High-Throughput Screening System To Identify Small Molecules That

Aug 20, 2014 - Masayuki Isa†, Daisuke Asanuma†, Shigeyuki Namiki†, Kazuo Kumagai‡, Hirotatsu Kojima‡, Takayoshi Okabe‡, Tetsuo Nagano‡, ...
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High-Throughput Screening System To Identify Small Molecules That Induce Internalization and Degradation of HER2 Masayuki Isa,† Daisuke Asanuma,*,† Shigeyuki Namiki,† Kazuo Kumagai,‡ Hirotatsu Kojima,‡ Takayoshi Okabe,‡ Tetsuo Nagano,‡ and Kenzo Hirose*,† †

Department of Neurobiology, Graduate School of Medicine and ‡Open Innovation Center for Drug Discovery, The University of Tokyo, Hongo, Tokyo 113-0033, Japan S Supporting Information *

ABSTRACT: Overexpression of growth factor receptors in cancers, e.g., human epidermal growth factor receptor 2 (HER2) in ovarian and breast cancers, is associated with aggressiveness. A possible strategy to treat cancers that overexpress those receptors is blockade of receptor signaling by inducing receptor internalization and degradation. In this study, we developed a cell-based high-throughput screening (HTS) system to identify small molecules that induce HER2 internalization by employing our recently developed acidic-pH-activatable probe in combination with protein labeling technology. Our HTS system enabled facile and reliable quantification of HER2 internalization with a Z′ factor of 0.66 and a signal-to-noise ratio of 44.6. As proof of concept, we used the system to screen a ∼155,000 small-molecule library and identified three hits that induced HER2 internalization and degradation via at least two distinct mechanisms. This HTS platform should be adaptable to other disease-related receptors in addition to HER2.

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(especially extensive washing steps) prior to measurement. For HTS applications, we require a homogeneous assay with a minimal number of steps. To address this issue, we aimed to develop a cell-based HTS system to identify small molecules that show activity to induce HER2 internalization (Figure 1). Our strategy for a homogeneous assay to quantify receptor internalization was to conjugate an acidic-pH-activatable fluorescence probe to the target receptor (Figure 1A). Internalized receptor molecules from the cell membrane are sorted to acidic vesicles, such as late endosomes and lysosomes, whose intravesicular milieu is acidic (pH ∼5−6)9 relative to the extracellular milieu (pH 7.4). Therefore, receptor-conjugated probe moieties will generate fluorescence signals only when the receptor is internalized, thereby enabling a homogeneous assay that does not require washout of residual extracellular probe. Acidic-pH-activatable probes have already been utilized for visualizing internalization of receptors, including HER210 and thyrotropin-releasing hormone receptor 1.11 However, the reported techniques have severe limitations as tools for chemical library screening. For instance, receptor labeling with probes using divalent antibodies10,11 causes receptor cross-linking, which can lead to receptor internalization. Also, noncovalent probe-receptor labeling mediated by antibodies might lead to dissociation of probes from receptors during assays. In addition, the reported probes DiEtNBDP10 and CypHer5E11 show poor photo-

nhanced expression of receptor tyrosine kinases (RTKs), leading to aberrant downstream signaling, is a feature of the development and progression of a wide range of cancers.1−3 In ovarian4,5 and breast cancers,6 for instance, HER2 is often overexpressed, and the overexpression is correlated with poor patient prognosis. For the treatment of RTK-overexpressing cancers, monoclonal antibodies directed against the ectodomains of the receptors are in widespread clinical use as molecular targeted therapies, and many antibodies are also under clinical trial.1−3 One of the mechanisms of antibodybased cancer therapy is considered to be blockade of receptor signaling via induction of receptor internalization, which is often followed by intracellular receptor degradation.3 However, large-molecular antibody-based biopharmaceuticals are well-known to have various disadvantages.3 For example, their poor cell membrane permeability means that their action is restricted to extracellular targets. Another issue is that they are very expensive; average daily treatment with a branded biopharmaceutical is estimated to be at least 20-fold more expensive than that with a branded small-molecular drug.7 Therefore, comprehensive exploration of small molecules with activity to induce target receptor internalization is an attractive strategy for developing new candidates for palliative receptor down-regulation. However, until now there has been no convenient method to screen chemical libraries for such activity. Methods are available to monitor the target receptor internalization, for instance, quantification of the cell-surface receptor by means of enzyme-linked immunosorbent assay (ELISA) (e.g., see ref 8), but they are unsuitable for an HTS format due to the requirement of a large number of steps © XXXX American Chemical Society

