MOL#39388 1 TITLE PAGE The molecular basis of high-affinity binding of the anti-arrhythmic

52 Pages·2007·1.56 MB·English

Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
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MOL#39388

TITLE PAGE

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The molecular basis of high-affinity binding of the anti-arrhythmic compound, ad
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vernakalant (RSD1235), to Kv1.5 channels d fro
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Jodene Eldstrom, Zhuren Wang,
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Hongjian Xu, Marc Pourrier, Alan Ezrin, Ken Gibson, & David Fedida h
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Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, tjo
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2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3; JE, ZW,HX, DF rn
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and .o
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Cardiome Pharma Corp., 6190 Agronomy Rd., Vancouver, BC, Canada V6T 1Z3; MP, AE, KG a
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Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.
Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
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MOL#39388

RUNNING TITLE PAGE

Running title: Vernakalant binding site in Kv1.5

Author for correspondence:
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Dr. David Fedida, Ph.D., B.M. B.Ch. n
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Professor and Associate Head, d fro
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Department of Anesthesiology, Pharmacology, and Therapeutics, University of
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British Columbia, 2176 Health Sciences Mall, Vancouver BC, Canada V6T 1Z3 rm
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[email protected] tjo
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List of nonstandard abbreviations: human embryonic kidney-293 (HEK293); Minimal Essential J
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Medium (MEM) ls o
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Number of text pages: 40 0
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Tables :2
Figures: 12
References: 35
Abstract: 240 words
Introduction: 597 words
Discussion: 1490 words.
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Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
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MOL#39388
ABSTRACT
Vernakalant (RSD1235) is an investigational drug recently shown to convert atrial
fibrillation rapidly and safely in patients (Roy et al., 2004). Here, the molecular mechanisms of
interaction of vernakalant with the inner pore of the Kv1.5 channel are compared with the Class
IC agent flecainide. Initial experiments showed that vernakalant blocks activated channels and
vacates the inner vestibule as the channel closes, and thus mutations were made, targeting
residues at the base of the selectivity filter and in S6, by drawing on studies of other Kv1.5- D
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selective blocking agents. Block by vernakalant or flecainide of Kv1.5 wild-type (WT) and ad
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mutants was assessed by whole cell patch clamp experiments in transiently transfected HEK293 m
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cells. The mutational scan identified several highly conserved amino acids, T479, T480, I502, h
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V505 and V508, as important residues for affecting block by both compounds. In general, sp
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mutations in S6 increased the IC for block by vernakalant, with I502A causing an extremely rn
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local 25-fold decrease in potency. Specific changes in the voltage-dependence of block with a
t A
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I502A supported the crucial role of this position. A homology model of the pore region of Kv1.5 E
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predicted that, of these residues, only T479, T480, V505 and V508 are potentially accessible for rn
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direct interaction, and that mutation at additional sites studied may therefore affect block through F
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allosteric mechanisms. For some of the mutations, the direction of changes in IC50 were opposite ry 1
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, 2
for vernakalant and flecainide, highlighting differences in the forces that drive drug-channel 0
2
3
interactions.

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Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
This article has not been copyedited and formatted. The final version may differ from this version.
MOL#39388

