Search

Background and aim Hydrocotyle bonariensis Comm ex Lamm (Araliaceae) is one of these plants sufficiently exploited in traditional African medicine for its hypotensive effect. However, the pharmacological effects of those plants on cardiac functions are not well known. The potassium currents IKs and IKr, responsible for the repolarization of cardiac cell action potential, strongly influence the human cardiac rhythm. Therefore, modulators of these currents have a beneficial or undesirable medical importance in relation to cardiac arrhythmias. In order to optimize the therapeutic use of this medicinal plant, we studied the effects of hydro-ethanolic leaf extract of Hydrocotyle bonariensis on both potassium currents. Experimental procedure The patch clamp experiments for IK currents recording were performed on the HEK 293 (Human Embryonic Kidney 293) cell line, stably transfected with either KCNQ1 and KCNE1 genes encoding the channel responsible for the "IKs" current (HEK293 IKs), or with hERG (human ether-a-go-go related gene) gene encoding "IKr" current (HEK293 IKr). Results and conclusion This study revealed that the hydro-ethanolic leaf extract of H. bonariensis significantly inhibits the slow potassium component (IKs) without altering the fast potassium component (IKr). The extract at 0.5 mg/ml decreases IKs conductance by 24 ± 4.1% (n = 6) without modifying its activation threshold suggesting a direct blockade of the slow potassium channel. This selective action of the extract on the IKs current reflects a class III anti-arrhythmic effect.

Drug isomers may differ in their proarrhythmia risk. An interesting example is the drug sotalol, an antiarrhythmic drug comprising d- and l- enantiomers that both block the hERG cardiac potassium channel and confer differing degrees of proarrhythmic risk. We developed a multi-scale in silico pipeline focusing on hERG channel - drug interactions and used it to probe and predict the mechanisms of pro-arrhythmia risks of the two enantiomers of sotalol. Molecular dynamics (MD) simulations predicted comparable hERG channel binding affinities for d- and l-sotalol, which were validated with electrophysiology experiments. MD derived thermodynamic and kinetic parameters were used to build multi-scale functional computational models of cardiac electrophysiology at the cell and tissue scales. Functional models were used to predict inactivated state binding affinities to recapitulate electrocardiogram (ECG) QT interval prolongation observed in clinical data. Our study demonstrates how modeling and simulation can be applied to predict drug effects from the atom to the rhythm for dl-sotalol and also increased proarrhythmia proclivity of d- vs. l-sotalol when accounting for stereospecific beta-adrenergic receptor blocking.

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.

Graphical abstract, Highlights • SAMiRNA-AREG is a modified siRNA nanoparticle for treatment of pulmonary fibrosis. • Safety pharmacology of SAMiRNA-AREG was evaluated using core battery studies. • No adverse effects on the neurobehavior, respiratory, and cardiovascular systems. • These data provide important safety data for clinical trials in humans., The present safety pharmacology core battery studies (neurobehavior, respiratory, cardiovascular system, and human ether a-go-go (hERG) channel current) investigated the potential harmful effects of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG). The SAMiRNA-AREG was administered by single intravenous injection at up to 300 mg/kg and 100 mg/kg in mice and monkeys, respectively. The hERG assay was performed in Chinese hamster ovary (CHO) cells at SAMiRNA-AREG concentrations of up to 200 μg/mL. In the evaluation on neurobehavior, a transient decrease in body temperature was found at 0.5 h (30 min) post-dose at both sexes in mice, with a single 300 mg/kg dose of SAMiRNA-AREG. However, these effects had returned to normal at 1 h post-dose. In the evaluation on hERG channel current, there were statistically significant differences in the inhibition of peak hERG potassium channel current between the 20, 100, and 200 μg/mL SAMiRNA-AREG treatment groups and the vehicle control group. However, these effects were less potent than that of E-4031, a positive control article. For the respiratory and cardiovascular systems, no treatment-related changes were observed in mice or monkeys. Thus, under these experimental conditions, these studies suggest that SAMiRNA-AREG showed no adverse effects on the neurobehavior, respiratory, and cardiovascular function.

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.

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.

The human ether-a-go-go-related gene (hERG) encodes a tetrameric potassium channel called Kv11.1. This channel can be blocked by certain drugs, which leads to long QT syndrome, causing cardiotoxicity. This is a significant problem during drug development. Using computer models to predict compound cardiotoxicity during the early stages of drug design will help to solve this problem. In this study, we used a dataset of 1865 compounds exhibiting known hERG inhibitory activities as a training set. Thirty cardiotoxicity classification models were established using three machine learning algorithms based on molecular fingerprints and molecular descriptors. Through using these models as the base classifier, a new cardiotoxicity classification model with better predictive performance was developed using ensemble learning method. The accuracy of the best base classifier, which was generated using the XGBoost method with molecular descriptors, was 84.8 %, and the area under the receiver-operating characteristic curve (AUC) was 0.876 in the five fold cross-validation. However, all of the ensemble models that we developed had higher predictive performance than the base classifiers in the five fold cross-validation. The best predictive performance was achieved by the Ensemble-Top7 model, with accuracy of 84.9 % and AUC of 0.887. We also tested the ensemble model using external validation data and achieved accuracy of 85.0 % and AUC of 0.786. Furthermore, we identified several hERG-related substructures, which provide valuable information for designing drug candidates.

The clinical utility of intravenous sotalol is limited due to an extended half-life combined with the potential to generate life-threatening arrhythmias. The authors developed a novel sotalol analogue, soestalol, with an ester linkage introduced to the molecule to shorten half-life. Their hypothesis was that soestalol, but not the acid metabolite, would inhibit the hERG potassium current. Whole-cell, voltage-clamp experiments were performed on cells expressing hERG. Soestalol inhibited outward IhERG tail current density in a manner similar to conventional sotalol. Additionally, soestalol right shifted the voltage dependence of activation. These results warrant further assessment of soestalol as a short-acting, Class III antiarrhythmic drug.

The human ether-a-go-go-related gene1 (hERG) ion channel has been the subject of fascination since it was identified as a target of long QT syndrome more than 20 years ago. In this Biophysical Perspective, we look at what makes hERG intriguing and vexingly unique. By probing recent high-resolution structures in the context of functional and biochemical data, we attempt to summarize new insights into hERG-specific function and articulate important unanswered questions. X-ray crystallography and cryo-electron microscopy have revealed features not previously on the radar—the “nonswapped” transmembrane architecture, an “intrinsic ligand,” and hydrophobic pockets off a pore cavity that is surprisingly small. Advances in our understanding of drug block and inactivation mechanisms are noted, but a full picture will require more investigation.

