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 DOI: 10.1177/1087057111427347

published online 15 November 2011J Biomol ScreenYong-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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by Anne Carpenter on November 29, 2011jbx.sagepub.comDownloaded from

Journal of Biomolecular ScreeningXX(X) 1 –7© 2011 Society for Laboratory Automation and ScreeningDOI: 10.1177/1087057111427347http://jbx.sagepub.com

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 reducing the number of candidates to be tested. 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.

427347 JBXXXX10.1177/1087057111427347Kwon et al.Journal of Biomolecular Screening

1Institut Pasteur-Korea, Gyeonggi-do, Korea2Department of Biochemistry, College of Science, Yonsei University, Seoul, South Korea3High 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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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 MethodsChemicals

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 l iga ted in the pCDNA3.1 (– ) vec to r (ADORA1:NM_000674, ADORA3:NM_000677 , E D N R A : A Y 2 7 5 4 6 2 , O P R K 1 : N M _ 0 0 9 1 2 . 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 ImagingCell-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 ScreeningCompounds in DMSO or H

2O 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% CO

2. 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% CO

2.

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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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-min incubation at 37 °C and 5% CO

2. 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 MetaXpress environment (Molecular Devices). 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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GPCR that gave the highest goodness of fit for agonist EC50

titrations, as determined by R2 as the metric for that assay.

Image analysis data were analyzed using PRISM (Graphpad, La Jolla, CA). Curve fitting was performed using sigmoidal dose-response fitting of triplicate titrations.

Results and DiscussionOur 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 EC

50 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: EC

50

19 nM CPA), adenosine type 3 receptor (A3AR/ADORA3: EC

50 1.6 nM IB-MECA), kappa opioid receptor (KOPR/

OPRK1: EC50

79 nM U69593), µ-opioid receptor (MOPR/OPRM1: EC

50 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: EC

50 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 (IC

50) 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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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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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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