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Assessing the magnitude of QTc prolongation is crucial in drug development due to its association with Torsades de Pointes. Inhibition of the hERG channel, pivotal in cardiac repolarization, is a key factor in evaluating this risk. In this study, the relationship between hERG inhibition and QTc prolongation magnitude was investigated, with the aim to derive simple guidance on the required hERG margin to avoid a large (>20 ms) QTc prolongation., Methods: Data from literature and FDA sources were searched for compounds with hERG IC 50 values alongside clinical QTc data with paired plasma concentrations, or compounds demonstrating a clinical concentration-QTc relationship. Relationships between hERG inhibition, hERG IC 50 margin to unbound plasma C max , and QTc prolongation magnitude were calculated., Results: Analysis of 148 clinical QTc observations from 98 compounds revealed that compounds associated with QTc prolongation >10 ms typically exhibited hERG margins of ≤33-fold, while those exceeding 20 ms were generally associated with margins of ≤24-fold. QTc increases above 10 ms were not observed at hERG margins >100-fold. Based on 53 clinical concentration-QTc datasets, modest hERG inhibition levels of ∼4-6 % correlated with a 10 ms QTc prolongation, while ∼10-13 % inhibition corresponded to a 20 ms prolongation., Conclusions: This study enhances understanding of the relationship between hERG inhibition and QTc prolongation magnitude, by conducting analysis across a wide range of 98 compounds. This information can be used to determine the optimal hERG margin, particularly for drug discovery projects with limited scope to completely design-out hERG activity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Alexander Harmer reports a relationship with AstraZeneca PLC that includes: employment and equity or stocks. Michael G Rolf reports a relationship with AstraZeneca PLC that includes: employment and equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Oral small-molecule GLP-1 receptor biased agonists exhibit promising treatment efficacy of type 2 diabetes and obesity. SAL0112 is a novel compound that has demonstrated remarkable efficacy in preclinical animal models. Herein, both in vitro and in vivo preclinical toxicity investigations were conducted to explore the safety profile of SAL0112. The HTRF assay and TR-FRET assay were utilized for cAMP detection. Patch clamp assay was employed for hERG potassium ion channel determination. Cynomolgus monkeys were used in a cardiovascular safety pharmacology study and a 13-week repeated dose toxicity study. The telemetry system was employed to detect cardiovascular indicators such as ECG, HR, and BP. During the repeated dose toxicity study, body weight, food intake, hematology, coagulation function test, serum biochemistry tests, and urine analysis were measured. Macroscopic and microscopic observations were conducted at the end of the study. TK studies were conducted on Day 1 and Day 91. SAL0112 exhibited a high degree of potency in activating the monkey GLP-1 receptor whereas had no effect on the rodent GLP-1 receptor. In contrast to Danuglipron, which demonstrated high potency on hERG with an IC 50 value of 6.9 μM, the IC 50 of SAL0112 on hERG was greater than 100 μM. Compared to the Vehicle Control group, no significant changes in cardiovascular indicators were observed in the cardiovascular safety pharmacology study after a single dose of SAL0112 up to 250 mg/kg (P > 0.05). A repeated dose toxicity study revealed moderate anorexigenic effects and a reduction in body weight, effects that were found to be reversible and not associated with any pathological changes. The NOAEL of SAL0112 is 150 mg/kg, providing an approximate safety margin of threefold. SAL0112 demonstrated a favorable safety profile in cynomolgus monkeys, with a substantial therapeutic window that supports the progression of this compound into clinical studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To design safe, selective, and effective new therapies, there must be a deep understanding of the structure and function of the drug target. One of the most difficult problems to solve has been resolution of discrete conformational states of transmembrane ion channel proteins. An example is KV11.1 (hERG), comprising the primary cardiac repolarizing current, I . hERG is a notorious drug anti-target against which all promising drugs are screened to determine potential for arrhythmia. Drug interactions with the hERG inactivated state are linked to elevated arrhythmia risk, and drugs may become trapped during channel closure. However, the structural details of multiple conformational states have remained elusive. Here, we guided AlphaFold2 to predict plausible hERG inactivated and closed conformations, obtaining results consistent with multiple available experimental data. Drug docking simulations demonstrated hERG state-specific drug interactions in good agreement with experimental results, revealing that most drugs bind more effectively in the inactivated state and are trapped in the closed state. Molecular dynamics simulations demonstrated ion conduction for an open but not AlphaFold2 predicted inactivated state that aligned with earlier studies. Finally, we identified key molecular determinants of state transitions by analyzing interaction networks across closed, open, and inactivated states in agreement with earlier mutagenesis studies. Here, we demonstrate a readily generalizable application of AlphaFold2 as an effective and robust method to predict discrete protein conformations, reconcile seemingly disparate data and identify novel linkages from structure to function.Kr . hERG is a notorious drug anti-target against which all promising drugs are screened to determine potential for arrhythmia. Drug interactions with the hERG inactivated state are linked to elevated arrhythmia risk, and drugs may become trapped during channel closure. However, the structural details of multiple conformational states have remained elusive. Here, we guided AlphaFold2 to predict plausible hERG inactivated and closed conformations, obtaining results consistent with multiple available experimental data. Drug docking simulations demonstrated hERG state-specific drug interactions in good agreement with experimental results, revealing that most drugs bind more effectively in the inactivated state and are trapped in the closed state. Molecular dynamics simulations demonstrated ion conduction for an open but not AlphaFold2 predicted inactivated state that aligned with earlier studies. Finally, we identified key molecular determinants of state transitions by analyzing interaction networks across closed, open, and inactivated states in agreement with earlier mutagenesis studies. Here, we demonstrate a readily generalizable application of AlphaFold2 as an effective and robust method to predict discrete protein conformations, reconcile seemingly disparate data and identify novel linkages from structure to function., Competing Interests: Competing Interest Statement: The authors declare no competing interests.

