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Bempedoic acid is an inhibitor of adenosine triphosphate–citrate lyase approved for use in adults with hypercholesterolemia. Nonclinical studies assessed binding to the human ether‐a‐go‐go–related gene (hERG) potassium channel in vitro and the effect of bempedoic acid on QT/QTc in cynomolgus monkeys. A randomized, double‐blind, parallel‐design clinical study assessed the effects of steady‐state bempedoic acid at a supratherapeutic dose (240 mg/day, 33.3% higher the180 mg/day therapeutic dose), placebo, and moxifloxacin (400 mg) in healthy subjects. In vitro binding potency for bempedoic acid to the hERG potassium channel was weak, with half‐maximal inhibition (IC50) estimated at greater than 1000 μM (>1670‐fold the bempedoic acid 180 mg/day steady‐state unbound maximum concentration). In monkeys, individual rate‐corrected QT intervals showed no time‐ or dose‐dependent changes up to 100 mg/kg of bempedoic acid. In human subjects, the upper 90% confidence interval (CI) for the difference in QTc interval, corrected using Fridericia’s formula (QTcF), between bempedoic acid and placebo was less than 5 msec at all time points. Concentration‐QTcF analysis showed that maximum bempedoic acid concentration at steady‐state was attained at a median 2.1 h postdose, and the predicted mean change (90% CI) in QTcF at the observed mean bempedoic acid concentration 2 h postdose was −0.5 (−5.0, 4.0) msec. The lower bound of the moxifloxacin 90% CI exceeded 5 msec at prespecified time points, establishing study sensitivity. Steady‐state bempedoic acid at a supratherapeutic dose of 240 mg was generally well‐tolerated and not associated with QTc prolongation in healthy subjects.

Aims The aim of this study was to determine the differences and potential mechanistic rationale for observed adverse drug reactions (ADRs) between four approved PARP inhibitors (PARPi). Methods The Medicines and Healthcare products Regulatory Authority (MHRA) Yellow Card drug analysis profiles and NHS secondary care medicines database enabled the identification of suspected ADRs associated with the PARPi in the UK from launch to 2020. The polypharmacology of the PARPi were data-mined from several public data sources. Results The overall ADRs per 100 000 Rx identified across the four PARPi are statistically significant (χ2 test, P rucaparib > olaparib tracked with the Vd trend. Hypertension is only associated with niraparib and could be explained by the therapeutically achievable inhibition of DYRK1A and/or transporters. Arrhythmia cases are potentially linked to the structural features of hERG ion-channel inhibition found in rucaparib and niraparib. Enhanced psychiatric/nervous disorders associated with niraparib can be interpreted from the diverse neurotransporter off-targets reported. Conclusions Despite their similar mode of action, the differential polypharmacology of PARP inhibitors influences their ADR profile.

For nearly 2 decades, regulators have adopted a harmonized approach to drug development, which has succeeded in bringing new pharmaceuticals to market without significant cardiac liability. Ushered in by technological advancements and better understanding of cellular electrophysiology, the initial paradigm detailed in the 2005 International Conference for Harmonization E14 and S7B documents has undergone evolutionary changes designed to streamline drug development and improve regulatory decision‐making and product labeling. The intent of this review is to summarize the new US Food and Drug Administration (FDA) Question and Answer update from August 2020 and key messaging from a subsequent FDA webinar describing best practices for preclinical and clinical data integration into a QT risk prediction model.

Drug‐mediated or medical condition‐mediated disruption of hERG function accounts for the main cause of acquired long‐QT syndrome (acLQTs), which predisposes affected individuals to ventricular arrhythmias (VA) and sudden death. Many Chinese herbal medicines, especially alkaloids, have risks of arrhythmia in clinical application. The characterized mechanisms behind this adverse effect are frequently associated with inhibition of cardiac hERG channels. The present study aimed to assess the potent effect of Rutaecarpine (Rut) on hERG channels. hERG‐HEK293 cell was applied for evaluating the effect of Rut on hERG channels and the underlying mechanism. hERG current (IhERG) was measured by patch‐clamp technique. Protein levels were analysed by Western blot, and the phosphorylation of Sp1 was determined by immunoprecipitation. Optical mapping and programmed electrical stimulation were used to evaluate cardiac electrophysiological activities, such as APD, QT/QTc, occurrence of arrhythmia, phase singularities (PSs), and dominant frequency (DF). Our results demonstrated that Rut reduced the IhERG by binding to F656 and Y652 amino acid residues of hERG channel instantaneously, subsequently accelerating the channel inactivation, and being trapped in the channel. The level of hERG channels was reduced by incubating with Rut for 24 hours, and Sp1 in nucleus was inhibited simultaneously. Mechanismly, Rut reduced threonine (Thr)/ tyrosine (Tyr) phosphorylation of Sp1 through PI3K/Akt pathway to regulate hERG channels expression. Cell‐based model unables to fully reveal the pathological process of arrhythmia. In vivo study, we found that Rut prolonged QT/QTc intervals and increased induction rate of ventricular fibrillation (VF) in guinea pig heart after being dosed Rut for 2 weeks. The critical reasons led to increased incidence of arrhythmias eventually were prolonged APD90 and APD50 and the increase of DF, numbers of PSs, incidence of early after‐depolarizations (EADs). Collectively, the results of this study suggest that Rut could reduce the IhERG by binding to hERG channels through F656 and Y652 instantaneously. While, the PI3K/Akt/Sp1 axis may play an essential role in the regulation of hERG channels, from the perspective of the long‐term effects of Rut (incubating for 24 hours). Importantly, the changes of electrophysiological properties by Rut were the main cause of VA.

The acidic and basic functional groups in a molecule strongly influence its physicochemical properties, affinity for a macromolecule, pharmacokinetics, and toxicity. For instance, basicity has been correlated with molecular promiscuity, hERG blockade, and phospholipidosis. Nonetheless, no systematic characterization of the acid/base profile of epigenomic databases has been reported. This study describes an analysis of the acidic ionization constant distribution of a library of 7820 compounds with reported activity against epigenetic targets. Furthermore, the epigenomics database's acid/base profile was compared to the reference libraries of food chemicals, natural products, and approved drugs. It was found that the acid/base profile of histone lysine demethylase ligands is more similar to previously approved drugs, and histone acetyltransferase ligands have acidic and basic functional groups largely found in food chemicals and natural products; this support the potential of these libraries for finding new epigenetic inhibitors.

