Trapping and dissociation of propafenone derivatives in HERG channels

Article abstract of DOI:10.1111/j.1476-5381.2010.01159.x

BACKGROUND AND PURPOSE Human ether-a-go-go related gene (HERG) channel inhibitors may be subdivided into compounds that are trapped in the closed channel conformation and others that dissociate at rest. The structural peculiarities promoting resting state

Full text of DOI:10.1111/j.1476-5381.2010.01159.x

DOI:10.1111/j.1476-5381.2010.01159.x
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Correspondence
RESEARCH PAPERbph_1159 1542..1552
Professor S Hering, Department
of Pharmacology and Toxicology,
Institute of Pharmacology,
University of Vienna,
Trapping and dissociation of
propafenone derivatives in
HERG channels
Althanstrasse 14, Vienna
A-1090, Austria. E-mail:
steffen.hering@univie.ac.at
----------------------------------------------------------------
Keywords
arrhythmia; drug trapping;
HERG; K-channels; propafenone;
QT syndrome; voltage clamp;
Xenopus oocyte
A Windisch1, EN Timin1, T Schwarz2, D Stork-Riedler1, T Erker2,
GF Ecker2 and S Hering1
----------------------------------------------------------------
Received
14 July 2010
Revised
1Department of Pharmacology and Toxicology University of Vienna, Vienna, Austria, and
2Department of Medicinal Chemistry, University of Vienna, Vienna, Austria
5 November 2010
Accepted
10 November 2010
BACKGROUND AND PURPOSE
Human ether-a-go-go related gene (HERG) channel inhibitors may be subdivided into compounds that are trapped in the
closed channel conformation and others that dissociate at rest. The structural peculiarities promoting resting state dissociation
from HERG channels are currently unknown. A small molecule-like propafenone is efficiently trapped in the closed HERG
channel conformation. The aim of this study was to identify structural moieties that would promote dissociation of
propafenone derivatives.
EXPERIMENTAL APPROACH
Human ether-a-go-go related gene channels were heterologously expressed in Xenopus oocytes and potassium currents were
recorded using the two-microelectrode voltage clamp technique. Recovery from block by 10 propafenone derivatives with
variable side chains, but a conserved putative pharmacophore, was analysed.
KEY RESULTS
We have identified structural determinants of propafenone derivatives that enable drug dissociation from the closed channel
state. Propafenone and four derivatives with ‘short’ side chains were trapped in the closed channel. Five out of six bulky
derivatives efficiently dissociated from the channel at rest. One propafenone derivative with a similar bulk but lacking an
H-bond acceptor in this region was trapped. Correlations were observed between molecular weight and onset of channel
block as well as between pKa and recovery at rest.
CONCLUSION AND IMPLICATIONS
The data show that extending the size of a trapped HERG blocker-like propafenone by adding a bulky side chain may impede
channel closure and thereby facilitate drug dissociation at rest. The presence of an H-bond acceptor in the bulky side chain is,
however, essential.
Abbreviations
E-4031, 1-[2-(6-methyl-2pyridyl)ethyl]-4-(4-methylsulfonyl aminobenzoyl)piperidine; HERG, human ether-a-go-go related
gene; MK-499, (+)-N-[1′-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-
benzopyran-2,4′-piperidin)-6-yl]methanesulfonamide] monohydrochloride; QT interval, QT interval of the
electrocardiogram; t, time constant; WT, wild type
channel nomenclature follows Alexander et al., 2009). In the
myocardium, this current accelerates the repolarization of the
Introduction
Human ether-a-go-go related gene (HERG) channels conduct
the rapid delayed rectifier K+ current (Sanguinetti et al., 1995;
action potential (Tseng, 2001) and HERG channel inhibition
delays repolarization and prolongs the cardiac action
© 2011 The Authors
British Journal of Pharmacology © 2011 The British Pharmacological Society
1542 British Journal of Pharmacology (2011) 162 1542–1552

Determinants of propafenone trapping in HERG
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potential and QT interval (Haverkamp et al., 2000). The
resulting prolongation of the QT interval can be associated
with syncope and sudden death due to torsades de pointes
ventricular tachycardia and ventricular fibrillation (Viskin,
1999; Keating and Sanguinetti, 2001). Several drugs have
been withdrawn from the market due to HERG channel inhi-
bition, including antiarrhythmics, antimicrobials, neurolep-
tics and antihistamines (Fermini and Fossa, 2003; Sanguinetti
and Tristani-Firouzi, 2006).
Compared with other voltage-activated potassium (Kv)
channels, HERG channels are remarkably sensitive to a large
variety of structurally diverse drugs (Mitcheson and Perry,
2003). This ‘pharmacological promiscuity’ (Mitcheson, 2008)
of HERG is apparently related to its structural and functional
peculiarities. Most drugs inhibit HERG channels more effi-
ciently in the open and/or inactivated state (for review, see
Zou et al., 1997 and Sanguinetti and Tristani-Firouzi, 2006).
