Toward specific cardiac IK1 modulators for in vivo application: Old drugs point the way

CREATIVE CONCEPTS

Toward specific cardiac IK1 modulators for in vivo application: Old drugs point the way Marcel A.G. van der Heyden, PhD,* José A Sánchez-Chapula, MD, PhD† From the *Department of Medical Physiology, Division Heart & Lungs, University Medical Center Utrecht, Utrecht, The Netherlands, and †Centro Universitario de Investigaciones Biomédicas de la Universidad de Colima, Colima, México. Current knowledge of cardiac electrophysiology depends heavily on specific manipulation of individual ion channels and transporters whose combined functioning results in action potential formation. Whereas molecular interference introduces opportunities to modify currents by transgenesis and null mutation in genetically accessible animals such as mice, electrophysiologic studies in large animal models still rely heavily on specific pharmaceutical compounds. Many drugs in use that address the role of the main cardiac ion channels, such as sodium (e.g., lidocaine), calcium (e.g., verapamil), transient outward (e.g., 4-aminopyridine), and delayed rectifier (e.g., dofetilide, chromanol 293B), have no doubt proven their scientific importance.1,2 Unfortunately, no specific compound currently available addresses the physiologic and pathophysiologic roles of KIR2.1, KIR2.2, and KIR2.3 carried cardiac inward rectifier current (IK1) in vivo. Existing blockers or activators of this ion current either are lethal when used in animal models or target other ion channels too. The primary functions of IK1 are to establish a negative and stable resting membrane potential and to contribute to the final phase of repolarization.3 Action potential modeling defines that action potential duration responses stronger to IK1 modulation than resting membrane potential and that the atrial action potential responses stronger to IK1 modulation than that of the ventricle (Figure 1). In general, IK1 channels are formed by homotetramerization or heterotetramerization of KIR2.x isoforms that each contributes their specific characteristics (e.g., single channel conductance, rectification strength).4,5 Whereas KIR2.1 is the strongest expressed isoKEYWORDS Chloroquine; Flecainide; Heart; Inward rectifier; Kir2.1; Kir2.2; Kir2.3 ABBREVIATIONS AF ⫽ atrial fibrillation (Heart Rhythm 2011;8: 1076 –1080) This work was supported by SEP-CONACYT (México) Grant CB2008-01-105941 to Dr. Sánchez-Chapula and by a grant from Top Institute Pharma (D2-101), Leiden, The Netherlands, to Dr. van der Heyden. Address reprint requests and correspondence: Dr. Marcel A.G. van der Heyden, University Medical Center Utrecht, Medical Physiology, Yalelaan 50, 3584 CM Utrecht, The Netherlands. E-mail address: [email protected]. (Received December 20, 2010; accepted January 26, 2011.)

Figure 1 Responses of ventricular (A) and atrial (B) action potentials to variations in IK1 density using computer simulations. Drawn line indicates normal IK1 density; dotted line indicates 0.75-fold IK1 density; dashed line indicates 1.5-fold IK1 density. Horizontal gray line depicts 0 mV; horizontal scale bar ⫽ 25 ms; vertical scale bar ⫽ 50 mV. Effects of variations in IK1 density on resting membrane potential (C) and action potential duration (D) in atrial (closed symbols) and ventricular (open symbols) cell models. Human atrial66 and ventricular67 cardiomyocyte cell models, paced at 1 Hz with decreased and increased IK1 densities, were used. In the ventricular model, IK1 decrease ⬎90% resulted in spontaneous activity and more severe action potential (AP) prolongation as seen experimentally (e.g., Miake et al13); 4-fold increased IK1 densities yielded strong AP shortening (⬎40 ms), in accordance with previous findings.68 The atrial cell model became unstable at IK1 fraction of 0.50, and spontaneous action potentials were generated. Our modeling did not include changes in rectification, which might have profound effects on repolarization.

form throughout the heart, KIR2.3 especially is a major contributor in the atrium and confers its weaker rectification to atrial IK1 mainly.5,6 In pulmonary vein cardiomyocytes, however, KIR2.3 levels are much lower, explaining the sensitivity of this tissue to reentrant arrhythmias compared to the working atrium.7 Under pathophysiologic conditions, expression levels of KIR2.1 are mainly altered (e.g., Gir-

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doi:10.1016/j.hrthm.2011.01.038

van der Heyden and Sánchez-Chapula

Cardiac IK1 Modulators

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Figure 2 Structure model of KIR2.1 ion channel. Negatively charged amino acid residues (D172, E224, F254, D255, D259) involved in polyamide-mediated rectification face the transmembrane and cytoplasmic pore regions, respectively. Color codes of amino acids are identical in the side view (left) and the surface representation of the cytoplasmic pore domain (right), which is viewed from the bottom. Figures generated as described previously.31 CPD ⫽ cytoplasmic pore domain; GG ⫽ G-gate; SF ⫽ selectivity filter; TMPD ⫽ transmembrane pore domain.