Received: August 19, 2014 Accepted: August 20, 2014

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inhibitor that induces HER2 internalization.17 After incubation, punctate fluorescence was observed inside the cells (Supplementary Figure S1A), colocalizing with acidic vesicles (Supplementary Figure S1B). Quantitative analysis revealed that 17-AAG treatment significantly increased cellular fluorescence intensity compared with that of the control (1722 ± 179 vs 917 ± 109, p < 0.01; Supplementary Figure S1C). The increase of fluorescence was reversed by intravesicular alkalization with NH4Cl (Supplementary Figure S1A and C). We also confirmed the absence of nonspecific staining with hRhP-M in the original SKOV3 cells (Supplementary Figure S1A). These results show that our proposed assay system is able to quantify HER2 internalization. Therefore, we used this approach to construct an HTS system by using a microplate reader with a 384-well plate format (Figure 1B). We adopted the fluorescence decrease of each well following NH4Cl treatment in the final step (ΔF; see Figure 1B) as the screening index to evaluate the fluorescence change due to HER2 internalization. In validation testing, this system showed a high Z′ factor of 0.66 (Supplementary Figure S2). The Z′ factor is a screening assay performance measure, and values of >0.5 are considered excellent.13 The assay also showed a high signal-to-noise ratio13 of 44.6. These results demonstrate that our assay system is suitable for HTS applications. Next, we screened a ∼155,000 small-molecule library using the developed HTS system. The top 150 compounds (∼0.1% of the total) were further validated in quadruplicate. We identified 16 compounds with >15% activity in terms of ΔF (%), calculated by setting ΔF of positive and negative controls as 100% and 0%, respectively (Supplementary Figure S3). As a counter assay, the amount of cell-surface HER2 after test compound treatment was quantified by specific labeling of cellsurface HER2 with membrane-impermeable Alexa647-HaloTag ligand (Figure 3A and Supplementary Figure S4). Finally, we obtained three hits, compounds a−c (Figure 2).

Figure 1. (A) Schematic representation for detecting HER2 internalization by using an acidic-pH-activatable fluorescence probe. When HER2 is internalized, the receptor-labeling probe moieties are transferred to acidic vesicles from the plasma membrane and consequently become highly fluorescent. (B) Schematic protocol for HTS. HaloTag-HER2 cells on each well of a 384-well plate are incubated with hRhP-M and an individual library compound for 5 h, followed by measurement of fluorescence intensity of the well (Fbefore). After intravesicular alkalization with NH4Cl, fluorescence intensity is measured again (Fafter). The fluorescence decrease, ΔF = Fbefore − Fafter, is used as the screening index.

stability,12 so their suitability for the assay is questionable. A Z′ factor13 of a similar HTS assay using CypHer5E for phagocytosis studies was determined to be 9fold) upon acidification (pH 7.4 to 5.0),12 being superior to other conventional pH-activatable probes in these respects. Moreover, RhP-M is bright in acidic environments (quantum yield >0.6 at pH 5.0),12 which would contribute to production of large fluorescence signals upon receptor internalization. Initially, we evaluated our proposed assay system by means of fluorescence microscopy. The human ovarian cancer cell line SKOV3 expressing HaloTag-HER2 fusion protein (SKOV3/ HaloTag-HER2) was simultaneously treated with a HaloTag ligand-bearing RhP-M, designated hRhP-M (see Supporting Information), and 17-AAG, a heat-shock protein (HSP) 90

Figure 2. Chemical structures of hit compounds.