There is evidence that drugs can be trapped in the channel
pore (Mitcheson et al., 2000; Kamiya et al., 2001; Perry et al.,
2004; Stork et al., 2007). The molecular mechanism of ‘drug
trapping’ in HERG channels is currently unknown.
recovered from block by amiodarone, cisapride, droperidol
and haloperidol (Stork et al., 2007). No correlation between
drug descriptors (size, volume, lipophilicity and accessible
surface area) and the observed differences in drug trapping
could, however, be established (Stork et al., 2007). We also
gained preliminary insights into the molecular basis of trap-
ping of the class 1C antiarrhythmic agent propafenone by
docking a small set of compounds into protein homology
models of the open and the closed state of the HERG channel
(Thai et al., 2010).
In Thai et al. (2010), we classified a small set of pro-
pafenone derivatives as ‘trapped’ or ‘non-trapped’, based on
theoretical considerations (i.e. ligand structures and docking
studies with a homology model). These studies suggested a
role of bulkiness in drug dissociation because the non-
trapped derivatives analysed in this investigation carried a
large substituent at the aniline nitrogen atom of the pipera-
zine ring. This data set was, however, too small for linking
drug trapping with molecular features of the compounds.
Here we have investigated the structural features of the
ligand that could be linked to drug trapping, by making use
of a systematically modified library of 10 propafenone deriva-
tives. Propafenone itself is efficiently trapped in the HERG
channel pore (Witchel et al., 2004). In particular, we studied
propafenone derivatives with variable side chains while
leaving the basic scaffold of the molecule (acylphenyloxypro-
panolamine) mostly unaffected (Figure 1).
In
a preceding study, we analysed the kinetics of
HERG channel inhibition and drug dissociation of eight
blockers. One group [1-[2-(6-methyl-2pyridyl)ethyl]-4-(4-
methylsulfonyl aminobenzoyl)piperidine (E-4031), domperi-
done, terfenadine and bepridil] did not dissociate from
the closed state at rest while channels almost completely
Figure 1
Chemical structures of propafenone and derivatives. All compounds were studied as hydrochlorides.
British Journal of Pharmacology (2011) 162 1542–1552 1543

A Windisch et al.
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By analysing the kinetics of HERG inhibition and recov-
ery from block we observed that more bulky derivatives tend
to block the channels slowly, in a ‘use-dependent’ manner. At
the same time, most bulky derivatives dissociated from the
closed state at rest while propafenone and its less bulky
derivatives were apparently trapped in the closed channel
pore (Witchel et al., 2004). A bulky derivative (SCT-AS03)
lacking the aniline-nitrogen in the side chain (i.e. carrying a
phenylpiperidine instead of phenylpiperazine) displayed a
slow onset of current inhibition and negligible recovery. Our
data suggest that increased bulkiness and the H-bonding
capabilities of the bulky moiety prevent appropriate channel
closure, thereby enabling dissociation and recovery of the
HERG channels from block.
IHERG,Drug
100 − A
=
+ A
n
H
IHERG,Control
C
1+
(
)
IC50
where IC50 is the concentration at which HERG inhibition is
half-maximal, C is the applied drug concentration, A is the
fraction of HERG current that is not blocked and nH is the Hill
coefficient.
Recovery from channel block
Channel block was induced by applying 1 Hz trains of 14
pulses (conditioning train). Recovery from block at rest
(holding potential -80 mV) was estimated after individual
conditioning trains and subsequent rest periods of 10, 30, 60,
120, 210 or 330 s.
Methods
Data analysis
Data are presented as mean Ϯ SEM from at least three oocytes
from Ն2 batches; statistical significance of differences was
defined as P < 0.05 in Student’s unpaired t-test.
Molecular biology
Preparation of stage V–VI oocytes from Xenopus laevis
(NASCO, Fort Atkinson, WI, USA), synthesis of capped run-
off complementary ribonucleic acid (cRNA) transcripts from
linearized complementary deoxyribonucleic acid (cDNA)
templates and injection of cRNA were performed as described
previously (Sanguinetti and Xu, 1999). Complementary
deoxyribonucleic acids of HERG (accession number
NP_000229) and the mutants F656A and Y652A were kindly
provided by Dr Sanguinetti (University of Utah, UT, USA).