The risk of fatal arrhythmias is the major concern for using chloroquine (CQ) or hydroxychloroquine (HCQ) to treat coronavirus disease 2019 (COVID‐19), but the reported number of life‐threatening arrhythmic events or deaths is relatively small. The objective of this study was to assess the arrhythmogenic risk of these two drugs using a multiscale heart simulation, which allows testing even at high concentrations, including those that cause fatal arrhythmias. We measured the inhibitory action of CQ, HCQ, and HCQ with 30 μM azithromycin (AZ) on six ion currents (fast [INa] and late [INa,L] components of the sodium current, L‐type calcium current [ICa,L], rapid [IKr/hERG], and slow [IKs] components of delayed rectifier potassium, and inward rectifier potassium [IK1]) over a wide range of concentrations using the automated patch‐clamp system. Using the concentration–inhibition relationship that was thus obtained, we simulated the drug effects while increasing the concentration until the life‐threatening arrhythmia, torsade de pointes (TdP), was observed. The obtained threshold concentrations for TdP were 12.5, 35, and 22.5 μM for CQ, HCQ, and HCQ with AZ, respectively. Adding therapeutic concentrations of mexiletine or verapamil successfully prevented the occurrence of TdP, and verapamil was more effective. CQ, HCQ, and HCQ with AZ thresholds for TdP were larger than both antiviral concentrations that were reported by in vitro experiments and free plasma concentrations that were attained by the clinically used dosage. The current simulation data provided a safety margin to the currently used clinical dose for CQ and HCQ/AZ. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Despite the potent in vitro antiviral effect, clinical trials have failed to show the therapeutic effects of chloroquine (CQ) and hydroxychloroquine (HCQ)/azithromycin (AZ) to treat coronavirus disease 2019. Torsadogenic potentials may limit the dosage of these drugs, but the reported incidence of fatal arrhythmias is rare. WHAT QUESTION DID THIS STUDY ADDRESS? Our objective was to assess the arrhythmogenicity of CQ and HCQ/AZ over a wide range of drug concentrations using a multiscale heart simulation. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? Our study showed that CQ and HCQ/AZ do not induce fatal arrhythmias even at concentrations much higher than in vitro antiviral half‐maximal effective concentration (EC50) values at which QT prolongation exceeds 150 ms. We also found that estimated free plasma concentrations of CQ and HCQ/AZ achieved by currently used dosing protocols are lower than the antiviral EC50 for these drugs. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Our simulation data provided a safety margin to the currently used clinical dose for CQ and HCQ/AZ.

BACKGROUND AND PURPOSE: The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor. EXPERIMENTAL APPROACH: A flavan series-based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM-11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM-11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae. KEY RESULTS: ORM-11372 inhibited human NCX 1.1 reverse and forward currents; IC(50) values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (I(Na) ) and hERG K(V) 11.1 currents (I(hERG) ) in a concentration-dependent manner; IC(50) values were 23.2 and 10.0 μM. ORM-11372 caused no changes in action potential duration; short-term variability and triangulation were observed for concentrations of up to 10 μM. ORM-11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose. CONCLUSION AND IMPLICATIONS: ORM-11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro-arrhythmic risk.

Defining an appropriate and efficient assessment of drug‐induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc‐prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14‐based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B‐based “double‐negative” nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high‐dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double‐negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

VEGF@A isacrucial factor of angiogenesis process in tumor cells. It mediates its biological function through VEGFR2. Therefore, VEGFR2 is a promising target to suppress angiogenesis. Till date, 8-FDA approved VEGFR2 inhibitors are available but correlated with serious side effects due to offtarget activity of compounds. Hence, these findings alarmed us to find the favorable features of compounds and association of compounds to the serious side effects. To address these question, an Atom-based 3D and fingerprint-based 2D QSAR models were constructed. The results of these models assisted us to design the hits 5 a and 6 a with favorable features from the inactive Compound 52 of diaryl urea analogs (IC50= 53 μM). The designed hits were subjected for docking, binding free energy calculation, ADME and hERG toxicity in comparison of Compound 52 of diaryl urea analogs. On the contrary to hit 5a and Compound 52 of diaryl urea analogs, hit 6 a possessed crucial interactions with ASP1046 and GLU885 along with acceptable ADMET profile which was affirmed by Molecular Dynamics simulation study. The best hit 6 a was also subjected first time to off-target analysis employing PoSSuM database.

A series of chemical optimizations, which was guided by in vitro affinity at histamine H3 receptor (H3 R), modulation of lipophilicity, ADME properties and preclinical efficacy resulted in the identification of 1-[2-(1-cyclobutylpiperidin-4-yloxy)-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-5-yl]propan-1-one (45 e) as a potent and selective (Ki =4.0 nM) H3 R inverse agonist. Dipsogenia induced by (R)-α-methylhistamine was dose dependently antagonized by 45 e, confirming its functional antagonism at H3 R. It is devoid of hERG and phospholipidosis issues. Compound 45 e has adequate oral exposures and favorable half-life in both rats and dogs. It has demonstrated high receptor occupancy (ED80 =0.22 mg/kg) and robust efficacy in object recognition task and, dose dependently increased acetylcholine levels in brain. The sub-therapeutic doses of 45 e in combination with donepezil significantly increased acetylcholine levels. The potent affinity, selectivity, in vivo efficacy and drug like properties together with safety, warrant for further development of this molecule for potential treatment of cognitive disorders associated with Alzheimer's disease.

