Role of hERG potassium channel assays in drug 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 potentially 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, ranging from high throughput binding assays on stably expressed recombinant channels to very time consuming electrophysiological examinations in cardiac myocytes. Depending on the number of compounds to be tested, binding assays or functional assays measuring membrane potential or Rb+ flux, combined with electrophysiology on a few compounds, can be used to efficiently develop the structure-function relationship of hERG interactions.     

Role of hERG potassium channel assays in drug 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 potentially 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, ranging from high throughput binding assays on stably expressed recombinant channels to very time consuming electrophysiological examinations in cardiac myocytes. Depending on the number of compounds to be tested, binding assays or functional assays measuring membrane potential or Rb(+) flux, combined with electrophysiology on a few compounds, can be used to efficiently develop the structure-function relationship of hERG interactions.     

Antidepressant-induced ubiquitination and degradation of the cardiac potassium channel hERG

The most common cause for adverse cardiac events by antidepressants is acquired long QT syndrome (acLQTS), which produces electrocardiographic abnormalities that have been associated with syncope, torsade de pointes arrhythmias, and sudden cardiac death. acLQTS is often caused by direct block of the cardiac potassium current I(Kr)/hERG, which is crucial for terminal repolarization in human heart. Importantly, desipramine belongs to a group of tricyclic antidepressant compounds that can simultaneously block hERG and inhibit its surface expression. Although up to 40% of all hERG blockers exert combined hERG block and trafficking inhibition, few of these compounds have been fully characterized at the cellular level. Here, we have studied in detail how desipramine inhibits hERG surface expression. We find a previously unrecognized combination of two entirely different mechanisms; desipramine increases hERG endocytosis and degradation as a consequence of drug-induced channel ubiquitination and simultaneously inhibits hERG forward trafficking from the endoplasmic reticulum. This unique combination of cellular effects in conjunction with acute channel block may explain why tricyclic antidepressants as a compound class are notorious for their association with arrhythmias and sudden cardiac death. Taken together, we describe the first example of drug-induced channel ubiquitination and degradation. Our data are directly relevant to the cardiac safety of not only tricyclic antidepressants but also other therapeutic compounds that exert multiple effects on hERG, as hERG trafficking and degradation phenotypes may go undetected in most preclinical safety assays designed to screen for acLQTS.     

A cellular test system expressing hERG K+ channels for assaying drug cardiotoxicity: Elaboration and analysis

Many drugs are cardiotoxic because they inhibit hERG K⁺ channels, thus prolonging the repolarization phase of the cardiomyocyte action potential giving rise to cardiac arrhythmias. Early detection of inhibiting effects of candidate drugs on the activity of K⁺ channels in cardiomyocytes is one of the main challenges in preclinical drug screening. The aim of this study was to obtain a cell line expressing recombinant hERG channels at a stable and reproducible level as a prerequisite to its further application as a test system.     

Evaluation of a high-throughput fluorescence assay method for HERG potassium channel inhibition

The number of projects in drug development that fail in late phases because of cardiac side effects such as QT prolongation can impede drug discovery and development of projects. The molecular target responsible for QT prolongation by a wide range of pharmaceutical agents is the myocardial hERG potassium channel. It is therefore desirable to screen for compound interactions with the hERG channel at an early stage of drug development. Here, the authors report a cell-based fluorescence assay using membrane potential-sensitive fluorescent dyes and stably transfected hERG channels from CHO cells. The assay allows semiautomated screening of compounds for hERG activity on 384-well plates and is sufficiently rapid for testing a large number of compounds. The assay is robust as indicated by a Z' factor larger than 0.6. The throughput is in the range of 10,000 data points per day, which is significantly higher than any other method presently available for hERG. The data obtained with the fluorescence assay were in qualitative agreement with those from patch-clamp electrophysiological analysis. There were no false-positive hits, and the rate of false-negative compounds is currently 12% but might be further reduced by testing compounds at higher concentration. Quantitative differences between fluorescence and electrophysiological methods may be due to the use- or voltage-dependent activity of the antagonists.     

