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ISSN: 1933-6950 (Print) 1933-6969 (Online) Journal homepage: http://www.tandfonline.com/loi/kchl20

Role of hERG potassium channel assays in drug

development

Birgit Priest, Ian M. Bell & Maria Garcia

To cite this article: Birgit Priest, Ian M. Bell & Maria Garcia (2008) Role of hERG potassium
channel assays in drug development , Channels, 2:2, 87-93, DOI: 10.4161/chan.2.2.6004

To link to this article: https://doi.org/10.4161/chan.2.2.6004

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[Channels 2:2, 87-93; March/April 2008]; ©2008 Landes Bioscience
Review
Role of hERG potassium channel assays in drug development
Birgit T. Priest,1,* Ian M. Bell2 and Maria L. Garcia1
1Department of Ion Channels; Merck Research Laboratories; Rahway, New Jersey USA;
Pennsylvania USA
Abbreviations: ECG, electrocardiogram; hERG, human ether-a-go-go related gene; LQTS, long QT syndrome; PPC, population patch
clamp
Key words: hERG, arrhythmia, drug development, torsades de pointes, potassium channel, assay development
Numerous structurally and functionally unrelated drugs block
the hERG potassium channel. HERG channels are involved in
cardiac action potential repolarization, and reduced function of
hERG lengthens ventricular action potentials, prolongs the QT
interval in an electrocardiogram, and increases the risk for poten‑
tially fatal ventricular arrhythmias. In order to reduce the risk
of investing resources in a drug candidate that fails preclinical
safety studies because of QT prolongation, it is important to
screen compounds for activity on hERG channels early in the lead
optimization process. A number of hERG assays are ­available,
ON
ranging from high throughput binding assays on stably expressed
.D
recombinant channels to very time consuming electrophysiological
examinations in cardiac myocytes. Depending on the number
CE
of compounds to be tested, binding assays or functional assays
measuring membrane potential or Rb+ flux, combined with electro‑
IEN
physiology on a few compounds, can be used to efficiently develop
the structure‑function relationship of hERG ­interactions.
SC
Introduction
BIO
In drug development, it is advantageous to discover potential
safety liabilities associated with a structural series before significant
resources have been invested. This process involves early counter
screening against related targets, such as a panel of kinases for a
ES
program targeting p38 kinase or other potassium channels for a
program directed against a member of this class of ion channels.
ND
Undesirable activities that are unrelated to the biological target of
interest are much more difficult to address at the early stages and are
LA
often only discovered during preclinical safety studies, after significant
resources have been devoted to the program. One off‑target activity
08
that is frequently encountered is block of hERG potassium channels,
which has the potential to cause life‑threatening arrhythmias. This is
20
a particular troublesome problem due to the fact that block of hERG
may be difficult to detect during routine preclinical and clinical
©
safety studies. In this work, we discuss the need for hERG screening
and the types of hERG assays that are currently available.
*Correspondence to: Birgit T. Priest; Department of Ion Channels; Merck Research
Laboratories; Rahway, New Jersey USA; Email: birgit_priest@merck.com
Submitted: 05/02/08; Accepted: 05/05/08
Previously published online as a Channels E-publication:
http://www.landesbioscience.com/journals/channels/article/6004
www.landesbioscience.com
hERG assays
2Department of Medicinal Chemistry; Merck Research Laboratories; West Point,
.
E
UT
RIB
What Is hERG?
IST
Technically, hERG is the name of a human gene and stands for
human ether‑a‑go‑go related gene. The name is based on homology to
a gene found in Drosophila melanogaster that was named ether‑a‑go‑go
D
(eag) because of the ether‑induced leg shaking observed in flies with
mutations in this gene.1 According to the new nomenclature, hERG
OT
is KCNH2. The protein encoded by this gene will be referred to as the
hERG channel, as is common practice, although the newer nomen-
clature refers to it as Kv11.1.2 The hERG channel is a voltage‑gated
potassium channel and, like other voltage‑gated potassium channels,
it is highly selective for potassium and is a tetramer formed of four
subunits, each containing six transmembrane domains (Fig. 1). Two
alternative splice forms, hERG1a and hERG1b, exist, which differ
only at the N‑terminal domain.3 The two splice forms can assemble
to form functional homo‑ or hetero‑tetramers with somewhat
different channel kinetics.4 Generally, hERG channels are involved
in action potential repolarization. In humans, the hERG channel
is expressed widely, including in the brain, adrenal gland, thymus,
retina, and in cardiac and smooth muscle tissues.1,5 In the heart,
ERG1 channels appear to be the only members of this potassium
channel family, whereas two related channels, ERG2 and ERG3, are
expressed in the nervous system.6 Although in heterologous expres-
sion systems, the hERG channel can associate with the β‑subunits
minK (KCNE1) and MiRP1 (KCNE2),7,8 the existence of such
complexes in native tissues remains a subject of debate.
HERG channels differ from other voltage‑gated potassium
channels in the relative kinetics of activation and inactivation.9 In
hERG channels, the development and reversal of inactivation is
fast compared to the activation and deactivation steps, leading to a
characteristic increase in current upon repolarization as illustrated in
Figure 2.
Why and When to Screen on hERG
In the heart, hERG channels are the molecular correlate of the IKr
current which, together with other potassium currents, is involved
in action potential repolarization.9,10 Reduced function of hERG
causes action potential prolongation, which in rare cases can lead
to the potentially fatal ventricular tachyarrhythmia Torsades de
Pointes.11,12 In a body surface electrocardiogram (ECG), ventricular
action potential prolongation manifests itself as a prolongation of
Channels 87

