Drug-induced inhibition of the human ether-à-go-go-related gene (hERG) ion channel has been related to the susceptibility of patients to potentially fatal ventricular tachyarrhythmia, torsade de pointes. In recent years, a number of FDA-approved drugs were withdrawn from the market due to their off-target effect on hERG. As a result, there has been an increasing need for identifying compounds that block the hERG channel at earlier stages in the drug discovery process. Here we present the utility of a new potassium assay kit on the FlexStation® 3 Multi-Mode Microplate Reader to investigate hERG compound activity. This assay exploits the permeation of thallium (Tl+) through potassium (K+) channels into the cytosol, which is then detected by a novel fluorescence indicator dye. Seven reference hERG blockers are examined in a medium-throughput cell-based assay, and the results are compared to values obtained using the FLIPR® Tetra System and the IonWorks Barracuda® Plus Automated Patch Clamp System.
Materials and methods
The FLIPR®Potassium Assay Kit (Figure 1) contains a thallium-sensitive indicator dye. During the initial dye-loading step, the Tl+ indicator dye in the form of an acetoxymethyl (AM) ester enters the cells through passive diffusion across the cell membrane. Cytoplasm esterases cleave the AM ester and relieve its active fluorogenic form. In addition, a proprietary masking dye is applied extracellularly to reduce background fluorescence. To activate the potassium channel, the cells are stimulated with either a mixture of K+ and Tl+ or a ligand in the presence of Tl+ . The increase in fluorescent signal represents the influx of Tl+ into the cell specifically through the potassium channel, and therefore represents a functional measurement of the potassium channel activity. The FLIPR Potassium Assay Explorer Kit (Molecular Devices Cat# R8222) contains Tl+ -sensitive dye, masking dye for homogenous operation, 200 mM K2SO4, 50 mM Tl2SO4, 5X chloride-free buffer, and HBSS + 20 mM HEPES buffer. The kit supports ten 96- or 384-well plates. The assay workflow is shown in Figure 2.
The hydrophobic property of compounds impacts the apparent potency values, likely through non-specific binding to labware. In this study, compounds were first diluted in 100% DMSO, followed by transfer with mixing to HBSS + 20 mM HEPES buffer in glass-lined polypropylene plates immediately prior to assay.
Chinese hamster ovary (CHO) cells stably transfected with human Kv11.1 (hERG) ion channel were provided by ChanTest Corporation (Cleveland, OH). Cells are plated at 25,000/well in a 96-well, blackwalled, clear bottom plate two days before the assay in growth medium including selection antibiotics and incubated at 37°C and 5% CO2. Twenty four hours prior to the assay, the growth media is exchanged for induction media containing tetracycline. Four hours prior to the assay, the cells are switched from 37°C to 28°C for enhanced membrane expression of hERG channel. Plates are incubated with dye for one hour at room temperature in the dark.
For pharmacology analysis, hERG channel blocking compounds are added first and incubated for 30 minutes at room temperature. Previously optimized stimulus buffer is added to each column of wells during detection on the FlexStation 3 reader. Using SoftMax® Pro Software, the FlexStation 3 reader excitation wavelength is set at 485 nm and the emission wavelength is set at 538 nm. Signal is acquired at 1.52 second intervals for approximately 120 seconds per column in a 96-well plate. For comparison, parallel experiments are performed using the FluxOR Assay Kit (Life Technologies) following the manufacturer’s assay protocol. Data analysis is performed using SoftMax Pro Software and GraphPad Prism.
Assay development is first carried out by determining optimal concentrations of thallium and potassium necessary for stimulation of the hERG channel. Plates are set up to test stimulating buffers containing various concentrations of K2SO4 and Tl2SO4. The Tl+ and K+ buffers are diluted in 1X chloride-free buffer. Since thallium sulfate and potassium sulfate have two equivalents of cation per mole, they are considered as having 2X their respective cation concentrations. The stimulus buffers are added to cells during detection. Signal traces are compared among the concentration combinations to determine the optimal concentrations that provide the largest signal. Using the FLIPR Tetra System, the optimal (final) Tl+ concentration that provided the largest signal for hERG is 1 mM and the optimal (final) K+ concentration is 10 mM1.
For comparison purposes, IC50 values for the same set of hERG channel blockers were collected using the IonWorks Barracuda Automated Patch Clamp System3 (Figure 3). To capture use-dependent compound effects, the voltage protocol is applied five times at 0.1 Hz before and after compound addition. Peak amplitude of hERG tail current at the fifth sweep is used to measure compound effects.
Seven known hERG blockers were evaluated using the FLIPR Potassium Assay Kit on the FlexStation 3 reader. The concentration response curves are shown in Figure 4. IC50 values compared to data obtained on the FLIPR Tetra System and on the IonWorks Barracuda System are shown in Table 1. The rank order of compound potency was preserved between the three assay systems and IC50 values were within one-half log with good correlation.
|Compound||IC50 (nM) FlexStation 3 reader||IC50 (nM) FLIPR Tetra System||IC50 (nM) IonWorks Barracuda System||Ratio FlexStation 3/ IonWorks Barracuda System|
In addition, four compounds were used to evaluate performance against a reference thallium-based assay kit, the FluxOR Potassium Ion Channel Assay (Figure 5). Except for cisapride, the IC50 values were similar between assays as shown in Table 2. The IC50 value for cisapride generated with the FLIPR Potassium Assay Kit was closer to the values generated with the FLIPR Tetra System and the IonWorks Barracuda System. The average signal window provided by the FLIPR Potassium Assay Kit was approximately 225% (determined as % baseline) which was significantly higher than the average value of 90% for the FluxOR kit. In each group, n = 8. For statistical analysis, the negative control was chosen at 4 µM terfenadine which completely blocked the hERG channel response, and the positive control was chosen as the stimulus buffer which elicited maximum hERG response. Lower well-to-well variability combined with a larger signal window contributed to the higher Z’ factor obtained with the FLIPR Potassium Assay Kit (Figure 6). This is likely due to the FLIPR Potassium Assay Kit being a truly homogeneous assay that does not require wash steps or media replacements and therefore shows less well-to-well variability.
|Compound||IC50 FLIPR Potassium Assay Kit||IC50 FluxOR Kit|
The FLIPR Potassium Assay Kit measures the functional activity of potassium channels using a homogeneous, no-wash protocol. In this study, we used a set of reference hERG blockers to demonstrate the tight correlation of results from the FlexStation 3 reader with data collected using the FLIPR Tetra System and electrophysiology methods.
In a separate set of experiments, the FLIPR Potassium Assay Kit displayed a significantly larger assay window and a higher Z’ factor when compared to a competitor product. The improved assay quality, in combination with either the medium-throughput capability of the FlexStation 3 Multi-Mode Microplate Reader or the high-throughput capability of the FLIPR Tetra System, provides a powerful platform for analyzing hERG liability during earlier phases of the drug discovery process.
Compatible with these Molecular Devices systems
- Characterization of hERG channel blockers using the FLIPR Potassium Assay Kit, Application Note, 2015.
- D. Rampe, et al, A mechanism for the proarrhythmic effects of cisapride (Propulsid): high affinity blockade of the human cardiac potassium channel HERG, FEBS Letters 1997; 417(1): 28-32.
- Karen Cook, James L. Costantin, and Xin Jiang, Validation of the IonWorks Barracuda System for hERG Ion Channel Assay, Application Note, 2011.