Blockade Of hERG Research Articles

HergSPred: Accurate Classification of hERG Blockers/Nonblockers with Machine-Learning Models.

The human ether-à-go-go-related gene (hERG) K+ channel plays an important role in cardiac action potentials. The inhibition of the hERG channel may lead to long QT syndrome (LQTS) and even sudden cardiac death. Due to severe hERG-related cardiotoxicity, many drugs have been withdrawn from the market. Therefore, it is necessary to estimate the chemical blockade of hERG in the early stage of drug discovery. In this study, we collected 12,850 compounds with hERG inhibition data from the literature and trained a series of hERG blocking classification models based on the MACCS and Morgan fingerprints. A consensus model named HergSPred was generated based on the individual models using voting principles. The accuracy of HergSPred is higher than previous models using identical training and test sets. Moreover, we analyzed the contribution of each input fingerprint to the prediction output to obtain intuitive chemical insights into the hERG inhibition, which allows visualization of warning substructures that may cause cardiotoxicity in the input compound. The model is available at http://www.icdrug.com/ICDrug/T.

Experimentally Validated Pharmacoinformatics Approach to Predict hERG Inhibition Potential of New Chemical Entities.

The hERG (human ether-a-go-go-related gene) encoded potassium ion (K+) channel plays a major role in cardiac repolarization. Drug-induced blockade of hERG has been a major cause of potentially lethal ventricular tachycardia termed Torsades de Pointes (TdPs). Therefore, we presented a pharmacoinformatics strategy using combined ligand and structure based models for the prediction of hERG inhibition potential (IC50) of new chemical entities (NCEs) during early stages of drug design and development. Integrated GRid-INdependent Descriptor (GRIND) models, and lipophilic efficiency (LipE), ligand efficiency (LE) guided template selection for the structure based pharmacophore models have been used for virtual screening and subsequent hERG activity (pIC50) prediction of identified hits. Finally selected two hits were experimentally evaluated for hERG inhibition potential (pIC50) using whole cell patch clamp assay. Overall, our results demonstrate a difference of less than ±1.6 log unit between experimentally determined and predicted hERG inhibition potential (IC50) of the selected hits. This revealed predictive ability and robustness of our models and could help in correctly rank the potency order (lower μM to higher nM range) against hERG.

Sensitivity of Flecainide Inhibition of hERG Channels to Channel Inactivation

hERG K+ channels are sensitive to pharmacological inhibition by many different drugs including Class I and Class III antiarrhythmics. These drugs exhibit differences regarding the reliance of hERG current (IhERG) blockade on channel inactivation: Class Ia (quinidine and disopyramide) and Ic (propafenone) agents are less dependent on inactivation than are Class III methanesulphonanilide compounds (dofetilide and E-4031) (1-3). Flecainide is a Class Ic agent that inhibits IhERG at clinically relevant concentrations (4) and its sensitivity to hERG inactivation has not been systematically investigated. This study was undertaken to evaluate the influence of inactivation on flecainide blockade of hERG, through the use of attenuated-inactivation mutants S631A, N588K and S631A/N588K (3). IhERG was recorded using whole-cell patch-clamp at 37oC from hERG-expressing HEK293 cells. IhERG tails were measured at −40mV after a 2sec depolarizing step from −80mV to +20mV (n≥5 at each concentration). Flecainide produced a concentration-dependent inhibition of wild-type (WT) IhERG with an IC50 of 1.49µM (CI:1.27-1.74). The IC50 for inhibition of S631A-IhERG [7.49µM (CI:6.33-8.87)] was ∼5-fold that for WT-IhERG, whilst that for N588K-IhERG [6.50µM (CI:5.37-7.88)] was ∼4.4-fold that of WT-IhERG. The S631A/N588K double mutant, which leads to a greater attenuation of IhERG inactivation than does either individual mutation (3), exhibited an IC50 [19.16µM (CI:15.43-23.80)] ∼12.9 fold that of WT-IhERG. Comparison of these data with prior work using these inactivation-mutants (3) is suggestive that flecainide inhibition of IhERG depends on inactivation to a slightly greater extent than Class Ia drugs and the Class Ic drug propafenone, but to a much lesser extent than does E-4031.Funded by the British Heart Foundation.1. Ficker et al. Circ Res 1998; 82:386-95.2. Perrin et al. Mol Pharmacol 2008; 74:1443-52.3. McPate et al. BJP 2008; 155:957-966.4. Paul et al. BJP 2002; 136:717-29.