Pharmacophore modeling for hERG channel facilitation

Human ether-a-go-go-related gene (hERG) channels play a critical role in cardiac action potential repolarization. The unintended block of hERG channels by compounds can prolong the cardiac action potential duration and induce arrhythmia. Several compounds not only block hERG channels but also enhance channel activation after the application of a depolarizing voltage step. This is referred to as facilitation. In this study, we tried to extract the property of compounds that induce hERG channel facilitation. We first examined the facilitation effects of structurally diverse hERG channel blockers in Xenopus oocytes. Ten of 13 assayed compounds allowed facilitation, suggesting that it is an effect common to most hERG channel blockers. We constructed a pharmacophore model for hERG channel facilitation. The model consisted of one positively ionizable feature and three hydrophobic features. Verification experiments suggest that the model well describes the structure-activity relationship for facilitation. Comparison of the pharmacophore for facilitation with that for hERG channel block showed that the spatial arrangement of features is clearly different. It is therefore conceivable that two different interactions of a compound with hERG channels exert two pharmacological effects, block and facilitation.     

Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243

Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.     

Physicochemical features of the HERG channel drug binding site

Blockade of hERG K(+) channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the two-dimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-pi interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltage-gated K(+) channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site.     

Structure Driven Design of Novel Human Ether-A-Go-Go-Related-Gene Channel (hERG1) Activators

One of the main culprits in modern drug discovery is apparent cardiotoxicity of many lead-candidates via inadvertent pharmacologic blockade of K⁺, Ca²⁺ and Na⁺ currents. Many drugs inadvertently block hERG1 leading to an acquired form of the Long QT syndrome and potentially lethal polymorphic ventricular tachycardia. An emerging strategy is to rely on interventions with a drug that may proactively activate hERG1 channels reducing cardiovascular risks. Small molecules-activators have a great potential for co-therapies where the risk of hERG-related QT prolongation is significant and rehabilitation of the drug is impractical. Although a number of hERG1 activators have been identified in the last decade, their binding sites, functional moieties responsible for channel activation and thus mechanism of action, have yet to be established. Here, we present a proof-of-principle study that combines de-novo drug design, molecular modeling, chemical synthesis with whole cell electrophysiology and Action Potential (AP) recordings in fetal mouse ventricular myocytes to establish basic chemical principles required for efficient activator of hERG1 channel. In order to minimize the likelihood that these molecules would also block the hERG1 channel they were computationally engineered to minimize interactions with known intra-cavitary drug binding sites. The combination of experimental and theoretical studies led to identification of functional elements (functional groups, flexibility) underlying efficiency of hERG1 activators targeting binding pocket located in the S4–S5 linker, as well as identified potential side-effects in this promising line of drugs, which was associated with multi-channel targeting of the developed drugs.     

Microfluidic cell culture and its application in high-throughput drug screening: cardiotoxicity assay for hERG channels

Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval and hence risk for life-threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, the authors demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. They validate their microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP), and polydimethylsiloxane (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG) and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. The authors demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. The authors further show that experimental procedures can be streamlined by using direct in-channel immunofluorescence staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with 5 different drugs suggests that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules.     

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K⁺ current, I_Kr is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I_Kr block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC₇₀ of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC₅₀ of dofetilide from 38.7 to 76.3 nm. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.     

3,5-Dialkoxypyridine analogues of bedaquiline are potent antituberculosis agents with minimal inhibition of the hERG channel

Bedaquiline is a new drug of the diarylquinoline class that has proven to be clinically effective against drug-resistant tuberculosis, but has a cardiac liability (prolongation of the QT interval) due to its potent inhibition of the cardiac potassium channel protein hERG. Bedaquiline is highly lipophilic and has an extremely long terminal half-life, so has the potential for more-than-desired accumulation in tissues during the relatively long treatment durations required to cure TB. The present work is part of a program that seeks to identify a diarylquinoline that is as potent as bedaquiline against Mycobacterium tuberculosis, with lower lipophilicity, higher clearance, and lower risk for QT prolongation. Previous work led to the identification of compounds with greatly-reduced lipophilicity compounds that retain good anti-tubercular activity in vitro and in mouse models of TB, but has not addressed the hERG blockade. We now present compounds where the C-unit naphthalene is replaced by a 3,5-dialkoxy-4-pyridyl, demonstrate more potent in vitro and in vivo anti-tubercular activity, with greatly attenuated hERG blockade. Two examples of this series are in preclinical development.     

Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library

High-throughput fluorescent intercalator displacement (HT–FID) was adapted to the semi-automated screening of a commercial compound library containing 60,000 molecules resulting in the discovery of cytotoxic DNA-targeted agents. Although commercial libraries are routinely screened in drug discovery efforts, the DNA binding potential of the compounds they contain has largely been overlooked. HT–FID led to the rapid identification of a number of compounds for which DNA binding properties were validated through demonstration of concentration-dependent DNA binding and increased thermal melting of A/T- or G/C-rich DNA sequences. Selected compounds were assayed further for cell proliferation inhibition in glioblastoma cells. Seven distinct compounds emerged from this screening procedure that represent structures unknown previously to be capable of targeting DNA leading to cell death. These agents may represent structures worthy of further modification to optimally explore their potential as cytotoxic anti-cancer agents. In addition, the general screening strategy described may find broader impact toward the rapid discovery of DNA targeted agents with biological activity.     

