High content screening identifies small molecules affecting GPCR activation and internalization

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Journal of Biomolecular Screening
http://jbx.sagepub.com/content/early/2011/11/15/1087057111427347
The online version of this article can be found at:
DOI: 10.1177/1087057111427347
published online 15 November 2011J Biomol Screen
Yong-Jun Kwon, Weontae Lee, Auguste Genovesio and Neil Emans
A High-Content Subtractive Screen for Selecting Small Molecules Affecting Internalization of GPCRs
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2. Journal of Biomolecular Screening
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© 2011 Society for Laboratory
Automation and Screening
DOI: 10.1177/1087057111427347
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Introduction
Phenotypic screens exploit automated cell imaging to identify
small molecules that can alter cellular protein localization and
are of increasing interest as high-throughput and forward
genetic screens for drug and pathway component identifica-
tion.1
In chemical genetics, small molecules active toward
cell-based assays are identified through cell-based screen-
ing with little or no a priori information on their targets.
Identifying these targets remains a challenge,2,3
as does
reducingthenumberofcandidatestobetested.Computational
methods have been developed to define classes of active
molecules relative to their activity toward single or poten-
tially multiple targets and their structure,4
but a cell-based
approach would exploit the assays themselves to select
promising candidates.
We present such an alternative: an experimental method
to select compounds specifically affecting activation of spe-
cific G-protein–coupled receptors (GPCRs). Using a panel
of fluorescent GPCR fusion proteins and a collection of
small-molecule inhibitors of cellular kinase and phospha-
tase activity, we constructed a selective exclusion screen.
This was designed to identify two classes of compounds:
those affecting housekeeping endocytic machinery, or
multiple GPCRs (pan-specific), and those affecting only
one (GPCR specific) in the same cellular genetic back-
ground. The screen was based on measurement of
agonist-induced receptor internalization using high-content
screening and cellular image analysis.
427347JBXXXX10.1177/1087057111427347Kwon et al.Journal of Biomolecular Screening
1
Institut Pasteur-Korea, Gyeonggi-do, Korea
2
Department of Biochemistry, College of Science,Yonsei University,
Seoul, South Korea
3
High Throughput Biology Group, Synthetic Biology Emerging Research
Area, CSIR, Pretoria, South Africa
Received June 20, 2011, and in revised form Sep 28, 2011. Accepted for
publication Sep 29, 2011.
Supplementary material for this article is available on the Journal of Biomo-
lecular Screening Web site at http://jbx.sagepub.com/supplemental./
Abbreviations: A1AR, adenosine receptor 1; A3AR, adenosine receptor
3; ETAR, endothelin A receptor; Et-1, endothelin-1; kOPr, kappa opioid
receptor; muOPr, µ-opioid receptor; M2AR, muscarinic acetylcholine
receptor 2; M5AR, muscarinic acetylcholine receptor.
Corresponding Author:
Neil Emans, High Throughput Biology Group, Synthetic Biology Emerging
Research Area, CSIR, Pretoria 0001, South Africa
Email: neilemans@gmail.com
A High-Content Subtractive Screen
for Selecting Small Molecules Affecting
Internalization of GPCRs
Yong-Jun Kwon1,2
, Weontae Lee2
, Auguste Genovesio1
,
and Neil Emans3
Abstract
G-protein–coupled receptors (GPCRs) are pivotal in cellular responses to the environment and are common drug targets.
Identification of selective small molecules acting on single GPCRs is complicated by the shared machinery coupling signal
transduction to physiology. Here, we demonstrate a high-content screen using a panel of GPCR assays to identify receptor
selective molecules acting within the kinase/phosphatase inhibitor family. A collection of 88 kinase and phosphatase inhibitors
was screened against seven agonist-induced GPCR internalization cell models as well as transferrin uptake in human
embryonic kidney cells. Molecules acting on a single receptor were identified through excluding pan-specific compounds
affecting housekeeping endocytosis or disrupting internalization of multiple receptors. We identified compounds acting
on a sole GPCR from activities in a broad range of chemical structures that could not be easily sorted by conventional
means. Selective analysis can therefore rapidly select compounds selectively affecting GPCR activity with specificity to one
receptor class through high-content screening.
Keywords
High-content screening, G-protein–coupled receptor, endocytosis, cell-based assay, image analysis, chemical biology
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3. 2 Journal of Biomolecular Screening XX(X)
GPCRs mediate signal transduction and physiological
homeostasis in systems as diverse as blood pressure regula-
tion, odor recognition, and visual perception.5
Given their
importance as regulators, GPCRs are the target of more
than 45% of all prescription drugs,6,7
many of which exploit
disruption of receptor-ligand interactions using antagonists
or artificial agonists.