Figure 1. Voltage-gated potassium channel family. (A) Diagram showing the predicted topology of an
individual potassium channel α and the minK-related β subunit (left), and the arrangement of the pore
forming subunits in a potassium channel tetramer (right). (B) Family tree for voltage-gated potassium
channels.
Figure 2. hERG channel gating. Conformational state diagram (top), exem-
plary current traces (middle) and voltage protocol (bottom) describing hERG
channel gating.
the QT interval or the period between the beginning of the QRS
complex and end of the T wave (Fig. 3).
A particular form of inherited long QT syndrome (LQTS), LQTS2,
has been linked to loss‑of‑function mutations in hERG.11,13,14 While
LQTS2 is typically not associated with any clinical symptoms,
except occasional fainting in some patients, it greatly increases an
­individual’s risk for Torsades de Pointes.
In analogy with the inherited syndrome, certain drugs that
inhibit hERG channels can cause LQTS (acquired or drug‑induced
88
hERG assays
LQTS) and an increased risk of Torsades de
Pointes. This was first demonstrated in the
late 1980s and 1990s for the histamine H1
receptor antagonist terfenadine (Fig. 4). In
1989, overdoses of terfenadine were shown
to prolong the QT interval.15 Terfenadine
use was linked to Torsades de Pointes in
1990,16 and terfenadine was subsequently
shown to inhibit the delayed rectifier
potassium current in isolated myocytes,
and hERG channels expressed in Xenopus
oocytes.17,18 The link between QT prolon-
gation and Torsades de Pointes is complex,
and not all drugs that prolong the QT
interval carry the same risk of arrhythmia.
However, QT interval prolongation is the
only known surrogate for an increased
risk for Torsades de Pointes.19 Based on
this increased risk, a significant number of
drugs that prolong the QT interval, ranging
from the antihistamines terfenadine and
astemizole to the antipsychotic droperidol
(Fig. 4), have been withdrawn from the
market, while others have received black
box warning labels.20 Although the correla-
tion between block of hERG and Torsades de Pointes is not perfect,
the potential for putting patients at risk means that an ­interaction of
a compound with the hERG channel must be taken very seriously.
The enormous cost of drug development, coupled with the desire
to avoid late‑stage compound failures, motivates pharmaceutical
companies to test compounds for inhibition of hERG early on in the
lead optimization process.
Medium and High Throughput hERG Assays
The ideal hERG assay provides a linear measure of channel
activity under physiologically relevant conditions. The closest to this
ideal would be voltage clamp recordings in cardiac myocytes using a
cardiac action potential waveform as the voltage command. However,
such a study is extremely laborious and only amenable to the detailed
characterization of very few selected compounds. As previously
noted, it is advantageous to screen compounds for hERG activity
early on in the lead evaluation and optimization process. However,
this approach requires testing of hundreds and potentially thousands
of compounds within a single drug discovery program. Although
the development of automated electrophysiology ­technologies has
improved the throughput of electrophysiological methods, radioli-
gand binding, fluorescent or ion flux assays may serve as efficient and
robust assays for examining the potential hERG liabilities of a large
number of compounds.
Electrophysiology. Electrophysiology can provide detailed and
quantitative information on the potency and mechanism of hERG
block by a test compound. One of the unique advantages of such
voltage clamp recordings is the ability to control membrane poten-
tial. Since activation and inactivation of hERG is dependent on
membrane potential, voltage clamp recordings can differentiate
between compounds that preferentially interact with different states
of the channel.
Channels 2008; Vol. 2 Issue 2
 hERG assays