Background and aims Mutations in KCNH2 cause long or short QT syndromes (LQTS or SQTS) predisposing to life‐threatening arrhythmias. Over 1000 hERG variants have been described by clinicians, but most remain to be characterised. The objective is to standardise and accelerate the phenotyping process to contribute to clinician diagnosis and patient counselling. In silico evaluation was also included to characterise the structural impact of the variants. Methods We selected 11 variants from known LQTS patients and two variants for which diagnosis was problematic. Using the Gibson assembly strategy, we efficiently introduced mutations in hERG cDNA despite GC‐rich sequences. A pH‐sensitive fluorescent tag was fused to hERG for efficient evaluation of channel trafficking. An optimised 35‐s patch‐clamp protocol was developed to evaluate hERG channel activity in transfected cells. R software was used to speed up analyses. Results In the present work, we observed a good correlation between cell surface expression, assessed by the pH‐sensitive tag, and current densities. Also, we showed that the new biophysical protocol allows a significant gain of time in recording ion channel properties and provides extensive information on WT and variant channel biophysical parameters, that can all be recapitulated in a single parameter defined herein as the repolarisation power. The impacts of the variants on channel structure were also reported where structural information was available. These three readouts (trafficking, repolarisation power and structural impact) define three pathogenicity indexes that may help clinical diagnosis. Conclusions Fast‐track characterisation of KCNH2 genetic variants shows its relevance to discriminate mutants that affect hERG channel activity from variants with undetectable effects. It also helped the diagnosis of two new variants. This information is meant to fill a patient database, as a basis for personalised medicine. The next steps will be to further accelerate the process using an automated patch‐clamp system., We developed a method for rapid analysis of the properties of new hERG channel variants that is helpful for clinicians to diagnose an arrhythmia and help counseling the patient. This method is rapid enough to provide a complete set of channel properties and will set the stage for a more extensive characterization in the future.

The β-site amyloid precursor protein cleaving enzyme 1 (BACE1, also known as β-secretase) is a promising target for the treatment of Alzheimer's disease. A pKa lowering approach over the initial leads was adopted to mitigate hERG inhibition and P-gp efflux, leading to the design of 6-CF3 dihydrothiazine 8 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-cyanopicolinamide). Optimization of 8 led to the discovery of 15 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-(fluoromethoxy)pyrazine-2-carboxamide) with an excellent balance of potency, hERG inhibition, P-gp efflux, and metabolic stability. Oral administration of 8 elicited robust Aβ reduction in dog even at 0.16 mg/kg. Reflecting the reduced hERG inhibitory activity, no QTc prolongation was observed at high doses. The potential for reactive metabolite formation of 15 was realized in a nucleophile trapping assay using [14 C]-KCN in human liver microsomes. Utilizing covalent binding (CVB) in human hepatocytes and the maximum projected human dosage, the daily CVB burden of 15 was calculated to be at an acceptable value of below 1 mg/day. However, hepatotoxicity was observed when 15 was subjected to a two-week tolerance study in dog, which prevented further evaluation of this compound.

Induced pluripotent stem cells (iPSCs) have shown promise in investigating donor-specific phenotypes and pathologies. The iPSC-derived cardiomyocytes (iPSC-CMs) could potentially be utilized in personalized cardiotoxicity studies, assessing individual proarrhythmic risk. However, it is unclear how closely iPSC-CMs derived from healthy subjects can recapitulate a range of responses to drugs. It is well known that QT-prolonging drugs induce subject-specific clinical response and that all healthy subjects do not necessarily develop arrhythmias or exhibit similar amounts of QT prolongation. We previously reported this variability in a study of four human ether-a-go-go-related gene (hERG) potassium channel-blocking drugs in which each subject underwent intensive pharmacokinetic and pharmacodynamic sampling such that subjects had 15 time-matched plasma drug concentration and electrocardiogram measurements throughout 24 hours after dosing in a phase I clinical research unit. In this study, iPSC-CMs were generated from those subjects. Their drug-concentration-dependent QT prolongation response from the clinic was compared with in vitro drug-concentration-dependent action potential duration (APD) prolongation response to the same two hERG-blocking drugs, dofetilide and moxifloxacin. Comparative results showed no significant correlation between the subject-specific APD response slopes and clinical QT response slopes to either moxifloxacin (P = 0.75) or dofetilide (P = 0.69). Similarly, no significant correlation was found between baseline QT and baseline APD measurements (P = 0.93). This result advances our current understanding of subject-specific iPSC-CMs and facilitates discussion into factors obscuring correlation and considerations for future studies of subject-specific phenotypes in iPSC-CMs.

The JTpeak interval has been proposed as a new biomarker to demonstrate mixed ion channel effects, potentially leading to reduced late-stage electrocardiogram (ECG) monitoring for mildly QT-prolonging drugs. ECG waveforms from the IQ-CSRC study were used. Twenty healthy subjects were enrolled with 6 subjects on placebo and 9 subjects on each of 5 mildly QT-prolonging drugs - moxifloxacin, dofetilide, ondansetron, dolasetron, and quinine - and 1 negative drug, levocetirizine. A vector magnitude lead was derived from 12-lead ECGs, and measurements were made on a median beat from three 10-second replicates. Data were analyzed using a linear concentration-response model with QTcF and heart rate corrected JTpeak (JTpeak_c) as dependent variables. For moxifloxacin, dofetilide, and ondansetron, all pure hERG blockers, slopes of the concentration (C)-QTcF and C-JTpeak_c relationships were positive and statistically significant. With the prespecified linear model, the predicted effects on ΔΔQTcF and ΔΔJTpeak_c were 11.4 and 9.4 milliseconds for moxifloxacin at the geometric mean Cmax on day 1, 9.0 and 11.7 milliseconds for dofetilide and 11.5, and 7.9 milliseconds for ondansetron, respectively. In contrast, dolasetron and quinine, both with additional ion channel effects, prolonged QTcF with a positive C-ΔQTcF slope and predicted ΔΔQTcF effect on day 1 of 6.2 and 11.4 milliseconds, whereas the C-ΔJTpeak_c slope and the predicted ΔΔJTpeak on day 1 were negative (-0.3 and -7.5 milliseconds per ng/mL). Pure hERG-blocking drugs prolonged both the QTc and the JTpeak_c intervals, whereas drugs with mixed ion channel effects, including peak sodium inhibition, prolonged QTcF but not the JTpeak_c interval.