Agonist-induced internalization is a property of many
GPCRs, such as the endothelin A and µ-opioid recep-
tors,8,9
and upon ligand binding, these GPCRs leave the
plasma membrane and are internalized. GPCR internaliza-
tion is physiologically important, for it controls signaling
via removal of receptors from the cell surface. Activated
receptors are rapidly desensitized from G-proteins through
phosphorylation by G-protein receptor kinases and the
subsequent recruitment of the arrestin adaptor pro-
teins.10
Arrestins promote the internalization of the
receptor into plasma membrane–derived vesicles, either
caveolae or clathrin-coated vesicles, which pinch off into
the cell interior to deliver the receptor into the endosomal
organelles.10,11
Once internalized, GPCRs have several
endocytic itineraries and may be transported for degrada-
tion, rapidly recycled to the cell surface, or resensitized
and recycled from the pericentriolar recycling compart-
ment. Receptor location is likely to be important for cell
responses and is suited to automated cell-based high-content
screening.
We demonstrate the usefulness of a selective screen for
selecting small molecules acting on a single GPCR path-
way (Fig. 1A). Starting from seven GPCR cell lines and 88
molecules, less than 10% of the molecules affected GPCR
internalization without affecting housekeeping endocytosis,
defined by transferrin internalization or pan-specific effects
on multiple receptors. In this selected set, single molecules
were identified that affected only the muscarinic acetylcho-
line receptor 2 (KN-93) and not other GPCRs in the panel.
This selective high-content screen provided a rapid means
to pare down potential candidates through exploiting the
cell biology of GPCRs and also adopting the genetic prin-
ciple of a selective screen.
Materials and Methods
Chemicals
All fine chemicals were purchased from Sigma-Aldrich (St.
Louis, MO). Fluorophores and their reactive forms were
purchased from Molecular Probes (Eugene, OR). DRAQ5
was from BioStatus (Shepshed, UK). Kinase and phospha-
tase inhibitors were purchased as 95% to 99% pure 10 mM
stock solutions in dimethylsulfoxide or water (Biomol,
Hamburg, Germany). Stock solutions and formatted assay
plates were stored at –20°C.
Cell Lines and Cell Culture
Human embryonic kidney (HEK) 293 cells were cultivated
in Dulbecco’s modified eagle’s medium/F12 (Invitrogen,
Carlsbad, CA) supplemented with 10% fetal calf serum, 1%
penicillin streptomycin, and 1% geneticin. GPCR receptor
cDNA clones (cDNA.org) were fused to the EGFP coding
sequence of the pEGFP-N1 vector (Clontech, Palo Alto,
CA) and ligated in the pCDNA3.1 (–) vector
(ADORA1:NM_000674, ADORA3:NM_000677,
EDNRA:AY275462, OPRK1:NM_00912.3,
OPRM1:NM_000914.3, CHRM2:NM_000739.2,
CHRM5:NM_012125.3). The resulting GPCR-EGFP DNA
was cloned, sequence verified, and used for transfection of
HEK293 cells using standard protocols. Each recombinant
clone was obtained through several cycles of selection and
limiting dilutions. For screening, cells were passed onto
fibronectin-coated coverslip-bottomed 96-well plates
(Greiner, Longwood, CO) at a density of 3 × 104
cells/well,
24 h in advance.
Cell Imaging
Cell-based assays were imaged using automated four-color
excitation/emission point scanning confocal systems
(ImageXpress ULTRA, Molecular Devices, Sunnyvale,
CA) with a 40× ELWD 0.6 NA Nikon objective. Images
were analyzed using metaXpress and Acuity express
(Molecular Devices). Typically, we acquired four images
per well/plate.
Cell-Based Screening
Compounds in DMSO or H2
O were diluted into 1% serum
1 mM HEPES pH 7.4 buffered media at the working con-
centration (0.1 nM to 10 µM) and warmed to 37 °C prior to
screening.
For the GPCR internalization assay, recombinant HEK
cells cultivated in fibronectin-coated 96-well plates were
washed in phosphate-buffered saline supplemented with cal-
cium and magnesium. The cells were then serum deprived
and compound treated by incubation for 120 min with 10
µM compounds in low serum (1% fetal bovine serum) assay
medium at 37 °C, 5% CO2
. The compound solutions were
then supplemented with agonist while maintaining com-
pound concentration at 10 µM. The cells were further incu-
bated for 60 min at 37 °C, 5% CO2
.
