Development of a Cell-Based,High-Throughput Screening Assay for ATM Kinase Inhibitors

Chemicals

Compound CP466722 was synthesized at the St. Jude Children’s Research Hospital by the condensation of 4-chloro-6,7-dimethoxyquinazoline with 3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine in the presence of Cs₂SO₃. Both reactants used for this final condensation, the quinazoline^13,14 and triazole, ¹⁵ were prepared according to reported procedures.

1H NMR (400 MHz, Chloroform-d) δ 9.21 (s, 1H), 8.93 (s, 1H), 8.79 (s, 1H), 8.16 (d, J = 7.9 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.38 (s, 2H), 7.06 (s, 2H), 4.15 (s, 3H), 4.10 (s, 3H). LC/MS (ESI): m/z 350.3 [M⁺ + H]⁺.

CP466722 was dissolved in DMSO at a concentration of 4 mM (its solubility limit). Ku55933 and Ku60019 were purchased from Sigma (St. Louis, MO) and were dissolved in DMSO at a concentration of 10 mM.

Cell Culture

MCF7 cells were obtained from ATCC (American Type Culture Collection, Manassas, VA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum at 37°C in a humidified atmosphere (5% CO₂).

Irradiation

Cells were irradiated using a ¹³⁷Cs-source (Gammacell 40-exactor, MDS Nordion, Ottawa, Ontario, Canada) at a dose of 10 Gy.

Western Blotting

Cells were harvested, lysed in RIPA buffer, quantitated, and prepared for Western blotting as previously described. ⁸ Primary antibodies were prepared in 1:1000 dilutions in 1× Tris-buffered saline with Tween (TBST) buffer containing 5% bovine serum albumin (BSA). Antibodies used in this experiment include anti–phospho-ATM(S1981) (cat. 2152-1; Epitomics, Burlingame, CA), anti–phospho-p53(S15) (cat. 9284L; Cell Signaling, Danvers, MA), anti–phospho-KAP1(S824) (cat. A300-767A; Bethyl Laboratories, Montgomery, TX), and anti–β-actin (cat. A5316; Sigma). One anti–phospho-ATM(S1981) ⁴ and one anti–phospho-SMC1(S957) ¹⁶ antibody were made by our laboratory.

ICW Assay for Screening of ATM Kinase Inhibitors

In total, 25 µL of a suspension of 4 × 10⁵ MCF7 cells/mL in DMEM was dispensed to each well in 384-well plates using a Wellmate liquid dispenser (Matrix Technologies, Maumee, OH). The plates were incubated overnight in a 5% CO₂, 37°C, 100% relative humidity atmosphere. After incubation, 25 nL of each test compound was transferred into the wells of the plates from stock plates (10 mM in DMSO) using a 384-pin array (V&P Scientific, San Diego, CA). DMSO was used as a negative control and CP466722, Ku55933, and Ku60019 as positive controls. A DNA DSB-inducing agent, etoposide, was then added using the 384-pin array to give a final concentration of 25 µM in each well to activate ATM kinase. The cells were incubated at 37°C, 5% CO₂, 100% relative humidity for 1 h and then fixed by dispensing 25 µL of 8% formaldehyde using a Wellmate liquid dispenser (Matrix Technologies) to a final concentration of 4% (vol/vol) and incubated for 20 min at room temperature. Then, the cells were permeabilized by washing the plates (3 × 50 µL) with a microplate washer (ELx405; BioTek, Winooski, VT) using phosphate-buffered saline (PBS) containing 0.1% Triton X-100. After permeabilization, the plates were washed three times with PBS containing 0.1% Tween-20 and then incubated overnight at 4°C with 20 µL of pKAP1 antibody (1:3000 dilution; Bethyl Laboratories) in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE). The next day, plates were washed three times with PBS containing 0.1% Tween-20. Subsequently, IRDye 800CW goat anti–rabbit secondary antibody solution (1:5000 dilution; LI-COR Biosciences) containing DNA stain DRAQ5 (1:5000 dilution) was added into each well. After a 1-h incubation at room temperature, the plates were washed three times with PBS containing 0.1% Tween-20. After the final wash, the washing buffer was discarded. The plates were then scanned on an Aerius automated Infrared Imaging system (LI-COR Biosciences) for quantitative analysis at 700 and 800 nm at 200 microns of scan resolution and 3.0 mm of focus offset. Images for DRAQ5 and pKAP1 staining were obtained by using the 700- and 800-nm channels of the Aerius Infrared imaging system, respectively. Fluorescence intensities were measured using the software provided with the imaging system.

For assay optimization, we optimized the washing parameters (height for aspiration and dispensing, washing cycles, and dispensing volume and rate) of the microplate washer to prevent cell loss. The timing after IR or etoposide treatment was also optimized to obtain maximum activation of pKAP1. To save cost on pKAP1 antibody, the dilutions of pKAP1 antibody were optimized to use minimal antibody without compromising the signal window.

