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Molecular dynamics was used to optimize the droperidol-hERG complex obtained from docking. To accommodate the inhibitor, residues T623, S624, V625, G648, Y652, and F656 did not move significantly during the simulation, while F627 moved significantly. Binding sites in cryo-EM structures and in structures obtained from molecular dynamics simulations were characterized using solvent mapping and Atlas ligands, which were negative images of the binding site, were generated. Atlas ligands were found to be useful for identifying human ether-á-go-go-related potassium channel (hERG) inhibitors by aligning compounds to them or by guiding the docking of compounds in the binding site. A molecular dynamics optimized structure of hERG led to improved predictions using either compound alignment to the Atlas ligand or docking. The structure was also found to be suitable to define a strategy for lowering inhibition based on the proposed binding mode of compounds in the channel.

The KCNH2 gene, encoding for a subunit of a voltage-gated potassium channel, has been identified as a key element of neuronal excitability and a promising novel therapeutic target for schizophrenia (SZ). Nonetheless, evidence highlighting the role of KCNH2 on cognitive and brain activity phenotypes comes mainly from studies based on healthy controls (HC). Therefore, we aimed to study the role of KCNH2 on the brain functional differences between patients with SZ and HC. The fMRI sample comprised 78 HC and 79 patients with SZ (matched for age, sex and premorbid IQ). We studied the effect of the polymorphism KCNH2-rs3800779 on attention and working memory-related brain activity, evaluated through the N-back task, in regions with detected diagnostic differences (regression model, controlled for age, sex and premorbid IQ, FEAT-FSL). We report a significant diagnosis x KCNH2 interaction on brain activity (1-back vs baseline contrast) at the medial superior prefrontal cortex (Zmax=3.55, p = 0.00861). In this region, patients with SZ carrying the risk genotype (AA) show a deactivation failure, while HC depict the opposite pattern towards deactivation. The brain region with significant diagnosis x KCNH2 interaction has been previously associated with SZ. The results of this study, in which the role of KCNH2 on fMRI response is analysed for the first time in patients, suggest that KCNH2 variability contributes to inefficient brain activity modulation during the N-back task in affected subjects. These data may pave the way to further understand how KCNH2 genetic variability is related to the pathophysiological mechanisms underlying schizophrenia.

Cardiotoxicity by tyrosine kinase inhibitors remains an important concern. Nilotinib and vandetanib clinically carry high proarrhythmic risk and the exact mechanism underlying arrhythmogenesis is not fully understood. In this study, we investigated the effects of nilotinib and vandetanib on the abundance of human ether-á-go-go-related gene (hERG) K

Drinking fresh grapefruit juice is associated with a significant prolongation of the QT segment on the electrocardiogram (ECG) in healthy volunteers. Among the prominent polyphenols contained in citrus fruits and primarily in grapefruit, the flavonoid naringenin is known to be a blocker of the human ether-a-go-go related gene (hERG) potassium channel. Here we hypothesized that naringenin could interfere with other major ion channels shaping the cardiac ventricular action potential (AP). To test this hypothesis, we examined the effects of naringenin on the seven channels comprising the Comprehensive in vitro Pro-Arrhythmia (CiPA) ion channel panel for early arrhythmogenic risk assessment in drug discovery and development. We used automated population patch-clamp of human ion channels heterologously expressed in mammalian cells to evaluate half-maximal inhibitory concentrations (IC