Ion channel behaviour can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing and inactivating) are commonly represented with Q10coefficients or an Eyring relationship. In this paper we assess the validity of these representations by characterising channel kinetics at multiple temperatures. We focus on the hERG channel, which is important in drug safety assessment and commonly screened at room temperature, so that results require extrapolation to physiological temperature. In Part I of this study we established a reliable method for high-throughput characterisation of hERG1a (Kv11.1) kinetics, using a 15 second information-rich optimised protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using CHO cells over-expressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch clamp platform, with temperature control. We characterise the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37 °C, and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q10coefficients: it broadly follows a generalised, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q10coefficients are protocol-dependent. Our results show that a direct fit using our 15 second protocol best represents hERG kinetics at any given temperature, and suggests that predictions from the Generalised Eyring theory may be preferentially used if no experimentally-derived data are available.Statement of SignificanceIon channel currents are highly sensitive to temperature changes. Yet because many experiments are performed more easily at room temperature, it is common to extrapolate findings to physiological temperatures through the use of Q10coefficients or Eyring rate theory. By applying short, information-rich protocols that we developed in Part I of this study we identify how kinetic parameters change over temperature. We find that the commonly-used Q10and Eyring formulations are incapable of describing the parameters’ temperature dependence, a more Generalised Eyring relationship works well, but remeasuring kinetics and refitting a model is optimal. The findings have implications for the accuracy of the many applications of Q10coefficients in electrophysiology, and suggest that care is needed to avoid misleading extrapolations in their many scientific and industrial pharmaceutical applications.

The human Ether-a-go-go Related Gene (hERG) encodes the pore forming subunit of the channel that conducts the rapid delayed rectifier potassium current IKr. IKr drives repolarization in the heart and when IKr is dysfunctional, cardiac repolarization delays, the QT interval on the electrocardiogram (ECG) prolongs and the risk of developing lethal arrhythmias such as Torsade de Pointes (TdP) increases. TdP risk is incorporated in drug safety screening for cardiotoxicity where hERG is the main target since the IKr channels appear highly sensitive to blockage. hERG block is also included as an important read-out in the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative which aims to combine in vitro and in silico experiments on induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to screen for cardiotoxicity. However, the hERG channel has some unique features to consider for drug safety screening, which we will discuss in this study. The hERG channel consists of different isoforms, hERG1a and hERG1b, which individually influence the kinetics of the channel and the drug response in the human heart and in iPSC-CMs. hERG1b is often underappreciated in iPSC-CM studies, drug screening assays and in silico models, and the fact that its contribution might substantially differ between iPSC-CM and healthy but also diseased human heart, adds to this problem. In this study we show that the activation kinetics in iPSC-CMs resemble hERG1b kinetics using Cs+ as a charge carrier. Not including hERG1b in drug safety testing might underestimate the actual role of hERG1b in repolarization and drug response, and might lead to inappropriate conclusions. We stress to focus more on including hERG1b in drug safety testing concerning IKr.

The use of recreational drugs, including new psychoactive substances (NPS), is paralleled by emergency department visits of drug users with severe cardiotoxicity. Drug-induced cardiotoxicity can be the (secondary) result of increased norepinephrine blood concentrations, but data on potential drug-induced direct effects on cardiomyocyte function are scarce. The presence of hundreds of NPS therefore calls for efficient screening models to assess direct cardiotoxicity.We investigated effects of four reference compounds (3-30 nM dofetilide, nifedipine and isoproterenol, and 1-10 mu M mexiletine) and six recreational drugs (0.01-100 mu M cocaine, 0.01-1000 mu M amphetamine, MDMA, 4-fluoroamphetamine, alpha-PVP and MDPV) on cardiomyocyte function (beat rate, spike amplitude and field potential duration (FPD approximate to QT interval in ECGs)), using Pluricyte (R) human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes cultured on ready-to-use CardioPlate (TM) multi-well microelectrode arrays (mwMEAs). Moreover, the effects of exposure to recreational drugs on cell viability were assessed.Effects of reference compounds were in accordance with the literature, indicating the presence of hERG potassium (dofetilide), sodium (mexiletine) and calcium (nifedipine) channels and alpha-adrenergic receptors (isoproterenol). All recreational drugs decreased the spike amplitude at 10-100 mu M. All amphetamine-type stimulants and alpha-PVP decreased the beat rate at 300 mu M, while cocaine and MDPV did so at 10 mu M and 30 mu M, respectively. All drugs increased the FPD, however at varying concentrations. MDMA, MDPV and amphetamine affected cardiomyocyte function at concentrations relevant for human exposure, while other drugs affected cardiomyocyte function only at higher concentrations ( >= 10 mu M). Cell viability was only mildly affected at concentrations well above the lowest concentrations affecting cardiomyocyte function.We demonstrate that MEA recordings of hiPSC-derived cardiomyocytes enable screening for acute, direct effects on cardiomyocyte function. Our data further indicate that tachycardia in patients exposed to recreational drugs is likely due to indirect drug effects, while prolonged repolarization periods (prolonged QT(c) interval) could (partly) result from direct drug effects on cardiomyocyte function.

Bedaquiline was approved by USFDA in 2012 for pulmonary MDR-TB. The IC50 value of bedaquiline was reported to be remarkably low (25 nM), effectively inhibiting mycobacterial ATP synthase. In addition to these obvious assets of bedaquiline, the potential disadvantages of bedaquiline include inhibition of the hERG (human Ether-a-go-related gene; KCNH2) potassium channel (concurrent risk of cardiac toxicity), hepatic toxicity and possibly phospholipidosis. The current review focuses primarily on the structural part of bedaquiline for the activity-toxicity optimization. This critical analysis of the structure of bedaquiline will help medicinal chemists to synthesize the better modified analouge of bedaquiline with reduced cardiotoxicity, hepatotoxicity potential and improved pharmacokinetics.

Through virtual screening, we identified the lead compound MCL1020, which exhibited modest CHK1 inhibitory activity. Then a series of 5-(pyrimidin-2-ylamino)picolinonitrile derivatives as CHK1 inhibitors were discovered by further rational optimization. One promising molecule, (R)-17, whose potency was one of the best, had an IC50 of 0.4 nM with remarkable selectivity (>4300-fold CHK1 vs. CHK2). Compound (R)-17 effectively inhibited the growth of malignant hematopathy cell lines especially Z-138 (IC50: 0.013 μM) and displayed low affinity for hERG (IC50 > 40 μM). Moreover, (R)-17 significantly suppressed the tumor growth in Z-138 cell inoculated xenograft model (20 mg/kg I.V., TGI = 90.29%) as a single agent with body weight unaffected. Taken together, our data demonstrated compound (R)-17 could be a promising drug candidate for the treatment of hematologic malignancies.