Poststimulation, cells were washed twice with ice-cold
phosphate-buffered saline supplemented with calcium and
magnesium and fixed in 4% (w/v) paraformaldehyde in
phosphate-buffered saline supplemented with calcium and
magnesium for 10 min. Cells were washed twice with
phosphate-buffered saline supplemented with calcium and
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4. Kwon et al. 3
Figure 1. (A) Compound subtractive screen scheme.Cell lines expressing G-protein–coupled receptors (GPCRs) are treated,stimulated
with small molecules, stimulated with agonist, imaged, and receptor internalization quantified by image analysis. Phenotypes are classified
as having no effect, blocking housekeeping endocytosis (transferrin uptake), and affecting multiple or single GCPRS. Selective molecules
are defined as not affecting housekeeping endocytosis, or more than one GPCR; all others are excluded as pan-specific. (B) Agonist-
induced GPCR-EGFP fusion protein internalization in cell lines expressing the A1 adenosine receptor (A1AR),A3 adenosine receptor
(A3AR), endothelin A receptor (ETAR), or the µ-opioid receptor (MOPR).To demonstrate internalization, cell lines were serum starved
for 120 min then stimulated with agonist or DMSO for 60 min and imaged. Scale bar = 50 µm.
magnesium and stained with 2.5 µM DRAQ5 (Biostatus,
Leicestershire, UK) prior to image acquisition.
Agonist EC50
titrations for each GPCR line were per-
formed on serum-deprived cells, as above.
Transferrin internalization was measured after 120 min
preincubation of wild-type HEK293 cells with compounds
and 10 µM Syto42, followed by the addition of 100 µg/mL
Alexa-633 transferrin in 1% serum assay medium and
60-minincubationat37°Cand5%CO2
.Postinternalization,
cells were washed and fixed as above without the addition
of DRAQ5 prior to imaging.
Curve Fitting and Analysis
Agonist-induced GPCR internalization was analyzed using
algorithms that recognize cell boundaries, segment the cells,
and perform cell-by-cell determination of the number, size,
and intensity of internal fluorescent compartments. These
algorithms are commercially available and run in the
MetaXpressenvironment(MolecularDevices).Measurements
were expressed as the mean and standard deviation for all
cells in four images per well, per treatment, and exported to
a spreadsheet. We selected the measurement output for each
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5. 4 Journal of Biomolecular Screening XX(X)
GPCR that gave the highest goodness of fit for agonist EC50
titrations, as determined by R2
as the metric for that assay.
ImageanalysisdatawereanalyzedusingPRISM(Graphpad,
La Jolla, CA). Curve fitting was performed using sigmoidal
dose-response fitting of triplicate titrations.
Results and Discussion
Our rationale to screen for molecules affecting a single GPCR
pathway was to test a kinase/phosphatase inhibitor collec-
tion against seven GPCR models and then select compounds
selectively affecting GPCR(s). The selection scheme is
shown in Figure 1A and exploited the principle of first
eliminating compounds with no effect, then those disturbing
endocytosis, and finally excluding pan-specific molecules
affecting more than one GPCR.
To evaluate this approach, we first constructed and vali-
dated cell lines expressing fluorescent GPCR fusion pro-
teins to the enhanced green fluorescent protein (Fig. 1B;
Supplementary Fig. 1). Cell lines showing cell surface
expression of a receptor were validated after agonist stimula-
tion and image analysis of agonist-induced receptor internal-
ization (Supplementary Fig. 1). Seven cell lines expressing
GPCR-EGFP fusion proteins were isolated that gave robust
EC50
measurements following image analysis (R2
> 0.85): the
endothelin A receptor (ETAR/ENDRA; EC50
0.48 nM endo-
thelin 1), adenosine type 1 receptor (A1AR/ADORA1: EC50
19 nM CPA), adenosine type 3 receptor (A3AR/ADORA3:
EC50
1.6 nM IB-MECA), kappa opioid receptor (KOPR/
OPRK1: EC50
79 nM U69593), µ-opioid receptor (MOPR/
OPRM1: EC50
0.36 µM DAMGO), muscarinic acetyl cho-
line receptor 2 (M2AR/CHRM2: EC50
carbachol 34 µM),
and the muscarinic acetyl choline receptor 5 (M5AR/
CHRM5: EC50
carbachol 0.47 µM; Fig. 1B; Supplementary
Fig. 1). All assays had a p value <0.0001 between stimulated
and control cells and a Z′ >0.6, indicating cell-based assays
suitable for screening. To quantify compound effects on
receptor-mediated housekeeping endocytosis, transferrin
internalization was measured through imaging internaliza-
tion of fluorophore-conjugated protein in wild-type cells and
controlled by measuring the additive increase in endosome
intensity with increased extracellular protein concentration
(Supplementary Fig. 1).