A major limitation of all automated

e­ lectrophysiology platforms is the high cost of the
instruments and consumables. Another concern
with regard to the automated platforms is the poten-
tial for results that are inconsistent with those from
conventional voltage clamp assays. Typically, hERG
potencies measured by automated and manual
electrophysiology are in good agreement; however,
some compounds are known to appear less potent
when tested by automated electrophysiology.23‑26
Discrepancies typically arise from hydrophobic
compounds being adsorbed to the surface of the plate
used to hold the compound or to the patch plate
itself. Glass‑coated compound plates can help to
minimize the nonspecific binding to the compound
plate but come at a significant cost. Multiple
compound additions can mitigate problems caused
by compounds sticking to the patch plate substrate
and are common practice.
Flux assays. An alternative to either manual or
automated electrophysiology is a functional assay that
measures ion flux across cell or vesicle membranes.
This assay offers higher throughput than the auto-
Figure 3. Electrocardiogram and cardiac action potential trace corresponding to one heart- mated voltage clamp protocols and takes advantage
beat. Adapted with permission from Macmillan Publishers Ltd: Nature Reviews: Drug Discovery of the ability of Rb+ to permeate through hERG
(Fermini & Fossa, 2003, Nature Rev Drug Disc 2:439–447), copyright 2003. channels.27 Typically, cells are loaded with Rb+
overnight and hERG‑dependent Rb+ efflux is initi-
Several higher throughput automated electrophysiological ated by an addition of high (50–60 mM) extracellular potassium
­instruments have been developed and typically use a planar substrate concentrations to depolarize the cell and open hERG channels. The
with holes, illustrated in Figure 5, that replaces the traditional patch amount of Rb+ efflux can be calculated by using 86Rb+ as a radioac-
pipette.21,22 Cells that seal to the substrate around a hole may be tive tracer or by flame atomic absorption spectrometry (FAAS).27,28
voltage clamped, if the section of plasma membrane covering the Such flux assays, which can operate in 96‑ and 384‑well formats,
hole can be ruptured manually, by applying negative pressure, or afford the testing of several thousand compounds per day. They
perforated using an antibiotic such as amphotericin, allowing elec- offer a robust measure of channel activity, but lack voltage control
trical access to the cell’s cytoplasm. Currently, the highest throughput and temporal resolution. In two reports, using either HEK‑293 or
devices available are the IonWorks HT and IonWorks Quattro. Both CHO‑K1 cells stably expressing recombinant hERG channels, IC50
instruments use a disposable 384‑well PatchPlate™ and a common values determined in a Rb+ flux assay were approximately 10‑fold
ground chamber and achieve increased throughput through synchro- higher than those determined by electrophysiology, but the rank
nous recording from 48 wells combined with simple and versatile order of compounds according to potency was similar for the two
software. Typical seal resistances are on the order of 100 mega‑Ohms assay types.29,30
for IonWorks HT and 50 mega‑Ohms for IonWorks Quattro. Whole Fluorescence‑based assays. The development of improved
cell access is obtained by perfusion with a membrane‑perforating ­fluorescent dyes and plate readers has provided another approach to
agent, typically amphotericin B. The IonWorks Quattro features a high throughput screening of ion channel activities. Fluorescent dyes
similar hardware and software design but uses an approach called which are sensitive to changes in membrane potential have proved
population patch clamp (PPC). PPC involves a modified PatchPlate especially useful for studying the pharmacology of many ion channel
with 64 holes per well and is especially advantageous when ­expression types.31,32 However, studying hERG by this approach ­presents
levels are not homogeneous from cell to cell. However, recording in a challenge since this channel does not typically control a cell’s
PPC mode requires a minimum success rate of sealing to holes and a resting membrane potential. It has been possible, however, to select
sufficiently high seal resistance to avoid short circuit currents through HEK‑293 and CHO‑K1 cell lines stably expressing recombinant
holes not occluded by a cell. hERG channels that give rise to a depolarization signal in response to
Other automated electrophysiology instruments, such as the addition of extracellular potassium (50–60 mM).29,30 At least in the
PatchXpress (Molecular Devices), the QPatch (Sophion Biosciences) HEK‑293 background, this signal is the sum of hERG‑dependent
and the Patchliner (Nanion Technologies GmbH) also use planar and hERG‑independent components; a complication that prob-
substrates, but record asynchronously from individual wells. ably contributes to the low signal‑to‑noise ratio observed in this
Currently, 16‑well or 48‑well formats are available. High resistance assay. In both cell lines, IC50 values were significantly right‑shifted
seals and flexible voltage protocols result in recordings that rival compared to those determined by electrophysiology, regardless of
traditional manual electrophysiology in quality. whether DiBAC4(3) (Invitrogen, Carlsbad, CA), FMP or Blue