INTRODUCTION LQTS is of great importance as it is the most common cause of sudden cardiac death in childhood. The diagnosis is made by the prolongation of the QTc interval on the ECG. However, clinical heterogeneity and non-diagnostic QTc intervals may cause a delay in the diagnosis. In such cases, genetic tests such as NGS panel analysis enable a definitive diagnosis. We present the first study that aimed to expand the LQTS's mutational spectrum by NGS panel analysis from Turkey. METHODS Fifty-seven unrelated patients with clinically diagnosed LQTS were investigated using an NGS panel that includes six LQTS-related genes. Clinical aspects, outcome, and molecular analysis results were reviewed. RESULTS Pathogenic(53%)/likely pathogenic(23%)/VUS(4%) variants were detected in any of the genes examined in 79% of the patients. Among all detected variants, KCNQ1(71%) was the most common gene, followed by SCN5A(11%), KCNH2(10%), CALM1(5%), and CACNA1C(3%). Twelve novel variants were detected. Among the variants in KCNQ1, the c.1097G>A variant was present in 42% of patients. This variant also composed 31% of the variants detected in all of the genes. CONCLUSION Our study expands the spectrum of the variations associated with LQTS with twelve novel variants in five genes. And also it draws attention to the frequency of the KCNQ1 c.1097G>A variant and forms the basis for new studies to determine the possible founder effect in the Turkish population. Furthermore, identifying new variants and clinical findings has importance in elaborating the roles of related genes in pathophysiology and determining the variable expression and incomplete penetration rates in this syndrome. This article is protected by copyright. All rights reserved.

Short QT syndrome (SQTS) is a rare but dangerous genetic disease. In this research, we conducted a comprehensive in silico investigation into the arrhythmogenesis in KCNH2 T618I-associated SQTS using a multi-scale human ventricle model. A Markov chain model of IKr was developed firstly to reproduce the experimental observations. It was then incorporated into cell, tissue, and organ models to explore how the mutation provided substrates for ventricular arrhythmias. Using this T618I Markov model, we explicitly revealed the subcellular level functional alterations by T618I mutation, particularly the changes of ion channel states that are difficult to demonstrate in wet experiments. The following tissue and organ models also successfully reproduced the changed dynamics of reentrant spiral waves and impaired rate adaptions in hearts of T618I mutation. In terms of pharmacotherapy, we replicated the different effects of a drug under various conditions using identical mathematical descriptions for drugs. This study not only simulated the actions of an effective drug (quinidine) at various physiological levels, but also elucidated why the IKr inhibitor sotalol failed in SQT1 patients through profoundly analyzing its mutation-dependent actions.

Cardiac autonomic dysfunction (CADF) is a major contributor to increased cardiac mortality in schizophrenia patients. The aberrant function of voltage-gated ion channels, which are widely distributed in the brain and heart, may link schizophrenia and CADF. In search of channel-encoding genes that are associated with both CADF and schizophrenia,First, we searched the literature for single-nucleotide polymorphisms (SNPs) inWe observed significantly increased QTvi in unmedicated patients with CADF-associated risk inWe propose a potential pleiotropic role for common variation in

Graphical abstract In this review, we outline the current trends in the field of machine learning-driven classification studies related to ADME (absorption, distribution, metabolism and excretion) and toxicity endpoints from the past six years (2015–2021). The study focuses only on classification models with large datasets (i.e. more than a thousand compounds). A comprehensive literature search and meta-analysis was carried out for nine different targets: hERG-mediated cardiotoxicity, blood–brain barrier penetration, permeability glycoprotein (P-gp) substrate/inhibitor, cytochrome P450 enzyme family, acute oral toxicity, mutagenicity, carcinogenicity, respiratory toxicity and irritation/corrosion. The comparison of the best classification models was targeted to reveal the differences between machine learning algorithms and modeling types, endpoint-specific performances, dataset sizes and the different validation protocols. Based on the evaluation of the data, we can say that tree-based algorithms are (still) dominating the field, with consensus modeling being an increasing trend in drug safety predictions. Although one can already find classification models with great performances to hERG-mediated cardiotoxicity and the isoenzymes of the cytochrome P450 enzyme family, these targets are still central to ADMET-related research efforts. Supplementary Information The online version contains supplementary material available at 10.1007/s11030-021-10239-x.

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.

