hERG Channel Research Articles

Generation of patient-specific induced pluripotent stem cell lines with Type 2 Long QT Syndrome and the KCNH2 c.379C > T pathogenic variant

Type 2 Long QT Syndrome (LQT2) is a rare genetic heart rhythm disorder causing life-threatening arrhythmias. We derived induced pluripotent stem cell (iPSC) lines from two patients with LQT2, aged 18 and 6, both carrying a heterozygous missense mutation on the 3rd and 11th exons of KCNH2. The iPSC lines exhibited normal genomes, expressed pluripotent markers, and differentiated into trilineage embryonic layers. These patient-specific iPSC lines provide a valuable model to study the molecular and functional impact of the hERG channel gene mutation in LQT2 and to develop personalized therapeutic approaches for this syndrome.

Inhibition of the hERG Potassium Channel by a Methanesulphonate-Free E-4031 Analogue.

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.

Synthesis of 1,3,5-triphenyl-1,2,4-triazole derivatives and their neuroprotection by anti-oxidative stress and anti-inflammation and protecting BBB

Acute ischemic stroke (AIS) is a serious cardiovascular and cerebrovascular disease; Oxidative stress and neuroinflammation are important factors which destroy blood-brain barrier (BBB) in AIS. In the study, a series of 1,3,5-triphenyl-1,2,4-triazole derivatives were designed and synthesized; the optimal compound 9 was obtained by screening their anti-oxidant and anti-inflammatory effects; the neuroprotection effect of compound 9 was evaluated with a rat middle cerebral artery occlusion (MCAO) model. Subsequently, the mechanism of neuroprotection were explored via Western blot. The results prompt compound 9 maybe exert anti-AIS neuroprotection by inhibiting oxidative stress and neuroinflammation inhibition by inhibiting Keap1, COX-2 and iNOS. At the same time, it can protect BBB by reducing glycocalyx degradation and matrix metallopeptidase-9 levels. Its LD50 > 1000 mg/kg on mice and hERG channel inhibition IC50 > 30 μM, which lower acute toxicity and hERG channel inhibition would make compound 9 a promising stroke treatment candidate.

Discovery of 2-Aminopyrimidine Derivatives as Potent Dual FLT3/CHK1 Inhibitors with Significantly Reduced hERG Inhibitory Activities.

FLT3 inhibitors as single agents have limited effects because of acquired and adaptive resistance and the cardiotoxicity related to human ether-a-go-go-related gene (hERG) channel blockade further impedes safe drugs to the market. Inhibitors having potential to overcome resistance and reduce hERG affinity are highly demanded. Here, we reported a dual FLT3/CHK1 inhibitor 18, which displayed potencies to overcome varying acquired resistance in BaF3 cells with FLT3-TKD and FLT3-ITD-TKD mutations. Moreover, 18 displayed high selectivity over c-KIT more than 1700-fold and greatly reduced hERG affinity, with an IC50 value of 58.4 μM. Further mechanistic studies demonstrated 18 can upregulate p53 and abolish the outgrowth of adaptive resistant cells. In the in vivo studies, 18 demonstrated favorable PK profiles and good safety, suppressed the tumor growth in the MV-4-11 cell inoculated mouse xenograft model, and prolonged the survival in the Molm-13 transplantation model, supporting its further development.

Synthesis and Validation of the First Cell-Impermeable Hsp90α-Selective Inhibitors.

Hsp90α is an isoform of the heat shock protein 90 (Hsp90) family of molecular chaperones and mediates the folding and activation of ∼400 client proteins. However, inhibition of intracellular Hsp90α has caused detrimental side effects and significantly hindered the clinical development of Hsp90 inhibitors. As an alternative strategy, 14 Hsp90α-selective inhibitors were synthesized to introduce permanently charged moieties onto the solvent-exposed portion of the Hsp90α binding site to produce cell-impermeable extracellular Hsp90α-selective inhibitors. The resulting lead compounds were cell-permeable dimethylamine 14 (NDNA3), with an affinity of 0.51 μM for Hsp90α and >196-fold selectivity over the other Hsp90 isoforms, and cell-impermeable quaternary ammonium 17 (NDNA4), with an affinity of 0.34 μM for Hsp90α and >294-fold selectivity. The permanently charged analogs were determined to have low membrane permeability, to be nontoxic against Ovcar-8 and MCF-10A cells, to avoid disruption of hERG channel maturation, and not to induce the heat shock response or Hsp90α-dependent client degradation.