www.landesbioscience.com Channels 89
 hERG assays
Figure 4. Structures of selected compounds that bind to the hERG channel.
Membrane Potential Indicator Dye (both from Molecular Devices,
Sunnyvale, CA) was used to monitor membrane potential. The
lack of sensitivity of these membrane potential assays resulted in
a high incidence of “false negatives”. Moreover, the rank order of
compounds by IC50 differed between the membrane potential assay
and ­electrophysiological determinations.
To avoid some of the problems arising from non‑hERG ­potassium
currents, it may be possible to take advantage of known activators, such
as PD‑307243,33 to open hERG channels under physiological condi-
tions which will yield a hERG‑dependent hyperpolarization. One
concern associated with this assay format is the potential ­interference
of the channel activator with binding of the test compound.
Yet another approach to hERG fluorescence‑based assays involves
dyes, such as FluxOR™ (Invitrogen, Carlsbad, CA), that are sensi-
tive to intracellular thallium concentrations, since thallium is known
to permeate through hERG channels. Such an assay provides a more
direct measurement of hERG activity and may be comparable to Rb+
flux assays, but with the benefit of being amenable to a 1536‑well
screening format.
In all fluorescent assays there is the potential for interactions
between the test compound and the dye that result in quenching of
the fluorescent signal, and this may limit the range of compound
concentrations that can be tested reliably.
Binding assays. Radioligand binding assays have been used
extensively to screen for interaction with the hERG channel. These
are non‑functional assays, usually performed using isolated cell
membranes, and they rely on competition or allosteric coupling
between the binding sites for the test compound and for the radioli-
gand. Although they do not provide a direct measure of IKr blockade,
such binding assays can test 50,000 to 100,000 compounds per day
90 Channels
Figure 5. Generalized scheme depicting the recording configurations used in
conventional (manual) and automated electrophysiology recordings.
and are relatively inexpensive, which is why they are commonly
used in most large pharmaceutical companies. A number of
well‑characterized radioligands for hERG have been described,
including [3H]‑dofetilide,34 [35S]‑MK‑499,35 [3H]‑astemizole,36 and
[125I]‑BeKm‑1.37 In general, binding assays using [3H]‑dofetilide,
[35S]‑MK‑499 and [3H]‑astemizole (Fig. 4) are very robust and
reproducible and IC50 values typically correlate well with those
determined in electrophysiology.30,36,38,39 These three non‑peptide
radioligands, in analogy with most small molecule hERG blockers,
are thought to bind in the large inner cavity of the hERG channel
and require channel opening to access their binding sites. In contrast,
the scorpion venom peptide BeKm‑1 preferentially blocks channels
in the closed state and binds to the extracellular S5‑pore linker of
the channel.40,41 The binding site for [125I]‑BeKm‑1 may be allos-
terically coupled to the inner cavity, since E‑4031, which is believed
to bind to the inner cavity, inhibits binding of [125I]‑BeKm‑1 to
­recombinant hERG channels expressed in HEK293 cells.37
Binding assays do not provide detailed information regarding the
nature of the interaction of the test compound with hERG, such
as the ability to block or activate the channel or a preference for a
particular state of the channel. Despite this limitation and the fact
that [35S]‑MK‑499 itself binds with a Kd of <  1 nM but blocks
hERG with an IC50 of approximately 30 nM, agreement between
compound potencies in [35S]‑MK‑499 binding and voltage clamp
assays is remarkably high (reviewed in ref. 38, personal observation).
Radioligand binding assays using isolated membranes differ from
whole cell functional assays in that they are amenable to a range of
assay conditions that may impact on the ability of test compounds to
bind.38 These include temperature, osmolarity, relative concentration
of organic solvent, and K+ concentration. In addition, binding assays
typically employ time periods of drug exposure significantly longer
than most functional assays, which may allow for a more accurate
2008; Vol. 2 Issue 2
 hERG assays
Figure 6. Homology model of hERG generated from the KcsA crystal structure
with MK-499 docked to its putative binding site.
determination of IC50 values for very potent compounds that require
a significant period of time to equilibrate.
Evaluation of Cardiac Risk
Measurements of action potential duration. As an alternative
to assays that utilize recombinant hERG channels expressed in
mammalian cell lines, it is possible to examine native hERG chan-
nels in myocardial cells from several species. The most common
tissue sources are isolated Purkinje fibers from guinea pig, rabbit or
dog.42‑45 The contribution of hERG to repolarization and action
potential prolongation in these tissues is similar to the human heart,