Background Long-QT syndrome type 2 (LQT2) is a common malignant hereditary arrhythmia. Due to the lack of suitable animal and human models, the pathogenesis of LQT2 caused by human ether-a-go-go-related gene (hERG) deficiency is still unclear. In this study, we generated an hERG-deficient human cardiomyocyte (CM) model that simulates ‘human homozygous hERG mutations’ to explore the underlying impact of hERG dysfunction and the genotype–phenotype relationship of hERG deficiency. Methods The KCNH2 was knocked out in the human embryonic stem cell (hESC) H9 line using the CRISPR/Cas9 system. Using a chemically defined differentiation protocol, we obtained and verified hERG-deficient CMs. Subsequently, high-throughput microelectrode array (MEA) assays and drug interventions were performed to characterise the electrophysiological signatures of hERG-deficient cell lines. Results Our results showed that KCNH2 knockout did not affect the pluripotency or differentiation efficiency of H9 cells. Using high-throughput MEA assays, we found that the electric field potential duration and action potential duration of hERG-deficient CMs were significantly longer than those of normal CMs. The hERG-deficient lines also exhibited irregular rhythm and some early afterdepolarisations. Moreover, we used the hERG-deficient human CM model to evaluate the potency of agents (nifedipine and magnesium chloride) that may ameliorate the phenotype. Conclusions We established an hERG-deficient human CM model that exhibited QT prolongation, irregular rhythm and sensitivity to other ion channel blockers. This model serves as an important tool that can aid in understanding the fundamental impact of hERG dysfunction, elucidate the genotype–phenotype relationship of hERG deficiency and facilitate drug development.

Objective To compare the frequency and impact on the channel function of KCNH2 variants in SUDEP patients with epilepsy controls comprising patients older than 50 years, a group with low SUDEP risk, and establish loss‐of‐function KCNH2 variants as predictive biomarkers of SUDEP risk. Methods We searched for KCNH2 variants with a minor allele frequency of 20% reduction in maximal amplitude, were observed in 8.9% (8/90) SUDEP patients compared to 3.3% (11/332) epilepsy controls suggesting about threefold enrichment (nominal p = 0.04). KCNH2 variants that did not change channel function occurred at a similar frequency in SUDEP (2.2%; 2/90) and epilepsy control (2.7%; 9/332) cohorts (p > 0.99). Rare KCNH2 variants (

Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.

The electrocardiogram (ECG) empowered clinician scientists to measure the electrical activity of the heart noninvasively to identify arrhythmias and heart disease. Shortly after the standardization of the 12-lead ECG for the diagnosis of heart disease, several families with autosomal recessive (Jervell and Lange-Nielsen Syndrome) and dominant (Romano-Ward Syndrome) forms of long QT syndrome (LQTS) were identified. An abnormally long heart rate-corrected QT-interval was established as a biomarker for the risk of sudden cardiac death. Since then, the International LQTS Registry was established; a phenotypic scoring system to identify LQTS patients was developed; the major genes that associate with typical forms of LQTS were identified; and guidelines for the successful management of patients advanced. In this review, we discuss the molecular and cellular mechanisms for LQTS associated with missense variants in

Background Brugada syndrome (Brs) and long QT syndrome (LQTs) are the most observed “inherited primary arrhythmia syndromes” and “channelopathies”, which lead to sudden cardiac death. Methods Detailed clinical information of Brs and LQTs patients was collected. Genomic DNA samples of peripheral blood were conducted for whole-exome sequencing on the Illumina HiSeq 2000 platform. Then, we performed bioinformatics analysis for 200 genes susceptible to arrhythmias and cardiomyopathies. Protein interaction and transcriptomic co-expression were analyzed using the online website and GTEx database. Results All sixteen cases of Brs and six cases of LQTs were enrolled in the current study. Four Brs carried known pathogenic or likely pathogenic of single-point mutations, including SCN5A p.R661W, SCN5A p.R965C, and KCNH2 p.R692Q. One Brs carried the heterozygous compound mutations of DSG2 p.F531C and SCN5A p.A1374S. Two Brs carried the novel heterozygous truncated mutations (MAF NEBL (p.R882X) and NPPA (p.R107X), respectively. Except for the indirect interaction between NEBL and SCN5A, NPPA directly interacts with SCN5A. These gene expressions had a specific and significant positive correlation in myocardial tissue, with high degrees of co-expression and synergy. Two Brs carried MYH7 p.E1902Q and MYH6 p.R1820Q, which were predicted as "damaging/possibly damaging" and "damaging/damaging" by Polyphen and SIFT algorithm. Two LQTs elicited the pathogenic single splicing mutation of KCNQ1 (c.922-1G > C). Three LQTs carried a single pathogenic mutation of SCN5A p.R1880H, KCNH2 p.D161N, and KCNQ1 p.R243S, respectively. One patient of LQTs carried a frameshift mutation of KCNH2 p. A188Gfs*143. Conclusions The truncated mutations of NEBL (p.R882X) and NPPA (p.R107X) may induce Brugada syndrome by abnormally affecting cardiac sodium channel. SCN5A (p.R661W, p.R965C and p.A1374S) and KCNH2 (p.R692Q) may cause Brugada syndrome, while SCN5A (p.R1880H), KCNQ1 (c.922-1G > C and p.R243S) and KCNH2 (p.D161N and p.A188Gfs*143) may lead to long QT syndrome.