4-phenylbutyric acid re-trafficking hERG/G572R channel protein by modulating the endoplasmic reticulum stress-associated chaperones and endoplasmic reticulum-associated degradation gene.

Long QT syndrome type 2 (LQT2) is caused by mutations in the KCNH2/human ether-à-go-go-related gene (hERG). Some hERG genetic mutation-associated diseases are alleviated by hERG-specific drug chaperones (glycerol, dimethyl sulfoxide, trimethylamine N-oxide, thapsigargin), delayed rectifier K+ current (IKr) blockers methanesulfonanilide E4031, the antihistamine astemizole, or the prokinetic drug cisapride, and the anti-arrhythmic drug quinidine. Meanwhile, many in vivo and in vitro studies have reported the efficacy of 4-phenylbutyric acid (4-PBA) in diseases with inherited genetic mutations. This study aims to explore potential therapeutic agents for hERG/G572R mutated ion channel. pcDNA3/hERG [wild type (WT)]-FLAG and pcDNA3/hERG (G572R)-FLAG plasmids were transfected into HEK293 cells. A western blot (WB) experiment was conducted to analyze protein expression. Quantitative real-time polymerase chain reaction (qPCR) was used to analyze the messenger RNA (mRNA) expression levels in the WT/G572R heterozygous HEK293 cell model treated with or without 4-PBA. The interaction between WT/G572R and BIP (GRP78), GRP94, and 3-hydroxy-3-methylglutaryl coenzyme A reductase degradation protein 1 (HRD1) was tested by co-immunoprecipitation (co-IP). To investigate the effect of 4-PBA on the WT/G572R channel current, we used electrophysiological assays (patch-clamp electrophysiological recordings). The results showed that WT/G572R activated the ATF6 pathway in the endoplasmic reticulum stress (ERS), the ERS response markers GRP78, GRP94, and calreticulin (CRT)/calnexin (CNX), and HRD1, which decreased after application of the ERS inhibitor 4-PBA. The results of co-IP confirmed that the ability of hERG interacted with GRP78, GRP94, and HRD1. Moreover, 4-PBA increased the current of WT/G572R and reversed the gating kinetics of the WT/G572R channel. 4-PBA corrects hERG channel transport defects by inhibiting excessive ERS and the endoplasmic reticulum-associated degradation (ERAD)-related gene E3 ubiquitin ligase HRD1. Additionally, 4-PBA improved WT/G572R channel current. 4-PBA is expected to be developed as a new treatment method for LQT2.

The role of the HERG channel in the secretion of glucagon-like peptide-1 (GLP-1) from murine intestinal L-cells

The HERG ion channel belongs to the voltage-gated potassium (Kv) channel family and is involved in potassium efflux during cellular repolarization. Mutations in HERG have been linked to long QT syndrome, which is associated with elevated secretion of glucagon-like peptide-1 (GLP-1). However, the precise contribution of HERG to GLP-1 secretion remains unclear. In this study, we demonstrate the expression of HERG in GLP-1-producing L-cells within the intestinal epithelium of rodents. Using a mouse L-cell model (GLUTag cell line), we observed that downregulation of HERG led to a significant prolongation of action potential duration, an increase in intracellular calcium concentration, and a stimulation of GLP-1 secretion following exposure to nutrients. These findings provide evidence that HERG plays a direct role in regulating GLP-1 secretion in the intestine and may hold promise as a potential target for the treatment of diabetes.

HERG agonists pose challenges to web-based machine learning methods for prediction of drug-hERG channel interaction

Pharmacological blockade of the IKr channel (hERG) by diverse drugs in clinical use is associated with the Long QT Syndrome that can lead to life threatening arrhythmia. Various computational tools including machine learning models (MLM) for the prediction of hERG inhibition have been developed to facilitate the throughput screening of drugs in development and optimise thus the prediction of hERG liabilities. The use of MLM relies on large libraries of training compounds for the quantitative structure-activity relationship (QSAR) modelling of hERG inhibition. The focus on inhibition omits potential effects of hERG channel agonist molecules and their associated QT shortening risk. It is instructive, therefore, to consider how known hERG agonists are handled by MLM. Here, two highly developed online computational tools for the prediction of hERG liability, Pred-hERG and HergSPred were probed for their ability to detect hERG activator drug molecules as hERG interactors. In total, 73 hERG blockers were tested with both computational tools giving overall good predictions for hERG blockers with reported IC50s below Pred-hERG and HergSPred cut-off threshold for hERG inhibition. However, for compounds with reported IC50s above this threshold such as disopyramide or sotalol discrepancies were observed. HergSPred identified all 20 hERG agonists selected as interacting with the hERG channel. Further studies are warranted to improve online MLM prediction of hERG related cardiotoxicity, by explicitly taking into account channel agonism as well as inhibition.

