thiophene-2-carbaldehyde, re-
spectively. For compounds 16aꢀe, acyl-substituted 3,4-dibro-
mothiophene 14aꢀe was first synthesized through a Friedelꢀ
Crafts acylation of 3,4-dibromothiophene (14) with acyl chlor-
ide. The ketone 14aꢀe then underwent a ring closure reaction
with ethyl mercaptoacetate, leading to ethyl 3-alkyl-6-bromo-
thieno[3,2-b]thiophene-2-carboxylate 15aꢀe. After hydrolysis,
compounds 16aꢀe was obtained (Scheme 1). Purity of all the
final compounds was found to be greater than 95%, as measured
by reverse phase HPLC on a C18 column.
’
RESULTS AND DISCUSSION
Screening GPR35 Agonists with DMR Assays. Because
human colorectal adenocarcinoma cell line HT29 endogenously
22
expresses functional GPR35, this cell line was chosen to screen
GPR35 agonists. The known GPR35 agonist zaprinast was used
as a reference agonist. Previously, we have showed that zaprinast
resulted in a robust DMR with an EC of 0.16 ( 0.02 μM and
50
22
internalization of GPR35 in HT29. Compound 17, a known
GPR35 antagonist, did not lead to any detectable DMR in HT29,
but dose-dependently blocked the zaprinast DMR with an IC50
of 10.5 ( 0.3 μM (n = 4) (Figure1). At the highest dose (64 μM),
compound 17 almost completely blocked the zaprinast DMR.
These results suggest that the zaprinast-induced DMR in HT29
is specific to the activation of GPR35.
To screen ligands for GPR35, we used a two-step DMR
desensitization assay. This assay begins with DMR agonist assay
to examine all compounds for their agonist activity in HT29,
followed by DMR desensitization assay to determine the ability
of each compound to block or desensitize the cells responding
to the repeated stimulation with zaprinast at a saturating dose
Figure 1. DMR characteristics of known GPR35 ligands in
HT29. (a) Structure of a known GPR35 antagonist compound 17.
(
1 μM). Both agonist and desensitization assays were monitored
in real time for 1 h using Epic system. The Epic system is a label-free
optical biosensor microtiter plate reader tailored to 384-well
resonant waveguide grating biosensor plates. A cellular response
was recorded as a shift in resonant wavelength in picometer
(pm). The adoption of such a two-step sequential assay format is
based on that (1) DMR assays are noninvasive, thus enabling
(
b) The DMR arising from zaprinast (500 nM), the antagonist
compound 17 (32 μM), and zaprinast (500 nM) + compound 17
32 μM). (c) The dose-dependent blockage of the zaprinast
(
DMR by compound 17. The data represents mean ( sd (standard
deviation) from two independent measurements, each in duplicate
(
n = 4).
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Figure 2. Screening GPR35 agonists. (a) The DMR amplitudes of compounds in the library as a function of compounds. (b) The DMR signals induced
by zaprinast after the cells were prestimulated with compounds in the library. (c) The correlation between the compound DMR in native cells and the
zaprinast DMR in the compound-pretreated cells.
Figure 4. Structures of thieno[3,2-b]thiophene-2-carboxylic acid deri-
vatives identified as GPR35 agonists.
Figure 3. Structures of 2-(4-methylfuran-2(5H)-ylidene)malononitrile
derivatives identified as GPR35 agonists.
control as a basis for hit selection, we found 65 hits that led to a
zaprinast DMR smaller than 160 pm (Figure2b). The correlation
multiple step assays, and (2) the cells pretreated with an agonist
become desensitized to the repeated stimulation with a second
agonist when two agonists activate the same receptor. Such a
homologous desensitization is common to almost all GPCR
signaling. The real-time kinetic responses of all compounds were
collected and analyzed.
To identify hits, the amplitudes at 8 min poststimulation of all
compound-induced responses were determined and used as a
basis for hit identification in the agonist screen. This is because
zaprinast at a saturating dose (1 μM) led to a maximal response
peaked at about 8 min poststimulation (246 ( 11 pm, n = 32).
Using the compound DMR amplitudes greater than 30% of the
zaprinast response as the hit identification criteria, 55 hits in total
were identified (Figure2a).
For the desensitization screen, the amplitudes of the zaprinast
DMR during the desensitization step were determined for each
compound. As a positive control, zaprinast in the dimethyl
sulfoxide (DMSO) treated cells gave rise to a DMR with an
amplitude of 238 ( 25 pm (n = 90). Using 3ꢁ σ of the positive
analysis suggests that 53 hits were common between the two
assays (Figure2c).
23
Structure and activity analysis led to identification of 20
compounds that belong to two chemical series, 2-(4-methylfur-
an-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-
carboxylic acid derivatives. Eighteen of these compounds were
further characterized in detail (Figures 3 and 4), given that there
were sufficient amounts available for follow-up studies. The
remaining 33 compounds were either orphan in structure or
contain aldehyde or ketone groups, thus excluded in further
studies.
The Agonist Specificity of Compounds to GPR35. Given
that many compounds may exhibit polypharmacology and DMR
measurement is integrative in nature, a compound-induced
DMR may contain contributions from multiple targets/pathways
with which the compound intervene. Thus, we first examined the
specificity of the 18 compound-induced DMR using DMR
antagonist assays. Each compound was assayed at a dose close
to its respective EC or EC in the presence of compound 17.