Diabetes (DM) patients have an increased risk (~50%) for sudden cardiac death (SCD), mostly as a result of ventricular arrhythmias. The molecular mechanisms involved remain partially defined. The potent proinflammatory lipid mediator leukotriene (LT) B4, is pathologically elevated in DM compared to nondiabetic patients, resulting in increased LTB4 accumulation in heart, leading to an increased risk for life-threatening proarrhythmic signatures. We used electrophysiology, immunofluorescence, and confocal microscopy approaches to evaluate LTB4 cellular effects in guinea pig heart and ventricular myocytes. We have observed that LTB4 is increased in multiple mouse models (C57BL/6 J/Lep ob/ob and PANIC-ATTAC) of DM, promotes profound cellular arrhythmogenesis (spontaneous beats and early after depolarizations, EADs), and severely depresses the rapidly activating delayed rectifier K current (hERG1/I Kr ) density in HEK293 cells and guinea pig ventricular myocytes. We have further found that guinea pigs challenged with LTB4 displayed a significantly prolonged QT interval, and that this can be prevented with LTB4R inhibition, suggesting that preventing such LTB4R effects may be therapeutically beneficial in DM. Our data suggests that a further elucidation of LTB4 vulnerable substrates, and how this leads to ventricular arrhythmias, is likely to lead to continued improvements in management options, and the development of new therapies for prevention of SCD in DM patients., (© 2024 The Author(s). Journal of Cellular Physiology published by Wiley Periodicals LLC.)

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG potassium channels by drugs as the major determinant for the potential to cause drug-induced Torsades de Pointes (TdP) cardiac arrhythmias. Here we report our findings on the next phase of CiPA: Determination of hERG inhibitory properties using the standard CiPA-defined data acquisition protocol, here called the standard protocol, at physiological temperature (37 degrees Celsius). To do this, we measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells by the small molecule verapamil, using manual whole-cell patch-clamp electrophysiology recordings with the standard protocol, which is characterized, in part, by a series of 10 s duration voltage steps to 0 mV, ultimately leading to a cumulative recording time of approximately 30 min. Using the standard protocol, we measured an IC 50 for verapamil of 225 nM, a Hill coefficient of 1, and time constant of inhibition at 0 mV of 0.64 s. But, using the standard protocol resulted in a very low (5 %) experimental success rate per cell, which had low practicality for future experiments. To address the 5 % success rate, we generated a revised protocol characterized, in part, by a series of 3 s duration voltage steps to 0 mV, leading to a cumulative recording time of approximately 10 min. Using the revised protocol, we found an IC 50 for verapamil of 252 nM, a Hill coefficient of 0.8, and time constant of inhibition at 0 mV of 0.67 s. The values measured with the revised protocol were similar to those measured using the standard protocol and, furthermore, our success rate using the revised protocol rose to 25 %, an increase of 5-fold over the standard protocol, and more in line with the success rate for biophysical studies. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised protocol with an increased success rate and an overall enhanced feasibility and practicality. We propose that the revised protocol may be more pragmatic for generation of some hERG channel drug inhibition data for CiPA and other regulatory sciences., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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

