HERG TESTING

hERG Screening

While arrhythmia, QT prolongation, and action potential parameters have been explicitly recommended as test criteria [ICH (Expert Working Group (Safety) of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use), 2005], the desire to assess and correct arrhythmogenic risk as early in the drug development programme as possible has led to an emphasis on extensive hERG testing. Originally hERG blockade was detected by manual electrophysiology, and because of the limitations of Xenopus oocytes, patch clamp of mammalian cell lines heterologously expressing hERG became the ‘gold standard’ test for hERG liability; recently the gold standard has been moving toward assays performed at physiological temperature as the issues of temperature-dependence of hERG blockade and the ability to make measurements at 37ºC become more prevalent [Witchel et al., 2003; Kirsch et al., 2004].

Heterologous hERG expression is generally preferred over ‘native’ IKr current due to the lack of contaminating currents, increased signal size, and comparability with extant data.  As high throughput drug development and combinatorial chemistry have multiplied the numbers of compounds tested during drug development, the need for information about hERG blockade by those compounds has escalated radically, and this need has driven the development of more rapid and less costly hERG assays.

Most high throughput assays are based around the multiwell plate format.  Because this was not suitable for traditional electrophysiology, initially other technologies were exploited to estimate hERG liability, including Rb+ efflux, radioligand displacement and fluorescence measurements of downstream hERG effects on membrane potential.  However, hERG current is both time-dependent and voltage-dependent, so in order to estimate drug blockade accurately, one must compare like-with-like, which is non-trivial for these substitute technologies because:

1. hERG function results in a current, and to measure current directly requires simultaneous electrical access to the intracellular compartment of the cell as well as to the extracellular space, and
2. measurements of hERG blockade require carefully timed and controlled changes in the transmembrane voltage.

hERG radioligand displacement (binding) assays have been developed to determine at what concentration test compounds can compete off 3H-dofetilide [Finlayson et al., 2001], 3H-astemizole [Chiu et al., 2004] or 35S-MK-499 [Wang et al., 2003]; these three drugs are high potency hERG-blockers that act inside the inner cavity of the channel [Mitcheson et al., 2000; Zhou et al., 1999; Kamiya et al., 2006].  The advantage of these radioligand binding assays is that they are high throughput and compare accurately with known TdP risk compared to electrophysiological hERG blockade [Webster et al., 2002].  The main disadvantage of the binding assays is underscored by the fact that they theoretically cannot distinguish agonists from antagonists.  The mechanism of the assays’ accuracy is unknown because the assays measure binding (a structure) rather than functional blockade; furthermore, for isolated membranes it is unknown what state (open, closed, inactivated) the channel is in or even what direction the channel is facing.  A further binding competition assay based on a radiolabelled scorpion toxin called BeKm-1 has also been developed [Angelo et al., 2003]; the mechanistic accuracy of this assay is even more controversial, since BeKm-1 binds to the extracellular S5-pore linker [Zhang et al., 2003] rather than to the canonical drug binding site inside the pore cavity.  All hERG binding displacement assays will have problems whenever a test compound can block hERG while acting at a different binding site from that of the radioligand.

By contrast, assays measuring the efflux of rubidium (Rb+, which is a monovalent cation acting as a surrogate for potassium) are functional rather than structural.  Rb+ is a trace element not normally present in quantity in physiological systems that is permeant in most K+ channels, so the cells can be preloaded with Rb+ and then Rb+ conducted into the extracellular medium can be measured either as radioactivity (when using 86Rb+) or by atomic absorption spectroscopy.  The problems with Rb+ efflux assays are that 1) Rb+ does not behave identically to K+ and that 2) changes in the transmembrane potential in these assays are typically induced with high K+ extracellular solution changes, which is not ideal for controlling intracellular voltage.  Rubidium efflux has been used to estimate hERG activity after drug blockade [Tang et al., 2001], but it is not as accurate as more recent high throughput electrophysiological methods [Sorota et al., 2005].

High throughput hERG screening assays have also been developed using fluorescence measurements with membrane potential sensitive dyes such as DiBAC4(3) or the FLIPR membrane potential dye (FMP) [Netzer et al., 2001].  In these assays changes in the transmembrane voltage are assumed to be dependent on whether the hERG channels in the cells remain permeable to K+ in the presence of the drug.  This assay suffers from being a highly indirect measure of hERG current; although it suffers in accuracy, its cost makes it feasible to use as a high throughput screening tool.

Planar patch clamp has been a tremendous advance for identifying compounds with hERG liability [Dubin et al., 2005] because it allows for a multiwell plate format and automation, which is much faster and less labour intensive than manual patch clamp for screening ion channels [Kiss et al., 2003].  In the short time since this technology has become commercially available, it has become very popular with drug companies that can afford the initial capital outlay and the large (compared with FLIPR technology) consumables costs; it could even be said that the needs of the hERG field have driven the technological development of rapid throughput electrophysiology.  The persistent and costly problem of gathering no data from wells in which debris has prevented any cell from reaching the “electrode” hole has been surmounted by using an array of holes (sixty-four of them) in each well and making ensemble current measurements from multiple cells in parallel [Finkel et al., 2006]; this is ideal for hERG liability assays, where the response of an individual cell would normally be combined with measurements from other cells to make a statistical mean.