Long QT syndrome type II (LQT2) is caused by loss-of-function mutations in the hERG K

Background: Accurately determining variant pathogenicity is critical in the diagnosis of cardiac channelopathies; however, it remains unknown how variant pathogenicity status changes over time. Our aim is to use a comprehensive analysis of ClinVar to understand the mutability of variant evaluation in channelopathy-associated genes to inform clinical decision-making around variant calling. Methods: We identified 10 genes ( RYR2, CASQ2, KCNQ1, KCNH2, SCN5A, CACNA1C, CALM1, CALM2, CALM3, TRDN ) strongly associated with cardiac channelopathies, as well as 3 comparison gene sets (disputed long QT syndrome, sudden unexpected death in epilepsy, and all ClinVar). We comprehensively analyzed variant pathogenicity calls over time using the ClinVar database with Rstudio. Analyses focused on the frequency and directionality of clinically meaningful changes in disease association, defined as a change from one of the following three categories to another: likely benign/benign, conflicting evidence of pathogenicity/variant of uncertain significance, and likely pathogenic/pathogenic. Results: In total, among channelopathy-associated genes, there were 9975 variants in ClinVar and 8.4% had a clinically meaningful change in disease association at least once over the past 10 years, as opposed to 4.9% of all ClinVar variants. The 3 channelopathy-associated genes with the most variants undergoing a clinically significant change were KCNQ1 (20.9%) , SCN5A (11.2%), and KCNH2 (10.1%). Ten of the 12 included genes had variant evaluations that trended toward diagnostic uncertainty over time. Specifically, channelopathy-associated gene variants with either pathogenic/likely pathogenic or benign/likely benign assignments were 5.6× and 2×, respectively, as likely to be reevaluated to conflicting/variant of uncertain significance compared to the converse. Conclusions: Over the past 10 years, 8.4% of variants in channelopathy-associated genes have changed pathogenicity status with a decline in overall diagnostic certainty. Ongoing clinical and genetic variant follow-up is needed to account for presence of clinically meaningful change in variant pathogenicity assignment over time.

We describe the discovery of histone deacetylase (HDACs) 1, 2, and 3 inhibitors with ethyl ketone as the zinc-binding group. These HDACs 1, 2, and 3 inhibitors have good enzymatic and cellular activity. Their serum shift in cellular potency has been minimized, and selectivity against hERG has been improved. They are also highly selective over HDACs 6 and 8. These inhibitors contain a variety of substituted heterocycles on the imidazole or oxazole scaffold. Compounds 31 and 48 stand out due to their good potency, high selectivity over HDACs 6 and 8, reduced hERG activity, optimized serum shift in cellular potency, and good rat and dog PK profiles.

Accumulation of amyloid β peptides (Aβ) is thought to be one of the causal factors of Alzheimer's disease (AD). The aspartyl protease β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting protease for Aβ production, and therefore, BACE1 inhibition is a promising therapeutic approach for the treatment of AD. Starting with a dihydro-1,3-thiazine-based lead, Compound J, we discovered atabecestat 1 (JNJ-54861911) as a centrally efficacious BACE1 inhibitor that was advanced into the EARLY Phase 2b/3 clinical trial for the treatment of preclinical AD patients. Compound 1 demonstrated robust and dose-dependent Aβ reduction and showed sufficient safety margins in preclinical models. The potential of reactive metabolite formation was evaluated in a covalent binding study to assess its irreversible binding to human hepatocytes. Unfortunately, the EARLY trial was discontinued due to significant elevation of liver enzymes, and subsequent analysis of the clinical outcomes showed dose-related cognitive worsening.