80
100
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Table 1. Compounds, Their Efficacy in DMR Assays or Tango Assays Relative to Zaprinast, and Potency (EC50 to Trigger DMR,
IC to Desensitize Cells upon Repeated Stimulation with 1 μM Zaprinast, IC of the Known GPR35 Antagonist Compound 17 to
50
50
Block the Agonist-Induced DMR, and EC50 to Trigger β-Arrestin Translocation in Tango Assays)
DMR assays
Tango assays
compd
% zaprinast
EC50 (μM)
desensitization IC50 (μM)
Antagonist IC50 (μM)
% zaprinast
EC50 (μM)
zaprinast
100 ( 5
97 ( 6
84 ( 9
99 ( 4
90 ( 6
90 ( 5
100 ( 4
106 ( 9
99 ( 7
87 ( 5
81 ( 4
99 ( 4
103 ( 3
118 ( 3
105 ( 3
103 ( 7
87 ( 9
77 ( 7
81 ( 11
inactive
0.16 ( 0.02
0.032 ( 0.002
3.35 ( 0.25
3.50 ( 0.21
6.6 ( 0.5
1.22 ( 0.10
0.060 ( 0.007
4.48 ( 0.36
1.94 ( 0.23
21.6 ( 3.1
10.3 ( 0.9
6.4 ( 1.1
10.5 ( 0.3
12.8 ( 0.8
14.7 ( 1.2
20.9 ( 1.5
5.1 ( 0.7
11.4 ( 1.3
13.3 ( 0.9
22.8 ( 1.7
8.2 ( 1.1
7.9 ( 0.5
6.3 ( 0.6
27.6 ( 3.5
16.0 ( 2.1
18.2 ( 1.7
6.9 ( 0.5
25.8 ( 1.9
23.8 ( 2.4
24.8 ( 1.5
20.2 ( 1.4
NA
100 ( 7
29 ( 5
inactive
14 ( 5
ND
4.20 ( 0.25
10.2 ( 0.91
inactive
>50
1
2
3
4
5
6
7
8
9
ND
11.1 ( 1.7
15.3 ( 2.1
45.9 ( 4.9
53.9 ( 5.4
17.1 ( 2.2
42.4 ( 3.7
3.44 ( 0.17
9.4 ( 0.7
ND
ND
inactive
weak
inactive
>50
16.4 ( 1.5
48.9 ( 3.7
12.1 ( 1.6
57.0 ( 4.1
1.49 ( 0.12
4.69 ( 0.09
6.07 ( 0.14
0.11 ( 0.01
1.05 ( 0.07
0.57 ( 0.05
1.87 ( 0.14
5.92 ( 0.67
NA
inactive
weak
inactive
>50
1
1
1
1
1
1
1
1
1
1
0
inactive
20 ( 4
9 ( 3
inactive
>50
1
2
>50
3
6.0 ( 1.2
19 ( 4
104 ( 5
8 ( 3
>50
6a
6b
6c
6d
6e
5a
0.064 ( 0.004
1.46 ( 0.17
1.89 ( 0.23
2.31 ( 0.35
4.56 ( 0.41
NA
15.0 ( 1.2
>50
55 ( 6
inactive
inactive
inactive
22.7 ( 3.1
inactive
inactive
inactive
Results showed that compound 17 dose-dependently attenuated
the DMR arising from all 18 compounds with similar potency
Fluorescent confocal imaging showed that GPR35 in the un-
stimulated 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).
(Table 1). However, compound 17 at 64 μM blocked the DMR
induced by most compounds, except for compounds 5, 7, 8, 11,
and 13, whose DMR were only partially suppressed (Figures 5
and 6). Together with the observed ability of these compounds
to desensitize the cells to the zaprinast stimulation, these results
suggest that the DMR induced by these compounds are mostly
specific to GPR35, although we can not rule out the possibility of
compounds 5, 7, 8, 11, and 13 to activate endogenous target(s)
besides GPR35.
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,
We further examined the specificity of these ligands using
DMR agonist assays after interference RNA (RNAi) knockdown
of GPR35. Results showed that knockdown of GPR35 using
short hairpin RNA (shRNA) significantly suppressed the DMR
signals induced by the five compounds examined (Figure.7aꢀe).
Western blot confirmed the efficiency of ShRNA knockdown at
least by 60% (Figure 7f). Noticeably, distinct compounds gave
rise to different sensitivity to RNAi knockdown. This may be due
to the difference in efficacy of these compounds. It is known that
partial agonists are generally more sensitive to the reduction of
24,25
functional receptors than full agonists.
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.
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 transloca-
tion 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
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Figure 5. The DMR characteristics of 2-(4-methylfuran-2(5H)-ylidene)malononitrile derivatives. (aꢀi) Compound 1 (250 nM), 2 (8 μM), 3 (4 μM),
(8 μM), 5 (16 μM), 6 (16 μM), 7 (32 μM), 8 (32 μM), and 9 (32 μM), respectively. Each compound-induced DMR (Control) was compared to its
4
corresponding DMR in the presence of 32 μM 17, the known GPR35 antagonist (Antagonist). The data represents mean ( sd from two independent
measurements, each with four replicates (n = 8).
demonstrated that the two chemical series compounds are
GPR35 agonists.
the bromo group at C6 and replacing the methyl group with
hexyl group led to compound 11, which exhibited about 54-fold
decrease in potency (Table 1). Removal of both bromo and
methyl groups resulted in compound 10, which decreased its
potency by over 600-fold (Table 1). Replacing the methyl group
with a long-chained n-alkyl group resulted in compound 16bꢀe
and also reduced both potency and efficacy. Further, convert-
ing the backbone from thieno[3,2-b]thiophene into dithieno-
[3,2-b:2 ,3 -d]thiophene led to compound 13, which also exhibited
a lower potency. These results clearly reveal the importance of 3-
and 6-position groups, carboxylic acid, and the size of backbone
for its potency and agonist activity of thieno[3,2-b]thiophene-2-
carboxylic acid derivatives. Compound 16a is a full agonist in
both DMR and Tango assays, while 11, 12, 13, 16b, and 16c are
partial agonists in Tango assays.