To characterize these hits, we analyzed their effects on cellsurface HER2. Compounds a−c each caused dose- and timedependent down-regulation of cell-surface HER2 (Figure 3). Notably, compound b (at 5 μM) rapidly decreased the cellsurface HER2 (∼1 h for 50% reduction), showing a 76.3 ± 0.4% decrease at 5 h (Figure 3B and C). At 15 μM, it induced a 95.3 ± 0.5% reduction of cell-surface HER2 (Figure 3D), with no apparent change in cell morphology (Figure 3E). Next, immunoblotting analysis was performed to examine whether compounds a−c induce HER2 degradation. All compounds caused time-dependent decreases in the amount of cellular HER2 (Figure 4; see representative immunoblots in Supplementary Figure S5). Compound a induced degradation of 65.8 ± 0.1% of cellular HER2 after 10 h of treatment. Compounds b and c exhibited moderate effects (degradation of 35.7 ± 5.1% and 43.3 ± 3.4% of cellular HER2 after 10 h, respectively). B

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Figure 4. Quantification of total cellular HER2. SKOV3 cells were treated with 5 μM compound (a, b, or c) or with vehicle. Cellular HER2 was quantified by immunoblotting of cell lysate with anti-HER2 antibody. A housekeeping protein, β-actin, was used as the standard for calculation of protein amount. Data are represented as mean ± SEM (n = 3).

compound b, which bears an adenine scaffold like ATP, nor compound c exhibited significant competition at the ATPbinding site of HSP90, even at 50 μM (Supplementary Figure S6). These compounds likely have molecular interaction(s) different from those of the classical HSP90 inhibition pathway for HER2 internalization/degradation. Further studies are planned to examine in detail the molecular target(s) of these compounds and also to investigate their structure−activity relationships with the aim of increasing the receptor internalization/degradation activities. In conclusion, we have developed a cell-based HTS system to identify small molecules that induce internalization and degradation of HER2 using our acidic-pH-activatable probe in combination with HaloTag labeling technology. Screening of a ∼155,000 small-molecule library yielded three hits. One belonged to a conventional class of resorcylic acid lactones that interact with HSP90. The other two induced HER2 internalization/degradation in a novel manner that did not involve competition at the ATP-binding site of HSP90. This result indicates that our cellular phenotype-based HTS system can identify small molecules with the desired activity among a chemical library, regardless of their molecular target. We believe this approach can be easily employed to develop HTS assays for other disease-related receptors, in addition to HER2.

Figure 3. Quantification of cell-surface HER2. (A) Schematic representation of specific labeling of cell-surface HER2 with a membrane-impermeable fluorescence probe. (B) SKOV3/HaloTagHER2 cells were treated with 5 μM hit compound (a, b, or c) or with vehicle for various times. Cell-surface HER2 was examined by labeling HaloTag-HER2 with Alexa647-HaloTag ligand (n = 4). Data are represented as mean ± SEM. (C) Representative fluorescence images of SKOV3/HaloTag-HER2 cells treated with 5 μM compound (a, b, or c) or with vehicle for 5 h. Cell-surface HER2 was labeled with Alexa647-HaloTag ligand. Scale bar represents 200 μm. (D) SKOV3/ HaloTag-HER2 cells were treated with various concentrations of hit compound (a, b, or c) or with vehicle (n = 4) for 5 h. Data are represented as mean ± SEM. (E) Representative bright field and fluorescence images of SKOV3/HaloTag-HER2 cells treated with 15 μM compound b or with vehicle for 5 h. Cell-surface HER2 was labeled with Alexa647-HaloTag ligand. Scale bar represents 200 μm.

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METHODS

Construction of HaloTag-HER2. A DNA fragment encoding a part of HER2 was obtained by ligation of four fragments, HER2ss-F1 (5′-GATCATGGAGCTGGCGGCCTTGTGCCGC-3′), HER2ss-F2 (5′-TGGGGGCTCCTCCTCGCCCTCTTGCCCCCCGGAGCCGCGAGCA-3′), HER2ss-R1 (5′-GAGGAGCCCCCAGCGGCACAAGGCCGCCAGCTCCAT-3′), and HER2ss-R2 (5′-AGCTTGCTCGCGGCTCCGGGGGGCAAGAGGGCGAG-3′), subcloned into pMX (a kind gift from Dr. T Kitamura, The University of Tokyo) at the BamHI/HindIII site, yielding pMX-HER2ss. A DNA fragment encoding HaloTag was obtained by polymerase chain reaction (PCR) with primers Halo-F (5′-CCCAAGCTTTACCCATACGATGTTCCAGATTACG-3′) and Halo-R (5′-CCGGTGCACACTTGGGTACCGGAAATCTCCAGAGTAGAC-3′). The DNA fragment encoding HER2 was first PCR-amplified from CMV HER2 WT (Addgene) using HER2-F (5′-GTCTACTCTGGAGATTTCCGGTACCCAAGTGTGCACCGGCA-3′) and HER2-R (5′-CCCGTCGACTCACACTGGCACGTCCAGAC-3′). The DNA fragment encoding HaloTag-HER2 was PCR-amplified from the above two templates at the HindIII/SalI site of pMX-HER2ss using Halo-F and HER2-R to afford pMX-HaloTag-HER2. Cell Culture. SKOV3 cells (ATCC) and SKOV3/HaloTag-HER2 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI1640, Nacalai Tesque) containing 10% fetal bovine serum (FBS: Sigma-Aldrich), 100 U/mL penicillin and 100 μg/mL