 

References

Angelo K, Korolkova YV, Grunnet M, Grishin EV, Pluzhnikov KA, Klaerke DA, Knaus HG, Moller M, Olesen SP (2003). A radiolabeled peptide ligand of the hERG channel, [125I]-BeKm-1. Pflügers Arch. 447:55-63.
Chiu PJ, Marcoe KF, Bounds SE, Lin CH, Feng JJ, Lin A, Cheng FC, Crumb WJ, Mitchell R (2004). Validation of a [3H] astemizole binding assay in HEK293 cells expressing HERG K+ channels. J. Pharmacol. Sci. 95:311-319.
Dubin AE, Nasser N, Rohrbacher J, Hermans AN, Marrannes R, Grantham C, Van Rossem K, Cik M, Chaplan SR, Gallacher D, Xu J, Guia A, Byrne NG, Mathes C (2005). Identifying modulators of hERG channel activity using the PatchXpress planar patch clamp. J. Biomol. Screen. 10:168-181.a
Finkel A, Wittel A, Yang N, Handran S, Hughes J, Costantin J (2006). Population patch clamp improves data consistency and success rates in the measurement of ionic currents. J. Biomol. Screen. 11:488-496.
Finlayson K, Turnbull L, January CT, Sharkey J, Kelly JS (2001). [3H]dofetilide binding to HERG transfected membranes: a potential high throughput preclinical screen. Eur. J Pharmacol. 430:147-148.
ICH (Expert Working Group (Safety) of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) (2005). The non-clinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals. FDA (Food and Drug Administration, USA): Rockville, Maryland, USA.
Kamiya K, Niwa R, Mitcheson JS, Sanguinetti MC (2006). Molecular determinants of HERG channel block. Mol. Pharmacol. 69:1709-1716.
Kirsch GE, Trepakova ES, Brimecombe JC, Sidach SS, Erickson HD, Kochan MC, Shyjka LM, Lacerda AE, Brown AM (2004). Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern. J. Pharmacol. Toxicol. Methods 50:93-101.
Kiss L, Bennett PB, Uebele VN, Koblan KS, Kane SA, Neagle B, Schroeder K (2003). High throughput ion-channel pharmacology: planar-array-based voltage clamp. Assay. Drug Dev. Technol. 1:127-135.
Mitcheson JS, Chen J, Lin M, Culberson C, Sanguinetti MC (2000). A structural basis for drug-induced long QT syndrome. Proc. Natl. Acad. Sci. U. S. A. 97:12329-12333.
Netzer R, Ebneth A, Bischoff U, Pongs O (2001). Screening lead compounds for QT interval prolongation. Drug Discov. Today. 6:78-84.
Sorota S, Zhang XS, Margulis M, Tucker K, Priestley T (2005). Characterization of a hERG screen using the IonWorks HT: comparison to a hERG rubidium efflux screen. Assay. Drug Dev. Technol. 3:47-57.
Tang W, Kang J, Wu X, Rampe D, Wang L, Shen H, Li Z, Dunnington D, Garyantes T (2001). Development and evaluation of high throughput functional assay methods for HERG potassium channel. J. Biomol. Screen. 6:325-331.
Wang J, Della PK, Wang H, Karczewski J, Connolly TM, Koblan KS, Bennett PB, Salata JJ (2003). Functional and pharmacological properties of canine ERG potassium channels. Am. J Physiol Heart Circ. Physiol 284:H256-H267.
Webster R, Leishman D, Walker D (2002). Towards a drug concentration effect relationship for QT prolongation and torsades de pointes. Curr. Opin. Drug Discov. Devel. 5:116-126.
Witchel HJ, Milnes JT, Mitcheson JS, Hancox JC (2003). Troubleshooting a heterologous potassium channel current used for in vitro screening for QT interval prolongation: HERG, mammalian cell lines and Xenopus oocytes. J. Pharmacol. Toxicol. Methods 48:65-80.
Zhang M, Korolkova YV, Liu J, Jiang M, Grishin EV, Tseng GN (2003). BeKm-1 is a HERG-Specific Toxin that Shares the Structure with ChTx but the Mechanism of Action with ErgTx1. Biophys. J. 84:3022-3036.
Zhou Z, Vorperian VR, Gong Q, Zhang S, January CT (1999). Block of HERG potassium channels by the antihistamine astemizole and its metabolites desmethylastemizole and norastemizole. J. Cardiovasc. Electrophysiol. 10:836-843.