Structure and Activity Analysis. To further examine struc-
ture and activity analysis, we determined the potency of all 18
compounds to trigger DMR signals in HT29 cells using DMR
assays. The results showed that all compounds gave rise to a clear
dose-dependent response (Figure10). All compounds led to a
saturable response at the dose ranges examined. However, there
are significant differences in potency, efficacy, and DMR char-
acteristics of these compounds (Table 1, Figures 5 and 6).
The DMR desensitization assays showed that all compounds
also dose-dependently desensitized the cells to the repeated
stimulation with 1 μM zaprinast, and the rank order of their
apparent IC50 values to desensitize the cells is similar to that of
their respective EC50 values (Figure11). This suggests that all
compounds activate GPR35 as zaprinast does. Of these, 2-(3-
cyano-5-(3,4-dichlorophenyl)-4,5-dimethylfuran-2(5H)-ylide-
ne)malononitrile (1, YE120) and 6-bromo-3-methylthieno[3,2-b]-
0
0
Next, we examined the structure and activity relationship of
2-(4-methylfuran-2(5H)-ylidene)malononitrile derivatives for
their agonist activity. Adding a chloro group at the 2-position of
the phenyl group of compound 1 leads to 5, which exhibited 350-
fold decrease in potency to trigger DMR signal and became
inactive in Tango assay. Replacing 3,4-dichlorophenyl group with
pentafluorophenyl group (2) or 2,4-bifluorophenyl group (8)
26
thiophene-2-carboxylic acid (16a, YE210) were the two most
potent agonists, and their respective EC50 was 32.5 ( 1.7 nM
(
n = 4) and 63.7 ( 4.1 nM (n = 4), both of which are more potent
than zaprinast (163 ( 19 nM, n = 4).
0
With the identification of potent compound 16a, we further
examined the functional groups critical to its agonist activity.
Changing the carboxylic acid to an ethyl carboxylate led to
compound 15a (Figure 8a). Compound 15a was found to be
inactive in HT-29 in DMR agonist assay (Figure 8b) and also
unable to trigger receptor internalization (Figure 8e). Removal of
or 4 -n-butylphenyl group (6) or methyl group (9) all led to
significant lost in its potency to trigger DMR as well as its efficacy
to trigger β-arrestin translocation. Replacing 3,4-dichlorophenyl
and methyl groups at the 5-position of its furan ring with phenyl
and spiro groups leads to compound 3, which exhibited lower
potency in both DMR and Tango assays. These results suggest
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Figure 6. The DMR characteristics of thieno[3,2-b]thiophene-2-carboxylic acid derivatives. (aꢀi) Compound 10 (32 μM), 11 (16 μM), 12 (16 μM),
3 (16 μM), 16a (250 nM), 16b (8 μM), 16c (8 μM), 16d (8 μM), and 16e (8 μM), respectively. Each compound induced DMR (Control) was
1
compared to its corresponding DMR in the presence of 32 μM compound 17, the known GPR35 antagonist (Antagonist). The data represents mean (
sd from two independent measurements, each with four replicates (n = 8).
Figure 7. shRNA knockdown of GPR35 significantly attenuated the DMR induced by GPR35 agonists identified. (a) Zaprinast, (b) 1, (c) 9, (d) 16a,
and (e) 13. All compounds were assayed at 10 μM. The data represents mean ( sd from four replicates (n = 4) for all compounds. The mock transfection
was used the positive control. (f) Western blotting showed the knockdown of GPR35 proteins by the shRNA.
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Figure 8. 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). (cꢀe) 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.
Figure 9. 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).
that the functional groups at the 5-position of its furan ring is
critical to the agonist activity of compound 1.
Finally, we examined the ligand-directed functional selectivity
found to be active in both DMR and Tango assays, with an EC50
of 0.16 and 1.47 μM, respectively. Compared to zaprinast, both 1
and 16a are more potent to trigger DMR signal (EC50 of 0.032
and 0.064 μM, respectively) but less potent to result in β-arrestin
of GPR35 agonists. The known GPR35 agonist zaprinast were
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Figure 10. Representative DMR signals of GPR35 agonists identified. (a) The dose-dependent DMR induced by 1, (b) the dose-dependent DMR
induced by 16a, (c) the amplitudes of the DMR induced by 2-(4-methylfuran-2(5H)-ylidene)malononitrile derivatives as a function of doses, and (d)
the amplitudes of the DMR induced by thieno[3,2-b]thiophene-2-carboxylic acid derivatives as a function of doses.
Figure 11. The dose-dependent desensitization by GPR35 agonists identified to the repeated stimulation with zaprinast (1 μM). (a,b) Real-time
zaprinast DMR after pretreatment with 1 (a) and 2 (b), respectively, and (c) and (d) dose-dependent desensitization by thieno[3,2-b]thiophene-2-
carboxylic acid derivatives (c) and thieno[3,2-b]thiophene-2-carboxylic acid derivatives (d), respectively.
translocation (EC50 of 10.2 and 15.0 μM, respectively). Further,
compared to zaprinast, 16a is also a full agonist in both DMR and
Tango assays, but 1 is only a full agonist to trigger DMR but a
partial agonist to result in β-arrestin translocation. Such an assay
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Journal of Medicinal Chemistry
ARTICLE
readout-dependent potency and efficacy suggests that different
GPR35 agonists display ligand-directed functional selectivity.