Protection of pancreatic islets from oxidative cell death by a peripherally-active morphinan with increased drug safety

ObjectiveDextromethorphan (DXM) is a commonly used antitussive medication with positive effects in people with type 2 diabetes mellitus, since it increases glucose tolerance and protects pancreatic islets from cell death. However, its use as an antidiabetic medication is limited due to its central nervous side effects and potential use as a recreational drug. Therefore, we recently modified DXM chemically to reduce its blood–brain barrier (BBB) penetration and central side effects. However, our best compound interacted with the cardiac potassium channel hERG (human ether-à-go-go-related gene product) and the μ-opioid receptor (MOR). Thus, the goal of this study was to reduce the interaction of our compound with these targets, while maintaining its beneficial properties. MethodsReceptor and channel binding assays were conducted to evaluate the drug safety of our DXM derivative. Pancreatic islets were used to investigate the effect of the compound on insulin secretion and islet cell survival. Via liquor collection from the brain and a behavioral assay, we analyzed the BBB permeability. By performing intraperitoneal and oral glucose tolerance tests as well as pharmacokinetic analyses, the antidiabetic potential and elimination half-life were investigated, respectively. To analyze the islet cell-protective effect, we used fluorescence microscopy as well as flow cytometric analyses. ResultsHere, we report the design and synthesis of an optimized, orally available BBB-impermeable DXM derivative with lesser binding to hERG and MOR than previous ones. We also show that the new compound substantially enhances glucose-stimulated insulin secretion (GSIS) from mouse and human islets and glucose tolerance in mice as well as protects pancreatic islets from cell death induced by reactive oxygen species and that it amplifies the effects of tirzepatide on GSIS and islet cell viability. ConclusionsWe succeeded to design and synthesize a novel morphinan derivative that is BBB-impermeable, glucose-lowering and islet cell-protective and has good drug safety despite its morphinan and imidazole structures.

Differential Effects of Remdesivir and Lumacaftor on Homomeric and Heteromeric hERG Channels.

The human ether-a-go-go-related gene (hERG) encodes for the pore-forming subunit of the channel that conducts the rapidly activating delayed K+ current (IKr) in the heart. The hERG channel is important for cardiac repolarization, and reduction of its expression in the plasma membrane due to mutations causes long QT syndrome type 2 (LQT2). As such, promoting hERG membrane expression is a strategy to rescue mutant channel function. In the present study, we applied patch clamp, western blots, immunocytochemistry, and quantitative reverse transcription polymerase chain reaction techniques to investigate the rescue effects of two drugs, remdesivir and lumacaftor, on trafficking-defective mutant hERG channels. As our group has recently reported that the antiviral drug remdesivir increases wild-type (WT) hERG current and surface expression, we studied the effects of remdesivir on trafficking-defective LQT2-causing hERG mutants G601S and R582C expressed in HEK293 cells. We also investigated the effects of lumacaftor, a drug used to treat cystic fibrosis, that promotes CFTR protein trafficking and has been shown to rescue membrane expression of some hERG mutations. Our results show that neither remdesivir nor lumacaftor rescued the current or cell-surface expression of homomeric mutants G601S and R582C. However, remdesivir decreased while lumacaftor increased the current and cell-surface expression of heteromeric channels formed by WT hERG and mutant G601S or R582C hERG. We concluded that drugs can differentially affect homomeric WT and heteromeric WT+G601S (or WT+R582C) hERG channels. These findings extend our understanding of drug-channel interaction and may have clinical implications for patients with hERG mutations. SIGNIFICANCE STATEMENT: Various naturally occurring mutations in a cardiac potassium channel called hERG can impair channel function by decreasing cell-surface channel expression, resulting in cardiac electrical disturbances and even sudden cardiac death. Promotion of cell-surface expression of mutant hERG channels represents a strategy to rescue channel function. This work demonstrates that drugs such as remdesivir and lumacaftor can differently affect homomeric and heteromeric mutant hERG channels, which have biological and clinical implications.

The Strength of hERG Inhibition by Erythromycin at Different Temperatures Might Be Due to Its Interacting Features with the Channels.