Cariprazine is a novel atypical antipsychotic drug that is widely used for the treatment of schizophrenia and bipolar mania/mixed disorder. We used the whole-cell patch-clamp technique to investigate the effects of cariprazine on hERG channels that are stably expressed in HEK cells. Cariprazine inhibited the hERG 1A and hERG 1A/3.1 tail currents at −50 mV in a concentration-dependent manner with IC50 values of 4.1 and 12.2 μM, respectively. The block of hERG 1A currents by cariprazine was voltage-dependent, and increased over a range of voltage for channel activation. Cariprazine shifted the steady-state inactivation curve of the hERG 1A currents in a hyperpolarizing direction and produced a use-dependent block. A fast application of cariprazine inhibited the hERG 1A currents elicited by a 5 s depolarizing pulse to +60 mV to fully inactivate the hERG 1A currents. During a repolarizing pulse wherein the hERG 1A current was deactivated slowly, cariprazine rapidly and reversibly blocked the open state of the hERG 1A current. However, cariprazine did not affect hERG 1A and hERG 1A/3.1 channel trafficking to the cell membrane. Our results indicated that cariprazine concentration-dependently inhibited hERG 1A and hERG 1A/3.1 currents by preferentially interacting with the open states of the hERG 1A channel, but not by the disruption of hERG 1A and hERG 1A/3.1 channel protein trafficking. Our study examined cariprazine's mechanism of action provides a biophysical profile that is necessary to assess the potential therapeutic effects of this drug.

Ion channel is a pore-forming membrane protein that allows ions to pass through the cell membrane, which is essential for the continuation of life. Analysis of ion channel characteristics at the single cell level will help to study the role of cellular heterogeneity in disease progression and cellular signaling processes. In this study, we fabricate a photocleavable and entropy-driven reaction based electrochemical biosensor for ultrasensitive detection of human ether-a-go-go related gene (hERG) potassium channel activity on HEK293 cell. We employed an antibody-DNA1 to conjugate the hERG channel in the cell membrane via antibody-antigen reaction. The release of the DNA1 by photocleavable reaction will trigger an amplification reaction by using the Exonuclease III (Exo III) to generate intermediate DNA. In addition, two hairpin DNA (DNA3 and DNA4) was employed for the signal amplification. We well designed a toehold on DNA3 for intermediate DNA hybridization to form double-strand DNA that opens the DNA3 hairpin. The free DNA3 exposed the relocked toehold domain to open the DNA4. After the entropy-driven toehold-mediated displacement amplification reaction by using intermediate DNA, DNA4 hybridized with DNA3 effectively, making the ferrocene labeled on the 5'-termini DNA4 close to the Au electrode surface to produce the electrochemical response. Then, the displaced intermediate DNA was released from the cell surface into solution for the next entropy-driven reaction. After two steps amplification reaction, one ion channel triggered thousands of DNA3/DNA4 duplex on the biosensor surface. By using this biosensor, electrochemical curve of hERG ion channels on a single cell was obtained.

Two emerging psychoactive substances, 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe) and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine (25C-NBOMe), are being abused, leading to fatal and non-fatal intoxications. However, most of their adverse effects have been reported anecdotally. In the present study, cardiotoxicity was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), and human ether-a-go-go-related gene (hERG) assay. Expression levels of p21 (CDC42/RAC)-activated kinase 1 (PAK1), one of known biomarkers for cardiotoxicity, were also analyzed. Both 25D-NBOMe and 25C-NBOMe at 100 μM reduced cell viability in MTT assay. At 2.0 mg/kg and 0.75 mg/kg, they prolonged QT intervals in rat ECG. PAK1 was down-regulated by treatment with these two test compounds. Furthermore, potassium channels were inhibited by 25D-NBOMe treatment in hERG assay. Taken together, these results suggest that both 25D-NBOMe and 25C-NBOMe have potential cardiotoxicity, especially regarding cardiac rhythm. Further studies are needed to confirm the relationship between PAK1 down-regulation and cardiotoxicity.

Ether-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation. Cryo-EM structures of two of them, K(V)10.1 and human ether-a-go-go-related gene (hERG or K(V)11.1), have revealed an original nondomain-swapped structure, suggesting that the mechanism of voltage-dependent gating of these two channels is quite different from the classical mechanical-lever model. Molecular aspects of hERG voltage-gating have been extensively studied, indicating that the S4-S5 linker (S4-S5(L)) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6(T)) and stabilizes it in a closed state. Moreover, the N-terminal extremity of the channel, called N-Cap, has been suggested to interact with S4-S5(L) to modulate channel voltage-dependent gating, as N-Cap deletion drastically accelerates hERG channel deactivation. In this study, using COS-7 cells, site-directed mutagenesis, electrophysiological measurements, and immunofluorescence confocal microscopy, we addressed whether these two major mechanisms of voltage-dependent gating are conserved in K(V)10.2 channels. Using cysteine bridges and S4-S5(L)–mimicking peptides, we show that the ligand/receptor model is conserved in K(V)10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in K(V)10.2 abolished the current and altered channel trafficking to the membrane, unlike for the hERG channel in which N-Cap and PAS domain truncations mainly affected channel deactivation. Our results suggest that EAG channels function via a conserved ligand/receptor model of voltage gating, but that the N-Cap and PAS domains have different roles in these channels.

N,N′-Diaryl-bishydrazones of [1,1′-biphenyl]-3,4′-dicarboxaldehyde, [1,1′-biphenyl]-4,4′-dicarboxaldehyde, and 4,4′-bisacetyl-1,1-biphenyl exhibited excellent antifungal activity against a broad spectrum of filamentous and non-filamentous fungi. These N,N′-diaryl-bishydrazones displayed no antibacterial activity in contrast to previously reported N,N′-diamidino-bishydrazones and N-amidino-N′-aryl-bishydrazones. The leading candidate, 4,4′-bis((E)-1-(2-(4-fluorophenyl)hydrazono)ethyl)-1,1′-biphenyl, displayed less hemolysis of murine red blood cells at concentrations at or below that of a control antifungal agent (voriconazole), was fungistatic in a time-kill study, and possessed no mammalian cytotoxicity and no toxicity with respect to hERG inhibition.