Comparison of Electrophysiological Data From Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes to Functional Preclinical Safety Assays

Human-induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) are a potential source to develop assays for predictive electrophysiological safety screening. Published studies show that the relevant physiology and pharmacology exist but does not show the translation between stem cell cardiomyocyte assays and other preclinical safety screening assays, which is crucial for drug discovery and safety scientists and the regulators. Our studies are the first to show the pharmacology of ion channel blockade and compare them with existing functional cardiac electrophysiology studies. Ten compounds (a mixture of pure hERG [E-4031 and Cisapride], hERG and sodium [Flecainide, Mexiletine, Quinidine, and Terfenadine], calcium channel blockers [Nifedipine and Verapamil], and two proprietary compounds [GSK A and B]) were tested, and results from hiPSC-CMs studied on multielectrode arrays (MEA) were compared with other preclincial models and clinical drug concentrations and effects using integrated risk assessment plots. All ion channel blockers produced (1) functional effects on repolarization and depolarization around the IC25 and IC50 values and (2) excessive blockade of hERG and/or blockade of sodium current precipitated arrhythmias. Our MEA data show that hiPSC-CMs demonstrate relevant pharmacology and show excellent correlations to current functional cardiac electrophysiological studies. Based on these results, MEA assays using iPSC-CMs offer a reliable, cost effective, and surrogate to preclinical in vitro testing, in addition to the 3Rs (refine, reduce, and replace animals in research) benefit.

Application of human stem cell-derived cardiomyocytes in safety pharmacology requires caution beyond hERG

Human embryonic stem cell-derived cardiomyocytes (hESC-CM) have been proposed as a new model for safety pharmacology. So far, a thorough description of their basic electrophysiology and extensive testing, and mechanistic explanations, of their overall pro-arrhythmic ability is lacking. Under standardized conditions, we have evaluated the sensitivity of hESC-CM to proarrhythmic provocations by blockade of hERG and other channels. Using voltage patch clamp, some ion current densities (pA/pF) in hESC-CM were comparable to adult CM: I(Kr) (-12.5 ± 6.9), I(Ks) (0.65 ± 0.12), I(Na,peak) (-72 ± 21), I(Na,late) (-1.10 ± 0.36), and I(Ca,L) (-4.3 ± 0.6). I(f) density was larger (-10 ± 1.1) and I(K1) not existent or very small (-2.67 ± 0.3). The low I(K1) density was corroborated by low KCNJ2 mRNA levels. Effects of pro-arrhythmic compounds on action potential (AP) parameters and provocation of early afterdepolarizations (EADs) revealed that Chromanol293B (100 μmol/l) and Bay K8644 (1 μmol/l) both significantly prolonged APD(90). ATX-II (<1 μmol/l ) and BaCl(2) (10 μmol/l ) had no effect on APD. The only compound that triggered EADs was hERG blocker Cisapride. Computer simulations and AP clamp showed that the immature AP of hESC-CM prevents proper functioning of I(Na)-channels, and result in lower peak/maximal currents of several other channels, compared to the adult situation. Lack of functional I(K1) channels and shifted I(Na) channel activation cause a rather immature electrophysiological phenotype in hESC-CM, and thereby limits the potential of this model to respond accurately to pro-arrhythmic triggers other than hERG block. Maturation of the electrical phenotype is a prerequiste for future implementation of the model in arrhythmogenic safety testing.

Computer Simulations of Structure–Activity Relationships for hERG Channel Blockers

The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.

Blockade of hERG K+ channel by antimalarial drug, primaquine

Lengthening of the Q-T interval and proarrhythmia are adverse effects associated with antimalarial agents. Also, lengthening of the Q-T interval is a definite outcome when patients are administered with an overdose of primaquine. Inhibition of potassium current I (Kr) and resultant QT prolongation is suggested as the reason behind drug-induced arrhythmias. The present study investigated the molecular mechanisms of voltage-dependent inhibition of human Ether-a-go-go Related Gene (hERG) delayed rectifier K(+) channels expressed in HEK-293 cells by primaquine. Primaquine inhibited hERG current in a concentration-dependent manner with the half-maximal inhibitory concentration (IC(50)) of 21.5 microM. The voltage-dependent inhibition of hERG current resulted in the activation curve to be shifted to a negative voltage after primaquine exposure in a dose-dependent manner. Blockade of hERG by primaquine was also found to be time-dependent, occurring rather rapidly. Blockade of wild-type hERG channel by primaquine was similar to those of both the S6 residue hERG mutants (F656A and Y652A) and the pore region mutants (T623A). In conclusion, these results indicate that primaquine preferentially inhibits the hERG potassium channel, but blockade of hERG channel by primaquine may not be related to the S6 residue or the pore region, but may be induced through other pathways such as binding other region or effect by drug binding receptor which indicates a need for further exploration.