in contrast to the rat heart, for which hERG does not contribute
significantly to repolarization.
Although measurements of action potential repolarization are very
labor intensive and can only be used to study a limited number of
compounds, they can provide valuable data since they examine effects
on native cardiac channels. This is potentially important since the
precise molecular composition of the channel that controls the IKr
current remains a matter of debate. Additionally, these assays can detect
effects on a number of cardiac channels in addition to hERG.43
In vivo ECG measurements. The most reliable measure of
cardiac safety is probably afforded by ECG recordings in conscious
or anesthetized animals,43,46,47 and new drug applications require
these data. ECG measurements are only practical for a very small
number of select compounds. At the same time, such in vivo studies
are the only methods of assessing cardiac safety that take into account
the safety liabilities of the test compound and all its metabolites
combined. They also allow a direct evaluation of other potential
complications, such as plasma protein binding, tissue distribution
and poly‑pharmacology.
The most commonly used species for these measurements is the
dog.47 Guinea pigs may also be used but are technically more chal-
lenging.48 If metabolite profiles are expected to differ between man
and dog, ECG measurements can also be made in primates.49,50 In
addition to the choice of species, the most important choice for ECG
www.landesbioscience.com
measurements is between the use of conscious animals implanted
with telemetry devices and anesthetized animals.51 Advantages of
the anesthetized animals include greater reproducibility of the data
and the potential for electrical pacing to study effects of heart rate.
Disadvantages include the preclusion of oral dosing and the potential
for interference of the anesthetic with normal cardiac function or
with metabolism of the test compound.
The effects of a test substance on QT interval can be obscured
by changes in heart rate since the QT interval is inversely correlated
with heart rate.52 Therefore, it is common to report QT intervals
corrected for the preceding RR interval (QTc). A number of correc-
tion methods exists that attempt to normalize QT intervals to an RR
interval of 1000 ms, corresponding to a heart rate of 60 beats per
minute.52‑55
While ECG measurements directly evaluate the effect of a
particular test compound on the QT interval, they may not be the
best choice to evaluate the potential off‑target activity of a class of
compounds on hERG channels since other factors may affect the
QT interval and either obscure an effect on IKr or complicate the
interpretation of the results.56
In Silico Modeling Predictions
Compounds representing a wide range of chemical structures
(Fig.  4) have been shown to block hERG. Alanine‑scanning muta-
genesis studies on the pore‑lining S6 segment and parts of the pore
helix identified a number of residues involved in conferring high
affinity for MK‑499,57 including threonine and valine (T623 and
V625) residues in the pore and glycine, tyrosine and phenylalanine
residues in S6 (G648, Y652, F656). The two aromatic residues in S6,
Y652 and F656, also contribute to the block of hERG by terfenadine,
cisapride, dofetilide and quinidine.57‑59 These residues are conserved
in ERG and EAG channels, but are not found in other voltage‑gated
potassium channels. Modeling studies have suggested that F656 may
be involved in π‑stacking interactions with aromatic rings in hERG
ligands, while Y652 may interact with the charged amine that is
present in many compounds that bind to this channel.60
A homology model based on the crystal structure of the ­bacterial
potassium channel KcsA suggested that the cavity lined by the
membrane‑spanning sections of hERG may be larger than the cavi-
ties found in most ion channels.57 Figure 6 shows a model of hERG
with MK‑499 docked using the FLOG (flexible ligands oriented
on grid) procedure. Together with the interactions afforded by the
unique aromatic residues in S6 of hERG, the larger size of the cavity
may explain the ability of hERG channels to accommodate binding
of a wide variety of structurally distinct compounds. While hERG
homology models can help to explain the promiscuity relative to
other potassium channels, they are less useful in predicting the ability
of test compounds to block hERG.
A different modeling approach involves quantitative
­structure‑activity relationship (QSAR) modeling.60 This approach
takes advantage of known hERG blockers that are used as a training
set to generate a pharmacophore model, which can then be tested
against a test set. Several pharmacophore models have been devel-
oped and can be used to prioritize compounds for hERG testing.
Since these models are generally better at predicting lack of hERG
activity, they can be used as a screening tool to reduce the cost of
hERG testing in the in vitro assays.
Channels 91