Two decades of research have proven the relevance of ion channel expression for tumor progression in virtually every indication, and it has become clear that inhibition of specific ion channels will eventually become part of the oncology therapeutic arsenal. However, ion channels play relevant roles in all aspects of physiology, and specificity for the tumor tissue remains a challenge to avoid undesired effects. Eag1 (KV 10.1) is a voltage-gated potassium channel whose expression is very restricted in healthy tissues outside of the brain, while it is overexpressed in 70% of human tumors. Inhibition of Eag1 reduces tumor growth, but the search for potent inhibitors for tumor therapy suffers from the structural similarities with the cardiac HERG channel, a major off-target. Existing inhibitors show low specificity between the two channels, and screenings for Eag1 binders are prone to enrichment in compounds that also bind HERG. Rational drug design requires knowledge of the structure of the target and the understanding of structure-function relationships. Recent studies have shown subtle structural differences between Eag1 and HERG channels with profound functional impact. Thus, although both targets' structure is likely too similar to identify leads that exclusively bind to one of the channels, the structural information combined with the new knowledge of the functional relevance of particular residues or areas suggests the possibility of selective targeting of Eag1 in cancer therapies. Further development of selective Eag1 inhibitors can lead to first-in-class compounds for the treatment of different cancers. ispartof: MEDICINAL RESEARCH REVIEWS vol:42 issue:1 pages:183-226 ispartof: location:United States status: published

The risk-benefit ratio associated with the use of repurposed drugs to treat severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-related infectious coronavirus disease 2019 (COVID-19) is complicated because benefits are awaited, not proven. A thorough literature search was conducted to source information on the pharmacological properties of 5 drugs and 1 combination (azithromycin, chloroquine, favipiravir, hydroxychloroquine, remdesivir, and lopinavir/ritonavir) repurposed to treat COVID-19. A risk assessment of drug-induced long QT syndrome (LQTS) associated with COVID-19 repurposed drugs was performed and compared with 23 well-known torsadogenic and 10 low torsadogenic risk compounds. Computer calculations were performed using pharmacokinetic and pharmacodynamic data, including affinity to block the rapid component of the delayed rectifier cardiac potassium current (IKr ) encoded by the human ether-a-go-go gene (hERG), propensity to prolong cardiac repolarization (QT interval) and cause torsade de pointes (TdP). Seven different LQTS indices were calculated and compared. The US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) database was queried with specific key words relating to arrhythmogenic events. Estimators of LQTS risk levels indicated a very high or moderate risk for all COVID-19 repurposed drugs with the exception for azithromycin, although cases of TdP have been reported with this drug. There was excellent agreement among the various indices used to assess risk of drug-induced LQTS for the 6 repurposed medications and 23 torsadogenic compounds. Based on our results, monitoring of the QT interval shall be performed when some COVID-19 repurposed drugs are used, as such monitoring is possible for hospitalized patients or with the use of biodevices for outpatients.

The voltage‐gated hERG (human‐Ether‐à‐go‐go Related Gene) K+ channel plays a fundamental role in cardiac action potential repolarization. Loss‐of‐function mutations or pharmacological inhibition of hERG leads to long QT syndrome, whilst gain‐of‐function mutations lead to short QT syndrome. A recent open channel cryo‐EM structure of hERG represents a significant advance in the ability to interrogate hERG channel structure‐function. In order to suppress protein aggregation, a truncated channel construct of hERG (hERGT) was used to obtain this structure. In hERGT cytoplasmic domain residues 141 to 350 and 871 to 1,005 were removed from the full‐length channel protein. There are limited data on the electrophysiological properties of hERGT channels. Therefore, this study was undertaken to determine how hERGT influences channel function at physiological temperature. Whole‐cell measurements of hERG current (IhERG) were made at 37°C from HEK 293 cells expressing wild‐type (WT) or hERGT channels. With a standard +20 mV activating command protocol, neither end‐pulse nor tail IhERG density significantly differed between WT and hERGT. However, the IhERG deactivation rate was significantly slower for hERGT. Half‐maximal activation voltage (V0.5) was positively shifted for hERGT by ~+8 mV (p, The hERGTtruncation mutant was made to derive the cryo‐EM structure that is employed to study hERG K+channel structure function. This paper shows that the hERGT mutant exhibits modest differences in hERG current kinetics from the wild‐type channel. Voltage‐dependent activation and “window current” were positively voltage‐shifted, deactivation was slowed and recovery from inactivation was faster for hERGT compared to the wild‐type channel.

Key points In the human ether-a-go-go related gene (hERG) channel, both the ether-a-go-go (EAG) domain in the N-terminal and the cyclic nucleotide (CN) binding homology (CNBH) domain in the C-terminal cytoplasmic region are known to contribute to the characteristic slow deactivation. Mutations of Phe860 in the CNBH domain, reported to fill the CN binding pocket, accelerate the deactivation and decrease the fluorescence resonance energy transfer (FRET) efficiencies between the EAG and CNBH domains. An electrostatic interaction between Arg696 and Asp727 in the C-linker domain, critical for HCN and CNG channels, is not formed in the hERG channel. Mutations of newly identified electrostatically interacting pair, Asp727 in the C-linker and Arg752 in the CNBH domains, accelerate the deactivation and decrease FRET efficiency. Voltage-dependent changes in FRET efficiency were not detected. These results suggest that the acceleration of the deactivation by mutations of C-terminal domains is a result of the lack of interaction between the EAG and CNBH domains. Abstract The human ether-a-go-go related gene (hERG) channel shows characteristic slow deactivation, and the contribution of both of the N-terminal cytoplasmic ether-a-go-go (EAG) domain and the C-terminal cytoplasmic cyclic nucleotide (CN) binding homology (CNBH) domain is well known. The interaction between these domains is known to be critical for slow deactivation. We analysed the effects of mutations in the CNBH domain and its upstream C-linker domain on slow deactivation and the interaction between the EAG and CNBH domains by electrophysiological and fluorescence resonance energy transfer (FRET) analyses using Xenopus oocyte and HEK293T cell expression systems. We first observed that mutations of Phe860 in the CNBH domain, which is reported to fill the CN binding pocket as an intrinsic ligand, accelerate deactivation and eliminate the inter-domain interaction. Next, we observed that the salt bridge between Arg696 and Asp727 in the C-linker domain, which is reported to be critical for the function of CN-regulated channels, is not formed. We newly identified an electrostatically interacting pair critical for slow deactivation: Asp727 and Arg752 in the CNBH domain. Their mutations also impaired the inter-domain interaction. Taking these results together, both mutations of the intrinsic ligand (Phe860) and a newly identified salt bridge pair (Asp727 and Arg752) in the hERG channel accelerated deactivation and also decreased the interaction between the EAG and CNBH domains. Voltage-dependent changes in FRET efficiency between the two domains were not detected. The results suggest that the CNBH domain contributes to slow deactivation of the hERG channel by a mechanism involving the EAG domain.