To identify kinase/phosphatase inhibitors disrupting
GPCR internalization, each of the panel of GPCR-GFP cell
lines was screened against a collection of 88 compounds
and trafficking inhibitors (Fig. 2A; Supplementary Fig. 2).
This included molecules directed against kinases in eight of
nine group/families described in the recent classification of
the human kinome and against three classes of phospha-
tases.12
Effects were measured in four images per well for
each plate, with a total of >200 000 individual cell-based
measurements in the entire screen. Raw screening data were
normalized to the positive control and center reduced, and
results for all assays are shown in Figure 2A as gray-scaled
bars for each compound. Active molecules (“hits”)
were defined as having effects greater than two standard
deviations from the mean of the positive controls (Fig. 2).
The hit rate was 7% ± 2.5% across all assays for single
GPCR screens, indicating the broad effect of these com-
pounds on GPCR activation and internalization. Ten mole-
cules were identified that affected transferrin
internalization, of which four had no significant effect on
GPCR internalization (PP1, quercetin dihydrate, for-
skolin, and HBDDE; Supplementary Fig. 2).
Molecules affecting GPCRs were selected using two cri-
teria: the first selection step removed molecules affecting the
housekeeping transferrin pathway by excluding any hit from
the screen that altered the transferrin assay by >0.6 standard
deviations; this excluded >65% of the hits for each GPCR
assay, for example, for the A3AR (Fig. 2A). The second
round then eliminated pan-specific molecules affecting more
than one GPCR (Fig. 1B, final panel) to ultimately identify
those acting on a single receptor. GPCR-specific molecules
were identified acting on the M2AR (KN-93, damnacanthal,
AG-879), ETAR (wortmannin), and KOPR (ML-7); pan-
specific molecules included the protein kinase C inhibitor
Gö6976 (A1AR, ETAR, MOPR, M5AR). Pan-specific
effects on more than three GPCR assays strongly correlated
with effects on transferrin internalization.
This analysis identified the majority of hits for individual
GPCR high-content screening as nonspecific/pan-specific.
The subtractive screen selectively reduced the hits by
excluding >90% overall and reduced the mean hit rate per
assay to about 1%. Molecules affecting single assays were
found for five of eight assays examined (four GPCRs and
transferrin) based on their phenotype, as shown in Figure 2,
for an example pan-specific inhibitor (Gö6976) as com-
pared with a molecule acting on the M2AR alone (KN93).
Analysis showed that >90% of molecules affecting a single
pathway also affected other assays, as shown for molecules
affecting >3 GPCRs that disrupted transferrin uptake in
>85% of cases. This yielded 10% of screened hits as active
toward a single assay. Single compound structures acting
on sole pathways were identified for the muscarinic acetyl
choline receptor 2 (KN93). KN-93 is a CAM kinase II
inhibitor and arrested muscarinic acetyl choline receptor 2
at the cell surface but did not affect the closely related mus-
carinic acetyl choline receptor 5. To exclude antagonism of
the receptor-agonist interaction, we determined the half
maximally inhibitory concentration (IC50
) as 0.6 µM for
KN-93 acting on the M2AR stimulated with 100 µM carba-
chol (Supplementary Fig. 3). This excludes a generic
antagonistic effect of KN-93 on agonist stimulation of
M2AR internalization and indicates it disrupts the M2AR
internalization machinery.