NMR and mass spectra of other compounds were included in Support-
ing Information.
27
2
-(3-Cyano-4,5,5-trimethylfuran-2(5H)-ylidene)malononitrile (9).
3
-Hydroxy-3-methylbutan-2-one (1.0 equiv), malononitrile (3.2 equiv),
’
CONCLUSIONS
We have screened an internally made compound library
and magnesium ethoxide (0.01 equiv) in ethanol were mixed and re-
fluxed overnight. The solution was concentrated by removing the
majority of the THF on a rotary evaporator under aspirator vacuum.
The remaining residue was taken up in methylene chloride and
washed with brine (2 times) and then DI water (2 times). The organic
layer was dried over anhydrous MgSO and filtered and the solvent
4
removed. The crude product was recrystallized from denatured alcohol
to afford 9. Yield: 29%. H NMR (300 MHz, acetone-d ): δ 2.36
(s, 3H), 1.63 (s, 6H). HRMS (EI) for C H N O: calcd, 199.07; found,
199.07 [M].
Thieno[3,2-b]thiophene-2-carboxylic Acid (10). 3-Bromothio-
phene-2-carbaldehyde (1.0 equiv) was mixed with K CO (4.0 equiv)
in dimethylformamide (DMF) in a three-neck flask equipped with
a condenser and addition funnel. After ethyl mercaptoacetate (1.0
equiv) was added dropwise at 60ꢀ70 °C, the mixture was heated
overnight until no starting materials were detected by GC/MS.
The mixture then was poured into water and the product was
extracted by diethyl ether. The organic phase was washed with brine
against a poorly characterized orphan receptor GPR35 using
DMR assays and have identified two chemical series, 2-(4-methyl-
furan-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-
carboxylic acid derivatives, which represent novel classes of
GPR35 agonists. The agonist activity of 18 compounds was
found to be specific to GPR35 based on DMR antagonist assays
using a known GPR35 antagonist compound 17, DMR desensi-
tization assays against zaprinast, receptor internalization, and
β-arrestin translocation assays. The structureꢀactivity relation-
ships of these ligands as GPR35 agonists have been also explored.
Notably, the carboxylic acid group of thieno[3,2-b]thiophene-2-
carboxylic acid derivatives was found to be critical to activate
GPR35. Because malononitrile is relatively acidic under physio-
logical conditions, it may also be important to the agonist activity
of 2-(4-methylfuran-2(5H)-ylidene)malononitrile derivatives. This
study expands current pharmacological tools to characterize the
biology and functions of GPR35 and presents a good starting
point to design and optimize new GPR35 ligands.
1
6
1
1
9 3
2
3
4
and dried over anhydrous MgSO . The resultant brownish crude ethyl
thieno[3,2-b]thiophene-2-carboxylate was obtained and found to be pure
enough for the next reaction. After dissolved in a mixture of THF
and methanol (1:1) in the presence of 1 M LiOH and refluxed over-
night, the mixture was poured into concentrated hydrochloric acid.
The acid mixture was then diluted with water. Solid was filtrated,
washed with water and methanol, and purified with flash column
’
EXPERIMENTAL SECTION
Chemistry. All reactions were performed in reagent grade solvent.
The progress of the reactions was checked by GC/MS (Agilent 7820A
GC/5875 MS) and analytical thin-layer chromatography (TLC). Plates
were visualized first with UV illumination, followed by charring with
phosphomolybdic acid. Flash column chromatography was performed
chromatography to provide the light-yellow solid of compound 10
1
(
(
89% overall yield). H NMR (300 MHz, acetone-d ): δ 8.12
s, 1H), 7.88 (d, 1H), 7.48 (d, 1H). HRMS (EI) for C H O S :
7 4 2 2
6
1
calcd, 183.96; found, 138.97 [M ꢀ COOH].