INTRODUCTION

Atrial fibrillation, the most common sustained cardiac arrhythmia, is associated with ~15% of all
strokes (Kannel et al., 1998; Go et al., 2001; Rockson and Albers, 2004) and occurs in about
30% of patients after cardiac surgery (Leung et al., 2004). Most drugs, currently in use for
treatment of atrial fibrillation, are indiscriminate, targeting channels in both atrial and ventricular D
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tissue and are associated with life-threatening (ventricular) arrhythmias as a consequence. ad
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Vernakalant (RSD1235) is a mixed voltage- and frequency-dependent Na+ and atria-preferred K+ from
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channel blocker (Roy et al., 2004; Fedida et al., 2005) under development for the acute h
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conversion of atrial fibrillation to sinus rhythm. In recent phase II and III clinical trials sp
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vernakalant has shown promise as an intravenous antiarrhythmic agent for rapid conversion of rn
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atrial fibrillation to sinus rhythm with an overall rate close to 52% within 90 min of infusion, a
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compared with a placebo conversion rate of just 3.8% (Roy et al., 2005; Pratt et al., 2006; Stiell E
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et al., 2006; Fedida, 2007). Previous studies have shown that one of the actions of vernakalant is rn
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block of the atrial specific IKur current (Fedida et al., 2005), which in human atria is thought to be Fe
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the result of expression of the KCNA5 gene and Kv1.5 protein (Fedida et al., 1993; Feng et al., ry
1
0
, 2
1997). Evidence suggests that the safety of this drug is in part related to the higher sensitivity of 0
2
3
atrial-specific Kv1.5 to block by vernakalant over other channels involved in ventricular
repolarization, such as hERG (Fedida et al., 2005) and KCNQ1 (unpublished data).
Several antiarrhythmic drugs in development, S0100176 (Decher et al., 2004), AVE0118
(Decher et al., 2006), AZD7009 (Persson et al., 2005), as well as some well-known agents,
quinidine (Snyders et al., 1992; Fedida, 1997), and flecainide (Grissmer et al., 1994), and the
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Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
This article has not been copyedited and formatted. The final version may differ from this version.
MOL#39388
local anesthetics bupivacaine (Franqueza et al., 1997) and benzocaine (Caballero et al., 2002),
have been shown to block Kv1.5 also. The potency of these agents is affected by introduction of
specific mutations in the S6 domain of Kv1.5, that lines the inner vestibule of the channel (V505,
T507, I508, L510, V512, V514), or mutations in the deep pore (T479 and T480) near the
selectivity filter (Caballero et al., 2002; Decher et al., 2004; Decher et al., 2006; Herrera et al.,
2005; Yeola et al., 1996; Franqueza et al., 1997). Many of these same residues are also
β
important sites of interaction for the Kv inactivation particle (Decher et al., 2005), for TEA D
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(Choi et al., 1993; Lopez et al., 1994), and for 4-AP block (Kirsch and Drewe, 1993). ad
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Flecainide is also a drug of choice for the acute conversion of atrial fibrillation to sinus m
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rhythm (Fuster et al., 2006), and is known to block Kv1.5 (Grissmer et al., 1994; Herrera et al., h
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2005), but with much lower potency than vernakalant. In the present study, we have investigated sp
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the binding site of vernakalant in the deep pore and S6 of Kv1.5 using both electrophysiology rn
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and site-directed mutagenesis. Flecainide has been used as a comparator compound to validate a
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our data against that already present in the literature, and also to extend studies of flecainide E
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block itself. The results demonstrate that I502 in the S6 domain is a key residue in the block of rn
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Kv1.5 by vernakalant, but less so for flecainide. We have interpreted our results in the context of F
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π ru
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the hydrophobicity, size, and potential for cation- interactions of the different substituted ry
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0
, 2
residues, and we have extended our analysis by carrying out homology-modeling and ligand 0
2
3
docking of vernakalant on Kv1.5 based on the published crystal structure of Kv1.2 (Long et al.,
2005).
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Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
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MOL#39388
MATERIALS AND METHODS
Cell Preparation
Stable lines of HEK293 cells expressing Kv1.5, or transient transfection of mutant Kv1.5
channels were used in all experiments. Primers to generate mutant channels were synthesized by
Integrated DNA Technologies, Inc. (IA, USA), and mutants were generated using the Stratagene
Quikchange kit (Stratagene, CA, USA). The presence of the mutation was confirmed by DNA
sequencing, and because of the size of the WT Kv1.5 clone, only the sequenced region harboring D
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the targeted nucleotides was subcloned as a fragment back into full-length Kv1.5. Transient ad
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transfections were performed with HEK 293 cells plated at 20-30 % confluency on sterile m
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coverslips in 25 mm Petri dishes one day prior to transfection. 0.1-4 µg of ion channel DNA was lph
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incubated with 1 µg of eGFP DNA (to enable detection of transfected cells) and 2 µl of sp
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Lipofectamine 2000 (Invitrogen) in 100 µl of Opti-MEM. This was added to the cells for rna
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overnight incubation after washing the cells with 900 µl of MEM with 10 % FBS. at A
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The cells were grown in Minimal Essential Medium (MEM) at 37°C in an air/5 % CO T
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incubator. Media contained 10 % bovine serum and 0.5 mg/ml geneticin for stable lines. On the als
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day before recording, stably-expressing cells were washed with MEM, treated with e
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trypsin/EGTA for one minute and plated on 25 mm2 coverslips. All cell culture supplies were ry 1
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, 2
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2
obtained from Invitrogen (Mississauga, ON, Canada). 3