The pharmacological space comprises all the dynamic events that determine the bioactivity (and/or the metabolism and toxicity) of a given ligand. The pharmacological space accounts for the structural flexibility and property variability of the two interacting molecules as well as for the mutual adaptability characterizing their molecular recognition process. The dynamic behavior of all these events can be described by a set of possible states (e.g., conformations, binding modes, isomeric forms) that the simulated systems can assume. For each monitored state, a set of state-dependent ligand- and structure-based descriptors can be calculated. Instead of considering only the most probable state (as routinely done), the pharmacological space proposes to consider all the monitored states. For each state-dependent descriptor, the corresponding space can be evaluated by calculating various dynamic parameters such as mean and range values.The reviewed examples emphasize that the pharmacological space can find fruitful applications in structure-based virtual screening as well as in toxicity prediction. In detail, in all reported examples, the inclusion of the pharmacological space parameters enhances the resulting performances. Beneficial effects are obtained by combining both different binding modes to account for ligand mobility and different target structures to account for protein flexibility/adaptability.The proposed computational workflow that combines docking simulations and rescoring analyses to enrich the arsenal of docking-based descriptors revealed a general applicability regardless of the considered target and utilized docking engine. Finally, the EFO approach that generates consensus models by linearly combining various descriptors yielded highly performing models in all discussed virtual screening campaigns., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Colorectal cancer (CRC) is one of the most common malignant cancers. Emodin is a lipophilic anthraquinone commonly found in medicinal herbs and known for its antitumor properties. However, its clinical utility has been hampered by low druggability. We designed and synthesized a new compound named Emodin succinimidyl ethyl ester (ESEE), which improves the bioavailability and preserves the original pharmacological effects of Emodin. In vitro, we have confirmed that ESEE induces apoptosis in colon cancer cells, suppresses cell proliferation, migration, and invasion, and inhibits the growth of subcutaneous transplantation tumors associated with colon cancer. And, in vivo, ESEE robustly inhibited tumor growth. Human Ether-a-go-go Related Gene (hERG) is aberrantly expressed in various cancer cells, where they play an important role in cancer progression. Focal adhesion kinase (FAK) is a tyrosine kinase overexpressed in cancer cells and plays an important role in the progression of tumors to a malignant phenotype. Mechanistically, the anti-CRC properties of ESEE are exerted through direct binding with hERG, which impedes the FAK/PI3K/AKT signaling axis-dependent apoptotic cascade., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

In silico modeling offers an opportunity to supplement and accelerate cardiac safety testing. With in silico modeling, computational simulation methods are used to predict electrophysiological interactions and pharmacological effects of novel drugs on critical physiological processes. The O'Hara-Rudy's model was developed to predict the response to different ion channel inhibition levels on cardiac action potential duration (APD) which is known to directly correlate with the QT interval. APD data at 30% 60% and 90% inhibition were derived from the model to delineate possible ventricular arrhythmia scenarios and the marginal contribution of each ion channel to the model. Action potential values were calculated for epicardial, myocardial, and endocardial cells, with action potential curve modeling. This study assessed cardiac ion channel inhibition data combinations to consider when undertaking in silico modeling of proarrhythmic effects as stipulated in the Comprehensive in Vitro Proarrhythmia Assay (CiPA). As expected, our data highlight the importance of the delayed rectifier potassium channel (I Kr ) as the most impactful channel for APD prolongation. The impact of the transient outward potassium channel (I to ) inhibition on APD was minimal while the inward rectifier (I K1 ) and slow component of the delayed rectifier potassium channel (I Ks ) also had limited APD effects. In contrast, the contribution of fast sodium channel (I Na ) and/or L-type calcium channel (I Ca ) inhibition resulted in substantial APD alterations supporting the pharmacological relevance of in silico modeling using input from a limited number of cardiac ion channels including I Kr , I Na , and I Ca , at least at an early stage of drug development., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The voltage-gated potassium ion channel K V 11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause long QT syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which pharmacological chaperones like E-4031 can rescue. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes of WT K V 11.1 or trafficking-deficient channel variants in the presence or absence of E-4031. We identified 572 core K V 11.1 protein interactors. Trafficking-deficient variants K V 11.1-G601S and K V 11.1-G601S-G965 ∗ had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We confirmed previous findings that the proteasome is critical for K V 11.1 degradation. Our report provides the first comprehensive characterization of protein quality control mechanisms of K V 11.1. We find extensive interactome remodeling associated with trafficking-deficient K V 11.1 variants and with pharmacological chaperone rescue of K V 11.1 cell surface expression. The identified protein interactions could be targeted therapeutically to improve K V 11.1 trafficking and treat LQTS., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Determination of a drug's potency in blocking the hERG channel is an established safety pharmacology study. Best practice guidelines have been published for reliable assessment of hERG potency. In addition, a set of plasma concentration and plasma protein binding fraction data were provided as denominators for margin calculations. The aims of the current analysis were five-fold: provide data allowing creation of consistent denominators for the hERG margin distributions of the key reference agents, explore the variation in hERG margins within and across laboratories, provide a hERG margin to 10 ms QTc prolongation based on several newer studies, provide information to use these analyses for reference purposes, and provide recommended hERG margin 'cut-off' values., Methods: The analyses used 12 hERG IC 50 'best practice' data sets (for the 3 reference agents). A group of 5 data sets came from a single laboratory. The other 7 data sets were collected by 6 different laboratories., Results: The denominator exposure distributions were consistent with the ICH E14/S7B Training Materials. The inter-occasion and inter-laboratory variability in hERG IC 50 values were comparable. Inter-drug differences were most important in determining the pooled margin variability. The combined data provided a robust hERG margin reference based on best practice guidelines and consistent exposure denominators. The sensitivity of hERG margin thresholds were consistent with the sensitivity described over the course of the last two decades., Conclusion: The current data provide further insight into the sensitivity of the 30-fold hERG margin 'cut-off' used for two decades. Using similar hERG assessments and these analyses, a future researcher can use a hERG margin threshold to support a negative QTc integrated risk assessment., Competing Interests: Declaration of competing interest The authors have listed their professional affiliation and have no competing conflicts of interest to declare., (Copyright © 2023. Published by Elsevier Inc.)