Jingzhaotoxin-I, -III, -IV, -XIII, and −35 (JZTX-I, -III, -IV, -XIII, and −35), gating modifier toxins isolated from the venom of the Chinese tarantula Chilobrachys Jingzhao, were reported to act on cardiac sodium channels and Kv channels. JZTX-I and JZTX-XIII inhibited the hERG channel with the IC50 value of 626.9 nM and 612.6 nM, respectively. JZTX-III, -IV, and −35 share high sequence similarity with JZTX-I and JZTX–XIII, but they showed much lower affinity on the hERG channel compared with JZTX-I and JZTX-XIII. The inhibitory potency of the above five toxins on the hERG channel was not in accordance with their affinity on the Nav1.5 and Kv2.1 channels, indicating that the bioactive surfaces of the five toxins interacting with hERG, Nav1.5 and Kv2.1 are at least in part different. Structure-function analysis of the gating modifier toxins suggested that the functional bioactive surface binding to the hERG channel consists of a conserved hydrophobic patch, surrounding acidic residues (Glu10 in JZTX-XIII, Glu11 in JZTX-I), and basic residues which may be different from residues binding to the Kv2.1 channel.

The usage of flavored electronic nicotine delivery systems (ENDS) is popular, specifically in the teen and young adult age-groups. The possible cardiac toxicity of the flavoring aspect of ENDS is largely unknown. Vaping, a form of electronic nicotine delivery, uses “e-liquid” to generate “e-vapor,” an aerosolized mixture of nicotine and/or flavors. We report our investigation into the cardiotoxic effects of flavored e-liquids. E-vapors containing flavoring aldehydes such as vanillin and cinnamaldehyde, as indicated by mass spectrometry, were more toxic in HL-1 cardiomyocytes than fruit-flavored e-vapor. Exposure of human induced pluripotent stem cell-derived cardiomyocytes to cinnamaldehyde or vanillin-flavored e-vapor affected the beating frequency and prolonged the field potential duration of these cells more than fruit-flavored e-vapor. In addition, vanillin aldehyde-flavored e-vapor reduced the human ether-à-go-go-related gene (hERG)-encoded potassium current in transfected human embryonic kidney cells. In mice, inhalation exposure to vanillin aldehyde-flavored e-vapor for 10 wk caused increased sympathetic predominance in heart rate variability measurements. In vivo inducible ventricular tachycardia was significantly longer, and in optical mapping, the magnitude of ventricular action potential duration alternans was significantly larger in the vanillin aldehyde-flavored e-vapor-exposed mice than in controls. We conclude that the widely popular flavored ENDS are not harm free, and they have a potential for cardiac harm. More studies are needed to further assess their cardiac safety profile and long-term health effects. NEW & NOTEWORTHY The use of electronic nicotine delivery systems (ENDS) is not harm free. It is not known whether ENDS negatively affect cardiac electrophysiological function. Our study in cell lines and in mice shows that ENDS can compromise cardiac electrophysiology, leading to action potential instability and inducible ventricular arrhythmias. Further investigations are necessary to assess the long-term cardiac safety profile of ENDS products in humans and to better understand how individual components of ENDS affect cardiac toxicity.

Background KCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that lead to decreased IKr have been associated with long QT syndrome type 2 (LQT2). The mechanism of LQT2 is most often induced loss of KV11.1 trafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would aid in understanding the deleterious nature of these variants; however, the volume of reported nonsynonymous KCNH2 variants precludes the use of conventional methods for functional study. Objective The purpose of this study was to report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in the resulting KV11.1 channel. Methods We developed a method to quantitate KV11.1 variant trafficking on a pilot region of 11 residues in the S5 helix. Results We generated trafficking scores for 220 of 231 missense variants in the pilot region. For 5 of 5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We further explored these results with planar patch electrophysiology and found that loss-of-trafficking variants do not produce IKr. Conversely, but expectedly, some variants that traffic normally were still functionally compromised. Conclusion We describe a new method for detecting KV11.1 trafficking-deficient variants in a multiplexed assay. This new method accurately generated trafficking data for variants in KV11.1 and is extendable both to all residues in KV11.1 and to other cell surface proteins.