HSP90 inhibitors such as 17-AAG and radicicol have been reported to induce HER2 internalization/degradation.17−20 Thus, as a preliminary examination of the molecular mechanism of HER2 internalization/degradation, we investigated whether compounds a−c inhibit Hsp90 through binding to its ATPbinding site. A competition assay using a fluorescence polarization probe21 (see Supplementary Methods) afforded IC50 values of compound a of 1.28 ± 0.03 μM for HSP90α and 0.97 ± 0.11 μM for HSP90β (Supplementary Figure S6). The observed competition might be due to the resorcylic acid lactone moiety, which interacts with HSP90 as a nucleotide mimic, as in the case of radicicol.22 However, neither C

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the indicated times at 37 °C in an atmosphere of 5% CO2 in air. After the incubation, the medium was exchanged to 1 μM Alexa647HaloTag ligand in HEPES-BSA, and the plate was incubated at RT for 15 min. The cells were washed with HEPES-BSA, and cells were fixed with 4% PFA in PBS for 10 min and then washed with HEPES-BSA. Fluorescence images were acquired using an inverted microscope equipped with a CCD camera (Andor) and a dry objective (×10, NA 0.30; Olympus). A set of filters (Semrock) was used: a 608−648 nm excitation filter, a 660 nm dichroic mirror, and a 672−712 nm emission filter. Image data were analyzed using ImageJ software and its plug-in. Immunoblotting. SKOV3 cells were treated with 5 μM test compounds or vehicle (DMSO, 0.25%) and incubated for 0, 1, 5, or 10 h at 37 °C in an atmosphere of 5% CO2 in air. After incubation, cells were washed with cold PBS and lysed with sodium dodecyl sulfate (SDS) sample buffer. The lysates were boiled for 5 min and separated on 8% SDS polyacrylamide gel. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, which was then blocked for 1 h in TBST (50 mM Tris-HCl, 150 mM NaCl, 2.68 mM KCl, 0.1% Tween, pH 7.4) containing 5% nonfat milk. Next, the membrane was incubated overnight with anti-HER2 antibody (no. 2165, 1:1000; Cell Signaling) or anti-β-actin antibody (no. A1978, 1:4000; SigmaAldrich) in TBST at 4 °C. After extensive washing with TBST and a final wash with TBS (50 mM Tris-HCl, 150 mM NaCl, 2.68 mM KCl, pH 7.4), immunoreactivity on the membranes was detected with antirabbit/mouse immunoglobulins conjugated to horseradish peroxidase (nos. 458 and 330, respectively; MBL), followed by a chemiluminescence reaction (ECL kit; PerkinElmer). The chemiluminescence signals were integrated with a CCD camera (Nakanishi Image Lab). Image data were analyzed using ImageJ software and its plug-in.