Drugs and Solutions
Control bath solution contained (in mM): 5 KCl, 135 NaCl, 2.8 sodium acetate, 1 MgCl , 10
2
HEPES and 1 CaCl , adjusted to pH 7.4 with NaOH. Patch pipettes contained (in mM): 130
2
KCl, 5 EGTA, 1 MgCl , 10 HEPES, 4 Na ATP, and 0.1 GTP, adjusted to pH 7.2 with KOH. All
2 2
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Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
This article has not been copyedited and formatted. The final version may differ from this version.
MOL#39388
chemicals used to make solutions were obtained from Sigma-Aldrich (Mississauga, ON,
Canada). Vernakalant (formerly known as RSD1235; 3-Pyrrolidinol, 1-[(1R,2R)-2-[2-(3,4-
dimethoxyphenyl)ethoxy]cyclohexyl]-,hydrochloride, (3R)), lot numbers DM-155-A and JL-78-
12) was synthesized by Cardiome Pharma Corp. (Vancouver, BC, Canada) and prepared as a
stock solution (50 mM) in H O. Flecainide (Sigma-Aldrich, lot number 094K4057) was
2
prepared as a stock solution (100 mM) in 100% DMSO. DMSO concentrations never exceeded
0.1% v/v in the final experimental solutions. D
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Whole-Cell Patch-Clamp Recordings m
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Coverslips with adherent cells plated on the surface were placed in a superfusion chamber h
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(volume 300 µl) containing the control bath solution at 22°C. Whole-cell current recording and sp
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analysis were carried out using an Axopatch 200B amplifier and pClamp10 software (Axon rn
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Instruments, CA, USA). Patch electrodes were pulled from thin-walled borosilicate glass (World a
t A
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Precision Instruments, FL, USA) on a horizontal micropipette puller (Sutter Instruments, CA, E
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Ω Jo
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USA). Electrodes had resistances of 1.0-3.0 M when filled with control filling solution. rn
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Analogue capacity compensation and 80% series resistance compensation were used during F
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whole cell measurements. Membrane potentials were not corrected for junction potentials that ry
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, 2
arise between the pipette and bath solution. For Kv1.5 current recordings, a holding potential of 0
2
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-80 mV was used. Data were sampled at 10-20 kHz and filtered at 5 to 10 kHz. To assess drug
block, half-log escalating concentrations of drug were added to the flowing bath solution and
current traces were recorded with 400 ms depolarizing pulses to +60 mV at a frequency of 0.1
Hz. Concentrations approximately equal to the 50 % inhibitory value for each drug on the WT
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Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
This article has not been copyedited and formatted. The final version may differ from this version.
MOL#39388
channel were used in the initial investigation of vernakalant and flecainide actions on mutated
channels.