The synthesis and biological evaluation as potential urotensin-II receptor antagonists of a series of 5-arylfuran-2-carboxamide derivatives 1, bearing a 4-(3-chloro-4-(piperidin-4-yloxy)benzyl)piperazin-1-yl group, are described. The results of a systematic SAR investigation of furan-2-carboxamides with C-5 aryl groups possessing a variety of aryl ring substituents led to identification of the 3,4-difluorophenyl analog 1y as a highly potent UT antagonist with an IC50 value of 6 nM. In addition, this substance was found to display high metabolic stability, and low hERG inhibition and cytotoxicity, and to have an acceptable PK profile.

Kinase irregularity has been correlated with several complex neurodegenerative tauopathies. Development of selective inhibitors of these kinases might afford promising anti-tauopathy therapies. While DAPK1 inhibitors halt the formation of tau aggregates and counteract neuronal death, CSF1R inhibitors could alleviate the tauopathies-associated neuroinflammation. Herein, we report the design, synthesis, biological evaluation, mechanistic study, and molecular docking study of novel CSF1R/DAPK1 dual inhibitors as multifunctional molecules inhibiting the formation of tau aggregates and neuroinflammation. Compound 3l, the most potent DAPK1 inhibitor in the in vitro kinase assay (IC50 = 1.25 μM) was the most effective tau aggregates formation inhibitor in the cellular assay (IC50 = 5.0 μM). Also, compound 3l elicited potent inhibition of CSF1R in the in vitro kinase assay (IC50 = 0.15 μM) and promising inhibition of nitric oxide production in LPS-induced BV-2 cells (55% inhibition at 10 μM concentration). Kinase profiling and hERG binding assay anticipated the absence of off-target toxicities while the PAMPA-BBB assay predicted potentially high BBB permeability. The mechanistic study and selectivity profile suggest compound 3l as a non-ATP-competitive DAPK1 inhibitor and an ATP-competitive CSF1R inhibitor while the in silico calculations illustrated binding of compound 3l to the substrate-binding site of DAPK1. Hence, compound 3l might act as a protein-protein interaction inhibitor by hindering DAPK1 kinase reaction through preventing the binding of DAPK1 substrates.

Cardiac safety assessment is challenging because a better understanding of torsadogenic mechanisms beyond hERG blockade and QT interval prolongation is necessary for patient safety. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a new human cell-based platform to assess cardiac safety in non-clinical testing during drug development. The multi-electrode array (MEA) platform is a promising electrophysiological technology to assess QT interval prolongation and proarrhythmic potential of drug candidates using hiPSC-CMs. The Japan iPS Cardiac Safety Assessment (JiCSA) has established an MEA protocol to evaluate the applicability of hiPSC-CMs for assessing the torsadogenic potential of compounds and completed a large-scale validation study using 60 compounds. During our study, an international multi-site study of hiPSC-CMs was performed by the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative using 28 compounds. We have comparatively analyzed our JiCSA datasets with those of CiPA using the CiPA logistical and ordinal linear regression model. Regardless of the protocol differences, the evaluation results of the 28 compounds were very similar and highly predictable for torsadogenic risks. Thus, an MEA-based approach using hiPSC-CMs would be a standard testing method to evaluate proarrhythmic potentials. This review paper would provide new insights into the hiPSC-CMs/MEA method required for its regulatory use. Keywords: Cardiac safety, CiPA, Human iPS cells, JiCSA, Standardization

Graphical abstract, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) continues to spread worldwide, with 25 million confirmed cases and 800 thousand deaths. Effective treatments to target SARS-CoV-2 are urgently needed. In the present study, we have identified a class of cyclic sulfonamide derivatives as novel SARS-CoV-2 inhibitors. Compound 13c of the synthesized compounds exhibited robust inhibitory activity (IC50 = 0.88 μM) against SARS-CoV-2 without cytotoxicity (CC50 > 25 μM), with a selectivity index (SI) of 30.7. In addition, compound 13c exhibited high oral bioavailability (77%) and metabolic stability with good safety profiles in hERG and cytotoxicity studies. The present study identified that cyclic sulfonamide derivatives are a promising new template for the development of anti-SARS-CoV-2 agents.

Understanding the effects of microgravity on human organs is crucial to exploration of low earth orbit, the moon, and beyond. Drosophila can be sent to space in large numbers to examine the effects of microgravity on heart structure and function, which is fundamentally conserved from flies to humans. Flies reared in microgravity exhibit reduced climbing ability, cardiac constriction with myofibrillar remodeling and diminished output. Isolated heart RNAseq revealed reduced expression of sarcomeric/ECM genes and increased proteasomal gene expression, consistent with a compromised, smaller heart and abnormal proteostasis. This was examined further on a second flight: we found dramatically elevated proteasome aggregates co-localizing with increased amyloid/polyQ deposits. Remarkably, in long-QT causing sei/hERG mutants, proteasomal gene expression at 1g was lower than wildtype, but nevertheless increased, albeit modestly, in microgravity. Therefore, cardiac remodeling and proteostatic stress may be a fundamental response of heart muscle

Similar to the opioid system, the endocannabinoid system which regulates immune response, memory and nociceptive thresholds, cell communication, appetite, and metabolism, also modulates (via Cannabinoid receptor type 1 - CB1R) the rewarding effects of natural and synthetic cannabinoids. Although currently there is significant controversy concerning the (legal) use of cannabis, it has been established that prolonged consumption of a large variety of chemicals from the cannabinoid family can lead to the development of psychiatric and cardiovascular pathologies. In addition to (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC or simply THC), the main addictive component of cannabis, 43 other synthetic cannabimimetic agents are currently classified as Schedule I drugs and their use in the U.S. is illegal (DEA-DCT-DIR-032-18 [1] ). In this study, three-dimensional spectrometric data-activity relationship (3D-SDAR) quantitative and categorical models aimed at better understanding the molecular mechanism of action and the structural features governing binding to CB1R were developed. The performance parameters for a consensus model constructed from composite PLS and KNN quantitative models achieved R2 of 0.703 for an external blind set and an accuracy of 0.846, sensitivity of 0.870, and specificity of 0.840 for binary classification. In addition to the classical A, B, C-tricyclic terpenoid system, other structural alerts were also discovered and reported. We also identified the presence of a hERG linked toxicophore in the molecular structures of a subset of diaryl-(piperidinyl)purines which are known to be selective and potent CB1R binders.

