IonWorks® HT

hERG Channel Screening Assay

 
A number of pharmaceuticals have been removed from the clinical market due to their propensity to cause LQT syndrome (acquired LQTS) that can, under certain circumstances, lead to potentially fatal cardiac arrhythmias. The vast majority of pharmaceuticals that cause acquired LQTS do so by blocking the human ether-à-go-go related gene (hERG) potassium channel that is responsible for the repolarization of the ventricular cardiac action potential. The IonWorks™ HT system can be used to perform hERG safety screening experiments to eliminate drug candidates with potentially adverse cardiac side effects early in the drug discovery process.

• hERG channel currents measured with the IonWorks HT system
• hERG pharmacology
• hERG channel safety screen
• Z-factor analysis of mock screening experiment
• Conclusions
• References and recommended reading

hERG channel currents measured with the IonWorks HT system

The hERG potassium channel has voltage-dependent gating properties that make it ideally suited to its principal role in controlling the repolarization phase, and therefore the duration, of the cardiac action potential. These include significant, fast inactivation coupled with slower activation when the voltage is rapidly stepped from a negative potential (-70 mV) to more depolarized potentials (+40 mV); e.g., during the upstroke of the action potential. The consequence is that little hERG current flows during the plateau (depolarizing) phases of the action potential. However as the membrane begins to hyperpolarize, hERG channels recover from inactivation, going through the open state prior to closure. This results in a transient increase in the outward flow of potassium ions (manifested as "tail currents" under voltage clamp), causing repolarization of the membrane and termination of the cardiac action potential. Figure 1 shows hERG currents recorded on the IonWorks HT system.

		Figure 1. hERG potassium channel currents measured with the IonWorks HT system. Pre-compound trace shown in black, blockade with 10 µM dofetilide shown in orange. Note the large "tail currents" at a simulated repolarization holding potential of -30 mV. Vertical bar is 500 pA, horizontal bar is 500 ms. Command voltage protocol shown below traces.

hERG pharmacology

The IonWorks HT system is ideal for rapidly performing high throughput dose response experiments. Eight-point dilution series of four known hERG channel antagonists were made in 96-well compound plate columns by performing 1:3 serial dilutions using a multi-channel pipettor. Concentration-response curves for dofetilide, E-4031, quinidine and sotalol were fitted to a four parameter logistic:

% of control = 100 (1 + ([drug]/IC₅₀)^p)^-1

where IC₅₀ is the concentration of the drug required to inhibit current by 50% and p is the Hill slope. IC₅₀ values for hERG antagonists are shown in Figure 2.

		Figure 2. Dose-response curves from single 45-minute runs. IC50 curves for dofetilide, E-4031, quinidine and sotalol. Mean  ± SEM; n <12 cells/concentration.

hERG channel safety screen

A mock hERG screening experiment was performed by randomly including hERG channel inhibitors (dofetilide, E-4031, terfenadine) among mock "unknowns." Six compound plates were used, each containing eight positive control wells (10 µM cisapride), eight negative control wells (buffer), and eighty wells of mock unknowns: three wells per plate contained mock hits (i.e., hERG inhibitors; see Panel B) and the remaining seventy-seven wells contained buffer. For each PatchPlate™ experiment, every compound was replicated four-fold. A total of six PatchPlate experiments were conducted on a single day (~5-6 hours), representing a 480-compound screen. Software snapshots of two PatchPlate experiments are shown in Figure 3.

		Figure 3. Screen snapshots from IonWorks HT software. Panel A. Pre- (purple) and post-compound (green) traces of hERG currents (in quadruplicate) from compound plate well D2 (shown in panel B) containing mock hit. Panel B. Compound Plate view from two PatchPlate experiments is shown; positive and negative control wells are in column 11 and 12, respectively. Mock hits were randomly placed in compound plate columns 1-10 and shown in yellow (positive control wells also shown in yellow). Each compound well was replicated in the PatchPlate four fold—traces from well D2 (black square) are shown in panel A. Numbers in each well show the number of successful recordings within the quadruplicate set. At the bottom of Panel B is a screen snapshot showing user-defined threshold setting for identifying a successful hit, in this case, hERG inhibition, or where the post-compound read current is reduced by more than 20% of the pre-compound read current.

Z-factor analysis of mock screening experiment

Z-factor statistical analysis of the mock hERG safety screen is summarized in Figure 4. Data was expressed as a percent of the pre-compound current at a -30 mV test potential used to elicit hERG tail currents.

Z-factor was calculated as:

		Figure 4. Mean (± sd) values for mock unknowns were plotted as percent of mean control currents (n=4 per compound) and Z-factor values were determined for each PatchPlate. Positive controls and mock hits were readily identified. Z-factors for each plate are shown at bottom.

conclusions

The IonWorks HT system is optimal for performing safety screens of candidate pharmaceuticals, and indeed has been successfully used in a pharmaceutical setting for hERG studies (Kiss, L. et al., 2003). IonWorks HT has daily throughput levels up to 3,000 successful recordings, or 100 eight-point IC₅₀ curves at n=4; and can be operated by technician-level personnel. For these reasons, the IonWorks HT system is a profound improvement over conventional electrophysiological methods for screening hERG channel blockers. For more details on experiments described on this page, download our application note entitled hERG safety screening assay using IonWorks HT (registration required).

references and recommended reading

Haverkamp, W., G. Breithardt, et al., (2000). The potential for QT prolongation and pro-arrhythmia by non-antiarrythmic drugs: clinical and regulatory implications. Report on a policy conference of the European Society of Cardiology. Eur. Heart. J., 21:1216-1231.

Fermini, B. and A.A. Fossa, (2003). The impact of drug-induced QT interval prolongation on drug discovery and development. Nat. Rev. Drug. Disc., 2:439-447.

Kiss, L., P.B. Bennett, et al., (2003). High throughput ion-channel pharmacology: Planar-array-based voltage clamp. Assay and Drug Dev Technol., 1:127-135.

Mitcheson, J.S., J. Chen, et al., (2000). A structural basis for drug-induced long QT syndrome. Proc. Natl. Acad. Sci., 97:12329-12333.

Roden, D.M., R. Lazzara, et al., (1996). Multiple mechanisms in the Long-QT Syndrome: Current knowledge, gaps, and future directions. Circulation, 94:1996-2012.

Sanguinetti, M.C., C. Jiang, et al., (1995). A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell, 81:299-307.

Ion Channels and Disease. Frances M. Ashcroft, Academic Press, San Diego, CA, 2000.