matsion et al8), thereby regionally changing IK1 density and characteristics. Interestingly, paroxysmal atrial fibrillation (AF) patients show a left-to-right IK1 gradient, further extending the complexity of regional IK1 distribution within the heart.9 Mechanistic insights into the pathophysiologic role of IK1 have been deduced from gain- and loss-offunction mutations in congenital AF, short QT syndrome type 3, catecholaminergic polymorphic ventricular tachycardia, and Andersen-Tawil syndrome patients.10,11 Experimentally, null mutation and overexpression of (dominant-negative) IK1 channel proteins (i.e., KIR2.1) in mice have been most informative.10 For example, cardiomyocytes isolated from KIR2.1 knockout mice displayed lengthening of action potential duration, and the majority of cells exhibited spontaneous beating activity.12 Furthermore, IK1 down-regulation in guinea pig ventricle by overexpression of dominant negative KIR2.1 induces ectopic pacemaker activity.13 On the other hand, Langendorff-perfused hearts from mice that overexpress KIR2.1 in their ventricles are more prone to stable, rotor-driven, ventricular tachycardia and fibrillation than are their wild-type littermates.14 Taken together, the clinical and experimental (in vivo as well as in vitro) findings support a role for IK1 in controlling cardiac excitability and leading to ventricular and/or atrial tachyarrhythmias when affected positively or negatively. Much still needs to be learned, and specific modifying compounds appear essential when it comes to investigating the role of IK1 in cardiac electrophysiology using larger animal models such as rabbit, dog, and pig. Moreover, IK1 may prove an attractive target for treatment of some forms of cardiac arrhythmias. Here we briefly discuss potential targets and currently available drugs that may point the way toward the development of specific cardiac IK1 modulators that

are clinically relevant and effective in the treatment of cardiac arrhythmias.

Targeting polyamine block Over the last two decades, enormous progress was made in unraveling the structure of the IK1 channel by means of crystallization15 and homology modeling16,17 approaches. In short, each KIR2.x monomer comprises a transmembrane part and a cytoplasmic part. The transmembrane domain consists of the two transmembrane helices (TM1 and TM2), a pore helix positioned between them, and a slide helix located in front of TM1. The cytoplasmic domain is formed by interaction of the amino-terminus and the carboxylterminus. Upon assembly in a tetramer, the four TM2 helices face the central pore in the transmembrane part with the potassium selectivity filter positioned in close proximity to the cellular exterior. It was hypothesized that because of the close spacing of the four extracellular protruding turrets, these channels are less prone to block by extracellular applied toxins compared to are other potassium channels.18 Tetramerization of cytoplasmic parts generates an extended pore region into the cytoplasm, separated from the transmembrane pore region by a narrow gate arranged by the so-called G-loops (G-gate). The characteristic inward rectification of the current is achieved by polyamines (e.g., spermine, spermidine) or Mg2⫹ entering the negatively charged cytoplasmic pore and proceeding through the Ggate to occupy and thereby block the transmembrane pore region.3 Upon reversal of the membrane potential to a hyperpolarized state, the polyamines rapidly release from the pore, allowing inward potassium flow. A number of residues within the cytoplasmic (E224, D259, E299, F254, D255) and the transmembrane pore regions (D172) are involved in polyamine– channel interactions (Figure 2).19 –22 We consider com-

1078 Table 1

Heart Rhythm, Vol 8, No 7, July 2011 Acute IK1 channel modulating drugs IK1 action (IC50/EC50)

Mechanism

Species

Additional currents (IC50)

References

Berberine

Inhibitor(⬃50 ␮M)*

Unknown

Cp

28,51,52

Celastrol Chloroethylclonidine Chlorpromazine

Inhibitor (⬎20 ␮M) Inhibitor (30 ␮M) Inhibitor (6.1 ␮M)

Unknown D172 dependent Unknown

Mm Mm, Rn Rn

Chloroquine

Inhibitor (0.3 -1.1 ␮M)

Plugs cytoplasmic pore

Cf, Cp, Hs, Mm

Flecainide

Activator (0.4-0.8 ␮M)

Lowers polyamine affinity

Cp, Hs,

Pentamidine

Inhibitor (⬃0.2 ␮M)

Cf, Hs, Mm

RP58866

Inhibitor (3.4 ␮M)