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.

1656-P: Glucagon-Like peptide-1 (GLP-1) Secretion from Intestinal L-Cells Is Mediated by hERG Potassium Channels

The hERG protein is a member of voltage-gated potassium (Kv) channels. Previous reports showed that patients with long QT syndrome due to mutations in hERG channel had higher responsive glucagon-like peptide-1 (GLP-1) secretion. However, the role of hERG in GLP-1 secretion remains uncertain. Here we report that hERG is co-expressed in GLP-1-expressing L cells in rodent intestinal epithelium (Fig. 1). In a murine L-cell model, GLUTag cell line, downregulation of hERG significantly prolonged action potential duration, increased intracellular calcium concentration ([Ca2+]i), and promoted GLP-1 secretion (Fig. 2) after stimulation of nutrients. These results reveal a direct role of hERG in GLP-1 secretion in murine L-cells, suggesting intestinal hERG is a potential target for the treatment of diabetes. Disclosure J.Yang: None. C.Liu: None. H.Wang: None. Y.Yuan: None. Funding National Natural Science Foundation of China (82170809, 81930019)

Acute osimertinib exposure induces electrocardiac changes by synchronously inhibiting the currents of cardiac ion channels.

Introduction: As the third generation of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), osimertinib has demonstrated more significant cardiotoxicity than previous generations of EGFR-TKIs. Investigating the mechanism of osimertinib cardiotoxicity can provide a reference for a comprehensive understanding of osimertinib-induced cardiotoxicity and the safety of the usage of this drug in clinical practice. Methods: Multichannel electrical mapping with synchronous ECG recording was used to investigate the effects of varying osimertinib concentrations on electrophysiological indicators in isolated Langendorff-perfused hearts of guinea pigs. Additionally, a whole-cell patch clamp was used to detect the impact of osimertinib on the currents of hERG channels transfected into HEK293 cells and the Nav1.5 channel transfected into Chinese hamster ovary cells and acute isolated ventricular myocytes from SD rats. Results: Acute exposure to varying osimertinib concentrations produced prolongation in the PR interval, QT interval, and QRS complex in isolated hearts of guinea pigs. Meanwhile, this exposure could concentration-dependently increase the conduction time in the left atrium, left ventricle, and atrioventricular without affecting the left ventricle conduction velocity. Osimertinib inhibited the hERG channel in a concentration-dependent manner, with an IC50 of 2.21 ± 1.29μM. Osimertinib also inhibited the Nav1.5 channel in a concentration-dependent manner, with IC50 values in the absence of inactivation, 20% inactivation, and 50% inactivation of 15.58 ± 0.83μM, 3.24 ± 0.09μM, and 2.03 ± 0.57μM, respectively. Osimertinib slightly inhibited the currents of L-type Ca2+ channels in a concentration-dependent manner in acutely isolated rat ventricular myocytes. Discussion: Osimertinib could prolong the QT interval; PR interval; QRS complex; left atrium, left ventricle, and atrioventricular conduction time in isolated guinea pig hearts. Furthermore, osimertinib could block the hERG, Nav1.5, and L-type Ca2+ channels in concentration-dependent manners. Therefore, these findings might be the leading cause of the cardiotoxicity effects, such as QT prolongation and decreased left ventricular ejection fraction.

The predictive ability of the QT response in supine-standing test for drug induced QT prolongation

