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The growing use of nitazene synthetic opioids heralds a new phase of the opioid crisis. However, limited information exists on the toxic effects of these drugs, aside from a propensity for respiratory depression. With restricted research availability of nitazenes, we used machine-learning-based tools to evaluate five nitazene compounds' interaction potential with the hERG potassium channel, a key drug antitarget in the heart. All nitazenes were predicted to inhibit hERG with low μM IC50 values. These findings indicate a potential for proarrhythmic hERG block by nitazene opioids, warranting detailed cardiac safety evaluations of these drugs.

Colorectal cancer (CRC) is one of the most common malignant cancers. Emodin is a lipophilic anthraquinone commonly found in medicinal herbs and known for its antitumor properties. However, its clinical utility has been hampered by low druggability. We designed and synthesized a new compound named Emodin succinimidyl ethyl ester (ESEE), which improves the bioavailability and preserves the original pharmacological effects of Emodin. In vitro, we have confirmed that ESEE induces apoptosis in colon cancer cells, suppresses cell proliferation, migration, and invasion, and inhibits the growth of subcutaneous transplantation tumors associated with colon cancer. And, in vivo, ESEE robustly inhibited tumor growth. Human Ether-a-go-go Related Gene (hERG) is aberrantly expressed in various cancer cells, where they play an important role in cancer progression. Focal adhesion kinase (FAK) is a tyrosine kinase overexpressed in cancer cells and plays an important role in the progression of tumors to a malignant phenotype. Mechanistically, the anti-CRC properties of ESEE are exerted through direct binding with hERG, which impedes the FAK/PI3K/AKT signaling axis-dependent apoptotic cascade.

Abstract Background Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the “opposite” voltage-sensing properties of hERG and HCN remains unknown. Results We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. Conclusions Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.

Sudden, unexpected deaths are extremely difficult for families, especially when the victim is a child. Most sudden deaths occur due to cardiovascular issues, and a smaller number (approximately one-quarter) are attributed to other causes, such as epilepsy. The medicinal and non-medicinal use of the synthetic opioid fentanyl, which can cause breathing problems, is frequently involved in these deaths. It is also being found more often in autopsies of sudden death cases, and the number of overdose deaths from illicit drugs containing fentanyl is increasing. There are cases in which it is mixed with other drugs. A gene known as the KCNH2 gene or human ether-a-go-go-related gene (hERG), involved in the heart’s electrical activity, can be related to abnormal heart rhythms. This gene, along with others, may play a role in sudden deaths related to fentanyl use. In response, we have examined the scientific literature on genetic variations in the KCNH2 gene that can cause sudden death, the impact of fentanyl on this process, and the potential benefits of genetic testing for the victims to offer genetic counseling for their family members.

Background: The rapid delayed rectifier potassium current (IKr) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting IKr, hERG channel, is not only permeable to K+ ions but also to Cs+ ions when present in equimolar concentrations inside and outside of the cell.Methods: In this study, IhERG was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs+ or K+ as the charge carrier. Equimolar Cs+ has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs+- and K+-based currents.Results: Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs+- and K+-mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs+-based conductance is larger compared to the known K+-based conductance, and the Cs+ hERG conductance can be inhibited similarly to the K+-based conductance.Conclusion: Using equimolar Cs+ instead of K+ for IhERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small IKr current densities in combination with small membrane capacitances.

Synthetic cannabinoid receptor agonists (SCRAs) have been associated with QT interval prolongation. Limited preclinical information on SCRA effects on cardiac electrogenesis results from the rapid emergence of new compounds and restricted research availability. We used two machine-learning-based tools to evaluate seven novel SCRAs' interaction potential with the hERG potassium channel, an important drug antitarget. Five SCRAs were predicted to have the ability to block the hERG channel by both prediction tools; ADB-FUBIATA was predicted to be a strong hERG blocker. ADB-5Br-INACA and ADB-4en-PINACA showed varied predictions. These findings highlight potentially proarrhythmic hERG block by novel SCRAs, necessitating detailed safety evaluations.

In March 2022, more than 600 million cases of Corona Virus Disease 2019 (COVID-19) and about 6 million deaths have been reported worldwide. Unfortunately, while effective antiviral therapy has not yet been available, chloroquine (CQ)/hydroxychloroquine (HCQ) has been considered an option for the treatment of COVID-19. While many studies have demonstrated the potential of HCQ to decrease viral load and rescue patients’ lives, controversial results have also been reported. One concern associated with HCQ in its clinical application to COVID-19 patients is the potential of causing long QT interval (LQT), an electrophysiological substrate for the induction of lethal ventricular tachyarrhythmias. Yet, the mechanisms for this cardiotoxicity of HCQ remained incompletely understood.

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Microelectrode array studies (MEAs) and voltage sensing optical technique on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were combined with ionic current measurements in mammalian cell lines, In-silico simulations of cardiac action potentials (APs) and statistic regression analysis. Proarrhythmic effects of 12 Evodia preparations, containing different amounts of the hERG inhibitors dehydroevodiamine (DHE) and hortiamine were analysed. Extracts produced different prolongation of the AP, occurrence of early after depolarisations and triangulation of the AP in hiPSC-CMs depending on the contents of the hERG inhibitors. DHE and hortiamine dose-dependently prolonged the field potential duration in hiPSC-CMs studied with MEAs. In-silico simulations of ventricular AP support a scenario where proarrhythmic effects of Evodia extracts are predominantly caused by the content of the selective hERG inhibitors. Statistic regression analysis revealed a high torsadogenic risk for both compounds that was comparable to drugs assigned to the high-risk category in a CiPA study.

Introduction: Given the direct association with malignant ventricular arrhythmias, cardiotoxicity is a major concern in drug design. In the past decades, computational models based on the quantitative structure–activity relationship have been proposed to screen out cardiotoxic compounds and have shown promising results. The combination of molecular fingerprint and the machine learning model shows stable performance for a wide spectrum of problems; however, not long after the advent of the graph neural network (GNN) deep learning model and its variant (e.g., graph transformer), it has become the principal way of quantitative structure–activity relationship-based modeling for its high flexibility in feature extraction and decision rule generation. Despite all these progresses, the expressiveness (the ability of a program to identify non-isomorphic graph structures) of the GNN model is bounded by the WL isomorphism test, and a suitable thresholding scheme that relates directly to the sensitivity and credibility of a model is still an open question.Methods: In this research, we further improved the expressiveness of the GNN model by introducing the substructure-aware bias by the graph subgraph transformer network model. Moreover, to propose the most appropriate thresholding scheme, a comprehensive comparison of the thresholding schemes was conducted.Results: Based on these improvements, the best model attains performance with 90.4% precision, 90.4% recall, and 90.5% F1-score with a dual-threshold scheme (active: 30μM). The improved pipeline (graph subgraph transformer network model and thresholding scheme) also shows its advantages in terms of the activity cliff problem and model interpretability.