Plugs cytoplasmic pore Unknown

Tamoxifen

Inhibitor (⬃0.9 ␮M)

Thiopental

Inhibitor (⬃30 ␮M)

IKv11.1 (75 ␮M), ICa-L (⬃30 ␮M)* ICa-T (⬃20 ␮M), ITO1 (⬎30 ␮M) IKr (4.1 ␮M) IKv11.1 (⬍10 ␮M) ␣-agonist IKATP (2-11 ␮M), ITO1 (⬎100 ␮M) IKr (⬃5-10 ␮M), IKv11.1 (1.5-21.6 ␮M), INa (0.7-3 ␮M) IKv11.1 (2.5-8.4 ␮M), INa (⬃10 ␮M) ICa-L (⬎10 ␮M), IKs (⬎10 ␮M) IKATP (⬃0.5 ␮M), IKAch (⬃0.4 ␮M) INa (7.4 ␮M, use-dependent), ITO1 (⬎10 ␮M), IKr (⬍10 ␮M) IKur.d (2.9 ␮M) inhibits trafficking of Kv11.1 and KIR2.1 IKr (⬃1 ␮M), IKAdo (⬃2 ␮M) ITO (2.3 ␮M) IKATP (⬃0.3 ␮M), IKAch (⬃0.2 ␮M) IKr (⬃2 ␮M), ICa-L (⬍10 ␮M) ITO (⬃10 ␮M), INa (⬃6 ␮M) IKATP (26 ␮M), IKIR1.1 (25 ␮M) IKs, ICa-L (28 ␮M)

Compound

Interferes in channel-PIP2 interaction Interferes in channel-PIP2 interaction

Cp Fc, Mm

Cp, Mm

30 26 24,53–56

27,35,36 32,40–43

31,38,39 23,57–59 60–62

63–65

Cf ⫽ dog; Cp ⫽ guinea pig; Fc ⫽ cat; Hs ⫽ human; Mm ⫽ mouse; Rn ⫽ rat. *IK1 and ICa-L inhibition was found in guinea pigs28,52 but not in cat51 cardiomyocytes.

pounds that mimic polyamine binding as potentially interesting blockers, whereas compounds that relieve polyamine-mediated rectification can be viewed as activators.

Lead compounds A number of compounds possess IK1 inhibiting or activating activity (e.g., RP58866,23 chlorpromazine,24 thiopental,25 chloroethylclonidine,26 chloroquine,27 berberine,28 tamoxifen,29 celastrol,30 pentamidine,31 flecainide32), but none of them is specific. Their general effects on the cardiovascular system include prominent inhibition of sodium, calcium, transient outward, or delayed rectifier channels (Table 1). The lipid binding drugs thiopental and tamoxifen most likely inhibit IK1 by their interference of channel–PIP2 interaction.33,34 Interestingly, chloroquine and pentamidine partially mimic polyamines and plug the cytoplasmic pore, whereas flecainide interferes in polyamine binding. Recently, in-depth analysis of chloroquine and pentamidine mediated IK1 inhibition and flecainide-induced activation has provided new avenues for further development of specific IK1 modulators. The antimalarial drug chloroquine inhibits the channel in a voltage-dependent fashion, that is, it produces stronger inhibition of outward than inward current.35 If applied from the outside of the channel, block is achieved relatively slowly, whereas application from the cytoplasmic side very rapidly inhibits the current. Alanine scans of the pore-lining residues identified amino acids E224, F254, D259, and E299 located in the cytoplasmic pore region as critical in chloroquine-mediated block. Subsequent molecular modeling provided further insight into electrostatic drug– channel interactions at such residues.35,36 Channel inhibition by the diamine antiprotozoal drug pentamidine is similar.31 Block is voltage dependent and achieved rapidly following cytoplasmic drug application, whereas extracellular application

strongly delays current inhibition. Molecular modeling predicted interactions with E224, D259, and E299, which were experimentally confirmed by alanine scanning. In contrast to chloroquine, F254 in the cytoplasmic pore was not involved in pentamidine-mediated block. Again, drug– channel interactions were established by electrostatic bonds. The exact mechanism of flecainide augmentation of IK1 is not fully understood.32 It was shown that flecainide interacted with C331 in the G-loop, thereby alleviating polyamine block by reducing spermidine’s affinity to the cytoplasmic pore residues E224, D259, and E299 as activity of mutant channels at these positions was unaffected by the drug. Moreover, based on pharmacokinetic data, the authors suggested that flecainide and spermidine did not compete for the same binding site. Finally, it was argued that an interaction site for flecainide within the cytoplasmic pore region was difficult to envision as it could hinder potassium flow through the channel.