After optimization, HTS was performed on a customized system developed by High Resolution Engineering (Woburn, MA) with integrated subsystems that exactly match those used for optimization. Plates were transferred from instruments to instruments by a Staübli T60 robot arm (Staübli, Duncan, SC).

Dose-Response Experiments

For dose-response experiments, screening compounds were diluted 3-fold over 10 steps using a Tecan Evo liquid handling system (Tecan, Männedorf, Switzerland), transferred from a compound plate to a 384-well assay plate using a Biomek FX (Beckman Coulter, Brea, CA) liquid handler equipped with a pin tool, and then assayed as described above.

Data Processing and Hit Selection

Primary screening data were analyzed using our in-house Robust Interpretation of Screening Experiments (RISE) application written in Pipeline Pilot (version 8.5; Accelrys, San Diego, CA) and the R program (R Development Core Team, Vienna, Austria). Percentage inhibition was calculated by first log₁₀-transforming the fluorescence intensity observed at 800 nm and then normalizing per plate to the 25% trimmed mean value of the positive control, CP466722, and the 25% trimmed mean value of the negative control, DMSO, according to the following formula: %inhibition = 100 * (screening compound – negative control)/(positive control – negative control). The top 1% of compounds from the primary screen were defined as “hits” and were selected for further study. Dose-response curves were fit with Prism (version 6; GraphPad Software, La Jolla, CA) using the standard four-parameter Hill equation.

Chemical Library and HTS

The screening library consisted of 7152 unique compounds with a minimum of 90% purity from commercial sources. The library includes Food and Drug Administration (FDA)–approved drugs, kinase inhibitors, CDK inhibitors, and other bioactive compounds. These compounds are stored at 1 to 10 mM in DMSO in 384-well plates.

ICW Assay Development for the ATM Signaling Pathway

Before setting up the ICW assay, we tested the cellular activities of some ATM kinase inhibitors obtained from earlier screens (CP466722, Ku55933, and Ku60019) in the MCF7 breast cancer cell line to determine if this cell line would be an adequate model for screening for ATM inhibitors. MCF7 cells were pretreated with these inhibitors or DMSO for 1 h before exposure to 10 Gy of IR. Then cells were incubated for 1 h after IR exposure and lysed for Western blotting analysis. As expected, IR induced ATM autophosphorylation at serine 1981 and ATM-mediated trans-phosphorylation of p53 and KAP1 ( Fig. 1A ). CP466722 and Ku55933 caused dose-dependent inhibition of phosphorylation of both ATM and its downstream targets p53 and KAP1 with potencies similar to those previously reported. Ku60019 is a more potent ATM kinase inhibitor than CP466722 and Ku55933, completely inhibiting ATM-dependent phosphorylation at 1 µM.

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Figure 1.

In-Cell Western (ICW) assay development for ATM (ataxia-telangiectasia, mutated) kinase inhibition. (A) MCF7 cells were pretreated 1 h with known ATM kinase inhibitors CP466722, Ku55933, and Ku60019 at a final concentration of 1, 5, or 10 µM. DMSO was used as a vehicle control. Cells were subjected to 10 Gy of ionizing irradiation (IR) or not (–IR control). One hour after IR, cells were lysed and Western blotting was performed by staining with anti-pATM, anti-pKAP1, anti-pp53, and anti-actin antibodies. (B) ICW assay in a 384-well plate for pKAP1 after MCF7 cells were treated with DMSO control (column 03) or various ATM kinase inhibitors CP466722 (column 06), Ku55933 (column 04), and Ku60019 (column 05) in 1:3 dilutions from rows A to L of the plate. The highest concentration for each compound is labeled at the top of its corresponding column. After a 1-h treatment, cells were subjected to 10 Gy of IR. The 800-nm image is for pKAP1 staining. The 700-nm image is for DRAQ5 staining, which is used for normalizing cell numbers in each well. (C) Dose-response curves for CP466722, Ku55933, and Ku60019. Their IC₅₀ values are listed underneath.

For the screening assay, cells were grown in 96- or 384-well plates, treated with test compounds, and then stimulated with DNA damaging agents. After treatment, cells were fixed with formaldehyde, permeabilized, and stained with phospho-specific primary antibodies that recognize target proteins. The cells were then incubated with an infrared fluorescent dye–labeled secondary antibody and a DNA binding dye, DRAQ5, for normalizing cell numbers in each well. The plates were scanned simultaneously at 700 nm (for signal of DRAQ5) and 800 nm (for signal of fluorescence dye labeled secondary antibody) with an Aerius instrument (LI-COR Biosciences), which has separate lasers and florescence detectors for each dye and is able to simultaneously detect two targets at 700 and 800 nm for two spectrally distinct dyes.