Data Analysis
Data are represented as mean ± SEM unless otherwise specified. For significance of differences,
* represents p < 0.05, ** p < 0.01; statistical analysis was conducted using Student’s t-test
(unpaired). Clampfit software (Axon Instruments, CA, USA) was used to analyze current traces. D
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Data from each test pulse (currents in the presence of drug) was normalized to control test pulse ad
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currents obtained before drug exposure. For drug potency studies of the mutant Kv1.5 channels m
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in the presence of vernakalant or flecainide (Figs. 3, 4), currents in the presence of different h
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concentrations of drug were normalized to the current in control and used to generate sp
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concentration-response curves for changes in steady state Kv1.5 current. The resulting rn
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concentration-response relations were computer-fitted to the Hill equation: a
t A
S
i = 1/[1 + (IC /[D]n)] [1] PE
50 T
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where i is the normalized current recorded (i = Idrug/Icontrol) at drug concentration [D]; IC50 is the rna
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concentration producing half-maximal inhibition; and n is the Hill coefficient. F
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, 2
The voltage dependence of block for vernakalant and flecainide was determined by the 0
2
3
calculation of the fractional block (f = 1-I /I ) at the potentials in the range of full channel
drug control
opening (between +10 to +60 mV) and fitting the data to the Woodhull equation:
f = [D]/([D]+K *e-δzFE/RT) [2]
d
where F, R, z and T have their usual meanings, δ represents the fractional electrical distance, i.e.,
the fraction of the transmembrane electrical field sensed by a single charge at the receptor site.
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Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
This article has not been copyedited and formatted. The final version may differ from this version.
MOL#39388
K * represents the binding affinity at the reference voltage (0 mV). The current amplitude (I
d drug
and I ) was measured at the end of a 400 ms depolarization.
control

Homology model
The Kv1.5 homology model was generated using "First Approach Mode" of SWISS-MODEL
(http://swissmodel.expasy.org) and the three-dimensional structure of rKv1.2 (Protein Data file
2A79; (Long et al., 2005)), which is believed to represent the open state of the channel. rKv1.2 D
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and hKv1.5 show 100% identity in S5 and S6 and 88% identity in the pore loop. The figure was ad
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generated using DeepView Swiss-PdbViewer (http://swissmodel.expasy.org/spdbv) and Adobe m
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Photoshop software. This homology model was used for docking of vernakalant using Chem h
a
rm
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Bio3D (Cambridge Scientific, MA, USA), AutoDock tools, and AutoDock4 (Scripps research sp
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tjo
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Inst, CA, USA; (Morris et al., 1998)). A lowest-energy conformation of vernakalant, protonated rn
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on the nitrogen atom as it would be at physiological pH, was used for docking. The channel a
t A
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macromolecule remained rigid during the docking computation, while vernakalant was flexible. E
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Molecular Pharmacology Fast Forward. Published on September 14, 2007 as DOI: 10.1124/mol.107.039388
This article has not been copyedited and formatted. The final version may differ from this version.
MOL#39388
RESULTS
Delayed closing of Kv1.5 channels in the presence of vernakalant at the inner pore
It was suggested from our previous experiments that block of Kv1.5 by vernakalant was
mediated after channel activation, as vernakalant caused a relatively rapid onset of block of
channel current upon depolarization (Fedida et al., 2005), but little evidence of resting or ‘tonic’
block of the channel. It is known that the activation gate of Kv channels lies on the intracellular
side of the pore (Holmgren et al., 1998), and thus a requirement for channel activation for block D
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to occur suggests intracellular access of the drug to the pore region of the channel. In our initial ad
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experiments aimed at localizing the site of binding of vernakalant within Kv1.5 we have m
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examined the ability of the drug to affect activation gate closure upon repolarization, after h
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channel opening (Fig. 1). Control currents in Figure 1A activate rapidly upon depolarization to sp
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+60 mV and show only limited inactivation during the 400 ms depolarization shown here. rn
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During repolarization to -40 mV a deactivation tail current is observed that reflects channel a
t A
µ S
P
closure. In the presence of 10 M vernakalant, rapid block is apparent after channel opening and E
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a steady-state current level is rapidly reached. Upon repolarization, the tail current amplitude is rn
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initially less than in control, but decays more slowly and crosses over the control tracing as it F
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declines to the baseline current level (see inset panel). The result suggests that vernakalant ry
1
0
, 2
delays closing of Kv1.5, and perhaps that the channels cannot close while vernakalant is bound, 0
2
3
but require drug expulsion from the inner vestibule before that can happen.
This possibility was tested in the experiment shown in Fig. 1B. Here, after equilibration at 0.1
Hz in control solutions, the cell was exposed to vernakalant as shown. This led to a progressive
reduction of steady-state current, and at 200 s, the cell was rested for 3 min at -80 mV and
vernakalant was washed from the bath. When the current was activated again by depolarizing
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