Blockade of hERG K+ channel by the Histamine H1 receptor antagonist, diphenhydramine

Blockade of hERG K+ channel by the Histamine H1 receptor antagonist, diphenhydramine

The Interactions Between hERG Potassium Channel and Blockers

The human ether-a-go-go related gene (hERG) potassium channel is critical to the QT interval in the human heart measured by the electrocardiogram (ECG). The blockade of hERG would induce undesired lethal arrhythmia, named torsades de pointes (TdP), a rare but life-threatening symptom. Although a large number of experimental studies on hERG have been conducted so far, knowledge of how known ligands bind to hERG still remains sketchy and has been a major hindrance in the effort to designing novel medicinal molecules devoid of hERG activity in the hope of improving drug safety. This review summarizes several studies on ligand-hERG interactions by in silico receptor-based and ligand-based modeling approaches during recent years. These efforts could aid tremendously in understanding the determinants of ligand binding to hERG channel and the molecular basis of hERG channel blockade, and offer a more rational approach for the prediction of QT-prolongation liability and for the development of novel and safe non-cardiac agents.

Oxidative Inactivation of the Lipid Phosphatase PTEN as a Novel Mechanism of Acquired Long QT Syndrome

The most common cause for cardiac side effects during therapy is drug-induced, acquired long QT syndrome due to direct blockade of the cardiac potassium channel hERG. However, antimony-based antileishmanial compounds and arsenic trioxide (As2O3), an anti-neoplastic drug, are pro-arrhythmic by an increase in cardiac calcium currents. In the present study we investigated the molecular mechanism(s) underlying the increase in calcium currents observed on incubation with As2O3. Calcium currents were recorded in guinea pig ventricular myocytes cultured overnight in the presence of As2O3. We found that arsenic-induced increases in calcium currents could be eliminated by co-incubation with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Similarly, As2O3 effects could be abolished by incubation with wortmannin, a structurally different PI3K inhibitor. When isoform-specific PI3K activities were evaluated in cardiomyocytes, we found PI3Kα to be most active. However, PI3Kα activity was not altered on incubation with As2O3. In marked contrast, the lipid phosphatase PTEN which degrades PI3K-produced PIP3, was oxidized on incubation with As2O3 as indicated on Western blots by the appearance of a fast migrating, inactive protein form. In line with this observation, intracellular applied PIP3 was able to increase cardiac calcium currents in guinea pig ventricular myocytes. This effect was specific and could not be observed upon intracellular application of PIP2. Furthermore, As2O3-induced increases of calcium currents were not abolished by inhibition of PKB/Akt, an important downstream kinase activated by PIP3, but instead appeared to require PKC and c-Src function. Taken together, our experiments establish the lipid phosphatase PTEN as a crucial target for oxidative inactivation by As2O3. We propose that an altered gain in PIP3-dependent cell signaling is responsible for the pro-arrhythmic increase in cardiac calcium currents seen with anti-neoplastic As2O3 during therapy.

Overcoming hERG Affinity in the Discovery of Maraviroc; A CCR5 Antagonist for the Treatment of HIV

Avoiding cardiac liability associated with blockade of hERG (human ether a go-go) is key for successful drug discovery and development. This paper describes the work undertaken in the discovery of a potent CCR5 antagonist, maraviroc 34, for the treatment of HIV. In particular the use of a pharmacophore model of the hERG channel and a high throughput binding assay for the hERG channel are described that were critical to elucidate SAR to overcome hERG liabilities. The key SAR involves the introduction of polar substituents into regions of the molecule where it is postulated to undergo hydrophobic interactions with the ion channel. Within the CCR5 project there appeared to be no strong correlation between hERG affinity and physiochemical parameters such as pKa or lipophilicity. It is believed that chemists could apply these same strategies early in drug discovery to remove hERG interactions associated with lead compounds while retaining potency at the primary target.