The evaluation of the inhibitory activities of drugs on multiple cardiac ion channels is required for the accurate assessment of proarrhythmic risks. Moreover, the in silico prediction of such inhibitory activities of drugs on cardiac channels can improve the efficiency of the drug-development process. Here, we performed molecular docking simulations to predict the complex structures of 25 reference drugs that were proposed by the Comprehensive in vitro Proarrhythmia Assay consortium using two cardiac ion channels, the human ether-a-go-go-related gene (hERG) potassium channel and human NaV1.5 (hNaV1.5) sodium channel, with experimentally available structures. The absolute binding free energy (ΔGbind) values of the predicted structures were calculated by a molecular dynamics-based method and compared with the experimental half-maximal inhibitory concentration (IC50) data. Furthermore, the regression analysis between the calculated values and negative of the common logarithm of the experimental IC50 values (pIC50) revealed that the calculated values of four and ten drugs deviated significantly from the regression lines of the hERG and hNaV1.5 channels, respectively. We reconsidered the docking poses and protonation states of the drugs based on the experimental data and recalculated their ΔGbind values. Finally, the calculated ΔGbind values of 24 and 19 drugs correlated with their experimental pIC50 values (coefficients of determination=0.791 and 0.613 for the hERG and hNaV1.5 channels, respectively). Thus, the regression analysis between the calculated ΔGbind and experimental IC50 data ensured the realization of an increased number of reliable complex structures.

Drug-induced cardiotoxicity represents one of the most critical safety concerns in the early stages of drug development. The blockade of the human ether-à-go-go-related potassium channel (hERG) is the most frequent cause of cardiotoxicity, as it is associated to long QT syndrome which can lead to fatal arrhythmias. Therefore, assessing hERG liability of new drugs candidates is crucial to avoid undesired cardiotoxic effects. In this scenario, computational approaches have emerged as useful tools for the development of predictive models able to identify potential hERG blockers. In the last years, several efforts have been addressed to generate ligand-based (LB) models due to the lack of experimental structural information about hERG channel. However, these methods rely on the structural features of the molecules used to generate the model and often fail in correctly predicting new chemical scaffolds. Recently, the 3D structure of hERG channel has been experimentally solved enabling the use of structure-based (SB) strategies which may overcome the limitations of the LB approaches. In this study, we compared the performances achieved by both LB and SB classifiers for hERG-related cardiotoxicity developed by using Random Forest algorithm and employing a training set containing 12789 hERG binders. The SB models were trained on a set of scoring functions computed by docking and rescoring calculations, while the LB classifiers were built on a set of physicochemical descriptors and fingerprints. Furthermore, models combining the LB and SB features were developed as well. All the generated models were internally validated by ten-fold cross-validation on the TS and further verified on an external test set. The former revealed that the best performance was achieved by the LB model, while the model combining the LB and the SB attributes displayed the best results when applied on the external test set highlighting the usefulness of the integration of LB and SB features in correctly predicting unseen molecules. Overall, our predictive models showed satisfactory performances providing new useful tools to filter out potential cardiotoxic drug candidates in the early phase of drug discovery.

Artificial lipid bilayer systems, such as vesicles, black membranes, and supported lipid bilayers (SLBs), are valuable platforms for studying ion channels at the molecular level. The reconstitution of the ion channels in an active form is a crucial process in studies using artificial lipid bilayer systems. In this study, we investigated the assembly of the human ether-a-go-go-related gene (hERG) channel prepared in a cell-free synthesis system. AFM topographies revealed the presence of protrusions with a uniform size in the entire SLB that was prepared with the proteoliposomes (PLs) incorporating the cell-free-synthesized hERG channel. We attributed the protrusions to hERG channel monomers, taking into consideration the AFM tip size, and identified assembled structures of the monomer that exhibited dimeric, trimeric, and tetrameric-like arrangements. We observed molecular images of the functional hERG channel reconstituted in a lipid bilayer membrane using AFM and quantitatively evaluated the association state of the cell-free synthesized hERG channel.

The hERG channel is one of the essential ion channels composing the cardiac action potential and the toxicity assay for new drug. Recently, the comprehensive in vitro proarrhythmia assay (CiPA) was adopted for cardiac toxicity evaluation. One of the hurdles for this protocol is identifying the kinetic effect of the new drug on the hERG channel. This procedure included the model-based parameter identification from the experiments. There are many mathematical methods to infer the parameters; however, there are two main difficulties in fitting parameters. The first is that, depending on the data and model, parametric inference can be highly time-consuming. The second is that the fitting can fail due to local minima problems. The simplest and most effective way to solve these issues is to provide an appropriate initial value. In this study, we propose a deep learning-based method for improving model fitting by providing appropriate initial values, even the right answer. We generated the dataset by changing the model parameters and trained our deep learning-based model. To improve the accuracy, we used the spectrogram with time, frequency, and amplitude. We obtained the experimental dataset from https://github.com/CardiacModelling/hERGRapidCharacterisation. Then, we trained the deep-learning model using the data generated with the hERG model and tested the validity of the deep-learning model with the experimental data. We successfully identified the initial value, significantly improved the fitting speed, and avoided fitting failure. This method is useful when the model is fixed and reflects the real data, and it can be applied to any in silico model for various purposes, such as new drug development, toxicity identification, environmental effect, etc. This method will significantly reduce the time and effort to analyze the data.