Human ether-a-go-go-related gene (hERG) channels are key regulators of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains with many long-QT syndrome (LQTS)-causing mutations located at the interface between these domains. Despite the importance of PAS/CNBH domain interactions, little is known about their affinity. Here, we used the surface plasmon resonance (SPR) technique to investigate interactions between isolated PAS and CNBH domains and the effects of LQTS-causing mutations R20G, N33T and E58D, located at the PAS/CNBH domain interface, on these interactions. We determined that the affinity of the PAS/CNBH domain interactions was ∼1.4 μM. R20G and E58D mutations had little effect on the domain interaction affinity, while N33T abolished the domain interactions. Interestingly, mutations in the intrinsic ligand, a conserved stretch of amino acids occupying the beta-roll cavity in the CNBH domain, had little effect on the affinity of PAS/CNBH domain interactions. Additionally, we determined that the isolated PAS domains formed oligomers with an interaction affinity of ∼1.6 μM. Co-expression of the isolated PAS domains with the full-length hERG channels or addition of the purified PAS protein inhibited hERG currents. These PAS/PAS interactions can have important implications for hERG function in normal and pathological conditions associated with increased surface density of channels or interaction with other PAS domain-containing proteins. Taken together, our study provides the first account of the binding affinities for wild-type and mutant hERG PAS and CNBH domains, and highlights the potential functional significance of PAS/PAS domain interactions.

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 μM–100 μM range with a 2–4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 μM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 μM and 30 μM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative is validating a new paradigm for assessing proarrhythmic potential of drugs that goes beyond hERG block and QT prolongation. Based on in vitro data of the drug's effects on multiple cardiac ion channel currents, CiPA's in silico model of the human cardiomyocyte will classify drugs as low, intermediate or high risk for torsade de pointes. Under CiPA, early phase 1 ECG data will be used to determine if there are unexpected ion channel effects in humans compared to the in vitro ion channel data. CiPA's ECG biomarker working group identified the heart rate corrected J-Tpeak interval (J-Tpeakc, from the end of the QRS to the peak of the T-wave) as the best of 12 ECG biomarkers to detect late sodium current block in presence of hERG block. While predominant hERG blockers prolonged QTc and J-Tpeakc, “balanced” ion channel blocking drugs (hERG + late sodium and/or calcium block) prolonged QTc without prolonging J-Tpeakc. This manuscript reviews the ECG component of CiPA and provides a description of the ECG methods used in the CiPA ECG validation clinical study.

α7-Nicotinic acetylcholine receptor (α7-nAChR) agonists are promising therapeutic drug candidates for treating the cognitive impairment associated with Alzheimer's disease (AD). Thus, a novel class of derivatives of 1,4-diazobicylco[3.2.2]nonane has been synthesized and evaluated as α7-nAChR ligands. Five of them displayed high binding affinity (Ki = 0.001–25 nM). In particular, the Ki of 14 was 0.0069 nM, which is superior to that of the most potent ligand that was previously reported by an order of magnitude. Four of them had high selectivity for α7-nAChRs over α4β2-nAChRs and no significant hERG (human ether-a-go-go-related gene) inhibition. Their agonist activity was also discussed preliminarily. One of the compounds, 15 (Ki = 2.98 ± 1.41 nM), was further radiolabeled with 18F to afford [18F]15 for PET imaging, which exhibited high initial brain uptake (11.60 ± 0.14%ID/g at 15 min post injection), brain/blood value (9.57 at 30 min post injection), specific labeling of α7-nAChRs and fast clearance from the brain. Blocking studies demonstrated that [18F]15 was α7-nAChR selective. In addition, micro-PET/CT imaging in normal rats further indicated that [18F]15 had obvious accumulation in the brain. Therefore, [18F]15 was proved to be a potential PET radiotracer for α7-nAChR imaging.

Long QT syndrome type 2 (LQT2) is a congenital disease characterized by loss of function mutations in hERG potassium channels (IKr). LQT2 is associated with fatal ventricular arrhythmias promoted by triggered activity in the form of early afterdepolarizations (EADs). We previously demonstrated that intracellular Ca2+ handling is remodeled in LQT2 myocytes. Remodeling leads to aberrant late RyR-mediated Ca2+ releases that drive forward-mode Na+-Ca2+ exchanger (NCX) current and slow repolarization to promote reopening of L-type calcium channels and EADs. Forward-mode NCX was found to be enhanced despite the fact that these late releases do not significantly alter the whole-cell cytosolic calcium concentration during a vulnerable period of phase 2 of the action potential corresponding to the onset of EADs. Here, we use a multiscale ventricular myocyte model to explain this finding. We show that because the local NCX current is a saturating nonlinear function of the local submembrane calcium concentration, a larger number of smaller-amplitude discrete Ca2+ release events can produce a large increase in whole-cell forward-mode NCX current without increasing significantly the whole-cell cytosolic calcium concentration. Furthermore, we develop novel insights, to our knowledge, into how alterations of stochastic RyR activity at the single-channel level cause late aberrant Ca2+ release events. Experimental measurements in transgenic LTQ2 rabbits confirm the critical arrhythmogenic role of NCX and identify this current as a potential target for antiarrhythmic therapies in LQT2.

Novel bacterial type II topoisomerase inhibitors (NBTIs) constitute a promising new class of antibacterial agents. We report a series of NBTIs with potent anti-staphylococcal activity and diminished hERG inhibition. Dioxane-linked compound 9 demonstrated MICs ≤1 μg/mL against both methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA), accompanied by reduced hERG inhibition as compared to cyclohexane- or piperidine-linked analogs.

The pharmacological activity of DSP-6952, a novel compound was investigated, compared to that of clinically efficacious gastrointestinal (GI) prokinetic 5-hydroxytryptamine 4 (5-HT 4 ) receptor agonists. DSP-6952 had a strong affinity of K i = 51.9 nM for 5-HT 4(b) receptor, and produced contraction in the isolated guinea pig colon with EC 50 of 271.6 nM and low intrinsic activity of 57%, similar to tegaserod and mosapride. In the development of the 5-HT 4 receptor agonists, cardiovascular risk was deliberately evaluated, because some related prokinetics were reported to cause with cardiovascular adverse events, such as ventricular arrhythmias or ischemia. DSP-6952 showed minimal effects up to 100 μM in human ether-a-go-go-related gene (hERG) channels or guinea pig cardiomyocytes. In telemetered conscious monkeys, DSP-6952 did not affect blood pressure or any electrocardiogram (ECG) up to 180 mg/kg, p.o.; however, DSP-6952 transiently increased heart rate, as well as in anesthetized dogs. The positive chronotropic effects of DSP-6952 were completely antagonized by a 5-HT 4 receptor antagonist, and another 5-HT 4 receptor agonist, TD-5108 also increased heart rate. These effects are considered a class effect seen in clinically developing and marketed 5-HT 4 receptor agonists, and have not been regarded as a critical issue in clinical use. DSP-6952 did not induce contraction in the rabbit coronary artery up to 100 μM, which differed from tegaserod or sumatriptan. These results show that DSP-6952 does not have cardiac ischemic risk via coronary vasoconstriction. In conclusion, DSP-6952 is a promising GI prokinetic compound with partial 5-HT 4 receptor agonistic activity as well as a favorable cardiovascular safety profile.