hERG Trafficking
More recently, effects on hERG trafficking have been identified as
a mechanism for drug‑induced LQTS.61 Drugs that reduce hERG/
IKr currents through interfering with hERG trafficking include
arsenic trioxide, pentamidine and the cardiac glycosides digoxin, and
ouabain.62‑64 A number of other drugs block hERG directly and
interfere with trafficking. These include the antidepressant fluoxetine
and the cholesterol‑lowering agent probucol.65,66 The mechanisms
by which drugs interfere with hERG trafficking are largely unknown.
In some cases, interactions with chaperones such as Hsp90 may
prevent proper folding of hERG;67 whereas in other cases drugs
appear to trap hERG in the endoplasmic reticulum.64 Most in vitro
and in vivo assays currently used to detect hERG inhibition and
QT prolongation focus on acute effects and are not likely to detect
drug effects on channel trafficking. Electrophysiology, Rb+ flux and
whole cell binding assays yield signals that are proportional to surface
channel expression and could therefore be adapted to detect drug‑in-
duced changes in hERG trafficking. In addition, an antibody‑based
chemiluminescent assay has been developed to specifically monitor
effects on hERG cell surface expression.68
hERG Activators
Recently, several hERG activators have been identified.69‑71 As
expected these compounds shorten the ventricular action ­potential
duration and the QT interval. Several forms of familial short QT
syndrome have been identified, one of which has been linked
to mutations in hERG.72 Familial short QT syndrome is associ-
ated with atrial and ventricular arrhythmias and sudden death,
suggesting that activation of hERG carries similar safety liabilities
as hERG blockade.
Summary
In order to reduce the risk of a drug candidate failing in preclinical
safety studies because of blockade of hERG channels and associated
QT prolongation, it is important to screen compounds for activity
on hERG channels early in the lead optimization process. However,
the earlier in the process hERG screening is to occur, the higher the
required assay throughput. Although binding assays do not measure
hERG function, they offer a rapid, robust and inexpensive measure
of the ability of compounds to interact with the hERG channel.
Combined with electrophysiology on a few benchmark compounds
to characterize the nature of the compound‑channel interaction,
binding assays can be used as an efficient means to develop the
­structure‑function relationship of hERG interactions within a
structural class of test compounds. This strategy should facilitate
the development of new small molecule drugs with improved
­cardiovascular safety profiles.
Acknowledgements
The authors thank Jill Williams and Chris Culberson for
­assistance in the preparation of figures.
92
hERG assays
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