-Hexylthieno[3,2-b]thiophene-2-carboxylic Acid (11). 1-(3-Bro-
mothiophen-2-yl)heptan-1-one (1.0 equiv) was mixed with K CO3
using silica gel (200ꢀ300 mesh). H NMR spectra were recorded by
3
Bruker-300 MHz NMR spectrometers in the indicated deuterated
2
solvents and are reported in parts per million (ppm) on the δ scale
(4.0 equiv) in DMF in a three-neck flask equipped with a condenser
relative to residual CD
acetone-d (δ 2.05). Splitting patterns are provided as apparent multi-
plicities: s (singlet), d (doublet), t (triplet), m (multiplet), and br
2 2
Cl (δ 5.32), THF-D7H (δ 3.58, 1.73), and
and addition funnel. After ethyl mercaptoacetate (1.0 equiv) was added
dropwise at 60ꢀ70 °C, the mixture was heated at 60ꢀ70 °C overnight
until no starting materials was detected by GC/MS. The mixture was
then poured into water and extracted by diethyl ether. The organic phase
6
13
(
broad). C NMR spectra were recorded in the indicated deuterated
solvents by Bruker-75 MHz and are reported in parts per million (ppm)
on the δ scale relative to residual CD Cl (δ 53.8) and DMSO-d
δ 39.5). High-resolution mass spectra (HRMS) were recorded on an
4
was washed with brine and dried over anhydrous MgSO . The residue
2
2
6
was the brownish crude ethyl 3-hexylthieno[3,2-b]thiophene-2-carbox-
ylate, which was directly dissolved into a mixture of THF and methanol
containing 1 M LiOH and refluxed overnight. The mixture was poured
into concentrated hydrochloric acid. The acid mixture was then diluted
with water. Solid was filtrated, washed with water and then methanol, and
purified by flash column chromatograph to provide the light-yellow solid
(
Agilent 1100 mass spectrometer using Matrix-assisted laser desorption
ionization (MALDI) or electron ionization (EI). We chose the negative
ion mode of operation for EI mass spectroscopy characterization of
fused thiophene molecules containing an acidic side group, which
resulted in the generation of the deprotonated molecular ion from these
molecules. The purity determination of all reported compounds was
performed with an Agilent 1100 equipped with Waters columns
1
of compound 11(83% overall yield). H NMR (300 MHz, TDF): δ 11.48
(
0
br, 1H), 7.68 (d, 1H), 7.31 (d, 1H), 3.19 (t, 2H), 1.42ꢀ1.31 (m, 8H),
.89 (t, 3H). The small peaks at 3.73 (s), and 2.80 (t) indicated that
(
5
H
2
Atlantis T3, 2.1 mm ꢁ 50 mm, 3 μm; or Atlantis dC18, 2.1 mm ꢁ
0 mm, 5 μm) eluted for >10 min with a gradient mixture of
Oꢀacetonitrile with formic or trifluoroacetic acid at wavelengths of
20, 254, and 280 nm. All compounds analyzed were >95% pure.
1
3
compound 11 was contaminated with 3ꢀ6% byproduct. C NMR (75
MHz, CD Cl ): 145.6, 142.4, 132.3, 126.9, 120.5, 32.0, 29.9, 29.7, 23.0,
4.2. HRMS(EI) for C H O S : calcd, 268.06; found, 267.13 [Mꢀ H].
2
2
2
1
1
3 16 2 2
6
-Hexylthieno[3,2-b]thiophene-2-carboxylic Acid (12). 3-Bromo-4-
hexylthiophene-2-carbaldehyde (1.0 equiv) was mixed with K CO
1.5 equiv) in DMF in a three-neck flask equipped with a condenser and
Synthesis and characterization of compound 1 (2-(3-cyano-5-(3,4-
dichlorophenyl)-4,5-dimethylfuran-2(5H)-ylidene)malononitrile, 2 2-
2
3
(
(3-cyano-4,5-dimethyl-5-(perfluorophenyl)furan-2(5H)-ylidene)ma lo-
an addition funnel. After ethyl mercaptoacetate (0.5 equiv) was added
dropwise at room temperature, the mixture was stirred at room tempera-
ture overnight until no starting materials were detected by GC/MS. The
mixture was then poured into water and extracted by ethyl acetate.
nonitrile), 3 (2-(3-cyano-4-methyl-5-phenyl-5-(trifluoromethyl)furan-
0 0 0
(5H)-ylidene)malononitrile), 4 (2-(4 -cyano-3 -methyl-5 H-spiro-
2
0
0
[
fluorene-9,2 -furan]-5 -ylidene)malononitrile), 5 (2-(3-cyano-4,5-di-
methyl-5-(2,3,4-trichlorophenyl)furan-2(5H)-ylidene)malononitrile), 6
2-(5-(4-butylphenyl)-3-cyano-4,5-dimethylfuran-2(5H)-ylidene)malono-
(
4
Organic extracts were washed by brine and dried over MgSO to yield
nitrile), 7 (2-(3-cyano-4-methyl-1-oxaspiro[4.5]dec-3-en-2-ylidene)-
malononitrile), and 8 (2-(3-cyano-5-(2,4-difluorophenyl)-4,5-di-
methylfuran-2(5H)-ylidene)malononitrile) were reported previously.
the brownish crude ethyl 6-hexylthieno[3,2-b]thiophene-2-carboxy-
late, which was then dissolved into a mixture of THF and 1 M LiOH.
This mixture was refluxed overnight and poured into concentrated
2
0
7
393
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Journal of Medicinal Chemistry
ARTICLE
1
hydrochloric acid. The acid mixture was then diluted with water. Solid
was filtrated and washed with water. The residue was recrystallized from
hexane to provide the light-yellow solid of 12 (46% overall yield). H
(s, 3H). H NMR (300 MHz, DMSO-d
6
): δ 8.07 (s, 1H), 2.60 (s, 3H).
1
3
C NMR (75 MHz, DMSO-d
102.1, 14.2. HRMS (EI) for C
M ꢀ H], 276.92 [M ꢀ H + 2], due to the presence of two isotopes of
bromide.
-Bromo-3-octylthieno[3,2-b]thiophene-2-carboxylic Acid (16b).
): 163.6, 140.7, 140.1, 137.7, 129.7, 129.2,
6
1
8 5
H
BrO : calcd, 275.89; found, 275.06
2 2
S
[
NMR (300 MHz, CD
.72ꢀ1.68 (m, 2H), 1.42ꢀ1.36 (m, 6H), 0.88 (t, 3H). HRMS (EI) for
C H O S : calcd, 268.06; found, 267.13 [M ꢀ H].