Materials
Propafenone and its derivatives GPV005, GPV009, GPV019,
GPV031, GPV062, GPV180, GPV574, GPV576, GPV929 and
SCT-AS03 (Figure 1; as hydrochlorides) were dissolved in dim-
ethyl sulphoxide to prepare 10 mM stock solutions that were
stored at -20°C. Drug stocks were diluted to the required
concentration in extracellular solution on the day of each
experiment. The maximal dimethyl sulphoxide concentra-
tion in the bath (0.1%) did not affect HERG currents.
Voltage clamp analysis
Currents through HERG channels were studied 1 to 4 days
after microinjection of the cRNA using the two-
microelectrode voltage clamp technique. The extra-
cellular recording solution contained: 96 mM Na
Synthesis of novel propafenone derivatives
Propafenone and derivatives GPV005, GPV009, GPV019,
GPV031, GPV062, GPV180, GPV574, GPV576 and GPV929
were synthesized as described previously (Chiba et al., 1996;
Klein et al., 2002). Synthesis of SCT-AS03 was achieved
2-(N-morpholino)ethanesulphonate,
2 mM
K
2-(N-
morpholino)ethanesulphonic acid, 2 mM CaCl2, 5 mM
HEPES and 1 mM MgCl2, pH adjusted to 7.6 with methane-
sulphonic acid (Sigma-Aldrich Chemie GmbH, Taufkirchen,
Germany).
as
follows:
1-(2-(oxiranylmethoxy)phenyl)-3-phenyl-1-
propanone (500 mg, 1.77 mmol) was dissolved in 2-propanol
(5 mL). Then 4-phenylpiperidine (291 mg, 1.8 mmol) was
added and the reaction mixture was stirred under reflux for
6 h. The solvent was evaporated and the residue was recrys-
tallized from ethyl alcohol (5 mL). To prepare the hydrochlo-
ride, the product was dissolved in diethyl ether followed by
addition of ethereal HCl (1 M). The precipitate was collected
by filtration and provided 417 mg of the orange product with
53% yield. Physical characteristics as follows; 1H-NMR
(200 MHz, CDCl3): d = 7.72 (dd, 1 H, J = 7.7, 1.7 Hz), 7.50–
7.39 (m, 1 H), 7.37–7.12 (m, 10 H), 7.07–6.93 (m, 2 H),
4.19–4.01 (m, 3 H), 3.67 (br s, 1 H), 3.45–3.30 (m, 2 H),
3.11–2.93 (m, 3 H), 2.85–2.70 (m, 1 H), 2.62–2.41 (m, 3 H),
2.40–2.21 (m, 1 H), 2.02–1.63 (m, 5 H). 13C-NMR(50 MHz,
CDCl3): d = 201.4 (Cq), 157.9 (Cq), 146.1 (Cq), 141.8 (Cq),
133.6 (CH), 130.6 (CH), 128.6 (CH), 128.6 (CH), 128.5 (CH),
128.4 (Cq), 126.9 (CH), 126.4 (CH), 126.0 (CH), 121.1 (CH),
112.8 (CH), 71.1 (CH2), 65.4 (CH), 61.3 (CH2), 56.1 (CH2),
52.9 (CH2), 45.8 (CH2), 42.4 (CH), 33.7 (CH2), 33.4 (CH2), 30.4
(CH2). MP: 102–103°C; MP (hydrochloride): 175–178°C. High
Resolution Mass Spectrometry: Calculated: 444.2539; found:
444.2533.
Voltage-recording and current-injecting microelectrodes
were filled with 3 M KCl and had resistances between 0.5 and
2 MW. Currents >3 mA were discarded to minimize voltage
clamp errors. Ionic currents were recorded with a Turbo Tec
03X Amplifier (npi electronic, GmbH, Tamm, Germany) and
digitized with a Digidata 1322A (Axon Instruments, Inc.,
Union City, CA, USA). The pClamp software package version
10.1 (Axon Instruments Inc.) was used for data acquisition.
Microcal Origin 7.0 was employed for analysis and curve
fitting.
A precondition for all measurements was the achievement
of stable peak current amplitudes over periods of 10 min after
an initial run-up period. All drugs were applied by means
of the ScreeningTool fast perfusion system (npi electronic
GmbH, Tamm, Germany) enabling solution exchange within
50–100 ms (Baburin et al., 2006). The oocytes were kept for
3 min at a holding potential of -80 mV to equilibrate drug
diffusion. Use-dependent HERG channel block was estimated
as peak tail current inhibition. The tail currents were measured
at -50 mV, after a step to +20 mV. Cumulative concentration–
inhibition curves were fitted using the Hill equation
1544 British Journal of Pharmacology (2011) 162 1542–1552

Determinants of propafenone trapping in HERG
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Results
Propafenone library
The propafenone derivatives used in the present study are
shown in Figure 1.