Macrocycles are defined as cyclic compounds with 12 or more members. In medicinal chemistry, they are categorized based on their core chemistry into cyclic peptides and macrocycles. Macrocycles are advantageous because of their structural diversity and ability to achieve high affinity and selectivity towards challenging targets that are often not addressable by conventional small molecules. The potential of macrocyclization to optimize drug-like properties while maintaining adequate bioavailability and permeability has been emphasized as a key innovation in medicinal chemistry. This review provides a detailed case study of the application of macrocyclization over the past 5 years, starting from the initial analysis of acyclic active compounds to optimization of the resulting macrocycles for improved efficacy and drug-like properties. Additionally, it illustrates the strategic value of macrocyclization in contemporary drug discovery efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Dihydroartemisinin-piperaquine is efficacious for the treatment of uncomplicated malaria and its use is increasing globally. Despite the positive results in fighting malaria, inhibition of the Kv11.1 channel (hERG; encoded by the KCNH2 gene) by piperaquine has raised concerns about cardiac safety. Whether genetic factors could modulate the risk of piperaquine-mediated QT prolongations remained unclear. Here, we first profiled the genetic landscape of KCNH2 variability using data from 141,614 individuals. Overall, we found 1,007 exonic variants distributed over the entire gene body, 555 of which were missense. By optimizing the gene-specific parametrization of 16 partly orthogonal computational algorithms, we developed a KCNH2 -specific ensemble classifier that identified a total of 116 putatively deleterious missense variations. To evaluate the clinical relevance of KCNH2 variability, we then sequenced 293 Malian patients with uncomplicated malaria and identified 13 variations within the voltage sensing and pore domains of Kv11.1 that directly interact with channel blockers. Cross-referencing of genetic and electrocardiographic data before and after piperaquine exposure revealed that carriers of two common variants, rs1805121 and rs41314375, experienced significantly higher QT prolongations (ΔQTc of 41.8 ms and 61 ms, respectively, vs 14.4 ms in controls) with more than 50% of carriers having increases in QTc >30 ms. Furthermore, we identified three carriers of rare population-specific variations who experienced clinically relevant delayed ventricular repolarization. Combined, our results map population-scale genetic variability of KCNH2 and identify genetic biomarkers for piperaquine-induced QT prolongation that could help to flag at-risk patients and optimize efficacy and adherence to antimalarial therapy., Competing Interests: V.M.L. is co-founder, CEO, and shareholder of HepaPredict AB, as well as co-founder and shareholder of PersoMedix AB.

Serotonin reuptake inhibition combined with the action targeting 5-hydroxytryptamine receptor subtypes can serve as a potential target for the development of antidepressant drugs. Herein a series of new aralkyl piperazines and piperidines were designed and synthesized by the structural modifications of the previously discovered aralkyl piperidine compound 1, targeting SSRI/5-HT 1A /5-HT 7 . The results exhibited that compound 5a showed strong binding to 5-HT 1A and 5-HT 7 (K i of 0.46 nM, 2.7 nM, respectively) and a high level of serotonin reuptake inhibition (IC 50 of 1.9 nM), all of which were significantly elevated compared to 1. In particular, compound 5a showed weaker inhibitory activity against hERG than 1, and demonstrated good stability in liver microsomes in vitro. The preliminary screening using FST indicated that orally administered 5a, at a high dose, could reduce immobility time in mice markedly, indicating potential antidepressant activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (K v 11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1 R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts., (© 2024 Federation of European Biochemical Societies.)