Methotrexate (MTX) has been widely used for the treatment of many types of autoimmune diseases, such as rheumatoid arthritis, psoriasis and dermatomyositis. However, its pharmacological mechanism is still unclear completely. In this study, we found that MTX is a potent and selective inhibitor of the Kv1.3 channel, a class of potassium channels highly associated with autoimmune diseases. Electrophysiological experiments showed that MTX inhibited human Kv1.3 channel with an IC50 of 41.5 ± 24.9 nM, and 1 μM MTX inhibited 32.6 ± 1.3% and 25.6 ± 2.2% of human Kv1.1 and Kv1.2 channel currents, respectively. These data implied the unique selectivity of MTX towards the Kv1.3 channel. Excitingly, using channel activation and chimeric experiments, we found that MTX bound to the outer pore region of Kv1.3 channel. Mutagenesis experiments in the Kv.3 channel extracellular pore region further showed that the Dsp371, Thr373 and His399 residues of outer pore region of Kv1.3 channel played important roles in MTX inhibiting activities. In conclusion, MTX inhibited Kv1.3 channel by targeting extracellular pore region, which is different form all the report small molecules, such as PAP-1 and 4-AP, but similar with many natural animal toxin peptides, such as ChTX, ShK and BmKTX. To the best of our knowledge, MTX is the first small molecular scaffold targeting the Kv1.3 channel extracellular pore region, suggesting its potential applications for designing novel Kv1.3 lead drugs and treating Kv1.3 channel-associated autoimmune diseases.

We assessed the torsadogenic effects of a novel remyelinating drug clemastine for multiple sclerosis using an in vivo proarrhythmia model of acute atrioventricular block rabbit, since the drug has been demonstrated to suppress the human ether-a-go-go related gene (hERG) K+ channels. Bradycardia was induced by atrioventricular node ablation in isoflurane-anesthetized New Zealand White rabbits (n = 5), and the ventricle was electrically driven at 60 beats/min throughout the experiment, except when extrasystoles appeared. Intravenous administration of clinically relevant dose of 0.03 mg/kg of clemastine and 10-times higher dose of 0.3 mg/kg hardly affected the QT interval or duration of the monophasic action potential (MAP) of the ventricle. Additional administration of clemastine at 3 mg/kg significantly increased the QT interval, MAP duration and the short-term variability of repolarization. Meanwhile, the premature ventricular contractions with R on T phenomenon were observed in 3 out of 5 animals, and torsades de pointes arrhythmias were detected in 1 out of 5 animals. These results suggest that the torsadogenic potential of clemastine is obviously observed in the acute atrioventricular block rabbit, which will not appear within the prescribed dose for multiple sclerosis.

Long QT syndrome is one of the most common hereditary arrhythmias. Mutations in KCNH2 can cause long QT syndrome type 2 (LQT2). In this study, we generated a human induced pluripotent stem cell line ZZUNEUi027-A from a LQT2 female patient with c. 128A → G in KCNH2 gene using non-integrative Sendai viral reprogramming technology. This cell line expresses pluripotency markers, exhibits a normal female karyotype (46, XX) and could differentiate into all three germ layers in vitro. ZZUNEUi027-A can serve as a cell disease model in the understanding of LQT2 pathogenesis.

Long-QT syndrome type 2 (LQT2) is a life-threatening Mendelian disease caused by genetic variants in KCNH2. Herein, we generated a human embryonic stem cell line (WAe009-A-88) carrying a LQT2 related mutation in KCNH2, c.1720 AG. The WAe009-A-88 line maintained stem cell-like morphology, expressed high levels of pluripotent markers, had a normal karyotype, and could differentiate into all three germ layers in vivo. The cell line can serve as valuable tools for modeling LQT2 in vitro and investigating the pathological mechanisms related to KCNH2 mutations.