Primary antibodies used in ICW assays need to be very specific to the studied target protein. To select the ideal antibodies for the ICW assay, we screened the specificity and efficiency of several antibodies that detect ATM-dependent phosphorylation events, including pATM(S1981), pp53(S15), pKAP1(S824), and pSMC1(S957) in immunoblot analyses (Suppl. Fig. S1). Among these, both the anti-pKAP1(S824) and anti-pp53 antibodies displayed the highest level of specificity, showing only one band on Western blots of total cellular protein. However, the anti-pKAP1 antibody had a much higher signal window than the anti-pp53 (Suppl. Fig. S1). Thus, KAP1 phosphorylation is a specific readout of DNA damage-induced activation of ATM in cells and should have sufficient signal to work in a cell-based screening assay. Therefore, the anti-pKAP1(S824) antibody was chosen as the optimal antibody to screen for inhibition of the ATM DNA damage response signaling pathway.

To evaluate whether the pKAP1 antibody worked in ICW assays, MCF7 cells were plated in a 384-well plate and incubated overnight at 37°C. The next day, cells were pretreated with CP466722, Ku55933, or Ku60019 at a final concentration of 10 µM or with DMSO as a control for 1 h before IR. Cells were then exposed to 10 Gy IR, incubated for 1 h after IR, and fixed and processed for the ICW assay. The phosphorylation signal (800 nm, green signal) of KAP1 showed a dose-dependent reduction in the presence of the inhibitors ( Fig. 1B , C ), indicating a dose-dependent inhibition of ATM signaling. These ATM inhibitors did not affect cell survival by themselves during the 2-h treatment since the DRAQ5 signals (700 nm, red signal) was consistent across all wells. The IC₅₀ values for CP466722, Ku55933, and Ku60019 in this cell-based assay were 0.41, 0.49, and 0.03 µM, respectively.

Use of Etoposide as the DNA Damaging Agent in the Screening Assay

Since irradiators are normally located in separate secured locations for safety and security reasons and are typically not part of HTS facilities, cell plates would have to be transferred between rooms or buildings to allow irradiation of this type of screening assay. Even if co-localized, at the very least, plates would have to be transferred between an irradiator and the plate reader, which would be suboptimal. Irradiation would thus become a bottleneck for HTS. For ease of screening, we explored the option of replacing the irradiation step with a DSB-inducing chemotherapeutic agent, such as etoposide, a topoisomerase II inhibitor.^17,18 The activation of ATM and pKAP1 in MCF7 cells treated with 25 µM etoposide peaked at 1 h after treatment, and pretreatment with 10 µM CP466722 inhibited both pATM and pKAP1 signals ( Fig. 2A ). Thus, the ATM specificity of the signaling pathway with this chemical agent is functionally equivalent to irradiation for this assay and makes etoposide a viable replacement for irradiation as the DSB-inducing agent. As confirmation of this conclusion, IC₅₀ values for the various inhibitors in the ICW assay with etoposide were similar to those seen following IR ( Fig. 2B , C ). Therefore, IR was replaced by etoposide as the DNA damaging agent in the HTS assay.

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Figure 2.

Evaluation of the activation of ATM (ataxia-telangiectasia, mutated) kinase by etoposide in MCF7 cells. (A) MCF7 cells were treated simultaneously with CP466722 at a final concentration of 10 µM and etoposide as a final concentration of 25 µM. Cells were then harvested at 0.5, 1, or 2 h after treatment and lysed for Western blotting analysis of pATM and pKAP1. (B) In-Cell Western (ICW) assay for pKAP1 after replacing ionizing irradiation (IR) with etoposide. MCF7 cells were treated with CP466722 (column 04), Ku55933 (column 02), or Ku60019 (column 03) in 1:3 dilutions from rows A to L of the plate with the highest concentration labeled at the top of its corresponding column. Etoposide was then added to reach a final concentration of 25 µM. The 800-nm image is for pKAP1 staining. The 700-nm image is for DRAQ5 staining, which is used for normalizing cell numbers in each well. (C) Dose-response curves for CP466722, Ku55933, and Ku60019. Their IC₅₀ values are listed underneath.