Background and Purpose: Drug-induced reduction of the rapid delayed rectifier potassium current carried by the human Ether-à-go-go-Related Gene (hERG) channel is associated with increased risk of arrhythmias. Recent updates to drug safety regulatory guidelines attempt to capture each drug's hERG binding mechanism by combining in vitro assays with in silico simulations. In this study, we investigate the impact on in silico proarrhythmic risk predictions due to uncertainty in the hERG binding mechanism and physiological hERG current model., Experimental Approach: Possible pharmacological binding models were designed for the hERG channel to account for known and postulated small molecule binding mechanisms. After selecting a subset of plausible binding models for each compound through calibration to available voltage-clamp electrophysiology data, we assessed their effects, and the effects of different physiological models, on proarrhythmic risk predictions., Key Results: For some compounds, multiple binding mechanisms can explain the same data produced under the safety testing guidelines, which results in different inferred binding rates. This can result in substantial uncertainty in the predicted torsade risk, which often spans more than one risk category. By comparison, we found that the effect of a different hERG physiological current model on risk classification was subtle., Conclusion and Implications: The approach developed in this study assesses the impact of uncertainty in hERG binding mechanisms on predictions of drug-induced proarrhythmic risk. For some compounds, these results imply the need for additional binding data to decrease uncertainty in safety-critical applications., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Among KCNH2 missense loss of function (LOF) variants, homozygosity -at any position in the Kv11.1/hERG channel - is very rare and generally leads to intrauterine death, while heterozygous variants in the pore are responsible for severe Type 2 long-QT syndrome (LQTS). We report a novel homozygous p.Gly603Ser missense variant in the pore of Kv11.1/hERG (KCNH2 c.1807G > A) discovered in the context of a severe LQTS., Methods: We carried out a phenotypic family study combined with a functional analysis of mutated and wild-type (WT) Kv11.1 by two-electrode voltage-clamp using the Xenopus laevis oocyte heterologous expression system., Results: The variant resulted in a severe LQTS phenotype (very prolonged corrected QT interval, T-wave alternans, multiple Torsades de pointes) with a delayed clinical expression in later childhood in the homozygous state, and in a Type 2 LQTS phenotype in the heterozygous state. Expression of KCNH2 p.Gly603Ser cRNA alone elicited detectable current in Xenopus oocytes. Inactivation kinetics and voltage dependence of activation were not significantly affected by the variant. The macroscopic slope conductance of the variant was three-fold less compared to the WT (18.5 ± 9.01 vs 54.7 ± 17.2 μS, p < 0.001)., Conclusions: We characterized the novel p.Gly603Ser KCNH2 missense LOF variant in the pore region of Kv11.1/hERG leading to a severe but viable LQTS in the homozygous state and an attenuated Type 2 LQTS in heterozygous carriers. To our knowledge we provide the first description of a homozygous variant in the pore-forming region of Kv11.1 with a functional impact but a delayed clinical expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of hERG and HCN remains unknown., Results: We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y., Conclusions: Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG., (© 2024. The Author(s).)

In vitro electrophysiological safety studies have become an integral part of the drug development process because, in many instances, compound-induced QT prolongation has been associated with a direct block of human ether-a-go-go-related gene (hERG) potassium channels or their native current, the rapidly activating delayed rectifier potassium current (I Kr ). Therefore, according to the ICH S7B guideline, the in vitro hERG channel patch-clamp assay is commonly used as an early screen to predict the ability of a compound to prolong the QT interval prior to first-in-human testing. The protocols described in this article are designed to assess the effects of acute or long-term exposure to new chemical entities on the amplitude of I Kr in HEK293 cells stably transfected with the hERG channel (whole-cell configuration of the patch-clamp technique). Examples of results obtained with moxifloxacin, terfenadine, arsenic, pentamidine, erythromycin, and sotalol are provided for illustrative purposes. © 2024 Wiley Periodicals LLC. Basic Protocol: Measurement of the acute effects of test items in the hERG channel test Alternate Protocol: Measurement of the long-term effects of test items in the hERG channel test., (© 2024 Wiley Periodicals LLC.)