Background and purpose Increase in high-frequency beat-to-beat heart rate oscillations by torsadogenic hERG blockers appears to be associated with signs of parasympathetic and sympathetic co-activation which cannot be assessed directly using classic methods of heart rate variability analysis. The present work aimed to find a translational model that would allow this particular state of the autonomic control of heart rate to be assessed. Experimental approach High-frequency heart rate and heart period oscillations were analysed within discrete 10 s intervals in a cohort of 200 healthy human subjects. Results were compared to data collected in non-human primates and beagle dogs during pharmacological challenges and torsadogenic hERG blockers exposure, in 127 genotyped LQT1 patients on/off β-blocker treatment and in subgroups of smoking and non-smoking subjects. Key results Three states of autonomic modulation, S1 (parasympathetic predominance) to S3 (reciprocal parasympathetic withdrawal/sympathetic activation), were differentiated to build a new model of heart rate variability referred to as high-frequency autonomic modulation. The S2 state corresponded to a specific state during which both parasympathetic and sympathetic systems were coexisting or co-activated. S2 oscillations were proportionally increased by torsadogenic hERG-blocking drugs, whereas smoking caused an increase in S3 oscillations. Conclusions and implications The combined analysis of the magnitude of high-frequency heart rate and high-frequency heart period oscillations allows a refined assessment of heart rate autonomic modulation applicable to long-term ECG recordings and offers new approaches to assessment of the risk of sudden death both in terms of underlying mechanisms and sensitivity.

Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics - the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fit a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells over-expressing hERG1a. The model is then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage clamps, and also a novel voltage clamp comprised of a collection of physiologically-relevant action potentials. We demonstrate that we can make predictive cell-specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell-cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches.

The long QT syndrome (LQTS) is a channelopathy that can lead to severe arrhythmia and sudden cardiac death. Pharmacologically induced LQTS is caused by interaction between drugs and potassium channels, especially the Kv11.1 channel. Due to such interactions, numerous drugs have been withdrawn from the market or are administered with precautions in human medicine. However, some compounds, such as trimethoprim–sulfonamide combinations are still widely used in veterinarian medicine. Therefore, we investigate the effect of trimethoprim–sulfadiazine (TMS), trimethoprim, sulfadiazine, and detomidine on equine-specific Kv11.1 channels. Kv11.1 channels cloned from equine hearts were heterologously expressed in Xenopus laevis oocytes, and whole cell currents were measured by two-electrode voltage-clamp before and after drug application. TMS blocked equine Kv11.1 current with an IC50 of 3.74 mm (95% CI: 2.95–4.73 mm) and affected the kinetics of activation and inactivation. Similar was found for trimethoprim but not for sulfadiazine, suggesting the effect is due to trimethoprim. Detomidine did not affect equine Kv11.1 current. Thus, equine Kv11.1 channels are also susceptible to pharmacological block, indicating that some drugs may have the potential to affect repolarization in horse. However, in vivo studies are needed to assess the potential risk of these drugs to induce equine LQTS.

BACKGROUND Use of the QT interval corrected for heart rate (QTc) on the electrocardiogram (ECG) to predict torsades de pointes (TdP) risk from culprit drugs is neither sensitive nor specific. The ratio of the half-maximum inhibitory concentration of the hERG channel (hERG IC50) to the peak serum concentration of unbound drug (Cmax ) is used during drug development to screen out chemical entities likely to cause TdP. PURPOSE To validate the use of the hERG IC50:Cmax ratio to predict TdP risk from a culprit drug by its correlation with TdP incidence. DATA SOURCES Medline (between 1966 and March 2017) was accessed for hERG IC50 and Cmax values from the antihistamine, fluoroquinolone, and antipsychotic classes to identify cases of drug-induced TdP. Exposure to a culprit drug was estimated from annual revenues reported by the manufacturer. STUDY SELECTION Inclusion criteria for TdP cases were provision of an ECG tracing that demonstrated QTc prolongation with TdP and normal serum values of potassium, calcium, and magnesium. Cases reported in patients with a prior rhythm disturbance and those involving a drug interaction were excluded. DATA EXTRACTION AND SYNTHESIS The Meta-Analysis of Observational Studies in Epidemiology checklist was used for epidemiological data extraction by two authors. MAIN OUTCOME AND MEASURE Negligible risk drugs were defined by an hERG IC50:Cmax ratio that correlated with less than a 5% chance of one TdP event for every 100 million exposures (relative risk [RR] 1.0). RESULTS The hERG IC50:Cmax ratio correlated with TdP risk (0.312; 95% confidence interval 0.205-0.476, p

Background and Purpose Translation of nonclinical markers of delayed ventricular repolarization to clinical QTc prolongation (a biomarker for Torsades de Pointes proarrhythmia) remains an issue in drug discovery and regulatory evaluations. A retrospective analysis of 150 submitted drug applications in a US-FDA database was conducted to determine the utility of established nonclinical in vitro iKr current (hERG), action potential duration (APD) and in vivo (QTc) repolarization assays to detect and predict clinical QTc prolongation. Experimental Approach The diagnostic and prognostic performance of three nonclinical assays were compared with clinical thorough QT study outcomes based on free clinical plasma drug concentrations using sensitivity and specificity, receiver operator curves (ROC), positive and negative predictive values, and likelihood ratios. Key Results Nonclinical assays demonstrated robust specificity (ability to identify drugs without clinical QTc prolongation) but poor sensitivity (ability to identify drugs eliciting clinical QTc prolongation) at low to intermediate (1x-30x) clinical exposure multiples. The QTc assay provided the most robust positive and negative predictive values (ability to predict clinical QTc prolongation). Receiver operator curves (defining overall test accuracy) and likelihood ratios (ability to influence post-test probabilities) demonstrated overall marginal performance for hERG and QTc assays (best results at 30x exposures) while the APD assay demonstrated minimal value. Conclusions and Implications The diagnostic and prognostic value of hERG, APD, and QTc assays varies, with drug concentrations strongly affecting translational performance. While useful in guiding preclinical candidates devoid of clinical QT prolongation, the hERG and QTc repolarization assays provide greater value compared to the APD assay.