The basic side chain of propafenone (GPV001) was prima-
rily altered by the replacement of the n-propylamino group
by di-isopropylamino (GPV009), by 1-piperidinyl (GPV005)
and by a series of 4-aryl-1-piperazinyl moieties with different
substitution pattern on the aryl ring (unsubstituted GPV019,
4-fluoro GPV031, 3,4-dimethyl GPV576). Furthermore, we
also used 4-phenyl-1-piperidinyl (SCT-AS03) and 4-hydroxy-
4-phenyl-1-piperidinyl (GPV062) analogues.
The scaffold of GPV005 was further modified by the
replacement of the 3-phenyl moiety by 1-naphthyl (GPV180)
and a change in the substitution pattern of the central aro-
matic ring, where the 3(-(1-napthyl)-1-propionyl in the ortho
position was shifted to the para position (GPV929). The scaf-
fold of GPV576 was further modified by the replacement of
the ortho-phenylpropionyl moiety by a para-acetyl group
(GPV574).
Potency of HERG inhibition by
propafenone derivatives
Human ether-a-go-go related gene channel inhibition by pro-
pafenone derivatives was studied in Xenopus oocytes by
means of the two-microelectrode voltage clamp technique
(see Methods). HERG channels were activated by a 300 ms
depolarization to 20 mV (see Figure 2A). A subsequent repo-
larization to -50 mV induced a large tail current due to rapid
recovery of HERG channels from inactivation. After pre-
incubation of the oocytes with a given drug concentration,
0.3 Hz pulse trains were applied until steady-state was
reached. Concentration–inhibition relationships for all tested
compounds were estimated by plotting the steady-state
values of current inhibition versus the cumulatively applied
drug concentrations (Figure 2B and C). IC50 values for pro-
pafenone and its derivatives ranged from 0.77 to 5.04 mM
(Table 1).
Onset of HERG inhibition by
propafenone derivatives
The onset of drug action was studied during trains of test
pulses (1 Hz) applied after a 3 min equilibrium period in
drug-containing solution, using the voltage protocol illus-
trated in the inset of Figure 2A. The development of HERG
channel inhibition by propafenone and derivative GPV009 is
illustrated in Figure 3A and B. The onset of channel inhibi-
tion was analysed by plotting the tail current amplitudes
versus time (Figure 3C). While propafenone induced about
90% of steady-state inhibition during the 1st pulse, the onset
of current inhibition by GPV009 was significantly slower
(only 47.0% of steady-state inhibition during the 1st pulse).
These differences prompted us to quantify the rate of onset of
block (well-fitted with a mono-exponential function as exem-
plified in Figure 3C). To analyse the block onset for different
derivatives, we applied different concentrations (ª 3·IC50) to
induce comparable current inhibition. This approach enabled
us to compare the time courses of the onsets of block by
Figure 2
A, superimposed current traces recorded in the absence (control)
and after attaining steady-state block with increasing concentrations
of GPV009 (indicated in the figure). Voltage protocol is shown in the
inset. B and C, concentration–response relationship for the block of
human ether-a-go-go related gene tail current by propafenone and
10 derivatives.
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A Windisch et al.
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Table 1
Experimental data for propafenone and derivatives
Concentration
(mM)
Time constant
of block (s)
Recovery after
330 s (%)
Time constant
of recovery (s)
Compound
IC50 (mM)
Propafenone
GPV005
GPV009
GPV019
GPV031
GPV062
GPV180
GPV574
GPV576
GPV929
SCT-AS03
3.79 Ϯ 0.240
1.34 Ϯ 0.04*
0.84 Ϯ 0.08*
1.42 Ϯ 0.18*
1.46 Ϯ 0.19*
1.05 Ϯ 0.05*
1.78 Ϯ 0.15*
5.04 Ϯ 0.540
1.84 Ϯ 0.24*
1.27 Ϯ 0.05*
0.77 Ϯ 0.11*
10
4
0.44 Ϯ 0.110
0.66 Ϯ 0.11*
1.39 Ϯ 0.14*
1.82 Ϯ 0.14*
1.65 Ϯ 0.18*
2.15 Ϯ 0.27*
0.87 Ϯ 0.15*
0.48 Ϯ 0.090
1.58 Ϯ 0.17*
1.00 Ϯ 0.13*
2.39 Ϯ 0.39*
05.43 Ϯ 12.240
05.68 Ϯ 10.010
01.67 Ϯ 07.220
59.00 Ϯ 02.77*
69.25 Ϯ 07.62*
46.41 Ϯ 02.35*
00.58 Ϯ 09.130
60.55 Ϯ 11.98*
61.03 Ϯ 06.91*
04.55 Ϯ 05.200
12.26 Ϯ 04.290
–
–
3
–
4
72.31 Ϯ 16.53
4
97.60 Ϯ 27.25
3
44.59 Ϯ 16.51
5
–
15
6
61.09 Ϯ 46.05
84.74 Ϯ 23.34
4
–
–
2
Data are given as means Ϯ SEM from at least three experiments. Drug concentrations (ª3·IC50) for studying onset of and recovery from HERG
channel block were chosen to induce substantial and comparable current inhibition.