Vinpocetine is a readily available nutritional supplement claimed to improve memory and weight loss. However, it blocks the Ikr current essential for cardiac action potential repolarisation and Ikr inhibition can cause “torsade de pointes” arrhythmias and sudden death. Moreover, Ikr blockers have exhibited teratogenic effects in reproductive toxicology studies, leading to increased birth defects and embryonic mortality. The FDA advises against vinpocetine use in pregnant and prospective mothers based on animal studies showing dose-dependent fetal mortality in rats and rabbits, and cardiovascular malformations in surviving fetuses. However, the mechanisms responsible for vinpocetine's fetal toxicity remain unclear.The present study used rat embryo culture to evaluate vinpocetine and its major metabolite, apovincaminic acid, on embryonic heart rate, a possible causative factor behind its adverse effects. Both compounds induced embryonic bradycardia in a concentration-dependent manner, with vinpocetine proving more potent.The minimum vinpocentine concentration to induce bradycardia was 100 nM, a level unlikely to be reached in humans following typical doses. Embryonic arrhythmias were also observed at the highest concentrations.These results suggest that the FDA's cautionary statement may generate undue anxiety, although re-evaluation of teratogenicity risk associated with vinpocetine should be revisited if a link to cardiac arrhythmias in adults is established.

Objective To investigate the role of Rer1 in hERG potassium channel protein transport,and to study the medicine Baf A1 that may restore abnormal hERG transport. Methods HEK293T cells were transiently transfected with constructed A561V mutant and WT plasmids, the wild-type,mutant and mixed transfection cell models were established.The expressions of hERG and Rer1 were detected by immunofluorescence and Western blot.siRNA was used to knockdown the expression of Rer1 and detect the expression of hERG;Bafilomycin A1,a V-ATPase inhibitor was incubated with 1 mmol/L for 6 hours.Western blot was used to detect the changes of hERG protein before and after the incubation.Patch clamp was used to measure the current of potential functional in mixed transfection cells. Results Compared with the WT group,A561V-mutation could cause trafficking deficient of hERG protein.The PM expression of hERG in mutant group was significantly reduced(P＜0.01).The expression of Rer1 in mutation and mixed transfection groups decreased significantly(P＜0.01).Knockdown of Rer1 promoted the forward transport of mature hERG.Besides,after incubation with Baf A1,the mature hERG protein in WT and mutant groups increased significantly(P＜0.05),while the immature hERG in the mixed transformation group was significantly increased(P＜0.05).Relative density of tail current was elevated following incubation with Baf A1 compared with control. Conclusions Rer1 is involved in the transport of hERG potassium channel and inhibits the forward transport of some abnormal hERG.Baf A1 significantly enhances the tail current density of mixed transfection cells.

Loss of function (LOF) mutations of voltage sensitive K+ channel proteins hERG (Kv11.1) and KCNQ1 (Kv7.1) account for the majority of instances of congenital Long QT Syndrome (cLQTS) with the dominant molecular phenotype being a mistrafficking one resulting from protein misfolding. We explored the use of Evolutionary Coupling (EC) analysis, which identifies evolutionarily conserved pairwise amino acid interactions that may contribute to protein structural stability, to identify regions of the channels susceptible to misfolding mutations. Comparison with published experimental trafficking data for hERG and KCNQ1 showed that the method strongly predicts “scaffolding” regions of the channel membrane domains and has useful predictive power for trafficking phenotypes of individual variants. We identified a region in and around the cytoplasmic S2-S3 loop of the hERG Voltage Sensor Domain (VSD) as susceptible to destabilising mutation, and this was confirmed using a quantitative LI-COR® based trafficking assay that showed severely attenuated trafficking in eight out of 10 natural hERG VSD variants selected using EC analysis. Our analysis highlights an equivalence in the scaffolding structures of the hERG and KCNQ1 membrane domains. Pathogenic variants of ion channels with an underlying mistrafficking phenotype are likely to be located within similar scaffolding structures that are identifiable by EC analysis.

Drug-induced cardiotoxicity is a common side effect of drugs in clinical use or under postmarket surveillance and is commonly due to off-target interactions with the cardiac human-ether-a-go-go-related (hERG) potassium channel. Therefore, prioritizing drug candidates based on their hERG blocking potential is a mandatory step in the early preclinical stage of a drug discovery program. Herein, we trained and properly validated 30 ligand-based classifiers of hERG-related cardiotoxicity based on 7,963 curated compounds extracted by the freely accessible repository ChEMBL (version 25). Different machine learning algorithms were tested, namely, random forest, K-nearest neighbors, gradient boosting, extreme gradient boosting, multilayer perceptron, and support vector machine. The application of 1) the best practices for data curation, 2) the feature selection method VSURF, and 3) the synthetic minority oversampling technique (SMOTE) to properly handle the unbalanced data, allowed for the development of highly predictive models (BAMAX = 0.91, AUCMAX = 0.95). Remarkably, the undertaken temporal validation approach not only supported the predictivity of the herein presented classifiers but also suggested their ability to outperform those models commonly used in the literature. From a more methodological point of view, the study put forward a new computational workflow, freely available in the GitHub repository (https://github.com/PDelre93/hERG-QSAR), as valuable for building highly predictive models of hERG-mediated cardiotoxicity.

hERG (human Ether-à-go-go Related Gene)-encoded potassium channels underlie the cardiac rapid delayed rectifier (IKr) potassium current, which is a major target for antiarrhythmic agents and diverse non-cardiac drugs linked to the drug-induced form of long QT syndrome. E-4031 is a high potency hERG channel inhibitor from the methanesulphonanilide drug family. This study utilized a methanesulphonate-lacking E-4031 analogue, “E-4031-17”, to evaluate the role of the methanesulphonamide group in E-4031 inhibition of hERG. Whole-cell patch-clamp measurements of the hERG current (IhERG) were made at physiological temperature from HEK 293 cells expressing wild-type (WT) and mutant hERG constructs. For E-4031, WT IhERG was inhibited by a half-maximal inhibitory concentration (IC50) of 15.8 nM, whilst the comparable value for E-4031-17 was 40.3 nM. Both compounds exhibited voltage- and time-dependent inhibition, but they differed in their response to successive applications of a long (10 s) depolarisation protocol, consistent with greater dissociation of E-4031-17 than the parent compound between applied commands. Voltage-dependent inactivation was left-ward voltage shifted for E-4031 but not for E-4031-17; however, inhibition by both compounds was strongly reduced by attenuated-inactivation mutations. Mutations of S6 and S5 aromatic residues (F656V, Y652A, F557L) greatly attenuated actions of both drugs. The S624A mutation also reduced IhERG inhibition by both molecules. Overall, these results demonstrate that the lack of a methanesulphonate in E-4031-17 is not an impediment to high potency inhibition of IhERG.