Assay Validation and Optimization of the High-Throughput Assay

The variability of the assay in 384-well plates was assessed by running triplicate plates with alternating max/min/mid signals on 3 days. After etoposide treatment, the maximum signal (cells treated with DMSO only), minimum signal (cells treated with 10 µM CP466722), and the mid-signal (cells treated with 0.8 µM CP466722) were plotted for these plates ( Fig. 3A ). We observed that the raw signal scattered the most for the DMSO-treated negative control cells and only minimally for cells treated with the highest concentration of the positive control. This is not unusual in a situation where the negative control represents pathway activation (maximal triggered signal, amplified by pathway) and can be corrected by applying a log₁₀-transform to the raw signal. This processing step equalizes variance across the dynamic range of the assay ( Fig. 3B ) and enables the use of parametric statistical methods for hit selection. Following data transformation, inter- and intraplate variability was consistent and reasonable: median Z′ (comparing the max and min signals) across the nine plates in the assay validation experiment was 0.61, with a minimum of 0.32 and maximum of 0.66. Assay drift was negligible: median column drift was 11% and median row drift was 7%. We did observe one instance each of row and column drift exceeding >20%, but in these cases, the values abruptly shifted for only a single row or column at the outer edge as opposed to gradually shifting across the plate ( Fig. 3C ). This variation did not correlate with cytotoxicity as measured by DRAQ5. We recommend that plate heatmaps for this assay be closely examined for row and column bias.

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Figure 3.

Validation of In-Cell Western (ICW) assay for ATM (ataxia-telangiectasia, mutated) kinase inhibitor in the high-throughput screening format. (A) Raw pKAP1 intensities showed asymmetric variance with higher scatter at the maximum signal (DMSO control). (B) Log₁₀-transform of A. Variance was equalized across the dynamic range of the assay, and Z′ improved. (C) The only example of column drift in the assay validation experiment. The rightmost column on the plate shows significant (>20%) drift for the minimum signal wells. However, in general, row and column drift was negligible. Both the raw pKAP1 signal in A and the log₁₀-transformed results in B were multiplied by −1 to be compatible with our assay validation template.

We had intended to normalize all data using the 700-nm cytotoxicity control channel, but it is clear that this data set is noisy relative to the antibody binding data and does not give reliable normalization. Instead, it can be used to filter the data, removing compounds with the 700-nm signal below the lower bound of the 95% confidence limit as potentially cytotoxic.

Primary Assay: HTS of a Compound Library

A relatively small collection of compounds (7152), including bioactive compounds, FDA-approved compounds, and kinase inhibitor collections, was used in a pilot screen with the assay. MCF7 cells were seeded in 384-well plates, cultured overnight, treated with compounds at a final concentration of 10 µM, and subsequently exposed to 25 µM etoposide. Then, 10 µM CP466722 was included as a positive control and DMSO was used as a negative control. The scatterplots for percentage inhibition of the library are shown in Figure 4A . The boxplot of log₁₀-transformed anti-pKAP1 fluorescence intensity values shows consistent and stable separation between positive and negative controls ( Fig. 4B ). Median Z′ was 0.52, with a minimum of 0.27 and a maximum of 0.82 ( Fig. 4C ). The top 1% of compounds tested, or 72 compounds, had percentage inhibition ≥43.4% and were defined as “hits” in the primary screen.

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Figure 4.

Results of the primary screen. (A) Scatterplot of percentage inhibition for screening compounds (black), negative controls (DMSO, red), and positive controls (10 µM CP466722, green). Orange and purple horizontal lines represent the 95th and 99th percentiles, respectively, for the screening compound distribution. (B) Boxplots of log₁₀-transformed anti-pKAP1 intensity values for the negative controls (red) and positive controls (green). (C) The Z′ for the primary screen.

Hit Validation

To validate the hits, two commercially available known kinase inhibitors ( Fig. 5A ) were repurchased as pure powders, reformulated, and tested in a dose-response assay to confirm the results of the primary screening. Both hits showed a dose-dependent inhibition of the pKAP1 signal with an IC₅₀ value of 0.48 µM and 4.72 µM, respectively ( Fig. 5B ).

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Figure 5.

Further identification of two hits from screening. (A) Two hits from screening. (B) The dose-response curves for SJ000573017 and SJ000573226 by the In-Cell Western (ICW) assay. (C) Western blotting analysis of SJ000573017 and SJ000573226 in MCF7 cells on their inhibition of ATM (ataxia-telangiectasia, mutated) phosphorylation and its downstream targets, pKAP1 and pp53. MCF7 cells were pretreated 1 h with SJ000573017, SJ000573226, or CP466722 at a final concentration of 1 and 10 µM. DMSO was used as a vehicle control. Cells were subjected to 10 Gy of ionizing irradiation (+IR) or not (–IR control). One hour after IR, cells were lysed and Western blotting was performed by staining with pATM, pKAP1, pp53, and actin antibodies. Ponceau staining of the blot was used as a loading control.

To further characterize the two initial positive hits, we tested them in confirmatory immunoblot assays. SJ000573017 (10 µM) inhibited IR-induced formation of pATM, pKAP1, and pp53, consistent with inhibition of the ATM signaling pathway. However, SJ000573226, which was ~10× less active than CP466722 based on IC₅₀ values in the ICW assay, only showed a partial inhibition on pATM and its downstream targets pKAP1 and pp53. Thus, the potencies observed in the immunoblot assays correlated well with those from the ICW results.