2 2
Cl ): δ 8.10 (s, 1H), 7.50 (s, 1H), 2.78 (t, 2H),
1
6
1
3 16 2 2
1
0
0
Overall yield: 86%. H NMR (300 MHz, TDF): δ 11.67 (br, 1H),
.79 (s, 1H), 3.18 (t, 2H), 1.45ꢀ1.33 (m, 12H), 0.92 (t, 3H). HRMS
EI) for C H BrO S : calcd, 374.00; found, 373.07 [M ꢀ H], 375.00
5
-Methyldithieno[3,2-b:2 ,3 -d]thiophene-2-carboxylic Acid (13). 3-
Bromo-6-methylthieno[3,2-b]thiophene-2-carbaldehyde (1.0 equiv) was
reacted with ethyl mercaptoacetate (1.0 equiv) in the presence of K CO
4.0 equiv) in DMF to provide the yellow solid ethyl 5-methyldithieno-
7
(
15 19
2 2
2
3
[
M ꢀ H + 2]
(
[
0 0
3,2-b:2 ,3 -d]thiophene-2-carboxylate, which was then (0.16 mol) dis-
6-Bromo-3-undecylthieno[3,2-b]thiophene-2-carboxylic Acid (16c).
1
Overall yield: 56%. H NMR (300 MHz, TDF): δ 11.61 (br, 1H), 7.73 (s,
H), 3.18 (t, 2H), 1.35ꢀ1.28 (m, 16H), 0.88 (t, 3H). HRMS (EI) for
25BrO
: calcd, 416.05; found, 415.15 [M ꢀ H], 417.01[M ꢀ H+2].
6-Bromo-3-tridecylthieno[3,2-b]thiophene-2-carboxylic Acid (16d).
solved in LiOH (10% in water), THF, and methanol (2:6:1). The mixture
wasrefluxedovernightandpouredintoconcentratedhydrochloricacid. The
residue was purified by flash column chromatography to provide the light-
1
18
C H
2 2
S
1
yellow powder 13 (73% overall yield); mp 289ꢀ290 °C. H NMR (300
1
1
Overall yield: 46%. H NMR (300 MHz, CD Cl ): δ 7.55 (s, 1H), 3.17 (t,
2 2
MHz, DMSO-d
6
):δ8.19 (s, 1H), 7.49 (s, 1H), 2.55 (s, 3H). HNMR(300
1
13
2H), 1.78ꢀ1.75 (t, 2H), 1.42ꢀ1.28 (m, 22H), 0.87 (t, 3H). H NMR
MHz, TDF): δ 8.04 (s, 1H), 7.25 (s, 1H), 2.37 (s, 3H). C NMR (75
(
(
300 MHz, TDF): δ 11.58 (br, 1H), 7.72 (s, 1H), 3.18 (t, 2H), 1.35ꢀ1.22
MHz, DMSO-d ): 163.1, 145.3, 140.0, 135.2, 134.1, 130.5, 129.2, 14.1.
6
m, 22H), 0.88 (t, 3H). HRMS (EI) for C20 29BrO : calcd, 444.08;
H
2 2
S
HRMS (EI) for C10
H
6
O
2
S
3
: calcd, 253.95; found, 253.04 [M ꢀ H].
found, 443.09 [M ꢀ H], 445.02 [M ꢀ H + 2]
General Procedure for the Synthesis of 3-Alkyl-thieno[3,2-b]thio-
phene-2-carboxylic Acid (16aꢀe). These compounds were synthesized
using the protocol shown in Scheme 1.
6
-Bromo-3-pentadecylthieno[3,2-b]thiophene-2-carboxylic Acid
1
(
1
16e). Overall yield 59%. H NMR (300 MHz, TDF): δ 11.63 (br,
H), 7.72 (s, 1H), 3.18 (t, 2H), 1.35ꢀ1.22 (m, 25H), 0.88 (t, 4H).
HRMS (EI) for C H BrO S : calcd, 472.11; found, 471.27 [M ꢀ H],
To a mixture of 3,4-dibromothiophene 14 (1.0 equiv) and AlCl
2.0 equiv) in CH Cl
3
(
2
2
at 0 °C, acetyl chloride (1.0 equiv) was added dropwise
22 33
2 2
4
73.13 [M ꢀ H + 2].
under a nitrogen stream. This mixture was stirred for 2ꢀ3 h until no
Compounds and Reagents. Zaprinast was obtained from Enzo
starting materials could be detected by GC/MS. The mixture was then
Life Sciences (Plymouth Meeting, PA). Compound 17 was obtained
from Ryan Scientific, Inc. (Mt. Pleasant, SC). Epic 384-well biosensor
microplates were obtained from Corning Inc. (Corning, NY, USA).
Cell Culture. Human colorectal adenocarcinoma HT-29 was ob-
tained from American Type Cell Culture (Manassas, VA, USA). The
cells were cultured in McCoy’s 5a Medium Modified supplemented with
10% fetal bovine serum, 4.5 g/L glucose, 2 mM glutamine, and anti-
2 2
poured into 6 M HCl and the organic was extracted with CH Cl and
dried over anhydrous MgSO . The mixture was then poured into 6 M HCl
4
and the organic was extracted with CH Cl and dried over anhydrous
2
2
4
MgSO . Evaporation of the solvent gave the crude acetyl-substituted 3,4-
dibromothiophene 14aꢀe.