*P < 0.05, significantly different from propafenone (unpaired Student’s t-test).
compounds with different potencies. The time constants for
all derivatives are given in Table 1.
Interestingly, a slower onset of channel inhibition was
associated with higher apparent affinity (lower IC50 value).
The mean fractions of recovered HERG currents after a
330 s rest at -80 mV are given in Figure 5A and in Table 1.
Negligible recovery (<13% after 330 s at rest) was observed in
the presence of propafenone, GPV005, GPV009, GPV180,
GPV929 and SCT-AS03 while substantial recovery was
observed for GPV019, GPV031, GPV574 and GPV576 (range
of recovery: 59.0–69.3%). An intermediate recovery value
(46.4 Ϯ 2.4%) was determined for GPV062.
Thus, SCT-AS03 displayed the slowest onset with a t
=
block
2.39 Ϯ 0.39 s corresponding to the highest apparent affinity
with an IC50 = 0.77 Ϯ 0.11 mM. The fastest block was induced
by propafenone (t
= 0.44 Ϯ 0.11 s) followed by GPV574
block
(t
= 0.48 Ϯ 0.09 s) with corresponding IC50 values of
block
3.79 Ϯ 0.24 mM and 5.04 Ϯ 0.54 mM respectively (see Table 1
for details). This inverse relation between the time constants
of block development and the apparent affinities is illustrated
in Figure 3D. As shown for sodium channels (Courtney,
1980), we observed a correlation with the size (molecular
weight) of the propafenone derivatives (Figure 3E).
Channel opening accelerates recovery from
block during ‘wash-out’
As illustrated in Figure 5A, propafenone and its derivatives
GPV005, GPV009, GPV180 GPV929 and SCT-AS03 did not
recover from block during 330 s at rest. To study whether this
lack of recovery results from drug trapping in the closed
channel pore, we attempted to induce drug wash-out by
repetitive channel opening (see Stork et al., 2007).
Human ether-a-go-go related gene currents were therefore
inhibited by conditioning trains (14 pulses at 1 Hz). Drug
wash-out was started immediately after the conditioning
train by means of a fast perfusion system (Baburin et al.,
2006). Repetitive pulsing enabled drug dissociation. This is
illustrated in Figure 5B where the application of 0.1 Hz pulses
after 8 min rest (without recovery) induced fast recovery from
block by GPV009. Furthermore, pulse-induced recovery was
frequency-dependent. Higher frequency pulsing (0.1 Hz)
induced faster restoration of HERG currents than lower fre-
quency pulsing (0.03 Hz, Figure 5B).
Recovery from block at rest
Recovery from HERG channel block by propafenone deriva-
tives (induced by 1 Hz pulse conditioning train, see Figure 4)
was determined by applying test pulses after rest periods of
10, 30, 60, 120, 210 and 330 s. Each data point was measured
after an individual conditioning train (see Methods). HERG
channels blocked by propafenone and derivatives GPV005,
GPV009, GPV180, GPV929 and SCT-AS03 did not recover at
rest. This is exemplified for GPV009 in Figure 4C and D.
Figure 4C illustrates three superimposed currents: (i) the
current during the 1st pulse of the conditioning train in the
presence of GPV009 (3 mM); (ii) a current during steady-state
inhibition (14th pulse of the conditioning train); and (iii) the
current after a 330 s rest period in the presence of drug.
Figure 4D illustrates the gradual decline of the peak currents
in GPV009 and the lack of recovery from block after 330 s.
Mono-exponential recovery from block at rest was
observed in the presence of GPV019, GPV031, GPV574 and
GPV576 (shown in Figure 4A and B for compound GPV031,
4 mM).
Evidence for interaction with putative binding
determinants F656 and Y652
To study if the compounds interact with the putative binding
pocket of propafenone, we analysed their action on selected
HERG channel mutants. Alanine scanning mutagenesis
revealed that the putative binding sites of most HERG
1546 British Journal of Pharmacology (2011) 162 1542–1552

Determinants of propafenone trapping in HERG
BJP
Figures 3
A–C, use-dependent block of human ether-a-go-go related gene currents by propafenone and GPV009. A and B, superimposed current traces
during repetitive stimulation at a frequency of 1 Hz under application of 3 mM propafenone (A) or 3 mM GPV009 (B). Control currents are shown
as dotted lines. C, peak tail current amplitudes are plotted versus time. D, exponential trend of the time constants of block development during
a train of pulses (see Figure 3C, Table 1) versus the apparent affinities (IC50) of the studied propafenone derivatives. E, correlation (R = 0.8, P < 0.03)
between the time constants of block development during a train of pulses and the size (molecular weight) of the studied propafenone derivatives.
inhibitors comprise three residues at the base of the selectiv-
ity filter (Thr623, Ser624 and Val625) and four on the S6
Discussion and conclusions
transmembrane helix (G648, Y652, F656 and V659) (San-
guinetti and Tristani-Firouzi, 2006; Mitcheson, 2008).