Drugs carry a proarrhythmic risk, which gets even greater when they are used in combination. In vitro assessment of the proarrhythmic potential of drugs is limited to one compound and thus neglects the potential of drug-drug interactions, including those involving active metabolites. Here we present the results of an in vitro study of potential drug-drug interactions at the level of the hERG channel for the combination of up to three compounds: loratadine, desloratadine and ketoconazole. Experiments were performed at room temperature on an automated patch-clamp device CytoPatch 2, with the use of heterogeneously, stably transfected HEK cells. Single drugs, pairs and triplets were used. The results provided as the inhibition of the IKr current for pairs were compared against the calculated theoretical interaction. Models applied to calculate the combined effect of inhibitory actions of simultaneously given drugs include: (1) simple additive model with a maximal inhibition limit of 1 (all channels blocked in 100%); (2) Bliss independence; and (3) Loewe additivity. The observed IC50 values for loratadine, desloratadine and ketoconazole were 5.15, 1.95 and 0.74 μm respectively. For the combination of drugs tested in pairs, the effect was concentration dependent. In lower concentrations, the synergistic effect was observed, while for the highest tested concentrations it was subadditive. To triple the effect, it was subadditive regardless of concentrations. The square root of sum of squares of differences between the observed and predicted total inhibition was calculated to assess the theoretical interaction models. For most of the drugs, the allotopic model offered the best fit.

Background and Purpose Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K+ current (IKr) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased IKr is involved in increased cardiac arrhythmogenicity. Stimulation of α1A-adrenoreceptors or angiotensin II AT1 receptors is known to inhibit IKr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of IKr by activation of these two different GPCRs. Experimental Approach The whole-cell patch-clamp technique was used to record IKr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α1A-adrenoreceptor or AT1 receptor genes. Key Results A broad spectrum PKC inhibitor Go6983 (not inhibiting PKCe), a selective cPKC inhibitor Go6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of IKr by the α1A-adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of IKr by activation of AT1 receptors, whereas the PKCe-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCe activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Conclusions and Implications Our results indicated that inhibition of IKr by activation of α1A-adrenoreceptors or AT1 receptors were mediated by PKCα and PKCe isoforms respectively, through different molecular mechanisms.

Background and purpose: We evaluated the concordance of results from two sets of nonclinical cardiovascular safety studies on pitolisant. Experimental approach: Nonclinical studies envisaged both in the ICH S7B guideline and Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were undertaken. CiPA-initiative studies included in vitro ion channels and stem cell-derived human ventricular myocyte studies as well as in silico modelling of results to simulate human ventricular electrophysiology. ICH S7B-recommended studies included in vitro hERG studies, in vivo dog study with follow-up investigations in rabbit Purkinje fibres and in vivo studies in the Carlsson rabbit proarrhythmia model. Key results: Both sets of nonclinical studies consistently excluded pitolisant from having clinically relevant QT-liability or proarrhythmic potential. CiPA studies revealed pitolisant to have modest calcium channel blocking and late I Na reducing activities at high concentrations, which resulted in reduction of dofetilide-induced early after-depolarisations (EADs) by pitolisantin ICH S7B studies. Studies in stem cell-derived human cardiomyocytes with dofetilide or E-4031 given alone and in combination with pitolisant confirmed these properties. In silico modelling confirmed that the measured ion channel effects are consistent with results from both the stem cell-derived cardiomyocyte and rabbit Purkinje fibre studies and categorised pitolisant as a drug with low torsadogenic potential. The results from the two sets of nonclinical studies correlated well with two clinical QT studies. Conclusions and implications: Our experience supports the CiPA initiative but suggests that sponsors should consider investigating drug effects on EADs and the use of proarrhythmia models when the results from CiPA studies are ambiguous.

The If channel inhibitor ivabradine is recommended for treatment of chronic heart failure. However, ivabradine also inhibits human ether-a-go-go (hERG) mediated potassium currents. The aim of the present study was to assess the electrophysiologic effects of ivabradine in an experimental model of short-QT-syndrome. 12 rabbit hearts were isolated and Langendorff-perfused. After obtaining baseline data, pinacidil, an IK-ATP channel opener, was infused (1μM). Eight endo- and epicardial monophasic action potentials and a 12-lead ECG showed a significant abbreviation of QT interval (-32ms,p

Human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium current (IKr) potassium channel, which is important for cardiac repolarization. Impairment of hERG function is the primary cause of acquired long QT syndrome, which predisposes individuals to cardiac arrhythmias and sudden death. Patients with hypoxia due to conditions such as cardiac ischemia or obstructive sleep apnea display increased incidence of cardiac arrhythmias and sudden death. We sought to understand the mechanisms that underlie hypoxia-associated cardiac arrhythmias. Using cell biology and electrophysiologic techniques, we found that hypoxic culture of hERG-expressing human embryonic kidney (HEK) cells and neonatal rat cardiomyocytes reduced hERG current/IKr and mature ERG channel expression with a concomitant increase in calpain expression. Calpain was actively released into the extracellular milieu and degraded cell-surface hERG. In contrast to hERG, the ether-a-go-go (EAG) channel was not reduced by hypoxic culture. By making chimeric channels between hERG and EAG, we identified that hypoxia-induced calpain degraded hERG by targeting its extracellular S5-pore linker. The scorpion toxin BeKm-1, which is known to selectively bind to the S5-pore linker of hERG, prevented hypoxia-induced hERG reduction. Our data provide novel information about hypoxia-mediated hERG dysfunction and may have biological and clinical implications in hypoxia-associated diseases.-Lamothe, S. M., Song, W., Guo, J., Li, W., Yang, T., Baranchuk, A., Graham, C. H., Zhang, S. Hypoxia reduces mature hERG channels through calpain up-regulation.