streptomycin (Nacalai Tesque). 293T cells (GenHunter) for retrovirus production were maintained in Dulbecco’ modified Eagle’s medium (Wako) with 10% FBS, 2 mM sodium pyruvate (Life Technologies) and 2 mM L-glutamine (Wako). Retrovirus Infection. For pMX-based retrovirus production, 293T cells were transfected with pMX-HaloTag-HER2, retroviral packaging plasmid pVPack-GP (Agilent) and envelope plasmid pMD2.G (Addgene) using Lipofectamine 2000 (Life Technologies), according to the manufacturers’ directions. Two days after plasmid transfection the supernatant containing viruses was collected and the virus particles were collected by centrifugation for 14 h at 8,000 g. SKOV3 cells were infected with the prepared retrovirus particles. Fluorescence Imaging. On the day before assay, SKOV3/ HaloTag-HER2 or SKOV3 cells were seeded at 2 × 104 cells per well in 96-well plates (Thermo Scientific). The assay was initiated by adding hRhP-M (30 nM final concentration) and 17-AAG (1 μM final concentration, Selleck Chemicals) or vehicle (DMSO, 0.01%) in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 1.8 mM KH2PO4, pH 7.4) containing 0.1% bovine serum albumin (BSA: Sigma-Aldrich) to the wells. The plate was incubated at 37 °C in an atmosphere of 5% CO2 in air for the indicated times. After the incubation, the cells were washed with PBS containing 0.1% BSA and alkalized with 10 μL of 200 mM NH4Cl in PBS. In the case of colocalization experiments, LysoTracker Green (Life Technologies) was added (100 nM final concentration) after the incubation, and the cells were further incubated for 1 h at 37 °C in an atmosphere of 5% CO2 in air and then washed with RPMI1640 containing 2% FBS. Fluorescence images were acquired using an inverted microscope equipped with a cooled charge coupled device (CCD) camera (Andor) and a water immersion objective (×40, NA 0.80; Olympus) or an oil immersion objective (×100, NA 1.40; Olympus). Sets of filters (Olympus) were used as follows: a 535−555 nm excitation filter, a 565 nm dichroic mirror, and a 570−625 nm emission filter for hRhPM and a 470−495 nm excitation filter, a 505 nm dichroic mirror, and a 510−550 nm emission filter for LysoTracker Green. Image data were analyzed using ImageJ software (NIH) and its plug-in. HTS. For HTS screening, a chemical library containing about ∼155,000 compounds was used (Open Innovation Center for Drug Discovery, The University of Tokyo). On the day before treatment with test compounds, SKOV3/HaloTag-HER2 cells were seeded at 1.0 × 104 cells in 50 μL of RPMI1640 containing 2% FBS per well in 384well plates (PerkinElmer). Solution handling, including cell seeding, was performed using a Biomek FX laboratory automation workstation (Beckman Coulter), unless otherwise noted. Into each well of 384-well plates (Greiner Bio-one) were dispensed 300 nL of 2 mM library compound or vehicle and 7.5 nL of 500 μM hRhP-M using an Labcyte Echo 555 acoustic liquid handler (Sunnyvale), followed by addition into each well of 19.7 μL of 6 mM HEPES, pH 7.4 containing 0.1% BSA. Assay was initiated by adding the solutions thus prepared (final concentrations: 5 μM library compound and 30 nM hRhP-M) to the cells on 384-well plates. These plates were incubated for 5 h at 37 °C in an atmosphere of 5% CO2 in air. After incubation, the medium was exchanged twice with 35 μL of HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 8.6). The fluorescence intensity (Fbefore) was measured with a PHERAstar plate reader (BMG Labtech, excitation at 540 nm and emission at 590 nm). The cells were alkalized with 10 μL of alkalization buffer (20 mM HEPES, 150 mM NaCl, 50 mM NH4Cl, 1 μM bafilomycin A1; Wako, pH 8.6). Then, fluorescence intensity (Fafter) was measured again. The screening index, ΔF, was obtained by subtracting Fafter from Fbefore. Assays were quality-controlled by evaluation of the Z′ factor, which was calculated by using 17-AAG and vehicle controls (16 wells for each control were arranged on every plate). Plates with a Z′ factor >0.50 were accepted for analysis, and those that did not meet this criterion were rejected and reassayed. Quantification of Cell-Surface HER2. On the day before assay, SKOV3/HaloTag-HER2 or SKOV3 cells were seeded at 2 × 104 cells per well in 96-well plates (Thermo Scientific). The assay was initiated by adding test compound (5 μM final concentration) in HEPES-BSA (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 25 mM glucose, 0.1% BSA) to the wells. The cells were incubated for

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was also supported in part by grants from SENTAN, JST (to K.H.), KAKENHI (Grant no. 26560441 to D.A.; 24115502, 24590313, and 25115704 to S.N.; and 24116004 to K.H.), Takeda Science Foundation (to K.H.), and Research Foundation for Opto-Science and Technology (to D.A.).

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REFERENCES

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