Abstract Funding Acknowledgements Type of funding sources: None. Introduction A significant cause of sudden cardiac death is arrhythmias induced by QT prolongation. The acquired long QT syndrome is much more common than the congenital form. A major cause of acquired long QT prolongation is drug exposure. Drug-induced QT prolongation is common when taking cardiac drugs such as antiarrhythmics but also common with other medication groups such as antibiotics, antihistamines, and psychiatric drugs. These medications prolong QT by inhibiting the HERG channel which controls the rectifier potassium current (IKr) and prolong depolarization. As HERG inhibition is multifactorial an individual’s effect of medications on QT is difficult to predict and thus leaving many patients with significant risk once they initiate treatment with such medications. In the last decade, tests that measure the QT reaction to sudden changes in heart rate while changing position for supine to standing have shown usefulness in assessing the possibility of congenital long QT in patients with equivocal QT interval, probably by measuring the repolarization reserve which is deficient in patients with congenital long QT syndrome. Lol Purpose We sought to identify whether the same tests can be useful in prediction of acquired QT prolongation after exposure to HERG potassium channel suppressing medications. Methods Fifty four healthy volunteers undergone a supine-standing test where QT changes: QT stretching, and QT stunning were examined after brisk standing. Afterward the volunteers received one 400 mg dose of moxifloxacin, a drug known to prolong QT by affecting the HERG channel. Results of the standing test were compared to the drug effects on QTc and predictive ability was assessed. Results There was no significant correlation between the maximal drug evoked QTc prolongation and either change in QTc during the stretching or stunning phase (R=0.090, p= 0.538 and R=0.224, p=0.176 accordingly). Both stunning and stretching tests results did not differ significantly between patient with a significant (>20msecs) QT prolongation in response to drugs and no such prolongation (p> 0.361). The area under the ROC curves of the QTc prolongation during both phases stunning phase did not show usefulness in predicting substantial QTc prolongation (>20msec) during drug therapy with an AUCs of 0.593 (95% CI 0.418-0.769) and 0.617 (95%CI 0.375-0.859) accordingly. Conclusions Repolarization reserve assessment using a simple brisk standing test did not efficiently aid in predict QT drug response in patients treated with HERG inhibiting drugs.

Evaluation of rosuvastatin-induced QT prolongation risk using real-world data, in vitro cardiomyocyte studies, and mortality assessment

Drug-induced QT prolongation is attributed to several mechanisms, including hERG channel blockage. However, the risks, mechanisms, and the effects of rosuvastatin-induced QT prolongation remain unclear. Therefore, this study assessed the risk of rosuvastatin-induced QT prolongation using (1) real-world data with two different settings, namely case–control and retrospective cohort study designs; (2) laboratory experiments using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM); (3) nationwide claim data for mortality risk evaluation. Real-world data showed an association between QT prolongation and the use of rosuvastatin (OR [95% CI], 1.30 [1.21–1.39]) but not for atorvastatin (OR [95% CI], 0.98 [0.89–1.07]). Rosuvastatin also affected the sodium and calcium channel activities of cardiomyocytes in vitro. However, rosuvastatin exposure was not associated with a high risk of all-cause mortality (HR [95% CI], 0.95 [0.89–1.01]). Overall, these results suggest that rosuvastatin use increased the risk of QT prolongation in real-world settings, significantly affecting the action potential of hiPSC-CMs in laboratory settings. Long-term rosuvastatin treatment was not associated with mortality. In conclusion, while our study links rosuvastatin use to potential QT prolongation and possible influence on the action potential of hiPSC-CMs, long-term use does not show increased mortality, necessitating further research for conclusive real-world applications.

Discovery of (S)-N-(2-Amino-4-fluorophenyl)-4-(1-(3-(4-((dimethylamino)methyl)phenyl)-6-oxopyridazin-1(6H)-yl)ethyl)benzamide as Potent Class I Selective HDAC Inhibitor for Oral Anticancer Drug Candidate.

A novel series of benzamide derivatives were successively designed and synthesized prepared from the pyridazinone scaffold. Among them, (S)-17b, demonstrated potent inhibitory activity in vitro toward human class I HDAC isoforms and human myelodysplastic syndrome (SKM-1) cell line. Also, (S)-17b strongly increased the intracellular level of acetyl-histone H3 and P21 simultaneously and effectively induced G1 cell cycle arrest and apoptosis. Through oral dosing in SKM-1 xenograft models, (S)-17b exhibited excellent in vivo antitumor activity. In addition, compound (S)-17b showed better antitumor efficacy on mouse models with intact immune system than those with thymus deficiencies. Furthermore, this compound displayed a favorable pharmacokinetic profile in ICR mice and SD rat, respectively, minimal metabolic property differences among hepatocytes from five species, and a low inhibition upon the human ether-a-go-go (hERG) channel with an IC50 value of 34.6 μΜ. This novel compound (S)-17b may serve as a new drug candidate for further investigation.

In silico prediction of hERG blockers using machine learning and deep learning approaches.