Erythromycin is one of the few compounds that remarkably increase ether-a-go-go-related gene (hERG) inhibition from room temperature (RT) to physiological temperature (PT). Understanding how erythromycin inhibits the hERG could help us to decide which compounds are needed for further studies. The whole-cell patch clamp technique was used to investigate the effects of erythromycin on hERG channels at different temperatures. While erythromycin caused a concentration-dependent inhibition of cardiac hERG channels, it also shifted the steady-state activation and steady-state inactivation of the channel to the left and significantly accelerated the onset of inactivation at both temperatures, although temperature itself caused a profound change in the dynamics of hERG channels. Our data also suggest that the binding pattern to S6 of the channels changes at PT. In contrast, cisapride, a well-known hERG blocker whose inhibition is not affected by temperature, does not change its critical binding sites after the temperature is raised to PT. Our data suggest that erythromycin is unique and that the shift in hERG inhibition may not apply to other compounds.

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.

Abstract Long QT syndrome type II (LQT2) is caused by loss‐of‐function mutations in the hERG K+ channel, leading to increased incidence of cardiac arrest and sudden death. Many genetic variants have been reported in the hERG gene with various consequences on channel expression, permeation, and gating. Only a small number of LQT2 causing variants has been characterized to define the underlying pathophysiological causes of the disease. We sought to determine the characteristics of the frameshift variant p.Thr1019ProfsX38 (T1019PfsX38) which affects the C‐terminus of the protein. This mutation was identified in an extended Omani family of LQT2. It replaces the last 140 amino acids of hERG with 37 unique amino acids. T1019 is positioned at a distinguished region of the C‐terminal tail of hERG, as predicted from the deep learning system AlphaFold v2.0. We employed the whole‐cell configuration of the patch‐clamp technique to study wild‐type and mutant channels that were transiently expressed in human embryonic kidney 293 (HEK293) cells. Depolarizing voltages elicited slowly deactivating tail currents that appeared upon repolarization of cells that express either wild‐type‐ or T1019PfsX38‐hERG. There were no differences in the voltage and time dependencies of activation between the two variants. However, the rates of hERG channel deactivation at hyperpolarizing potentials were accelerated by T1019PfsX38. In addition, the voltage dependence of inactivation of T1019PfsX38‐hERG was shifted by 20 mV in the negative direction when compared with wild‐type hERG. The rates of channel inactivation were increased in the mutant channel variant. Next, we employed a step‐ramp protocol to mimic membrane repolarization by the cardiac action potential. The amplitudes of outward currents and their integrals were reduced in the mutant variant when compared with the wild‐type variant during repolarization. Thus, changes in the gating dynamics of hERG by the T1019PfsX38 variant contribute to the pathology seen in affected LQT2 patients.

Sdox is a hydrogen sulfide (H2S)-releasing doxorubicin effective in P-glycoprotein-overexpressing/doxorubicin-resistant tumor models and not cytotoxic, as the parental drug, in H9c2 cardiomyocytes. The aim of this study was the assessment of Sdox drug-like features and its absorption, distribution, metabolism, and excretion (ADME)/toxicity properties, by a multi- and transdisciplinary in silico, in vitro, and in vivo approach. Doxorubicin was used as the reference compound. The in silico profiling suggested that Sdox possesses higher lipophilicity and lower solubility compared to doxorubicin, and the off-targets prediction revealed relevant differences between Dox and Sdox towards several cancer targets, suggesting different toxicological profiles. In vitro data showed that Sdox is a substrate with lower affinity for P-glycoprotein, less hepatotoxic, and causes less oxidative damage than doxorubicin. Both anthracyclines inhibited CYP3A4, but not hERG currents. Unlike doxorubicin, the percentage of zebrafish live embryos at 72 hpf was not affected by Sdox treatment. In conclusion, these findings demonstrate that Sdox displays a more favorable drug-like ADME/toxicity profile than doxorubicin, different selectivity towards cancer targets, along with a greater preclinical efficacy in resistant tumors. Therefore, Sdox represents a prototype of innovative anthracyclines, worthy of further investigations in clinical settings.

The endocannabinoid system (ECS) plays a very important role in numerous physiological and pharmacological processes, such as those related to the central nervous system (CNS), including learning, memory, emotional processing, as well pain control, inflammatory and immune response, and as a biomarker in certain psychiatric disorders. Unfortunately, the half-life of the natural ligands responsible for these effects is very short. This perspective describes the potential role of the inhibitors of the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL), which are mainly responsible for the degradation of endogenous ligands in psychic disorders and related pathologies. The examination was carried out considering both the impact that the classical exogenous ligands such as Δ9-tetrahydrocannabinol (THC) and (−)-trans-cannabidiol (CBD) have on the ECS and through an analysis focused on the possibility of predicting the potential toxicity of the inhibitors before they are subjected to clinical studies. In particular, cardiotoxicity (hERG liability), probably the worst early adverse reaction studied during clinical studies focused on acute toxicity, was predicted, and some of the most used and robust metrics available were considered to select which of the analyzed compounds could be repositioned as possible oral antipsychotics.

Bacterial DNA gyrase and topoisomerase IV inhibition has emerged as a promising strategy for the cure of infections caused by antibiotic-resistant bacteria. Small molecule antibacterials inhibiting bacterial topoisomerases have been previously exploited by the successful fluoroquinolone class. The Novel Bacterial Topoisomerase Inhibitors (NBTIs) bind to a different site to that of the fluoroquinolones with novel mechanism of action to evade the existing target-mediated bacterial resistance associated with fluoroquinolones. This review comprehensively summarizes various efforts with respect to structural modifications of inhibitors and their impact on their general physicochemical and biological properties.

Voltage-gated sodium (Na+) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na+ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na+ channel (Nav1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Nav1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K+ channels on the AP shape, we co-expressed either Kv1.2 or hERG with one of the Nav1.5-ChR2 fusions. As expected, both delayed rectifier K+ channels shortened AP duration significantly. Kv1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na+ current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na+ channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.