The binding pocket for propafenone comprises F656 and
Y652. Alanine substitution of these residues markedly
reduced HERG current inhibition by propafenone (Witchel
et al., 2004). We compared the current inhibition of wild type
(WT) and mutants F656A and Y652A by all compounds.
Figure 6 shows representative currents recorded from oocytes
expressing WT (Figure 6B), Y652A (Figure 6C) and F656A
(Figure 6D) in the absence and presence of 15 mM GPV009.
Figure 6E illustrates the effects of the propafenone derivatives
on mutants Y652A and F656A. The drug concentrations were
chosen to induce Ն90% inhibition of WT HERG channels
(range: 15–70 mM). The mutant F656A channel almost com-
pletely lost sensitivity to all propafenone derivatives, while
the Y652A channel showed a reduced sensitivity to pro-
pafenone and its derivatives.
Recovery of voltage-gated ion channels from block after
membrane repolarization is usually slower than the accumu-
lation process and ranges from several seconds to several
minutes. Physicochemical properties of the blocking mol-
ecule such as molecular weight (or size) (Courtney, 1980;
Campbell, 1983), lipid solubility (Hille, 1977), stereo-
specificity and the distribution of the molecule’s charged
groups have been shown to modulate recovery of sodium
channels from block (Hille, 2001). Yeh and TenEick (1987)
have shown that recovery from block in sodium channels can
be accelerated by frequent pulsing to open the channels indi-
cating that blockers can be trapped in the channel.
Evidence for drug trapping in HERG channels comes from
ultra-slow recovery (or lack of recovery) at rest (e.g. Carme-
liet, 1992; Mitcheson et al., 2000; Kikuchi et al., 2005; Kamiya
et al., 2006). Stork et al. (2007) analysed the recovery kinetics
of HERG channels from block by eight known HERG
British Journal of Pharmacology (2011) 162 1542–1552 1547

A Windisch et al.
BJP
Figure 4
Recovery of human ether-a-go-go related gene channel from block at rest, in the continued presence of drug. Channel block was induced by 1 Hz
pulse trains. Fourteen conditioning pulses were applied to reach steady state of inhibition by the particular compound. Single test pulses were
applied after the indicated resting time at -80 mV. A and C, superimposed current traces of first and last (‘steady state’) pulse during a
conditioning train after application of 4 mM GPV031 (A) and 3 mM GPV009 (C). Recovery current resulting from a single test pulse after 330 s
resting time is depicted as dotted line. C and D, mean normalized tail current amplitudes in the presence of 4 mM GPV031 (B) and 3 mM GPV009
(D) are plotted against time. The section ‘block’ shows the development of inhibition during a 1 Hz pulse train. The grey highlighted section
‘recovery’ maps the amount of recovery after 330 s resting time as black bar, and additionally the recovery kinetics of GPV031 (B).
inhibitors that either dissociated at rest (amiodarone,
cisapride, haloperidol and droperidol) or were completely
trapped (bepridil, terfenadine, E-4031 and domperidone).
Frequent depolarization during wash-out or reopening at
hyperpolarization (mutant D540K) allowed the trapped drug
molecules to escape from the channel (Stork et al., 2007).
The aim of the present study was to investigate whether
structural changes in propafenone (a drug that is trapped in
the closed channel, Witchel et al., 2004) would affect drug
dissociation. To gain insights into determinants of drug trap-
ping, propafenone was modified both in the vicinity of the
basic nitrogen atom and at the central aromatic ring system
(Figure 1).
Y652A in the channel protein, suggesting that these com-
pounds bind to the previously identified binding pocket of
propafenone (Figure 6E, Witchel et al., 2004; Thai et al.,
2010). Inhibition of mutant F656A by GPV929 and GPV574
was stronger than that by propafenone and the other
derivatives. It is tempting to speculate that the ortho-
phenylpropanone structure affects the orientation in the
binding pocket, thereby making the drug-Phe656 interaction
more favourable.
Open channel block by
propafenone derivatives
All compounds inhibited HERG channels, with potencies
(IC50) ranging from 0.77 Ϯ 0.11 mM (SCT-AS03) to 5.04 Ϯ
0.54 mM (GPV574) (Figure 2, Table 1). Structural modifica-
tions of propafenone affected the onset of HERG channel
inhibition and recovery (Figures 2 and 5A, Table 1). Channel
block by all derivatives was affected by mutations F656A and
Use-dependent HERG channel inhibition by propafenone
derivatives is a hallmark of state-dependent drug binding.