The human ether-à-go-go-related gene (hERG) is associated with drug cardiotoxicity. If the hERG channel is blocked, it will lead to prolonged QT interval and cause sudden death in severe cases. Therefore, it is important to evaluate the hERG-blocking property of compounds in early drug discovery. In this study, a dataset containing 4556 compounds with IC50 values determined by patch clamp techniques on mammalian lineage cells was collected, and hERG blockers and non-blockers were distinguished according to 3 single-thresholds and 2 binary thresholds. Four machine learning (ML) algorithms combining four molecular fingerprints and molecular descriptors as well as graph convolutional neural networks (GCN) were used to construct a series of binary classification models. The results showed that the best models varied for different thresholds. The ML models implemented by support vector machine and random forest performed well based on Morgan fingerprints and molecular descriptors, with AUCs ranging from 0.884 to 0.950. GCN showed superior prediction performance with AUCs above 0.952, which might be related to its direct extraction of molecular features from the original input. Meanwhile, the classification of binary threshold was better than that of single threshold, which could provide us with a more accurate prediction of hERG blockers. At last, the applicability domain for the model was defined, and seven structural alerts that might generate hERG blockage were identified by information gain and substructure frequency analysis. Our work would be beneficial for identifying hERG blockers in chemicals.

Hydroxychloroquine induces long QT syndrome by blocking hERG channel

Abstract Objective 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. Materials and methods Adult New Zealand white rabbits were used for investigating the effects of HCQ on cardiac electrophysiology and expression of ion channel genes. HEK-293T cells with sustained overexpression of human-ether-a-go-go-related gene (hERG) K+ channels were used for whole-cell patch-clamp recordings of hERG K+ channel current (IhERG). Quantitative RT-PCR analysis and Western blot analysis were employed to determine the expression of various genes at mRNA and protein levels, respectively. Results electrocardiogram (ECG) recordings revealed that HCQ prolonged QT and RR intervals and slowed heart rate in rabbits. Whole-cell patch-clamp results showed that HCQ inhibited the tail current of hERG channels and slowed the reactivation process from inactivation state. HCQ suppressed the expression of hERG and hindered the formation of the heat shock protein 90 (Hsp90)/hERG complex. Moreover, the expression levels of connexin 43 (CX43) and Kir2.1, the critical molecular/ionic determinants of cardiac conduction thereby ventricular arrythmias, were decreased by HCQ, while those of Cav1.2, the main Ca2+ handling proteins, remained unchanged and SERCA2a was increased. Conclusion HCQ could induce LQT but did not induce arrhythmias, and whether it is suitable for the treatment of COVID-19 requires more rigorous investigations and validations in the future.

Structure-Activity relationships of replacements for the triazolopyridazine of Anti-Cryptosporidium lead SLU-2633

Cryptosporidiosis is a diarrheal disease particularly harmful to children and immunocompromised people. Infection is caused by the parasite Cryptosporidium and leads to dehydration, malnutrition, and death in severe cases. Nitazoxanide is the only FDA approved drug but is only modestly effective in children and ineffective in immunocompromised patients. To address this unmet medical need, we previously identified triazolopyridazine SLU-2633 as potent against Cryptosporidium parvum, with an EC50 of 0.17 µM. In the present study, we develop structure–activity relationships (SAR) for the replacement of the triazolopyridazine head group by exploring different heteroaryl groups with the aim of maintaining potency while reducing affinity for the hERG channel. 64 new analogs of SLU-2633 were synthesized and assayed for potency versus C. parvum. The most potent compound, 7,8-dihydro-[1,2,4]triazolo[4,3-b]pyridazine 17a, was found to have a Cp EC50 of 1.2 µM, 7-fold less potent than SLU-2633 but has an improved lipophilic efficiency (LipE) score. 17a was found to decrease inhibition in an hERG patch-clamp assay by about two-fold relative to SLU-2633 at 10 µM despite having similar inhibition in a [3H]-dofetilide competitive binding assay. While most other heterocycles were significantly less potent than the lead, some analogs such as azabenzothiazole 31b, have promising potency in the low micromolar range, similar to the drug nitazoxanide, and represent potential new leads for optimization. Overall, this work highlights the important role of the terminal heterocyclic head group and represents a significant extension of the understanding of the SAR for this class of anti-Cryptosporidium compounds.

Calcium- and sodium-activated potassium channels (K<sub>Ca</sub>, K<sub>Na</sub>) in GtoPdb v.2023.1

Calcium- and sodium- activated potassium channels are members of the 6TM family of K channels which comprises the voltage-gated KV subfamilies, including the KCNQ subfamily, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 6 or 7TM) and the Ca2+- and Na+-activated SK subfamily (nomenclature as agreed by the NC-IUPHAR Subcommittee on Calcium- and sodium-activated potassium channels [126]). As for the 2TM family, the pore-forming a subunits form tetramers and heteromeric channels may be formed within subfamilies (e.g. KV1.1 with KV1.2; KCNQ2 with KCNQ3).