Clomipramine block of the hERG K + channel: Accessibility to F656 and Y652

Clomipramine is a tricyclic antidepressant for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. Since blockade of cardiac human ether- a- go- go-related gene (hERG) channels is an important cause of acquired long QT syndrome, we investigated the acute effects of clomipramine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of clomipramine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Clomipramine induced a concentration-dependent decrease in the current amplitude at the end of the voltage steps and hERG tail currents. The IC50 for clomipramine needed to block the hERG current in Xenopus oocytes decreased progressively relative to the degree of depolarization. The fractional electrical distance was estimated to be δ = 0.83. The IC50 for the clomipramine-induced blockade of the hERG currents in HEK293 cells at 36 °C was 0.13 μM at + 20 mV. Clomipramine affected the channels in the activated and inactivated states but not in the closed states. The clomipramine-induced blockade of hERG was found to be use-dependent, exhibiting a more rapid onset and a greater steady-state block at the higher frequencies of activation. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG-current blockade. These results suggest that clomipramine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of clomipramine.

Identification of “toxicophoric” features for predicting drug-induced QT interval prolongation

Drugs delaying cardiac repolarization by blockade of hERG K + channel generally prolong the QT interval of the electrocardiogram, an effect regarded as a cardiac risk factor with the potential to cause ‘ torsade des pointes’-type arrhythmias in humans. The present study applied a homology building technique and molecular dynamics simulations to model the pore of hERG K + channel. A docking analysis was then performed on selected ligands which were classified as QT-prolonging or non-prolonging after experimental measurements in in vivo anesthetized guinea pig. The results of this structural analysis provided a “toxicophoric” model that was further exploited to inspect a dataset of known QT-prolonging/non-prolonging molecules. The emerging major chemical features to be avoided, in order to obtain cardiac safe therapeutic agents, comprise the simultaneous presence of (i) a protonated nitrogen atom within an observed range of distances from a heteroatom; (ii) aromatic groups capable of interacting within an area defined by Gly657 residues of the pore or within an area located at the top of the longitudinal axis of the pore. Moreover, additional hydrophobic moieties interacting with one of the equatorial cavities located in the area near-by Tyr652 residues and/or with a hydrophobic ring defined by Phe656 residues should be avoided.

Computer-aided prediction of QT-prolongation1

Drug-induced cardiac arrhythmia is acknowledged as a serious obstacle in successful development of new drugs. Several methods for in silico prediction of acquired long QT syndrome (LQTS) caused by the pharmacological blockade of human hERG K+ channels are discussed in literature. We propose to use the computer program PASS, which estimates the probabilities of about 3000 biological activities, not only for prediction of hERG blockade and QT-prolongation but also for the analysis of indirect mechanisms of these actions. After addition in the PASS training set of 163 compounds with data on QT-Prolongation and re-training, it was shown that accuracy of prediction was 87.1% and 81.8% for hERG blockade and QT-prolongation, respectively. Using computer program PharmaExpert we found that in the predicted biological activity spectra there was a certain correlation between the hERG blockade and some other molecular mechanisms of action. Possible role of 1-phosphatidylinositol-4-phospate 5-kinase, dimethylargininase and progesterone 11 alpha-monooxygenase inhibition in hERG blockade was discussed. 1Presented at CMTPI 2007: Computational Methods in Toxicology and Pharmacology Integrating Internet Resources (Moscow, Russia, September 1–5, 2007).

Mechanism of Block of the hERG K + Channel by the Scorpion Toxin CnErg1

The scorpion toxin CnErg1 binds to human ether-a-go-go related gene (hERG) K + channels with a 1:1 stoichiometry and high affinity. However, in contrast to other scorpion toxin-ion channel interactions, the inhibition of macroscopic hERG currents by high concentrations of CnErg1 is incomplete. In this study, we have probed the molecular basis for this incomplete inhibition. High concentrations of CnErg1 had only modest effects on hERG gating that could not account for the incomplete block. Furthermore, the residual current in the presence of 1 μM CnErg1 had normal single channel conductance. Analysis of the kinetics of CnErg1 interaction with hERG indicated that CnErg1 binding is not diffusion-limited. A bimolecular binding scheme that incorporates an initial encounter complex and permits normal ion conduction was able to completely reproduce both the kinetics and steady-state level of CnErg1-hERG binding. This scheme provides a simple kinetic explanation for incomplete block; that is, relatively fast backward compared to forward rate constants for the interconversion of the toxin-channel encounter complex and the blocked toxin-channel complex. We have also examined the temperature-dependence of CnErg1 binding to hERG. The dissociation constant, K d, for CnErg1 increases from 7.3 nM at 22°C to 64 nM at 37°C (i.e., the affinity decreases as temperature increases) and the proportion of binding events that lead to channel blockade decreases from 70% to 40% over the same temperature range. These temperature-dependent effects on CnErg1 binding correlate with a temperature-dependent decrease in the stability of the putative CnErg1 binding site, the amphipathic α-helix in the outer pore domain of hERG, assayed using circular dichroism spectropolarimetry. Collectively, our data provides a plausible kinetic explanation for incomplete blockade of hERG by CnErg1 that is consistent with the proposed highly dynamic conformation of the outer pore domain of hERG.

