A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel

Abstract

Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.

Fig. 1

(A) Schematic diagram of the FluxOR thallium assay for the measurement of hERG channel activity. Cells that express hERG channels are loaded with dye from the FluxOR thallium assay kit and then washed to remove the dye from medium. When the cells are stimulated, thallium ions enter the cells through open hERG channels and bind to the dye, yielding an increase in green fluorescence (530 nm) on excitation at 480 nm. (B) Excitation and emission spectra of FluxOR dye (red and black solid lines, respectively) and Red 40 absorbance (black dotted line). FluxOR dye was deesterified by treating with PBS (pH 10.0) for 3 days, followed by the addition of stimulation buffer. Measurements were taken on a Tecan monochrometer. RFU, relative fluorescence units. (C) Titration curve of Red 40 absorbance as measured on a Tecan monochrometer. Molecular structure of Red 40 presented on right. (D) Schematic diagram of traditional wash assay. Cells are loaded with FluxOR dye. As the dye crosses the cell membrane, intracellular esterases cleave the ester group, trapping the dye within the cell. Extracellular deesterified dye is present due to efflux as well as extracellular esterase activity. After loading, the dye is removed by an aspiration step and cells are loaded with assay buffer. Stimulation of channel opening and subsequent signaling are accomplished by the addition of stimulation buffer containing thallium. False signal is generated by extracellular deesterified dye. (E) Schematic diagram of quenching effect of Red 40. The cell-permeable FluxOR dye enters cells and becomes deesterified. Thallium ion binds to the dye that forms an excitable complex emitting at 530 nm. Cell-impermeable Red 40 quenches the fluorescence caused by extracellular dye that had leaked out of cells or had become deesterified by extracellular esterases. A bottom reading plate reader will collect the fluorescence signal inside cells that is generated only by the cells attached to the bottom of a plate.

Fig. 2

(A) Time course of hERG channel stimulation in U-2 OS cells in the presence of haloperidol dilutions. The max/min ratio was calculated from time of stimulation over 1-s intervals. The linear range of response within 30 s of stimulation was used to calculate slope. (B) Comparison of transduced and nontransduced cells stimulated in the presence and absence of 50 μM haloperidol. All transduced cells were treated with a 100:1 multiplicity of infection virus-to-cell ratio. Each data point represents the mean + S.E.M. (n = 4).

Fig. 3

Concentration-dependent inhibition of haloperidol on hERG channel activity in the transduced cells in comparison with that in untransduced cells. (A) U-2 OS cells. The IC₅₀ value of haloperidol was 257 nM in the transduced cells, whereas haloperidol had no effect on the untransduced U-2 OS cells. hERG channel activity is measured as slope. (B–D) HEK293 (B), HeLa (C), and CHO-K1 (D) exhibited relatively low levels of stimulation and blocking by haloperidol. Cells were transduced with a multiplicity of infection of 100:1. Each data point represents the mean + S.E.M. (n = 4).

Fig. 4

(A) Cell number titration of U-2 OS hERG cells treated with or without haloperidol. The signal-to-basal (S/B) ratios defined by with (+) and without (−) haloperidol in the thallium flux assay for 500, 1000, 2000, and 4000 cells/well were 2.7-, 2.6-, 3.1-, and 3.1-fold, respectively. (B) Comparison of wash and no-wash protocols. For the wash protocol, cells in suspension were loaded in Opti-MEM medium containing 2% serum, FluxOR dye, and probenecid for 60 min at room temperature. Cells were then pelleted, washed, and plated at a density of 2000 cells/3 μl in assay buffer containing probenecid. Immediately after plating, cells were treated with 50 μM haloperidol or DMSO. For the no-wash protocol, cells were plated at a density of 2000 cells/3 μl in Opti-MEM containing 2% serum and allowed to adhere to the bottom of the plate for 4 h at 37 °C. Cells were then loaded with 1 μl of dye mixture and incubated at room temperature prior to being assayed. The S/B ratios were 3.1 and 4.2 for wash and no-wash, respectively. Each data point represents the mean + S.E.M. (n = 4).

Fig. 5

(A) Concentration responses of 10 known hERG inhibitors determined in the thallium flux assay using the hERG channel-transduced U-2 OS cells. Each data point represents the mean + S.E.M. (n = 6). (B) Correlation of IC₅₀ values from thallium flux assay with those determined in manual patch clamp data from Cerep. Goodness-of-fit between patch clamp and HTS had an R² value of 0.80. Each data point represents the mean + S.E.M. (n = 5).