The crude acetyl-substituted 3,4-dibromothiophene (14aꢀe)
(
2 3
1.0 equiv) was mixed with K CO (5.0 equiv) and DMF in a three-
neck flask equipped with a condenser and addition funnel. After ethyl
mercaptoacetate (1.0 equiv) was added dropwise at 60ꢀ70 °C, a catalytic
amount of 18-crown-6 was added. The mixture was heated at 60ꢀ70 °C
overnight until no starting materials were detected by GC/MS. The mixture
was then poured into water.
biotics at 37 °C under air/5% CO . Tango GPR35-bla U2OS cells were
2
purchased from Invitrogen. The cells were cultured according to the
protocols recommended by the supplier. Briefly, the cells were passed
using McCoy’s 5A medium (Invitrogen 16600ꢀ082) supplemented
with 10% dialyzed fetal bovine serum, 0.1 μM NEAA, 25 μM Hepes
(pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL
streptomycin, 200 μg/mL zeocin, 50 μg/mL hygromycin, and
Solid was filtrated, washed with water and then methanol, and
afforded crude ethyl 3-alkyl-6-bromothieno[3,2-b]thiophene-2-carbox-
ylate 15aꢀe.
100 μg/mL geneticin in a humidified 37 °C/5% CO incubator.
2
Ethyl 3-alkyl-6-bromothieno[3,2-b]thiophene-2-carboxylate (15aꢀe)
DMR Assays Using Epic System. All DMR assays were per-
formed using Epic system (Corning Inc., Corning, NY). Epic is a
wavelength interrogation reader system tailored for resonant waveguide
grating biosensors in microtiter plates. This system consists of a
temperature-control unit (26 °C), an optical detection unit, and an
on-board liquid handling unit with robotics. The detection unit is
centered on integrated fiber optics and enables kinetic measures of
cellular responses with a time interval of ∼15 s. Cells were directly
seeded in Epic plates and cultured overnight to form a confluent
monolayer in the cell culture medium. After being washed twice, the
cells were maintained with Hank’s Balanced Salt Solution and further
incubated inside the system for 1 h. For agonist screen, a 2 min baseline
was then established. Immediately after the compound addition using
the onboard liquid handler, the cellular responses were recorded. For
desensitization assays, cells were initially treated with compounds for
1 h, followed by stimulation with zaprinast at a fixed dose. The cellular
responses were recorded throughout the assays. All EC50 or IC50
described in the main text were calculated based on the amplitudes of
DMR signals at 8 min post agonist stimulation. Because all GPR35
agonists led to a sustained positive-DMR (P-DMR) signal, the ampli-
tudes at 50 min post stimulation were also used to determine kinetics
dependent potency and efficacy of all ligands.
(
(
0.16 mol) dissolved in LiOH (10% in water), THF, and methanol
2:6:1). The mixture was refluxed overnight until no starting materials
were detected by TLC. THF was evaporated, and the residue was acidified
with 6N HCl to afford 3-alkyl-thieno[3,2-b]thiophene-2-carboxylic acid
1
6aꢀe.
1
-(3,4-Dibromothiophen-2-yl)ethanone (14a). Crude yields: 95%
1
mp 75ꢀ78 °C. H NMR (300 MHz, CD
2
Cl
2
): δ 7.67 (s, 1H), 2.69 (s,
13
3
2
H). C NMR (75 MHz, CD Cl ): 189.6, 140.6, 130.2, 118.0, 117.3,
9.7. HRMS (EI) for C
and 282.91 [M ꢀ H + 2]. The dual molecular ion species are chara-
cteristic of a single bromide due to the presence of two isotopes of
bromide.
2
2
6
H
4
Br
2
OS: calcd, 281.83 found, 280.97 [M ꢀ H]
Ethyl 6-Bromo-3-methylthieno[3,2-b]thiophene-2-carboxylate
15a). 15a was purified with flash column chromatography. Yields: 95%
(
1
mp 91ꢀ92 °C. H NMR (300 MHz, CD Cl ): δ 7.48 (s, 1H), 4.36 (q,
2
2
13
2H), 2.63 (s, 3H), 1.38 (t, 3H). C NMR (75 MHz, CD Cl ): 163.2,
2 2
1
C
[
41.7, 141.4, 138.9, 129.5, 127.9, 103.5, 61.6, 14.9. HRMS (EI) for
BrO
: calcd, 303.92; found, 303.20 [M ꢀ H] and 305.13
M ꢀ H + 2].
-Bromo-3-methylthieno[3,2-b]thiophene-2-carboxylic Acid (16a).
10
H
7
2 2
S
6
1
Overall yield: 86%. mp 280ꢀ282 °C. H NMR (300 MHz, TDF): δ 7.68
1
2 2
(s, 1H), 2.69 (s, 3H). H NMR (300 MHz, CD Cl ): δ 7.67 (s, 1H), 2.69
7
394
dx.doi.org/10.1021/jm200999f |J. Med. Chem. 2011, 54, 7385–7396

Journal of Medicinal Chemistry
ARTICLE
For the RNAi knockdown of GPR35, HT29 cells were seeded on Epic
plates at 5000 cells/well and allowed to incubate at 37 °C for 24 h (day 1).
On day 2, cells were washed with fresh growth media of HT29 once,
then leave cells with 30 μL/well of growth media (McCoy’s 5a media,
enhanced chemiluminescence detection with ECL plus (GE Healthcare
Life Science). Equivalent gel loading was confirmed by probing with β-
actin antibody (Santa Cruz).
Tango β-Arrestin Translocation Gene Reporter Assays.
Tango GPR35-bla U2OS cells was used. This cell line stably expresses
two fusion proteins: human GPR35 linked to a TEV protease site and a
Gal4-VP16 transcription factor 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 proteins to the activated GPR35. As a result, the protease cleaves
the Gal4-VP16 transcription factor from the receptor, which then
translocates to the nucleus and activates the expression of β-lactamase.