Arias et al. (2003) concluded that propafenone binds to the
open and the inactivated states of the channel. Later studies
revealed, however, that the non-inactivating HERG mutants
S631A and S628C/S631C were also efficiently inhibited by
1548 British Journal of Pharmacology (2011) 162 1542–1552

Determinants of propafenone trapping in HERG
BJP
Figure 5
A, summary of the 330 s recovery fractions for the indicated compounds (GPV001 = propafenone). *Indicates statistically significant (unpaired
t-test, P < 0.05) differences compared to propafenone. B, repetitive stimulation accelerates wash-out of GPV009 (and other trapped compounds,
not shown in this figure). Human ether-a-go-go related gene channels were inhibited by a 1 Hz pulse train, as described in Figure 3. After reaching
steady state of inhibition, the drug was washed out. During the wash-out process, pulses were applied with the indicated frequencies, starting
either simultaneously with the beginning of wash-out or after 8 min of wash-out. Peak tail currents were normalized to control currents (amplitude
before drug application) and plotted against time. More frequent stimulation (0.1 Hz) led to faster recovery during wash-out. Without pulsing (‘no
stimulation’, -80 mV) channels barely recovered from block during wash-out.
propafenone indicating a less essential role of inactivation
(Witchel et al., 2004).
and the size of the molecules is in line with previous obser-
vations on sodium channels (Courtney, 1980).
With the exception of propafenone and GPV574, HERG
inhibition gradually accumulated during the pulse trains.
Pronounced first pulse inhibition observed for propafenone
(Figure 3A and C) and GPV574 (data not shown) may reflect
either resting state inhibition (initial block occurring during
pre-incubation in drug) or fast inhibition of open channels.
Our data support the second scenario where drug molecules
rapidly enter and occlude the open channel. Hence, recovery
from block by propafenone, GPV005, GPV009, GPV180
GPV929 and SCT-AS03 is induced by repetitive channel open-
ings during wash-out (exemplified in Figure 5B for GPV009).
These data suggest that access and dissociation of these com-
pounds occurs via the open channel pore.
Structural modifications of propafenone
facilitate recovery of HERG channels
from block
Ultraslow recovery from block at rest as indication for drug
trapping was first observed in cardiac myocytes for rapid
delayed rectifier K+ current block by methanesulfonanilides
(dofetilide, MK-499) (Carmeliet, 1992). Direct evidence for
drug trapping was provided by Mitcheson et al. (2000) using
the HERG mutant D540K which reopens during pronounced
hyperpolarization, thereby accelerating recovery of channels
from block by MK-499. Similar observations were made for
nifekalant, bepridil, E-4031, dofetilide (Kamiya et al., 2006)
and propafenone (Witchel et al., 2004).
Relation between onset of channel
In a preceding study we reported that trapped com-
pounds, such as bepridil, terfenadine, E-4031 and domperi-
done, cannot be washed out during 330 s (Stork et al., 2007)
while repetitive stimulation during wash-out induced a rapid
recovery of HERG channels from block (see also Figure 5B).
These data support the hypothesis that trapped drugs may
leave the channel through the open gate. From the study of
Stork et al. (2007), it was not clear which physicochemical
properties promoted drug dissociation from closed channels
at rest. To get insights into the structure–dissociation rela-
tionship, we made use of systematically modified pro-
pafenone derivatives (a compound known to be trapped in
HERG channels, Witchel et al., 2004). Propafenone and the
five derivatives GPV005, GPV009, GPV180, GPV929 and SCT-
AS03 (Figure 1) did not dissociate at rest (<13% during 330 s
at -80 mV, Figures 4C,D and 5A). In contrast, channels recov-
ered from block by GPV019, GPV031, GPV574 or GPV576
almost completely during a 330 s rest period (Figures 4A,B
and 5A). In this context it would be conceivable that the
block and IC50
Figure 3 illustrates substantial differences in block develop-
ment induced by propafenone and GPV009. The initial rates
of onset of HERG inhibition were comparable for both drugs
(Figure 3C), while the steady-state level of block was deeper
for GPV009. This pattern may reflect a decrease in the rate
constant for GPV009 dissociation from the open channel
state: a deeper level of channel inhibition (KD = k-/k+) at
comparable initial rates of the reaction (k+[D]). An inverse
relation between potencies (IC50) and the rate of block devel-
opment was observed for most compounds (Figure 3D). At
concentrations ª3 ¥ IC50 inducing comparable levels of
channel block (see Table 1), current inhibition by SCT-AS03
(the most potent inhibitor, IC50 = 0.77 Ϯ 0.11 mM) occurred
slowly (t block = 2.39 Ϯ 0.39 s) compared with the least potent
inhibitor GPV574 (IC50 = 5.04 Ϯ 0.54 mM) which induced fast
HERG inhibition (t
= 0.48 Ϯ 0.09 s). The inverse relation
block
(Figure 3E) between the time constants of block development
British Journal of Pharmacology (2011) 162 1542–1552 1549

A Windisch et al.