Blockade of HERG human K + channel and IKr of guinea pig cardiomyocytes by prochlorperazine

Prochlorperazine, a drug for the symptomatic control of nausea, vomiting and psychiatric disorders, can induce prolonged QT, torsades de pointes and sudden death. We studied the effects of prochlorperazine on human ether- a- go- go-related gene ( HERG) channels expressed in Xenopus oocytes and also in the delayed rectifier K + current of guinea pig cardiomyocytes. Prochlorperazine induced a concentration-dependent decrease in current amplitudes at the end of the voltage steps and tail currents of HERG. The IC 50 for a prochlorperazine block of HERG current in Xenopus oocytes progressively decreased relative to the degree of depolarization, from 42.1 μM at − 40 mV to 37.4 μM at 0 mV to 22.6 μM at + 40 mV. The block of HERG by prochlorperazine was use-dependent, exhibiting a more rapid onset and a greater steady-state block at higher frequencies of activation, while there was partial relief of the block with reduced frequencies. In guinea pig ventricular myocytes, bath applications of 0.5 and 1 μM prochlorperazine at 36 °C blocked rapidly activating delayed rectifier K + current by 38.9% and 76.5%, respectively, but did not significantly block slowly activating delayed rectifier K + current. Our findings suggest that the arrhythmogenic side effects of prochlorperazine are caused by a blockade of HERG and the rapid component of the delayed rectifier K + current rather than by a blockade of the slow component.

Effects of Antipsychotic Drugs on Ito, INa, Isus, IK1, and hERG: QT Prolongation, Structure Activity Relationship, and Network Analysis

To evaluate in vitro and computationally model the effects of selected antipsychotic drugs on several ionic currents that contribute to changes in the action potential in cardiac tissue. Fourteen antipsychotic drugs or metabolites were examined to determine whether QT interval prolongation could be accounted for by an effect on one or more myocardial ion channels [I(to), I(Na), I(sus), I(K1), and human ether-a-go-go related gene (hERG)]. Using the patch clamp technique, drug effects on these human cardiac currents were tested. All molecules had little inhibitory effect on ion channels (blocking at concentrations >5 microM) other than hERG. A significant correlation was observed between the estimated hERG blockade and the increase in corrected QT for five of the antipsychotics. Molecular modeling identified hydrophobic features related to the interaction with hERG and correctly rank-ordered the test set molecules olanzapine and its metabolites. A network analysis of ligand and protein interactions around hERG using MetaCore (GeneGo Inc., St. Joseph, MI, USA) was used to visualize antipsychotics with affinity for this channel and their interactions with other proteins in this database. The antipsychotics do not inhibit the ion channels I(to), I(Na), I(sus), I(K1) to any appreciable extent; however, blockade of hERG is a likely mechanism for the prolongation of the QT interval.

Prediction of hERG potassium channel affinity by the CODESSA approach

The problem of predicting torsadogenic cardiotoxicity of drugs is afforded in this work. QSAR studies on a series of molecules, acting as hERG K + channel blockers, were carried out for this purpose by using the CODESSA program. Molecules belonging to the analyzed dataset are characterized by different therapeutic targets and by high molecular diversity. The predictive power of the obtained models was estimated by means of rigorous validation criteria implying the use of highly diagnostic statistical parameters on the test set, other than the training set. Validation results obtained for a blind set, disjoined from the whole dataset initially considered, confirmed the predictive potency of the models proposed here, so suggesting that they are worth to be considered as a valuable tool for practical applications in predicting the blockade of hERG K + channels.

A model for identifying HERG K + channel blockers

Acquired long QT syndrome (LQTS) occurs frequently as a side effect of blockade of cardiac HERG K + channels by commonly used medications. A large number of structurally diverse compounds have been shown to inhibit K + current through HERG. There is considerable interest in developing in silico tools to filter out potential HERG blockers early in the drug discovery process. We describe a binary classification model that combines a 2D topological similarity filter with a 3D pharmacophore ensemble procedure to discriminate between HERG actives and inactives with an overall accuracy of 82%, with false negative and false positive rates of 29% and 15%, respectively. This model should be generally applicable in virtual library counterscreening against HERG.

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.