Briefly, 10000 cells per well were seeded in 384-well, black-wall, clear-
bottom assay plates with low fluorescence background (Corning)
and cultured in Dulbecco’s Modified Eagle Medium (Invitrogen,
10569ꢀ010) supplemented with 10% dialyzed fetal bovine serum,
0.1 μM nonessential amino acids, 25 μM Hepes (pH 7.3), 100 U/mL
penicillin, and 100 μg/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:fluorescein ratio
was measured using Tecan Safire II microplate reader (M €a nnedorf,
Switzerland). In the absence of β-lactamase expression (i.e., no GPR35
activation), cells generated green fluorescence. In the presence of β-
lactamase expression upon receptor activation, the substrate was cleaved
and the cells generated blue fluorescence. The coumarin:fluorescein
ratio was used as a normalized reporter response.
10ꢁ PBS, 1ꢁ Pen/Strap, all from Invitrogen, San Diego, CA, USA).
Short hairpin RNA (shRNA) constructs for targeting human GPR35
mRNA were obtained from Origene (Rockville, MD, USA) and are in
pGFP-V-RS vector which express green fluorescent protein (GFP) for
easy determination of transfection efficiency. Transient transfection of
shRNA plasmids and control plasmids was performed using Effectene
transfection system from Qiagen according to manufacturer’s instruc-
tions (Qiagen, Valencia, CA, USA). Briefly, 0.05 μg of plasmid DNA
were mixed with Enhancer and Buffer EC for 20 min at room
temperature followed by the addition of diluted Effectene solution.
The final mixture were incubated at room temperature for 30 min before
being dispensed to each 384-well for 20 μL/well. The Epic plates
containing HT29 cells and shRNA constructs in transfection reagents
were centrifuged at 500 rpm for 1 min and put back in 37 °C for
incubation. On day 3, media of HT29 cells were replaced with fresh
growth media at 40 μL/well and cells were allowed to incubate for
another 24 h. On day 4, about 48 h post-transfection, HT29 cells were
subjected to DMR assays in agonist mode as described above. Zaprinast
was obtained from Tocris Chemical Co. (St. Louis, MO, USA). All
compounds were stocked in DMSO at 10 mM and were diluted directly
in the assay buffer (1ꢁ Hanks’ Balanced Salt Buffer, 20 mM Hepes, pH
7.1; HBSS) to the indicated concentrations. Epic 384-well biosensor
microplates were obtained from Corning Inc.
Receptor Internalization Assays. HT29 cells were plated on an
8
-well chamber slide (Nalge Nunc International, Rochester, NY, USA)
with a seeding density of 10000 cells per well and incubated at 37 °C for
4 h. Next day, cells were stimulated with a compound or equal amount
’
ASSOCIATED CONTENT
2
of DMSO at 37 °C for 1 h. Afterward, cells were fixed with 4%
formaldehyde in 1ꢁ PBS for 15 min, followed by blocking and
permeabilization in a buffer containing 4% goat serum, 0.1% bovine
serum albumin (BSA), 0.1% Triton X100 in 1ꢁ PBS for 2 h. After 5 min
wash with PBS, fixed cells were incubated with the anti-GPR35 (1:500)
S
b
Supporting Information. Characterization of compounds
10, 11, 12, 13, 15a, 16a (YE210), 16b, 16c, 16d, and 16e,
respectively, using NMR and mass spectroscopy. This material is
available free of charge via the Internet at http://pubs.acs.org.
(
anti-GPR35, T-14, intracellular domain) (Santa Cruz Biotechnology,
’
AUTHOR INFORMATION
Santa Cruz, CA, USA) in 3% BSA/PBS buffer for 24 h, followed by
incubation with secondary antibody Alexa Fluor 594 donkey antigoat
IgG (H + L) (1:500) (Invitrogen) in 3% BSA/PBS for 1 h at room
temperature. Cells were finally washed once with PBS and sealed with
1
4
Corresponding Author
*
Phone: +1-607-9747203. E-mail: fangy2@corning.com.
ABBREVIATIONS USED
.5 mm thick glass coverslip (Corning, NY). Dried slides were stored at
°C until imaging. Confocal imaging was performed with Zeiss confocal
’
microscope Axiovert 40. The collected images were analyzed using
MacBiophotonics Image J software (http://www.macbiophotonics.ca/
downloads.htm).
GPR35, G protein-coupled receptor 35; GPCR, G protein-
coupled receptor; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate;
DMR, dynamic mass redistribution; RNAi, interference RNA; shRNA,
short hairpin RNA; FRET, fluorescence resonance energy transfer;
GC/MS, gas chromatographyꢀmass spectroscopy; TLC, thin-layer
chromatography; MALDI, matrix-assisted laser deposition ionization;
EI, electron ionization; HRMS, high-resolution mass spectra; GFP,
green fluorescent protein
Western Blotting. Transfection of shRNA constructs were per-
formed in 6-well tissue culture treated plate (Corning) with Effectene
transfection system according to manufacturer’s instructions (Qiagen).
Two days following transfection, cells were washed with PBS once and
lysed with NP40 lysis buffer (Invitrogen) containing 1ꢁ protease
inhibitor cocktail (Fisher Scientific) and the whole cell lysate was
harvested and centrifuged at 20000 rpm for 20 min at 4 °C. Supernatant
’
REFERENCES
(cytoplasmic fraction) of the cell lysate was collected and subjected to
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