Starting from 4-amino-8-quinoline carboxamide lead 1a and scaffold hopping to the chemically more tractable quinazoline, a systematic exploration of the 2-substituents of the quinazoline ring, utilizing structure activity relationships and conformational constraint, resulted in the identification of 39 novel CD38 inhibitors. Eight of these analogs were 10–100-fold more potent human CD38 inhibitors, including the single digit nanomolar inhibitor 1am. Several of these molecules also exhibited improved therapeutic indices relative to hERG activity. A representative analog 1r exhibited suitable pharmacokinetic parameters for in vivo animal studies, including moderate clearance and good oral bioavailability. These inhibitor compounds will aid in the exploration of the enzymatic functions of CD38, as well as furthering the study of the therapeutic implications of NAD enhancement in metabolic disease models.

Fluid movement within the heart generates substantial shear forces, but the effect of this mechanical stress on the electrical activity of the human heart has not been examined. The fast component of the delayed rectifier potassium currents responsible for repolarization of the cardiac action potential, Ikr, is encoded by the human ether-a-go-go related gene (hERG) channel. Here, we exposed hERG1a channel–expressing HEK293T cells to laminar shear stress (LSS) and observed that this mechanical stress increased the whole-cell current by 30–40%. LSS shifted the voltage dependence of steady-state activation of the hERG channel to the hyperpolarizing direction, accelerated the time course of activation and recovery from inactivation, slowed down deactivation, and shifted the steady-state inactivation to the positive direction, all of which favored the hERG open state. In contrast, the time course of inactivation was faster, favoring the closed state. Using specific inhibitors of focal adhesion kinase, a regulator of mechano-transduction via the integrin pathway, we also found that the LSS-induced modulation of the whole-cell current depended on the integrin pathway. The hERG1b channel variant, which lacks the Per-Arnt-Sim (PAS) domain, and long QT syndrome–associated variants having point mutations in the PAS domain were unaffected by LSS, suggesting that the PAS domain in hERG1a channel may be involved in sensing mechanical shear stress. We conclude that a mechano-electric feedback pathway modulates hERG channel activity through the integrin pathway, indicating that mechanical forces in the heart influence its electrical activity.

Abuse of the common anti-diarrheal loperamide is associated with QT interval prolongation as well as development of the potentially fatal arrhythmia torsades de pointes. The mechanism underlying this cardiotoxicity is high affinity inhibition of the human ether-a-go-go-related gene (hERG) cardiac K+ channel. N-Desmethyl loperamide is the major metabolite of loperamide and is a close structural relative of the parent molecule. To date no information is available regarding the affinity of N-desmethyl loperamide for human cardiac ion channels. The effects of N-desmethyl loperamide on various cloned human cardiac ion channels including hERG, KvLQT1/mink and Nav1.5 were studied and compared to that of the parent. N-Desmethyl loperamide was a much weaker (7.5-fold) inhibitor of hERG compared to loperamide. However, given the higher plasma levels of the metabolite relative to the parent, it is likely that N-desmethyl loperamide can contribute, at least secondarily, to the cardiotoxicity observed with loperamide abuse. We used the recently solved cryo-EM structure of the hERG channel together with previously published inhibitors, to understand the basis of the interactions as well as the difference that a single methyl plays in the hERG channel blocking affinities of these two compounds.

Introduction Theileria equi, an etiologic agent of equine piroplasmosis, is a tick-transmitted hemoprotozoan of the phylum Apicomplexa. Recent outbreaks of piroplasmosis in the United States have renewed interest in safe and effective treatment options. Although imidocarb dipropionate (IMD) is the drug of choice for clearance of T. equi, adverse reactions and recently documented resistance support the need for alternative therapeutic strategies. The recently described bumped kinase inhibitors (BKIs) are a new class of compounds that could potentially be used as safe and effective alternatives to IMD. In an initial effort to evaluate this potential, herein we determined the T. equi growth inhibitory activity of 11 BKIs relative to that of IMD and the previously tested BKI 1294. Because some BKIs have known human ether-a-go-go related gene (hERG) channel activity, we also assessed the hERG activity of each compound with the goal to identify those with the highest potency against T. equi coupled with the lowest potential for cardiotoxicity. Results Six BKIs inhibited T. equi growth in vitro, including the previously evaluated BKI 1294 which was used as a positive control. All six compounds were significantly less potent (higher 50% effective concentration (EC50)) than IMD. Two of those compounds were more potent than BKI 1294 control but had similar hERG activity. Although the remaining three compounds had similar to lower potency than BKI 1294, hERG EC50 was higher for three of them (BKI 1735, BKI 1369 and BKI 1318). Conclusions The BKI compounds evaluated in this study inhibited T. equi in vitro and had diverse hERG activity. Based on these considerations, three compounds would be suitable for further evaluation. While these results provide a foundation for future work, in vivo pharmacokinetic, pharmacodynamics, and safety studies are needed before BKI compounds can be recommended for clinical use in T. equi infected horses.

Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC50 = 2.31 mM, LiEC50 = 6.14 mM, TcEC50 = 1.31 mM) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4'-dimethoxy-[1.1'-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide (A12) and (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene)hydrazine carbothioamide (A14) were able to potentiate the anti-parasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei, yielding a 6-fold and 4-fold respectively Dose Reduction Index for MTX. The toxicity profile against four human cell lines and a panel of in vitro early-toxicity assays (comprising hERG, Aurora B, five cytochrome P450 isoforms and mitochondrial toxicity) demonstrated the low toxicity for the thosemicarbazones class in comparison with known drugs. The results confirmed thiosemicarbazones as a suitable chemical scaffold with potential for the development of properly decorated new anti-parasitic drugs.