Inhibition of the human ether-à-go-go (hERG) channel may lead to QT prolongation and fatal arrhythmia. While pharmaceutical drug candidates that exhibit potent hERG channel inhibition often fail early in development, many drugs with both cardiac and non-cardiac indications proceed to market. In this study, the relationship between in vitro hERG channel inhibition and published occupational exposure limit (OEL) was evaluated. A total of 23 cardiac drugs and 44 drugs with non-cardiac indications with published hERG channel IC

BACKGROUND: Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previous study of the human ether-à-go-go related gene (hERG)–encoded K(+) channel (K(v)11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERG(T613M)) and RNF207 frameshift (RNF207(G603fs)) mutations. However, the underlying mechanistic underpinning remains unknown. OBJECTIVE: The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K(+) channel subunits. METHODS: Whole-cell patch-clamp experiments were performed in human embryonic kidney (HEK 293) cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) together with immunofluorescent confocal and high resolution microscopy, auto-ubiquitinylation assays, and co-immunoprecipitation experiments to test the functional interactions between hERG and RNF207. RESULTS: Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERG(T613M) proteins for degradation. RNF207(G603fs) exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERG(T613M) subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERG(A614V), a known dominant-negative mutant subunit. Finally, the presence of RNF207(G603fs) with hERG(T613M) results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell–derived cardiomyocytes. CONCLUSION: Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K(+) channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and consequently cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in patients with LQTS.

[EN] Background: Assessment of drug cardiac safety is critical in the development of new compounds and is commonly addressed by evaluating the half-maximal blocking concentration of the potassium human ether-à-go-go related gene (hERG) channels. However, recent works have evidenced that the modelling of drug-binding dynamics to hERG can help to improve early cardiac safety assessment. Our goal is to de- velop a methodology to automatically generate Markovian models of the drug-hERG channel interactions. Methods: The training and the test sets consisted of 20800 and 5200 virtual drugs, respectively, dis- tributed into 104 groups with different affinities and kinetics to the conformational states of the chan- nel. In our system, drugs may bind to any state (individually or simultaneously), with different degrees of preference for a conformational state and the change of the conformational state of the drug bound channels may be restricted or allowed. To model such a wide range of possibilities, 12 Markovian chains are considered. Our approach uses the response of the drugs to our three previously developed voltage clamp protocols, which enhance the differences in the probabilities of occupying a certain conformational state of the channel (open, closed and inactivated). The computing tool is comprised of a classifier and a parameter optimizer and uses linear interpolation, support vector machines and a simplex method for function minimization. Results: We propose a novel methodology that automatically generates dynamic drug models using Markov model formulations and that elucidates the states where the drug binds and unbinds and the preferential binding state using data obtained from simple voltage clamp protocols that captures the preferential state-dependent binding properties, the relative affinities, trapping and non-trapping dynam- ics and the onset of I Kr block. Overall, the tool correctly predicted the class of 92.04% of the drugs and the model provided by the tool accurately fitted the response of the target compound, the mean accu- racy being 97.53%. Moreover, generation of the dynamic model of an I Kr blocker from its response to our voltage clamp protocols usually takes less than an hour on a common desktop computer. Conclusion: Our methodology could be very useful to model and simulate dynamic drug¿hERG channel interactions. It would contribute to the improvement of the preclinical assessment of the proarrhythmic risk of drugs that inhibit I Kr and the efficacy of antiarrhythmic I Kr blockers., This work was the Spanish Ministerio de Ciencia, Innovacion y Universidades [grant "Formacion de Profesorado Universitario" FPU19/02200; grant PID2019-104356RB-C41 funded by MCIN/AEI/10.13039/50110 0 011033 ]; the European Union's Horizon 2020 research and innovation program [grant agreement No 101016496 (SimCardioTest)]; and the Direccion General de Politica Cientifica de la Generalitat Valenciana [grant PROMETEO/2020/043]. Patenting of the proposed system/software is under consideration