Expression of the voltage-gated potassium channel KV10.1 (Eag1) has been detected in over 70% of human cancers, making the channel a promising new target for new anticancer drug discovery. A new structural class of KV10.1 inhibitors was prepared by structural optimisation and exploration of the structure–activity relationship of the previously published hit compound ZVS-08 (1) and its optimised analogue 2. The potency and selectivity of the new inhibitors between KV10.1 and hERG were investigated using whole-cell patch-clamp experiments. We obtained two new optimised KV10.1 inhibitors, 17a and 18b, with improved nanomolar IC50 values of 568 nM and 214 nM, respectively. Compound 17a exhibited better ratio between IC50 values for hEAG1 and hERG than previously published diarylamine inhibitors. Compounds 17a and 18b moderately inhibited the growth of the KV10.1-expressing cell line MCF-7 in two independent assays. In addition, 17a and 18b also inhibited the growth of hERG-expressing Panc-1 cells with higher potency compared with MCF-7 cells. The main obstacle for newly developed diarylamine KV10.1 inhibitors remains the selectivity toward the hERG channel, which needs to be addressed with targeted drug design strategies in the future.

Abstract Many volatile chemicals inhaled for a recreational high have a chemical structure similar to chloroform and may lead to Ikr blockade and subsequent torsades de pointes. This is one potential mechanism of action for huffing‐associated sudden death.

Abstract In this paper, we explore the impact of combining different in silico prediction approaches and data sources on the predictive performance of the resulting system. We use inhibition of the hERG ion channel target as the endpoint for this study as it constitutes a key safety concern in drug development and a potential cause of attrition. We will show that combining data sources can improve the relevance of the training set in regard of the target chemical space, leading to improved performance. Similarly we will demonstrate that combining multiple statistical models together, and with expert systems, can lead to positive synergistic effects when taking into account the confidence in the predictions of the merged systems. The best combinations analyzed display a good hERG predictivity. Finally, this work demonstrates the suitability of the SOHN methodology for building models in the context of receptor based endpoints like hERG inhibition when using the appropriate pharmacophoric descriptors.

Context: Discovery of pharmacologically active natural products as starting points for drug development remains important and, for reasons of consumer safety, the identification of toxicologically relevant compounds in herbal drugs. Objective: To explain, with the aid of relevant examples from our own research, how these goals can be achieved. Methods: An in-house technology platform comprising pre-formatted extract libraries in 96-well format, miniaturized tracking of activity in extracts via HPLC-activity profiling, structure elucidation with microprobe NMR, and in vitro and in vivo pharmacological methods were used. Results: Piperine was identified as a new scaffold for allosteric GABAA receptor modulators with in vivo activity that interacts at a benzodiazepine-independent binding site. Selectivity and potency were improved by iterative optimization towards synthetic piperine analogues. Dehydroevodiamine and hortiamine from the traditional Chinese herbal drug Evodiae fructus were identified as potent hERG channel blockers in vitro. The compounds induced torsades de pointes arrhythmia in animal models. Conclusions: The allosteric binding site for piperine analogues remains to be characterized and cardiac risks of herbal drugs need to be further evaluated to ensure consumer safety.

Background: In the absence of SARS-CoV-2 specific antiviral treatments, various repurposed pharmaceutical approaches are under investigation for the treatment of COVID-19. Antiviral drugs considered for this condition include atazanavir, remdesivir, lopinavir-ritonavir, and favipiravir. Whilst the combination of lopinavir and ritonavir has been previously linked to prolongation of the QTc interval on the ECG and risk of torsades de pointes arrhythmia, less is known in this regard about atazanavir, remdesivir, and favipiravir. Unwanted abnormalities of drug-induced QTc prolongation by diverse drugs are commonly mediated by a single cardiac anti-target, the hERG potassium channel. This computational modeling study was undertaken in order to explore the ability of these five drugs to interact with known determinants of drug binding to the hERG channel pore.Methods: Atazanavir, remdesivir, ritonavir, lopinavir and favipiravir were docked to in silico models of the pore domain of hERG, derived from cryo-EM structures of hERG and the closely related EAG channel.Results: Atazanavir was readily accommodated in the open hERG channel pore in proximity to the S6 Y652 and F656 residues, consistent with published experimental data implicating these aromatic residues in atazanavir binding to the channel. Lopinavir, ritonavir, and remdesivir were also accommodated in the open channel, making contacts in a model-dependent fashion with S6 aromatic residues and with residues at the base of the selectivity filter/pore helix. The ability of remdesivir (at 30 μM) to inhibit the channel was confirmed using patch-clamp recording. None of these four drugs could be accommodated in the closed channel structure. Favipiravir, a much smaller molecule, was able to fit within the closed channel and could adopt multiple binding poses in the open channel, but with few simultaneous interactions with key binding residues. Only favipiravir and remdesivir showed the potential to interact with lateral pockets below the selectivity filter of the channel.Conclusions: All the antiviral drugs studied here can, in principle, interact with components of the hERG potassium channel canonical binding site, but are likely to differ in their ability to access lateral binding pockets. Favipiravir's small size and relatively paucity of simultaneous interactions may confer reduced hERG liability compared to the other drugs. Experimental structure-function studies are now warranted to validate these observations.

A full-length three-dimensional structure of the tetrameric potassium ion channel (hERG Kv11.1) including the N- and C-terminal domains was built, with a diameter of 6 Å and 12 Å between the K + selectivity filter and the pore cavity residue Tyr652 of opposite subunits. Further docking studies with a set of 233 structurally known blockers have shown that compounds bind near the inner vestibule of the pore channel, as well as the helix-IV region of the voltage sensor domain (VSD) in the alpha subunit. The residues of hERG, Gly626, Phe627, Gly628, Tyr652 and Phe656 of the pore channel and Arg488 of VSD plays an important role in ligand binding and hERG blockage. The conducted QuaSAR model is statistically significant, with R2 of 0.72 in predicting the hERG blocking activity. Furthermore, QuaSAR descriptors employing computer-assisted multiple regression procedure reveal that increase in hydrophobicity with higher number of aromatic rings are favorable for the binding affinity of hERG blockers. Additionally, the pIC50 values of 25 commercial compounds screened using structure-based pharmacophore model also show binding to the selectivity filter and pore cavity of hERG potassium channel like the known hERG blockers with a wide range of inhibition from weak to strong blockage predicting to have proarrhythmic potential.