The authors demonstrate the feasibility of technological innovation for personalized medicine in the context of drug-induced arrhythmia. The authors use atomistic-scale structural models to predict rates of drug interaction with ion channels and make predictions of their effects in digital twins of induced pluripotent stem cell-derived cardiac myocytes. The authors construct a simplified multilayer, 1-dimensional ring model with sufficient path length to enable the prediction of arrhythmogenic dispersion of repolarization. Finally, the authors validate the computational pipeline prediction of drug effects with data and quantify drug-induced propensity to repolarization abnormalities in cardiac tissue. The technology is high throughput, computationally efficient, and low cost toward personalized pharmacologic prediction., Competing Interests: Funding Support and Author Disclosures This work was supported by National Heart, Lung, and Blood Institute (NHLBI) grants R01HL128537 (to Drs Clancy, Santana, and Vorobyov), R01HL152681 (to Drs Santana and Clancy) and U01HL126273 (to Drs Clancy and Yarov-Yarovy), NIH Common Fund Grant OT2OD026580 (to Drs Clancy and Santana), American Heart Association Career Development Award 19CDA34770101 and Oracle for Research fellowship (to Dr Vorobyov). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The rapid delayed rectifier potassium current (I Kr ) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting I Kr , hERG channel, is not only permeable to K + ions but also to Cs + ions when present in equimolar concentrations inside and outside of the cell. Methods: In this study, I hERG was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs + or K + as the charge carrier. Equimolar Cs + has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs + - and K + -based currents. Results: Using equimolar Cs + solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs + - and K + -mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs + solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs + -based conductance is larger compared to the known K + -based conductance, and the Cs + hERG conductance can be inhibited similarly to the K + -based conductance. Conclusion: Using equimolar Cs + instead of K + for I hERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small I Kr current densities in combination with small membrane capacitances., Competing Interests: Author FS was employed by Nanion Technologies GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Bloothooft, Verbruggen, Seibertz, Heyden, Voigt and de Boer.)

Arrhythmias account for over 300,000 annual deaths in the United States, and approximately half of all deaths are associated with heart disease. Mechanisms underlying arrhythmia risk are complex; however, work in humans and animal models over the past 25 years has identified a host of molecular pathways linked with both arrhythmia substrates and triggers. This chapter will focus on select arrhythmia pathways solved by linking human clinical and genetic data with animal models., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Ion channels are transmembrane proteins essential for cellular functions and are important drug targets. Surface plasmon resonance (SPR) is a powerful technique for investigating protein-protein and protein-small molecule ligand interactions. SPR has been underutilized for studies of ion channels, even though it could provide a wealth of information on the mechanisms of ion channel regulation and aid in ion channel drug discovery. Here we provide a detailed description of the use of SPR technology for investigating inter-domain interactions in KCNH potassium-selective and voltage-gated ion channels., (© 2024. The Author(s).)

A mutation to serine of a conserved threonine (T634S) in the hERG K + channel S6 pore region has been identified as a variant of uncertain significance, showing a loss-of-function effect. However, its potential consequences for ventricular excitation and arrhythmogenesis have not been reported. This study evaluated possible functional effects of the T634S-hERG mutation on ventricular excitation and arrhythmogenesis by using multi-scale computer models of the human ventricle. A Markov chain model of the rapid delayed rectifier potassium current (I Kr ) was reconstructed for wild-type and T634S-hERG mutant conditions and incorporated into the ten Tusscher et al . models of human ventricles at cell and tissue (1D, 2D and 3D) levels. Possible functional impacts of the T634S-hERG mutation were evaluated by its effects on action potential durations (APDs) and their rate-dependence (APDr) at the cell level; and on the QT interval of pseudo-ECGs, tissue vulnerability to unidirectional conduction block (VW), spiral wave dynamics and repolarization dispersion at the tissue level. It was found that the T634S-hERG mutation prolonged cellular APDs, steepened APDr, prolonged the QT interval, increased VW, destablized re-entry and augmented repolarization dispersion across the ventricle. Collectively, these results imply potential pro-arrhythmic effects of the T634S-hERG mutation, consistent with LQT2., Competing Interests: All authors declare that there are no conflicts of interest., (© 2023 The Authors.)