Introduction Genetic mutations in KCNQ2 which encodes hERG, the alpha subunit of the potassium channel responsible for the IKr current, cause Long QT Syndrome, an inherited cardiac arrhythmia disorder. Electrophysiology techniques are used to correlate genotype with molecular phenotype to determine which mutations identified in patients diagnosed Long QT Syndrome are disease causing, and which are benign. These investigations are usually done using heterologous expression in cell lines, and often, epitope fusion tags are used to enable isolation and identification of the protein of interest. Methods and results Here, we demonstrate through electrophysiology techniques and immunochemistry, that both N-terminal and C-terminal myc fusion tags may perturb hERG protein channel expression and kinetics of the IKr current. We also characterize the impact of two previously reported inadvertent cDNA variants on hERG channel expression and half-life. Conclusion Our results underscore the importance of careful characterization of the impact of epitope fusion tags and conformational sequencing prior to genotype-phenotype studies for ion channel proteins such as hERG. This article is protected by copyright. All rights reserved

Background and Purpose hERG channel activators have potential utility in the treatment of acquired and congenital long QT syndrome. In this article, we describe a new hERG activator, 5-(((1H-indazol-5-yl)oxy)methyl)-N-(4-(trifluoromethoxy)phenyl)pyrimidin-2-amine (ITP-2), with a chemical structure distinct from the previously reported compounds. Experimental Approach We investigated the effect of ITP-2 on hERG1a and hERG1a/1b channels expressed heterologously in HEK-293 cells using conventional electrophysiological methods. Key Results ITP-2 selectively increased test pulse currents (EC50 1.0μM) and decreased tail currents. ITP-2 activated hERG1a homomeric channels primarily by causing large depolarizing shifts in the midpoint of voltage-dependent inactivation and hyperpolarizing shifts in the voltage-dependence of activation. In addition, ITP-2 slowed rates of inactivation and made recovery from inactivation faster. hERG1a/1b heteromeric channels showed reduced sensitivity to ITP-2 and their inactivation properties were differentially modulated. Effects on midpoint of voltage-dependent inactivation and rates of inactivation were less pronounced for hERG1a/1b channels. Effects on voltage-dependent activation and activation kinetics were no different from hERG1a channels. Interestingly, hERG1b channels were inhibited by ITP-2. Inactivation impairing mutations completely abolished activation by ITP-2 and caused inhibition of hERG channels. ITP-2 exerted agonistic effect from extracellular side of the membrane and could activate one of the arrhythmia associated trafficking deficient LQT2 mutants. Conclusions and Implications ITP-2 may serve as another novel lead molecule for designing robust hERG activators. hERG1a/1b gating kinetics were differentially modulated by ITP-2 leading to altered sensitivity. ITP-2 is capable of activating an LQT2 mutant and hence potentially useful in the development of LQT2 therapeutics.

Author(s): Yang, Pei-Chi; Perissinotti, Laura L; Lopez-Redondo, Fernando; Wang, Yibo; DeMarco, Kevin R; Jeng, Mao-Tsuen; Vorobyov, Igor; Harvey, Robert D; Kurokawa, Junko; Noskov, Sergei Y; Clancy, Colleen E | Abstract: KEY POINTS:This study represents a first step toward predicting mechanisms of sex-based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening. We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias. Using the recently solved cryo-EM structure for the Eag-family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S). Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore. ABSTRACT:Female sex is a risk factor for inherited and acquired long-QT associated torsade de pointes (TdP) arrhythmias, and sympathetic discharge is a major factor in triggering TdP in female long-QT syndrome patients. We used a combined experimental and computational approach to predict 'the perfect storm' of hormone concentration, IKr block and sympathetic stimulation that induces arrhythmia in females with inherited and acquired long-QT. More specifically, we developed mathematical models of acquired and inherited long-QT syndrome in male and female ventricular human myocytes by combining effects of a hormone and a hERG blocker, dofetilide, or hERG mutations. These 'male' and 'female' model myocytes and tissues then were used to predict how various sex-based differences underlie arrhythmia risk in the setting of acute sympathetic nervous system discharge. The model predicted increased risk for arrhythmia in females when acute sympathetic nervous system discharge was applied in the settings of both inherited and acquired long-QT syndrome. Females were predicted to have protection from arrhythmia induction when progesterone is high. Males were protected by the presence of testosterone. Structural modelling points towards two plausible and distinct mechanisms of oestrogen action enhancing torsadogenic effects: oestradiol interaction with hERG mutations in the pore loop containing G604 or with common TdP-related blockers in the intra-cavity binding site. Our study presents findings that constitute the first evidence linking structure to function mechanisms underlying female dominance of arousal-induced arrhythmias.

Background Pilsicainide, classified as a relatively selective Na + channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K + current ( I Kr ) through human ether-a-go-go-related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild-type (WT) hERG proteins and WT-hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system. Methods HEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch-clamp experiments. Results Acute exposure to pilsicainide at 0.03–10μM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03–10μM pilsicainide for 48h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3μM pilsicainide for 48h delayed degradation of WT-hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane-impermeant pilsicainide derivative did not influence the expression of WT-hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF-1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking-defective mutant hERG, G601S and R752W. Conclusions Pilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.

This Minireview describes a presentation made at the XXIV National Meeting in Medicinal Chemistry (NMMC) held in Perugia (Italy), September 11-14, 2016. It relates to the discovery of novel templates of the so-called "amino" region of dopamine D3 receptor antagonists. Moving from the early scaffolds, which were modified in the amine portion, this review discusses the variations that led to the discovery of new systems published in 2016, which allowed the identification of compounds endowed with great selectivity over the dopamine D2 receptor and the human ether-a-go-go-related gene (hERG) ion channel. The main efforts in characterizing these compounds were devoted not only to determining their potency and selectivity relative to closely associated targets (e.g., the dopamine D2 receptor), but to ensure a large therapeutic window versus liability points such as hERG. In particular, we present examples of derivatives with selectivities greater than 2000-fold. Furthermore, much focus is devoted to the overall developability of the scaffolds, ensuring that appropriate physicochemical and pharmacokinetic parameters are present in all compounds progressing through the screening cascade.

Drug-induced blockade of human ether-a-go-go-related gene (hERG) remains a major impediment in delivering safe drugs to the market. Several drugs have been withdrawn from the market due to their severe cardiotoxic side effects triggered by their off-target interactions with hERG. Thus, identifying the potential hERG blockers at early stages of lead discovery is fast evolving as a standard in drug design and development. A number of in silico structure-based models of hERG have been developed as a low-cost solution to evaluate drugs for hERG liability, and it is now agreed that the hERG blockers bind at the large central cavity of the channel. Nevertheless, there is no clear convergence on the appropriate drug binding modes against the channel. The proposed binding modes differ in their orientations and interpretations on the role of key residues in the channel. Such ambiguities in the modes of binding remain to be a significant challenge in achieving efficient computational predictive models and in saving many important already Food and Drug Administration approved drugs. In this review, we discuss the spectrum of reported binding modes for hERG blockers, the various in silico models developed for predicting a drug's affinity to hERG, and the known successful optimization strategies to avoid off-target interactions with hERG.