Abstract 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

Increasing evidence point out the important roles of ion channels in the physiopathology of cancers, so that these proteins are now considered as potential new therapeutic targets and biomarkers in this disease. Indeed, ion channels have been largely described to participate in many hallmarks of cancers such as migration, invasion, proliferation, angiogenesis, and resistance to apoptosis. At the molecular level, the development of cancers is characterised by alterations in transduction pathways that control cell behaviors. However, the interactions between ion channels and cancer-related signaling pathways are poorly understood so far. Nevertheless, a limited number of reports have recently addressed this important issue, especially regarding the interaction between ion channels and one of the main driving forces for cancer development: the Wnt/β-catenin signaling pathway. In this review, we propose to explore and discuss the current knowledge regarding the interplay between ion channels and the Wnt/β-catenin signaling pathway in cancers.

Chemical investigation of the leaves of Manniophyton fulvum led to the isolation of seven flavonoid glycosides: myricetin-3-O-β-Dd-rhamnoside (1), kaempferol-3-O-β-d-rhamnoside (2), quercetin-3-O-β-d-glucoside (3), quercetin-3-O-β-d-rhamnoside (4), quercetin-3-O-β-d-galactoside (5), rutin (6) and quercetin (7). The structures of the compounds were established by spectroscopic analyses as well as by comparison with published data. Some of the compounds showed strong antioxidant activity which validates the traditional use of the plant. An attempted correlation between the computed HOMO-LUMO energies and the measured antioxidant activities was established. We have also estimated the cardiotoxicity of the compounds by calculating the predicted logarithm of the human Ether-`a-go-go Related Gene (loghERG) using the QikProp program. These purified flavonoids are new potential lead compounds for the development of antioxidant drugs.

The COVID-19 pandemic is an unprecedented challenge and will require novel therapeutic strategies. Affected patients are likely to be at risk of arrhythmia due to underlying comorbidities, polypharmacy and the disease process. Importantly, a number of the medications likely to receive significant use can themselves, particularly in combination, be pro-arrhythmic. Drug-induced prolongation of the QT interval is primarily caused by inhibition of the hERG potassium channel either directly and/or by impaired channel trafficking. Concurrent use of multiple hERG-blocking drugs may have a synergistic rather than additive effect which, in addition to any pre-existing polypharmacy, critical illness or electrolyte imbalance, may significantly increase the risk of arrhythmia and Torsades de Pointes. Knowledge of these risks will allow informed decisions regarding appropriate therapeutics and monitoring to keep our patients safe.

The cardiac action potential is critical to the production of a synchronized heartbeat. This electrical impulse is governed by the intricate activity of cardiac ion channels, among them the cardiac voltage-gated potassium (Kv) channels KCNQ1 and hERG as well as the voltage-gated sodium (Nav) channel encoded by SCN5A. Each channel performs a highly distinct function, despite sharing a common topology and structural components. These three channels are also the primary proteins mutated in congenital long QT syndrome (LQTS), a genetic condition that predisposes to cardiac arrhythmia and sudden cardiac death due to impaired repolarization of the action potential and has a particular proclivity for reentrant ventricular arrhythmias. Recent cryo-electron microscopy structures of human KCNQ1 and hERG, along with the rat homolog of SCN5A and other mammalian sodium channels, provide atomic-level insight into the structure and function of these proteins that advance our understanding of their distinct functions in the cardiac action potential, as well as the molecular basis of LQTS. In this review, the gating, regulation, LQTS mechanisms, and pharmacological properties of KCNQ1, hERG, and SCN5A are discussed in light of these recent structural findings.

Abstract The macrolide antibiotic erythromycin has been associated with QT interval prolongation and inhibition of the hERG‐encoded channels responsible for the rapid delayed rectifier K+ current I(Kr). It has been suggested that low concentrations of erythromycin may have a protective effect against hERG block and associated drug‐induced arrhythmia by reducing the affinity of the pore‐binding site for high potency hERG inhibitors. This study aimed to explore further the notion of a potentially protective effect of erythromycin. Whole‐cell patch‐clamp experiments were performed in which hERG‐expressing mammalian (Human Embryonic Kidney; HEK) cells were preincubated with low to moderate concentrations of erythromycin (3 or 30 µM) prior to whole‐cell patch clamp recordings of hERG current (IhERG) at 37°C. In contrast to a previous report, exposure to low concentrations of erythromycin did not reduce pharmacological sensitivity of hERG to the antipsychotic thioridazine and antihistamine terfenadine. The IC50 value for IhERG tail inhibition by terfenadine was decreased by ~32‐fold in the presence of 3 µM erythromycin (p .05 vs. no preincubation). The effects of low concentrations of erythromycin were investigated for a series of pore blocking drugs, and the results obtained were consistent with additive and/or synergistic effects. Experiments with the externally acting blocker BeKm‐1 on WT hERG and a pore mutant (F656V) were used to explore the location of the binding site for erythromycin. Our data are inconsistent with the use of erythromycin for the management of drug‐induced QT prolongation.

The hERG (human-ether-à-go-go-related gene) channel underlies the rapid delayed rectifier current, Ikr, in the heart, which is essential for normal cardiac electrical activity and rhythm. Slow deactivation is one of the hallmark features of the unusual gating characteristics of hERG channels, and plays a crucial role in providing a robust current that aids repolarization of the cardiac action potential. As such, there is significant interest in elucidating the underlying mechanistic determinants of slow hERG channel deactivation. Recent work has shown that the hERG channel S4 voltage sensor is stabilized following activation in a process termed relaxation. Voltage sensor relaxation results in energetic separation of the activation and deactivation pathways, producing a hysteresis, which modulates the kinetics of deactivation gating. Despite widespread observation of relaxation behaviour in other voltage-gated K+ channels, such as Shaker, Kv1.2 and Kv3.1, as well as the voltage-sensing phosphatase Ci-VSP, the relationship between stabilization of the activated voltage sensor by the open pore and voltage sensor relaxation in the control of deactivation has only recently begun to be explored. In this review, we discuss present knowledge and questions raised related to the voltage sensor relaxation mechanism in hERG channels and compare structure-function aspects of relaxation with those observed in related ion channels. We focus discussion, in particular, on the mechanism of coupling between voltage sensor relaxation and deactivation gating to highlight the insight that these studies provide into the control of hERG channel deactivation gating during their physiological functioning.