Bruton's tyrosine kinase (Btk) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) and Fcγ receptor (FcR) signaling pathways, which makes it a uniquely attractive target for the treatment of autoimmune diseases, such as rheumatoid arthritis (RA). We reported a series of compounds bearing 7H-pyrrolo [2,3-d]pyrimidin-4-amine scaffold that potently inhibited Btk in vitro. Analysis of the structure-activity relationships (SAR) and drug-like profiles led to the discovery of the optimal compound B16. B16 preferentially inhibited Btk (IC50 = 21.70 ± 0.82 nM) over closely related kinases with moderate selectivity. Cell-based tests also confirmed that B16 significantly inhibited Btk Y223 auto-phosphorylation and PLCγ2 Y1217 phosphorylation. MTT revealed that B16 displayed weak suppression against normal LO2, HEK293 and THP-1 cell lines with IC50 values over 30 μM. Moreover, B16 showed very weak potential to block the hERG channel (IC50 = 11.10 μM) in comparison to ibrutinib (IC50 = 0.97 μM). Owing to its favorable physicochemical properties (ClogP = 2.53, aqueous solubility ≈ 0.1 mg/mL), pharmacokinetic profiles (F = 49.15%, t1/2 = 7.02 h) and reasonable CYP450 profile, B16 exhibited potent anti-arthritis activity and similar efficacy to ibrutinib in reducing paw thickness in CIA mice. In conclusion, B16 is a potent, selective and durable inhibitor of Btk and has the potential to a safe and efficacious treatment for arthritis.

Inhibition of S. aureus diapophytoene desaturase (CrtN) could serve as an alternative approach for addressing the tricky antibiotic resistance by blocking the biosynthesis of carotenoid pigment which shields the bacterium from host oxidant killing. In this study, we designed and synthesized 44 derivatives with piperonyl scaffold targeting CrtN and the structure-activity relationships (SARs) were examined extensively to bring out the discovery of 21b with potent efficacy and better hERG safety profile compared to the first class CrtN inhibitor benzocycloalkane derivative 2. Except the excellent pigment inhibitory activity against wild-type S. aureus, 21b also showed excellent pigment inhibition against four pigmented MRSA strains. In addition, H2O2 killing and human whole blood killing assays proved 21b could sensitize S. aureus to be killed under oxidative stress conditions. Notably, the murine study in vivo validated the efficacy of 21b against pigmented S. aureus Newman, vancomycin-intermediate S. aureus Mu50 and linezolid-resistant S. aureus NRS271.

The human ether-a-go-go-related gene (hERG) encodes the α subunit of a rapidly activating delayed-rectifier potassium (IKr) channel. Mutations of the hERG cause long QT syndrome type 2 (LQT2). Acetylation of lysine residues occurs in a subset of non-histone proteins and this modification is controlled by both histone acetyltransferases and deacetylases (HDACs). The aim of this study was to clarify effects of HDAC(s) on wild-type (WT) and mutant hERG proteins. WThERG and two trafficking-defective mutants (G601S and R752W) were transiently expressed in HEK293 cells, which were treated with a pan-HDAC inhibitor Trichostatin A (TSA) or an isoform-selective HDAC6 inhibitor Tubastatin A (TBA). Both TSA and TBA increased protein levels of WThERG and induced expression of mature forms of the two mutants. Immunoprecipitation showed an interaction between HDAC6 and immature forms of hERG. Coexpression of HDAC6 decreased acetylation and, reciprocally, increased ubiquitination of hERG, resulting in its decreased expression. siRNA against HDAC6, as well as TBA, exerted opposite effects. Immunochemistry revealed that HDAC6 knockdown increased expression of the WThERG and two mutants both in the endoplasmic reticulum and on the cell surface. Electrophysiology showed that HDAC6 knockdown or TBA treatment increased the hERG channel current corresponding to the rapidly activating delayed-rectifier potassium current (IKr) in HEK293 cells stably expressing the WT or mutants. Three lysine residues (K116, K495 and K757) of hERG were predicted to be acetylated. Substitution of these lysine residues with arginine eliminated HDAC6 effects. In HL-1 mouse cardiomyocytes, TBA enhanced endogenous ERG expression, increased IKr, and shortened action potential duration. These results indicate that hERG is a substrate of HDAC6. HDAC6 inhibition induced acetylation of hERG which counteracted ubiquitination leading its stabilization. HDAC6 inhibition may be a novel therapeutic option for LQT2.

A series of novel aralkyl piperazine derivatives were synthesized, and evaluated for their serotonin reuptake inhibitory and 5-HT1A/5-HT7 receptors affinities activity. Antidepressant activities in vivo of the compounds were screened using the forced swimming test (FST) and tail suspension test (TST). The results indicated that compounds 21k (RUI, IC50 = 31 nM; 5-HT1A, 5-HT7, ki = 62, 12 nM) and 21n (RUI, IC50 = 25 nM; 5-HT1A, 5-HT7, ki = 28, 3.3 nM) exhibited high affinities for the 5-HT1A/5-HT7 receptors coupled with potent serotonin reuptake inhibition. Specifically, the most promising compound 21n possessed a good oral pharmacokinetic properties and an acceptable hERG profile, and showed potent antidepressant-like effect in the FST and TST models.

Despite the importance of the hERG channel in drug discovery and the sizable number of antagonist molecules discovered, only a few hERG agonists have been discovered. Here we report a novel hERG agonist; SKF-32802 and a structural analog of the agonist NS3623, SB-335573. These were discovered through a similarity search of published hERG agonists. SKF-32802 incorporates an amide linker rather than NS3623's urea, resulting in a compound with a different mechanism of action. We find that both compounds decrease the time constant of open channel kinetics, increase the amplitude of the envelope of tails assay, mildly increased the amplitude of the IV curve, bind the hERG channel in either open or closed states, increase the plateau of the voltage dependence of activation and modulate the effects of the hERG antagonist, quinidine. Neither compound affects inactivation nor deactivation kinetics, a property unique among hERG agonists. Additionally, SKF-32802 induces a leftward shift in the voltage dependence of activation. Our structural models show that both compounds make strong bridging interactions with multiple channel subunits and are stabilized by internal hydrogen bonding similar to NS3623, PD-307243 and RPR26024. While SB-335573 binds in a nearly identical fashion as NS3623, SKF-32802 makes an additional hydrogen bond with neighboring threonine 623. In summary, SB-335573 is a type 4 agonist which increases open channel probability while SKF-32802 is a type 3 agonist which induces a leftward shift in the voltage dependence of activation.

Treatment of hyperglycemia with drugs that block renal glucose reabsorption via inhibition of sodium-dependent glucose cotransporter 2 (SGLT2) is a novel approach to diabetes management. In this study, twenty-seven aryl C-glycosides bearing a C=N/C−N linkage at the glucosyl C6 position were designed, synthesized and evaluated for their inhibitory activity against human SGLT2 (hSGLT2). Compounds with good hSGLT2 inhibition were further investigated to determine their selectivity over hSGLT1. Of these, five representative aryl C-glycosides were chosen for pharmacokinetic analysis. Oxime 2a was determined to have the most promising pharmacokinetic properties and was selected for in vivo glucosuria and plasma glucose level studies, which found it to exhibit comparable efficacy to dapagliflozin (1). Furthermore, 2a was not found to exhibit either significant cytotoxicity (CC50 > 50 μM) or human ether-a-go-go related gene (hERG) inhibition (2% inhibition at 10 μM). Taken together, these efforts culminated in the discovery of oxime 2a as a potential SGLT2 inhibitor.

Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late INa and on IK1, with IC50 of tens of μM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the β-escin-perforated method, inhibited IKr, ICaL, INa peak, but had no effect on If. In current-clamp they caused action potential prolongation. Our data and those from literature for Ito were used to compute proarrhythmogenic risk predictors Bnet (Mistry HB, 2018) and Qnet (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group., Graphical abstract Image 1

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.

Astemizole is a H 1 -antagonist endowed with antimalarial activity, but has hERG liabilities. Systematic structural modifications of astemizole led to the discovery of analogues that display very potent activity as inhibitors of the growth of the Plasmodium parasite, but show a decreased hERG inhibition, when compared to astemizole. These compounds can be used as starting point for the development of a new class of antimalarials.

Voltage-activated human ether-á-go-go–related gene (hERG) potassium channels are critical for the repolarization of cardiac action potentials and tune-spike frequency adaptation in neurons. Two isoforms of mammalian ERG1 channel subunits, ERG1a and ERG1b, are the principal subunits that conduct the IKr current in the heart and are also broadly expressed in the nervous system. However, there is little direct evidence that ERG1a and ERG1b form heteromeric channels. Here, using electrophysiology, biochemistry, and fluorescence approaches, we systematically tested for direct interactions between hERG1a and hERG1b subunits. We report 1) that hERG1a dominant-negative subunits suppress hERG1b currents (and vice versa), 2) that disulfide bonds form between single cysteine residues experimentally introduced into an extracellular loop of hERG1a and hERG1b subunits and produce hERG1a–hERG1b dimers, and 3) that hERG1a and hERG1b subunits tagged with fluorescent proteins that are FRET pairs exhibit robust energy transfer at the plasma membrane. Thus, multiple lines of evidence indicated a physical interaction between hERG1a and hERG1b, consistent with them forming heteromeric channels. Moreover, co-expression of variable ratios of hERG1a and hERG1b RNA yielded channels with deactivation kinetics that reached a plateau and were different from those of hERG1b channels, consistent with a preference of hERG1b subunits for hERG1a subunits. Cross-linking studies revealed that an equal input of hERG1a and hERG1b yields more hERG1a–hERG1a or hERG1a–hERG1b dimers than hERG1b–hERG1b dimers, also suggesting that hERG1b preferentially interacts with hERG1a. We conclude that hERG1b preferentially forms heteromeric ion channels with hERG1a at the plasma membrane.

Background Ventricular tachycardia (VT), which is related to haemodynamic and electrophysiological alterations, is an important complication in repaired tetralogy of Fallot (rTOF) patients. We defined the role of implantable cardioverter defibrillator (ICD) therapy after pulmonary valve replacement (PVR) and the implications of coexisting long QT gene mutations/polymorphisms. Methods From 2003 to 2016, rTOF patients with VT who received ICD implantation were enrolled. rTOF patients without VT served as a control group. We performed long QT gene mutation analysis through the direct sequencing method. Results In total, 12 (male/female, 8/4) patients with VT received ICD implantation. The mean age at TOF repair and ICD implantation was 4.4±4.5years and 27.1±11.5years, respectively. All but one patient received PVR for severe pulmonary regurgitation or repair for a residual ventricular septal defect. After 5.4±4.0years of follow-up, six patients (50%) had at least one episode of appropriate shock: two had received PVR with intraoperative arrhythmia ablation and two had received PVR only. Compared with 121 patients without VT, the ICD patients had a higher frequency of long QT gene mutation/polymorphisms (10/12 vs. 54/121, p =0.014), especially in the hERG and SCN5A genes. The mechanism of VT in rTOF was discussed thoroughly. Conclusion In rTOF patients who received ICD as secondary prevention, the appropriate shock rate remained high despite PVR and intraoperative arrhythmia ablation. Coexisting long QT gene mutations/polymorphisms may be associated with ventricular arrhythmia.

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [3H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [3H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [3H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [3H]DA uptake at VMAT2, Ki changes in the nanomolar range (Ki = 0.014–0.073 µM). Compound 15d exhibited the highest affinity (Ki = 0.014 µM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (Ki = 0.073 µM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2.

Introduction Several drugs can induce QT prolongation by altering cardiac ion currents, notably IKr. The cardiotoxic response to a drug is however extremely variable between individuals, ranging from no impact to life-threatening ventricular arrhythmias. The molecular mechanisms underlying this variability remain unclear. Previously, we reported higher expression levels of Cavin1, an essential protein for caveolae formation, in cardiomyocytes (CM) derived from patients with high susceptibility to develop sotalol-induced long QT. Purpose We aimed to explore whether Cavin1 could sway the cardiac repolarization response to drugs. Method We used induced pluripotent stem cells-derived CM (iPS-CM) from two subjects with the highest sensitivity (HS) to sotalol (ie, those with the highest increase in QTc) and two individuals with the lowest sensitivity to sotalol (LS) to perform molecular and electrophysiological investigations. Results At baseline, no differences in terms of repolarization durations, IKr amplitude, and ion channel expression were noted between the HS- and the LS-iPS-CM. However, the HS-iPS-CM displayed significant prolongation in action potential and field potential (FPD) durations after the application of 30 and 50μM of sotalol. They also displayed higher sensitivity to other drugs targeting hERG (E4031, vandetanib, and clarithromycin). Cavin1 mRNA and protein levels were increased by 2 fold in the HS-iPS-CM as compared to the LS-iPS-CM. Overexpressing Cavin1 in the LS-iPS-CM using adenovirus induced a higher sensitivity to sotalol as only 10μM was sufficient to induce 12% longer FPD. Consistently, the infection with Cavin1 increased the sensitivity of LS-CM to the other hERG blockers. In contrast, siRNA against Cavin1 reduced its expression by 60% in HS iPS-CM and switched their phenotype to a lower sensitivity to sotalol and the other hERG blockers. Interestingly, in line with the shortening of the FPD in LS-iPS-CM, overexpressing Cavin1 increased hERG expression in isolated membrane fraction from HEK cells stably expressing KCNH2. This suggests a role of Cavin1 in modulating hERG trafficking/recycling to the membrane and thereby the number of channels that can be blocked by drugs. Conclusion Altogether, this work identified Cavin1 as a novel modulator of cardiomyocyte response to hERG blockers and a potential risk factor for diLQT. Further understanding of its contribution may pave the way for personalized drug prescription to avoid undesirable cardiotoxicity. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Leducq