BACKGROUND AND PURPOSE Recent studies showed that clemizole has a potential therapeutic effect for hepatitis C infection and also potently inhibits TRPC5 channels. The aim of the present study was to investigate whether clemizole blocks cardiac K+ currents and affects cardiac repolarization. EXPERIMENTAL APPROACH Whole-cell patch technique was employed to examine the effects of clemizole on hERG channel current, IKs and Kv1.5 current in HEK 293 cell expression systems as well as on ventricular action potentials of guinea pig hearts. The ex vivo guinea pig hearts were used to determine the effect on electrocardiogram. KEY RESULTS Clemizole decreased hERG current by blocking both open and closed states of the channel in a concentration-dependent manner (IC50: 0.07 μM). The S631A, S636A, Y652A and F656V hERG mutant channels reduced the inhibitory effect of clemizole (IC50: 0.82, 0.89, 1.49 and 2.98 μM, respectively), suggesting that clemizole is a pore blocker of hERG channels. Clemizole also moderately decreased IKs and hKv1.5 current. Moreover, clemizole increased ventricular action potential duration of guinea pig hearts and the QTc interval in perfused ex vivo guinea pig hearts in a concentration dependent manner (0.1-1.0 μM). CONCLUSION AND IMPLICATIONS Our results demonstrate the first evidence that clemizole potently blocks hERG channels, moderately inhibits cardiac IKs, delays cardiac repolarization, and thereby prolongs QT intervals. Thus, caution should be taken when clemizole is used as a TRPC5 channel blocker or for treating hepatitis C infection.

Background and purpose A second-generation antihistamine, terfenadine, is known to induce arrhythmia by blocking hERG channels. In this study, we have shown that terfenadine also inhibits the activity of G-protein-gated inwardly rectifying K+ (GIRK) channels, which regulate the excitability of neurons and cardiomyocytes. To clarify the underlying mechanism(s), we examined the effects of several antihistamines on GIRK channels and identified the structural determinant for the inhibition. Experimental approach Electrophysiological recordings were made in Xenopus oocytes and rat atrial myocytes to analyse the effects of antihistamines on various GIRK subunits (Kir 3.x). Mutagenesis analyses identified the residues critical for inhibition by terfenadine and the regulation of ion selectivity. The potential docking site of terfenadine was analysed by molecular docking. Key results GIRK channels containing Kir 3.1 subunits heterologously expressed in oocytes and native GIRK channels in atrial myocytes were inhibited by terfenadine and other non-sedating antihistamines. In Kir 3.1 subunits, mutation of Phe137, located in the centre of the pore helix, to the corresponding Ser in Kir 3.2 subunits reduced the inhibition by terfenadine. Introduction of an amino acid with a large side chain in Kir 3.2 subunits at Ser148 increased the inhibition. When this residue was mutated to a non-polar amino acid, the channel became permeable to Na+ . Phosphoinositide-mediated activity was also decreased by terfenadine. Conclusion and implications The Phe137 residue in Kir 3.1 subunits is critical for inhibition by terfenadine. This study provides novel insights into the regulation of GIRK channels by the pore helix and information for drug design.

BACKGROUND AND PURPOSE: The citrus flavanone hesperetin has been proposed for the treatment of several human pathologies, but its cardiovascular actions remain largely unexplored. Here, we evaluated the effect of hesperetin on cardiac electrical and contractile activities, on aortic contraction, on the wild-type voltage-gated NaV 1.5 channel, and on a channel mutant (R1623Q) associated with lethal ventricular arrhythmias in the long QT syndrome type 3 (LQT3). EXPERIMENTAL APPROACH: We used cardiac surface ECG and contraction force recordings to evaluate the effects of hesperetin in rat isolated hearts and aortic rings. Whole-cell patch clamp was used to record NaV 1.5 currents (INa ) in rat ventricular cardiomyocytes and in HEK293T cells expressing hNaV 1.5 wild-type or mutant channels. KEY RESULTS: Hesperetin increased the QRS interval and heart rate and decreased the corrected QT interval and the cardiac and aortic contraction forces at concentrations equal or higher than 30 μmol·L-1 . Hesperetin blocked rat and human NaV 1.5 channels with an effective inhibitory concentration of ≈100 μmol·L-1 . This inhibition was enhanced at depolarized holding potentials and higher stimulation frequency and was reduced by the disruption of the binding site for local anaesthetics. Hesperetin increased the rate of inactivation and preferentially inhibited INa during the slow inactivation phase, these effects being more pronounced in the R1623Q mutant. CONCLUSIONS AND IMPLICATIONS: Hesperetin preferentially inhibits the slow inactivation phase of INa , more markedly in the mutant R1623Q. Hesperetin could be used as a template to develop drugs against lethal cardiac arrhythmias in LQT3. ispartof: BRITISH JOURNAL OF PHARMACOLOGY vol:176 issue:8 pages:1090-1105 ispartof: location:England status: published

miRNAs Regulate hERGBackground The human ether-a-go-go-related gene (hERG) is the major molecular component of the rapidly activating delayed rectifier K+ current (Ikr). Impairment of hERG function is believed to be a mechanism causing long-QT syndromes (LQTS). Growing evidences have shown that microRNAs (miRNAs) are involved in functional modulation of the hERG pathway. The purpose of this study was to screen and validate miRNAs that regulate the hERG pathway. The miRNAs identified in this study will provide new tools to assess the mechanism of LQTS. Methods Six miRNAs were selected by algorithm predictions based on potential interaction with hERG. The effects of each miRNA on hERG were assessed by use of the Dual-Luciferase Reporter assay system, qRT-PCR, Western blotting, and confocal fluorescence microscopy. Furthermore, whole-cell patch clamp technique was used to validate the effect of miR-103a-1 on the electrophysiological characteristic of the Ikr of the hERG protein channel. Results miR-134, miR-103a-1, miR-143, and miR-3619 significantly downregulated luciferase activity (P < 0.05) in a reporter test system. These 4 miRNAs significantly suppressed expression of hERG mRNA and protein in U2OS cells (P < 0.05).Corresponding AMOs rescued expression of hERG mRNA and protein. Confocal microscopy showed that all 4 miRNAs reduced the expression of both immature and mature hERG protein. miR-103a-1 decreased the maximum current and tail current amplitudes of hERG channel. Conclusions Expression and functions of hERG are regulated by specific miRNAs.