Capsule networks (CapsNets), a new class of deep neural network architectures proposed recently by Hinton et al., have shown a great performance in many fields, particularly in image recognition and natural language processing. However, CapsNets have not yet been applied to drug discovery-related studies. As the first attempt, we in this investigation adopted CapsNets to develop classification models of hERG blockers/nonblockers; drugs with hERG blockade activity are thought to have a potential risk of cardiotoxicity. Two capsule network architectures were established: convolution-capsule network (Conv-CapsNet) and restricted Boltzmann machine-capsule networks (RBM-CapsNet), in which convolution and a restricted Boltzmann machine (RBM) were used as feature extractors, respectively. Two prediction models of hERG blockers/nonblockers were then developed by Conv-CapsNet and RBM-CapsNet with the Doddareddy's training set composed of 2,389 compounds. The established models showed excellent performance in an independent test set comprising 255 compounds, with prediction accuracies of 91.8 and 92.2% for Conv-CapsNet and RBM-CapsNet models, respectively. Various comparisons were also made between our models and those developed by other machine learning methods including deep belief network (DBN), convolutional neural network (CNN), multilayer perceptron (MLP), support vector machine (SVM), k-nearest neighbors (kNN), logistic regression (LR), and LightGBM, and with different training sets. All the results showed that the models by Conv-CapsNet and RBM-CapsNet are among the best classification models. Overall, the excellent performance of capsule networks achieved in this investigation highlights their potential in drug discovery-related studies.

The human voltage-sensitive K+ channel hERG plays a fundamental role in cardiac action potential repolarization, effectively controlling the QT interval of the electrocardiogram. Inherited loss- or gain-of-function mutations in hERG can result in dangerous “long” (LQTS) or “short” QT syndromes (SQTS), respectively, and the anomalous susceptibility of hERG to block by a diverse range of drugs underlies an acquired LQTS. A recent open channel cryo-EM structure of hERG should greatly advance understanding of the molecular basis of hERG channelopathies and drug-induced LQTS. Here we describe an update of recent research that addresses the nature of the particular gated state of hERG captured in the new structure, and the insight afforded by the structure into the molecular basis for high affinity drug block of hERG, the binding of hERG activators and the molecular basis of hERG's peculiar gating properties. Interpretation of the pharmacology of natural SQTS mutants in the context of the structure is a promising approach to understanding the molecular basis of hERG inactivation, and the structure suggests how voltage-dependent changes in the membrane domain may be transmitted to an extracellular “turret” to effect inactivation through aromatic side chain motifs that are conserved throughout the KCNH family of channels.

Chemical inhibition of the human ether-a -go-go-related gene (hERG) potassium channel leads to a prolonged QT interval that can contribute to severe cardiotoxicity. The adverse effects of hERG inhibition are one of the principal causes of drug attrition in clinical and pre-clinical development. Preliminary studies have demonstrated that a wide range of environmental chemicals and toxicants may also inhibit the hERG channel and contribute to the pathophysiology of cardiovascular (CV) diseases. As part of the US federal Tox21 program, the National Center for Advancing Translational Science (NCATS) applied a quantitative high throughput screening (qHTS) approach to screen the Tox21 library of 10,000 compounds (~7871 unique chemicals) at 14 concentrations in triplicate to identify chemicals perturbing hERG activity in the U2OS cell line thallium flux assay platform. The qHTS cell-based thallium influx assay provided a robust and reliable dataset to evaluate the ability of thousands of drugs and environmental chemicals to inhibit hERG channel protein, and the use of chemical structure-based clustering and chemotype enrichment analysis facilitated the identification of molecular features that are likely responsible for the observed hERG activity. We employed several machine-learning approaches to develop QSAR prediction models for the assessment of hERG liabilities for drug-like and environmental chemicals. The training set was compiled by integrating hERG bioactivity data from the ChEMBL database with the Tox21 qHTS thallium flux assay data. The best results were obtained with the random forest method (~92.6% balanced accuracy). The data and scripts used to generate hERG prediction models are provided in an open-access format as key in vitro and in silico tools that can be applied in a translational toxicology pipeline for drug development and environmental chemical screening.

Background/Aims: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation. Methods: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting. Results: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1. Conclusions: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.

Abstract Background Blockage of some ion channels and in particular, the hERG (human Ether-a’-go-go-Related Gene) cardiac potassium channel delays cardiac repolarization and can induce arrhythmia. In some cases it leads to a potentially life-threatening arrhythmia known as Torsade de Pointes (TdP). Therefore recognizing drugs with TdP risk is essential. Candidate drugs that are determined not to cause cardiac ion channel blockage are more likely to pass successfully through clinical phases II and III trials (and preclinical work) and not be withdrawn even later from the marketplace due to cardiotoxic effects. The objective of the present study is to develop an SAR (Structure-Activity Relationship) model that can be used as an early screen for torsadogenic (causing TdP arrhythmias) potential in drug candidates. The method is performed using descriptors comprised of atomic NMR chemical shifts (13C and 15N NMR) and corresponding interatomic distances which are combined into a 3D abstract space matrix. The method is called 3D-SDAR (3-dimensional spectral data-activity relationship) and can be interrogated to identify molecular features responsible for the activity, which can in turn yield simplified hERG toxicophores. A dataset of 55 hERG potassium channel inhibitors collected from Kramer et al. consisting of 32 drugs with TdP risk and 23 with no TdP risk was used for training the 3D-SDAR model. Results An artificial neural network (ANN) with multilayer perceptron was used to define collinearities among the independent 3D-SDAR features. A composite model from 200 random iterations with 25% of the molecules in each case yielded the following figures of merit: training, 99.2%; internal test sets, 66.7%; external (blind validation) test set, 68.4%. In the external test set, 70.3% of positive TdP drugs were correctly predicted. Moreover, toxicophores were generated from TdP drugs. Conclusion A 3D-SDAR was successfully used to build a predictive model for drug-induced torsadogenic and non-torsadogenic drugs based on 55 compounds. The model was tested in 38 external drugs.

The data presented in this article are related to the research article entitled “The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression” (Y.F. Teah, M.A. Abduraman, A. Amanah, M.I. Adenan, S.F. Sulaiman, M.L. Tan) [1], which the possible hERG blocking properties of deoxyelephantopin were investigated. This article describes the construction of human embryonic kidney 293 (HEK293) cells overexpressing HERG potassium channel and verification of the presence of hERG mRNA and protein expression in this recombinant cell line.

Background: Pilsicainide, classified as a relatively selective Na+ channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K+ current (IKr) 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 0.03–10 μM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03–10 μM pilsicainide for 48 h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3 μM pilsicainide for 48 h 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.