IK1 modulation and arrhythmias A recent chloroquine follow-up study elegantly indicated the scientific potential of IK1 blockers for basic science but also their clinical relevance.36 Chloroquine displayed antifibrillary action in a mouse model of augmented KIR2.1 activity. Moreover, chloroquine-mediated termination of pinacidil (IKATP opener) induced ventricular fibrillation in the rabbit and rapid pacing-induced AF in the presence of acetylcholine (IKACh) in the sheep heart model further emphasized the relevance of IK1 block in treatment of specific forms of tachyarrhythmias. In general, chloroquine inhibited KIR2.1, KIR3.1, and KIR6.2 channels at comparable effective concentrations. Nevertheless, molecular modeling of drug– channel interactions showed specific differences between the channels, which indicated that channel type– specific pharmacophores can be designed.36

van der Heyden and Sánchez-Chapula

Cardiac IK1 Modulators

As mentioned earlier, chloroquine, pentamidine, and flecainide are not specific for KIR2.1-3 carried IK1. Chloroquine blocks cardiac IKATP, IKACh, IKr, IKs, INa, and ICa-L channels27,35,36 and inhibits lysosomal protein degradation of many proteins, including KIR2.1.37 Pentamidine inhibits forward trafficking of Kv11.1, resulting in reduced IKr38,39 and KIR2.1 protein expression by an unknown mechanism.31,38 Finally, flecainide is a well-known INa blocker that also possesses ITO1, IKr, IKur.d, and ryanodine receptor blocking activity.40 – 43 Notwithstanding these off-target effects, at least when considering IK1 modulation, we are convinced that these drugs can be regarded as “lead” compounds for development of specific blockers and activators, respectively.

Further directions and prospects A first essential component in rationalized development of specific drugs would be generation of comprehensive structure–activity relationships for all lead compounds, preferably in combination with molecular modeling of drug– channel interactions. Based on these results, new derivatives must be designed; alternatively, existing derivatives explored in these primary studies should be tested further for efficacy and specificity. It may take a number of cycles before the ideal candidate compound emerges that can be analyzed in relevant cell, animal, and eventually clinical studies for toxicity, efficacy, and specificity. When successful, a specific cardiac IK1 modulator will be invaluable for in vivo experimental studies. However, pharmaceutical and commercial interest will only be achieved when the potential clinical application of such a drug is supported by solid basic science and translational data. Gain-of-function mutations in KIR2.1 have been associated with short QT syndromes and with familial AF,10 although their occurrence is rare. Interestingly, IK1 and KIR2.1 densities are affected in the process of cardiac remodeling. Rapid pacing–induced heart failure decreases IK1 and KIR2.1 levels in canine ventricle and rabbit atrium.44,45 In humans with heart failure, increased KIR2.1 expression is observed in the ventricle.46 Furthermore, in nonfailing valvular heart disease patients with AF, IK1 densities and KIR2.1 levels are clearly increased.8,47,48 Finally, the rabbit with chronic AV block displays increased IK1 densities.49 Therefore, both increase and decrease of IK1 are associated with severe cardiac arrhythmias. When we succeed in producing isoform specific drugs, the possibilities for regional IK1 modulation would allow increasing and stabilizing resting membrane potential at one location while leaving other regions in the heart unaffected. Finally, as KIR2.x channels are expressed in many excitable tissues,10 a specific modulating drug may find additional applications in neurologic as well as muscular and vascular diseases. The other side of the coin is that application of such a modulator will be hampered when issues of tissue specificity have not been addressed properly.

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Conclusion and outlook High-resolution crystal structures of KIR channels have produced detailed information determining the structural features to which a highly efficient IK1 channel modulator must comply. Resolving the mechanistic properties of three nonspecific IK1 modulators has indicated clear avenues for further development of compounds that either relieve polyamine block, thereby activating the channel, or, by imitating polyamine binding, may yield specific and efficient blockers. For the former, flecainide and its derivatives should be further explored. For the latter, chloroquine and pentamidine may be considered as lead compounds. Specific compounds that targets KIR2.1, KIR2.2 and KIR2.3 based IK1 will definitely find their way into state-of-the art cardiac physiology research and beyond. From a clinical perspective, a well-tolerated IK1 blocker or activator should provide new therapeutic options for AF,50 short QT syndrome, and potentially other, yet unidentified patient groups.

Acknowledgments We thank Dr. Jose Jalife (University of Michigan) for careful reading of this manuscript, Dr. Teun de Boer (UMC Utrecht) for action potential modeling, and Dr. Anna StaryWeinzinger (University of Vienna) for KIR2.1 structure modeling.

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