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.     

Identifying modulators of hERG channel activity using the PatchXpress planar patch clamp

The authors used the PatchXpress 7000A system to measure compound activity at the hERG channel using procedures that mimicked the "gold-standard" conventional whole-cell patch clamp. A set of 70 compounds, including hERG antagonists with potencies spanning 3 orders of magnitude, were tested on hERG302-HEK cells using protocols aimed at either identifying compound activity at a single concentration or obtaining compound potency from a cumulative concentration dependence paradigm. After exposure to compounds and subsequent washout of the wells to determine reversibility of the block, blockade by a reference compound served as a quality control. Electrical parameters and voltage dependence were similar to those obtained using a conventional whole-cell patch clamp. Rank order of compound potency was also comparable to that determined by conventional methods. One exception was flunarizine, a particularly lipophilic compound. The PatchXpress accurately identified the activity of 29 moderately potent antagonists, which only weakly displace radiolabeled astemizole and are false negatives in the binding assay. Finally, no false hits were observed from a collection of relatively inactive compounds. High-quality data acquisition by PatchXpress should help accelerate secondary screening for ion channel modulators and the drug discovery process.     

Ligand-Based Virtual Screening to Identify New T-Type Calcium Channel Blockers

T-type calcium channels are involved in the generation of rhythmical firing patterns in the mammalian central nervous system and in various pathological alterations of neuronal excitability such as in epilepsy or neuropathic pain. In the search for new T-type calcium channel blockers that would help to treat these disorders, we have followed a bi-dimensional pharmacophore-based virtual screening approach to identify new inhibitors. Nineteen molecules extracted from AurSCOPE Ion Channels knowledgebase were used as query molecules to screen an external database. This in silico approach was then validated using electrophysiology. Interestingly, 16 compounds out of 38 distinct molecules tested showed more than 50% blockade of the CaV3.2 mediated T-type current. Two series of compounds show chemical originality compared with known T-type calcium channel blockers.     

Development of recombinant cell line co-expressing mutated Nav1.5, Kir2.1, and hERG for the safety assay of drug candidates

To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K(+) channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na(+) channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K(+) channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K(+) channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC(50) values to induce the cell death were 3 μM, 19 nM, 17 nM, 74 nM, and 3 μM, respectively, whereas 10 μM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K(+) channel safety assay.     

Effect of sibutramine HCl on cardiac hERG K+ channel

Common clinically used drugs block the delayed rectifier K(+) channels and prolong the cardiac action potential duration associated with long QT syndrome. Here, we investigated the mechanism of hERG K(+) channel current (I(hERG)) blockade expressed in HEK-293 cells by sibutramine HCl, a serotonin-norepinephrine reuptake inhibitor. Sibutramine HCl inhibited I (hERG) in a concentration-dependent manner with the half-maximal inhibitory concentration (IC(50)) value of 2.5 microM at -40 mV. I(hERG) inhibition by sibutramine HCl showed weak voltage dependency, but the time-dependence of I(hERG) inhibition was developed relatively rapidly on membrane depolarization. On hERG channel gating for the S6 and pore regions, the S6 residue hERG mutant Y652A and F656A largely reduced the blocking potency of I(hERG), unlike the pore-region mutants T623A and S624A. These results indicate that sibutramine HCl preferentially inhibits the hERG potassium channel through the residue Y652 and F656, in a supratherapeutic concentration should be avoided by patients with high susceptibility for cardiac arrhythmia.     

Novel roles for hERG K+ channels in cell proliferation and apoptosis

The human ether-a-go-go-related gene potassium channel (hERG, Kv11.1, KCNH2) has an essential role in cardiac action potential repolarization. Electrical dysfunction of the voltage-sensitive ion channel is associated with potentially lethal ventricular arrhythmias in humans. hERG K⁺ channels are also expressed in a variety of cancer cells where they control cell proliferation and apoptosis. In this review, we discuss molecular mechanisms of hERG-associated cell cycle regulation and cell death. In addition, the significance of hERG K⁺ channels as future drug target in anticancer therapy is highlighted.