BJP
bulky residue of these derivatives affects the closure of the
channel gate according to a ‘foot in the door’ type mecha-
nism of channel blockade. This mechanism was first hypoth-
esized by Yeh and Armstrong (1978), and shown to be
applicable to the block of HERG channels by chloroquine
(Sanchez-Chapula et al., 2002) and Kv1.5 (Decher et al., 2006).
We have therefore asked the question: What renders a com-
pound a ‘foot’?
Role of physicochemical properties
in drug trapping
The common structural modification of these compounds is
the extension of the propafenone pharmacophore by a phe-
nylpiperazine residue (Figure 1). An obvious feature of the
phenylpiperazine residue is its bulkiness compared with the
propylamino and piperidine side chains of propafenone and
the other trapped derivatives. However, while the onset of
block development correlated with the molecular weight
(Figure 3E), no correlation between recovery from block and
molecular weight was observed (Figure 7A; see Table 2 for
physico-chemical values). Hence, the relatively small GPV574
was able to dissociate from the closed state while the bulky
SCT-AS03 was efficiently trapped. This finding suggests that
bulkiness per se does not explain recovery. Also no correlation
between recovery and log P could be established (Figure 7B).
Our data revealed, however, a significant correlation between
dissociation of a given compound from the closed state
(recovery at rest) and its pKa value (Figure 7C). This empha-
sizes that also the protonation state and/or the H-bond accep-
tor capabilities in this region of the molecules play a role.
Figure 6
A, voltage pulse protocol for investigating wild type (WT)-, Y652A-
and F656A-HERG channels. B–D, representative current traces of
HERG and mutant HERG channels in the absence (Con) and presence
(GPV009) of 15 mM GPV009. E, summary of mean current remaining
with 50 mM propafenone (=GPV001), 15 mM GPV005, 15 mM
GPV009, 15 mM GPV019, 15 mM GPV031, 15 mM GPV062, 15 mM
GPV180, 70 mM GPV574, 25 mM GPV576, 15 mM GPV929 or
15 mM SCT-AS03. Inhibition of Y652A- and F656A-HERG by the
indicated compounds was significantly weaker (unpaired t-test, P <
0.05) than inhibition of WT-HERG. HERG, Human ether-a-go-go
related gene.
Summary and outlook
An important finding of this study is that the extension of the
propafenone pharmacophore by a phenylpiperazine moiety
(Figure 1; GPV019, GPV031, GPV574 and GPV576) facilitates
drug dissociation from the closed channel state (Figure 5A).
Our data suggest, however, that either the aniline-
nitrogen atom or the basicity of N-1 of the piperazine plays a
key role. Modification of the phenylpiperazine extension, for
example, replacement of the aniline-nitrogen by a carbon
Figure 7
Correlations between the % of recovery after 330 s (Table 1) and different physicochemical properties (Table 2). A, no significant correlation with
molecular weight. B, no correlation with log P. C, significant correlation (R = -0.96, P < 0.0001) with pKa.
1550 British Journal of Pharmacology (2011) 162 1542–1552

Determinants of propafenone trapping in HERG
BJP
Table 2
Conflicts of interest
Physicochemical properties of propafenone and derivatives
None.
Molecular
weight
Compound
(g·mol-1
)
log P
pKa
References
Propafenone
GPV005
GPV009
GPV019
GPV031
GPV062
GPV180
GPV574
GPV576
GPV929
SCT-AS03
341.44
367.48
383.52
444.57
462.56
459.58
417.54
382.50
472.62
417.54
443.58
3.93 Ϯ 0.30
5.00 Ϯ 0.41
5.12 Ϯ 0.39
5.48 Ϯ 0.50
5.57 Ϯ 0.79
4.49 Ϯ 0.44
6.24 Ϯ 0.41
4.10 Ϯ 0.49
6.40 Ϯ 0.50
6.24 Ϯ 0.49
6.09 Ϯ 0.41
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8.83 Ϯ 0.20
9.82 Ϯ 0.28
5.99 Ϯ 0.70
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8.83 Ϯ 0.20
6.51 Ϯ 0.40
6.51 Ϯ 0.40
8.83 Ϯ 0.20
8.45 Ϯ 0.40
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