BIIB131, a small molecule, is currently in Phase 2 for the treatment of acute ischemic stroke. Safety and metabolism of BIIB131 were evaluated following intravenous administration to rats and monkeys. Exposure increased dose-proportionally in rats up to 60 mg/kg and more than dose-proportionally in monkeys at greater than 10 mg/kg accompanied by prolonged half-life and safety findings. The BIIB131 was poorly metabolized in microsomes with no inhibition of CYPs. BIIB131-glucuronide, formed by UGT1A1, accounted for 21.5% metabolism in human hepatocytes and 28-40% in rat bile. In rats, excretion was primarily via the bile. BIIB131 inhibited the hERG and Nav1.5 cardiac channels by 39% but showed no effect on cardiovascular parameters in monkeys. Toxicology findings were limited to reversable hematuria, changes in urinary parameters and local effects. A MTD of 30 mg/kg was established in monkeys, the most sensitive species, at total plasma C max and AUC of 6- and 14-fold, respectively, greater than the NOAEL. The Phase 1 study started with intravenous 0.05 mg/kg and ascended to 6.0 mg/kg which corresponded to safety margins of 147- to 0.9-fold (for C max ) within the linear drug exposure. Thus, the preclinical profile of BIIB131 has been appropriately characterized and supports its further clinical development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Keiko Hasegawa reports financial support was provided by Japan Science and Technology Agency. Keiji Hasumi reports financial support was provided by Japan Science and Technology Agency. Naoko Nishimura reports financial support was provided by Japan Science and Technology Agency. Vic Kostrubsky reports a relationship with Biogen Inc that includes: employment and equity or stocks. Chungang Gu reports a relationship with Biogen Inc that includes: employment and equity or stocks. Cathy Muste reports a relationship with Biogen Inc that includes: employment and equity or stocks. Melissa Kirkland reports a relationship with Biogen that includes: employment and equity or stocks. Ying Liu reports a relationship with Biogen Inc that includes: employment and equity or stocks. Long Yuan reports a relationship with Biogen Inc that includes: employment and equity or stocks. Keiko Hasegawa reports a relationship with TMS Co Ltd that includes: employment and equity or stocks. Keiji Hasumi reports a relationship with TMS Co Ltd that includes: employment and equity or stocks. Naoko Nishimura reports a relationship with TMS Co Ltd that includes: consulting or advisory, employment, and equity or stocks., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Synthetic cannabinoid receptor agonists (SCRAs) have been associated with QT interval prolongation. Limited preclinical information on SCRA effects on cardiac electrogenesis results from the rapid emergence of new compounds and restricted research availability. We used two machine-learning-based tools to evaluate seven novel SCRAs' interaction potential with the hERG potassium channel, an important drug antitarget. Five SCRAs were predicted to have the ability to block the hERG channel by both prediction tools; ADB-FUBIATA was predicted to be a strong hERG blocker. ADB-5Br-INACA and ADB-4en-PINACA showed varied predictions. These findings highlight potentially proarrhythmic hERG block by novel SCRAs, necessitating detailed safety evaluations., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Aims: Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243., Methods and Results: The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness., Conclusion: Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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

Vinpocetine is a readily available nutritional supplement claimed to improve memory and weight loss. However, it blocks the I kr current essential for cardiac action potential repolarisation and Ikr inhibition can cause "torsade de pointes" arrhythmias and sudden death. Moreover, Ikr blockers have exhibited teratogenic effects in reproductive toxicology studies, leading to increased birth defects and embryonic mortality. The FDA advises against vinpocetine use in pregnant and prospective mothers based on animal studies showing dose-dependent fetal mortality in rats and rabbits, and cardiovascular malformations in surviving fetuses. However, the mechanisms responsible for vinpocetine's fetal toxicity remain unclear. The present study used rat embryo culture to evaluate vinpocetine and its major metabolite, apovincaminic acid, on embryonic heart rate, a possible causative factor behind its adverse effects. Both compounds induced embryonic bradycardia in a concentration-dependent manner, with vinpocetine proving more potent. The minimum vinpocentine concentration to induce bradycardia was 100 nM, a level unlikely to be reached in humans following typical doses. Embryonic arrhythmias were also observed at the highest concentrations. These results suggest that the FDA's cautionary statement may generate undue anxiety, although re-evaluation of teratogenicity risk associated with vinpocetine should be revisited if a link to cardiac arrhythmias in adults is established., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023 Published by Elsevier B.V.)

The ICH E14/S7B Questions and Answers (Q&As) guideline introduces the concept of a "double negative" nonclinical scenario (negative hERG assay and negative in vivo QTc study) to demonstrate that a drug does not produce a clinically relevant QT prolongation (i.e., no QT liability). This nonclinical "double negative" data package, along with negative Phase 1 clinical QTc data, may be sufficient to substitute for a clinical Thorough QT (TQT) study in some specific cases. While standalone GLP in vivo cardiovascular studies in non-rodent species are standard practice during nonclinical drug development for small molecule programs, a variety of approaches to the design, conduct, analysis and interpretation are utilized across pharmaceutical companies and contract research organizations (CROs) that may, in some cases, negatively impact the stringent sensitivity needed to fulfill the new Q&As. Subject matter experts from both Pharma and CROs have collaborated to recommend best practices for more robust nonclinical cardiovascular telemetry studies in non-rodent species, with input from clinical and regulatory experts. The aim was to increase consistency and harmonization across the industry and to ensure delivery of high quality nonclinical QTc data to meet the proposed sensitivities defined within the revised ICH E14/S7B Q&As guideline (Q&As 5.1 and 6.1). The detailed best practice recommendations presented here cover the design and execution of the safety pharmacology cardiovascular study, including optimal methods for acquiring, analyzing, reporting, and interpreting the resulting QTc and pharmacokinetic data to allow for direct comparison to clinical exposures and assessment of safety margin for QTc prolongation., Competing Interests: Declaration of Competing Interest All authors are employed by pharmaceutical companies or contract research organizations., (Copyright © 2023 Elsevier Inc. All rights reserved.)