Together, these data demonstrate that although screens
on one receptor target may identify many molecules, most
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6. Kwon et al. 5
Figure 2. (A) Seven stable cell lines expressing G-protein–coupled receptor (GPCR)–green fluorescent protein (GFP) fusion proteins
to each of the following GPCRs:A1AR,A3AR, ETAR, KOPR, MOPR, M5AR, and M2AR, and an assay of wild-type cell transferrin uptake
was treated with a collection of 88 kinase/phosphatase inhibitors then agonist stimulated. Data were normalized, center reduced, and
plotted against a linear scale from black (minimum) to white (maximum).Hits affecting GPCRs were defined as greater than two standard
deviations from the mean (phenotypic selection:red overlay),and any hits also affecting transferrin (>0.6 standard deviations,blue overlay
panel) were excluded (∉ housekeeping endocytosis panel). Molecules affecting more than one GPCR were excluded (∉pan-specific
hits) to identify molecules affecting single GPCRs (pathway selective hits).The number of hits in each step of the subtractive screen is
indicated at the base of the screening panels.The pan-specific hit Gö6976 (∉) and the M2AR-specific hit KN-93 (∉) are indicated. (B)
Confocal images of the effects of a pan-specific compound Gö6976 and a selected M2AR-specific compound KN-93 are shown for the
entire assay panel after agonist stimulation. Neither molecule affected housekeeping endocytosis (right panel). Gö6976 is defined as pan-
specific because it affected more than half of the assays (blue), whereas the selected molecule KN-93 affected only internalization of the
M2AR (red). Scale bar = 50 µm.
of these are pan-specific, but these can be efficiently sorted
into specific versus pan-specific hits through a subtractive
screen examining multiple GPCR responses.
Phenotypic screening has the advantage that small mole-
cules can be identified that perturb cell phenotypes with
little or no a priori information on their targets or mode of
action.2
High-content screening13,14
has been applied to
several cell pathways such as p53 signaling,15
neurite
outgrowth,16
TGF signaling,17
and correction of the cystic
fibrosis defect.18
High-content screens often operate on
single pathways, but screening results are convolved by
off-target side effects of compounds that are therapeuti-
cally undesirable.
Here, we demonstrated how molecules could be selected
that act on a sole GPCR in a high-content screen. The ratio-
nale applied the principle of a subtractive screen, as used in
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7. 6 Journal of Biomolecular Screening XX(X)
classical genetics, to high-content compound screening. By
excluding compounds affecting multiple GPCRs, a handful
of compounds were identified affecting specific single
GPCRs with no a priori knowledge on the compound-
receptor interaction.
To demonstrate the usefulness of this screening rationale
(Fig. 1A), a panel of cell-based assays was screened against
a collection of 88 kinase and phosphatase inhibitors using
high-content image analysis. A screening strategy based on
the exclusion of molecules that act on housekeeping path-
ways or similar pathways permits the removal of those mol-
ecules with the highest likelihood of pan-specific modes of
action. “Hits” were identified as those that affected one
GPCR without affecting transferrin uptake. When the assays
are all performed in the same cell background, where each
molecule acts against the same set of proteins, the assay
itself acts as a discriminate filter, for it defines where these
interact with a given set of signaling pathways.
Classical phenotypic screens examine compound effects
on one cell-based assay, and the data indicate that second-
ary screening hits against multiple biologically related
assays greatly increases the likelihood that hits are selec-
tive and do not affect housekeeping or common machinery
shared between GPCRs.
GPCR biology provided a set of similar assays that per-
mit correlated screening to identify hits. Given the similar-
ity of the assays analyzed here and the low number of
molecules, using a subtractive screen was a metric for iso-
lating the most selective molecules from screening experi-
ments, with the highest relative activity toward the biological
pathways resolved by any single given assay. The majority
of the molecules identified in single screens (greater than
90%) affected either basic endocytic housekeeping or were
pan-specific (e.g., Gö6976). This indicated that subtractive
screening greatly increases the potential value of the mole-
cules selected.
Using existing kinase and phosphatase inhibitors created
a challenging test case for this analysis: these molecules
presumably act on the same protein pathways in each assay
studied for they are exposed to the same cellular back-
ground (HEK cells) and have not been chemically
optimized for selectivity toward an assay, so they have
pleiotropic effects. Surprisingly, we did find compound
selectivity for half of the assays when they were compared
with the entire panel tested.
This approach is of interest for drug discovery using
high-content screening because unbiased small-molecule
selection will result in the isolation of candidate molecules
that indirectly affect several pathways. Combining several
assays with subtractive secondary screening provided a
rapid approach for compound selection and furthermore for
structural optimization from a confined set of starting com-
pounds, an approach that may benefit drug development.
Pathway-selective analysis may have applications in the
field of phenotypic chemical genetic screening, as a method
for isolating the most pathway-specific molecules from cell-
based screening. This may improve the identification of
small-molecule targets from phenotypic screens by signifi-
cantly narrowing the number of molecules to be examined
and increasing the efficiency of high-content screening
campaigns.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
This work was supported by the National Research Foundation of
Korea (NRF) grant funded by the Korean government (MEST;
No. 2011-00244), Gyeonggi-do, KISTI, and by parliamentary
grant and Department of Science and Technology support in
South Africa (to N.E.).
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