Background: The rapid delayed rectifier K+ current (IKr), carried by the hERG protein, is one of the main repolarising currents in the human heart and a reduction of this current increases the risk of ventricular fibrillation. α1-adrenoceptors (α1-AR) activation reduces IKr but, despite the clear relationship between an increase in the sympathetic tone and arrhythmias, the mechanisms underlying the α1-AR regulation of the hERG channel are controversial. Thus, we aimed to investigate the mechanisms by which α1-AR stimulation regulates IKr. Methods: α1-adrenoceptors, hERG channels, auxiliary subunits minK and MIRP1, the non PIP2-interacting mutant D-hERG (with a deletion of the 883-894 amino acids) in the C-terminal and the non PKC-phosphorylable mutant N-terminal truncated-hERG (NTK-hERG) were transfected in HEK293 cells. Cell membranes were extracted by centrifugation and the different proteins were visualized by Western blot. Potassium currents were recorded by the patch-clamp technique. IKr was recorded in isolated feline cardiac myocytes. Results: Activation of the α1-AR reduces the amplitude of IhERG and IKr through a positive shift in the activation half voltage, which reduces the channel availability at physiological membrane potentials. The intracellular pathway connecting the α1-AR to the hERG channel in HEK293 cells includes activation of the Gαq protein, PLC activation and PIP2 hydrolysis, activation of PKC and direct phosphorylation of the hERG channel N-terminal. The PKC-mediated IKr channel phosphorylation and subsequent IKr reduction after α1-AR stimulation was corroborated in feline cardiac myocytes. Conclusions: These findings clarify the link between sympathetic nervous system hyperactivity and IKr reduction, one of the best characterized causes of torsades de pointes and ventricular fibrillation.

Obesity mechanisms that make atrial tissue vulnerable to arrhythmia are poorly understood. Voltage-dependent potassium (IK, IKur, and IK1) and L-type calcium currents (ICa,L) are electrically relevant and represent key substrates for modulation in obesity. We investigated whether electrical remodeling produced by high-fat diet (HFD) alone or in concert with acute atrial stimulation were different. Electrophysiology was used to assess atrial electrical function after short-term HFD-feeding in guinea pigs. HFD atria displayed spontaneous beats, increased IK (IKr + IKs) and decreased ICa,L densities. Only with pacing did a reduction in IKur and increased IK1 phenotype emerge, leading to a further shortening of action potential duration. Computer modeling studies further indicate that the measured changes in potassium and calcium current densities contribute prominently to shortened atrial action potential duration in human heart. Our data are the first to show that multiple mechanisms (shortened action potential duration, early afterdepolarizations and increased incidence of spontaneous beats) may underlie initiation of supraventricular arrhythmias in obese guinea pig hearts. These results offer different mechanistic insights with implications for obese patients harboring supraventricular arrhythmias.

The proarrhythmic risk is a major concern in drug development. The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative has proposed the JTpeak interval on electrocardiograms (ECGs) and qNet, an in silico metric, as new biomarkers that may overcome the limitations of the hERG assay and QT interval. In this study, we simulated body-surface ECGs from patch-clamp data using realistic models of the ventricles and torso to explore their suitability as new in silico biomarkers for cardiac safety. We tested seven drugs in this study: dofetilide (high proarrhythmic risk), ranolazine, verapamil (QT increasing, but safe), bepridil, cisapride, mexiletine, and diltiazem. Human ventricular geometry was reconstructed from computed tomography (CT) images, and a Purkinje fiber network was mapped onto the endocardial surface. The electrical wave propagation in the ventricles was obtained by solving a reaction-diffusion equation using finite-element methods. The body-surface ECG data were calculated using a torso model that included the ventricles. The effects of the drugs were incorporated in the model by partly blocking the appropriate ion channels. The effects of the drugs on single-cell action potential (AP) were examined first, and three-dimensional (3D) body-surface ECG simulations were performed at free Cmax values of 1×, 5×, and 10×. In the single-cell and ECG simulations at 5× Cmax, dofetilide, but not verapamil or ranolazine, caused arrhythmia. However, the non-increasing JTpeak caused by verapamil and ranolazine that has been observed in humans was not reproduced in our simulation. Our results demonstrate the potential of 3D body-surface ECG simulation as a biomarker for evaluation of the proarrhythmic risk of candidate drugs.

Short QT syndrome variant 1 (SQT1) arises due to gain-of-function mutations to the human Ether-à-go-go-Related Gene (hERG), which encodes the α subunit of channels carrying rapid delayed rectifier potassium current, IKr. In addition to QT interval shortening and ventricular arrhythmias, SQT1 is associated with increased risk of atrial fibrillation (AF), which is often the only clinical presentation. However, the underlying basis of AF and its pharmacological treatment remain incompletely understood in the context of SQT1. In this study, computational modeling was used to investigate mechanisms of human atrial arrhythmogenesis consequent to a SQT1 mutation, as well as pharmacotherapeutic effects of selected class I drugs–disopyramide, quinidine, and propafenone. A Markov chain formulation describing wild type (WT) and N588K-hERG mutant IKr was incorporated into a contemporary human atrial action potential (AP) model, which was integrated into one-dimensional (1D) tissue strands, idealized 2D sheets, and a 3D heterogeneous, anatomical human atria model. Multi-channel pharmacological effects of disopyramide, quinidine, and propafenone, including binding kinetics for IKr/hERG and sodium current, INa, were considered. Heterozygous and homozygous formulations of the N588K-hERG mutation shortened the AP duration (APD) by 53 and 86 ms, respectively, which abbreviated the effective refractory period (ERP) and excitation wavelength in tissue, increasing the lifespan and dominant frequency (DF) of scroll waves in the 3D anatomical human atria. At the concentrations tested in this study, quinidine most effectively prolonged the APD and ERP in the setting of SQT1, followed by disopyramide and propafenone. In 2D simulations, disopyramide and quinidine promoted re-entry termination by increasing the re-entry wavelength, whereas propafenone induced secondary waves which destabilized the re-entrant circuit. In 3D simulations, the DF of re-entry was reduced in a dose-dependent manner for disopyramide and quinidine, and propafenone to a lesser extent. All of the anti-arrhythmic agents promoted pharmacological conversion, most frequently terminating re-entry in the order quinidine > propafenone = disopyramide. Our findings provide further insight into mechanisms of SQT1-related AF and a rational basis for the pursuit of combined IKr and INa block based pharmacological strategies in the treatment of SQT1-linked AF.