hERG ( human Ether-à-go-go Related Gene )-encoded potassium channels underlie the cardiac rapid delayed rectifier (I Kr ) 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 (I hERG ) were made at physiological temperature from HEK 293 cells expressing wild-type (WT) and mutant hERG constructs. For E-4031, WT I hERG was inhibited by a half-maximal inhibitory concentration (IC 50 ) 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 I hERG 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 I hERG .

Interleukin-1 receptor associated kinase-4 (IRAK4) has emerged as a therapeutic target for inflammatory and autoimmune diseases. Through reversing the amide of CA-4948 and computer aided structure-activity relationship (SAR) studies, a series of IRAK4 inhibitors with oxazolo[4,5-b]pyridine scaffold were identified. Compound 32 showed improved potency (IC 50  = 43 nM) compared to CA-4948 (IC 50  = 115 nM), but suffered from hERG inhibition (IC 50  = 5.7 μM). Further optimization led to compound 42 with reduced inhibition of hERG (IC 50  > 30 μM) and 13-fold higher activity (IC 50  = 8.9 nM) than CA-4948. Importantly, compound 42 had favorable in vitro ADME and in vivo pharmacokinetic properties. Furthermore, compound 42 significantly reduced LPS-induced production of serum TNF-α and IL-6 cytokines in the mouse model. The overall profiles of compound 42 support it as a lead for the development of IRAK4 inhibitors for the treatment of inflammatory and autoimmune disorders., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Shuwei Wu reports financial support was provided by China Postdoctoral Science foundation. Xiaohu Zhang, Sudan He reports financial support was provided by National Natural Science Foundation of China. Xiaohu Zhang reports financial support was provided by the Priority Academic Program Development of the Jiangsu Higher Education Institutes. Xiaohu Zhang reports financial support was provided by the Jiangsu Key Laboratory of Neuropsychiatric Diseases. Sudan He reports financial support was provided by the CAMS Innovation Fund for Medical Sciences. Sudan He reports financial support was provided by Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences. Xiaohu Zhang, Sudan He reports financial support was provided by Accro Bioscience. Xiaohu Zhang, Sudan He has patent pending to Accro Bioscience(Shuwei Wu, Xiaohu Zhang, Sudan He)]., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Long QT syndrome type 2 (LQT2) is a genetic disorder caused by mutations in the KCNH2 gene, also known as the human ether-a-go-go-related gene (HERG). More than 30% of HERG mutations result in a premature termination codon that triggers a process called nonsense-mediated messenger RNA (mRNA) decay (NMD), where the mRNA transcript is degraded. NMD is a quality control mechanism that removes faulty mRNA to prevent the translation of truncated proteins. Recent advances in antisense oligonucleotide (ASO) technology in the field of cystic fibrosis (CF) have yielded significant progress, including the ASO-mediated comprehensive characterization of key NMD factors and exon-skipping therapy. These advances have contributed to our understanding of the role of premature termination codon-containing mutations in disease phenotypes and have also led to the development of potentially useful therapeutic strategies. Historically, studies of CF have provided valuable insights for the research on LQT2, particularly concerning increasing the expression of HERG. In this article, we outline the current state of knowledge regarding ASO, NMD, and HERG and discuss the introduction of ASO technology in the CF to elucidate the pathogenic mechanisms through targeting NMD. We also discuss the potential clinical therapeutic benefits and limitations of ASO for the management of LQT2. By drawing on lessons learned from CF research, we explore the potential translational values of these advances into LQT2 studies., (Copyright © 2023 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

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.

The congenital short QT syndrome (SQTS) is a rare condition characterized by abbreviated rate-corrected QT (QTc) intervals on the electrocardiogram and by increased susceptibility to both atrial and ventricular arrhythmias and sudden death. Although mutations to multiple genes have been implicated in the SQTS, evidence of causality is particularly strong for the first three (SQT1-3) variants: these result from gain-of-function mutations in genes that encode K + channel subunits responsible, respectively, for the I Kr , I Ks and I K1 cardiac potassium currents. This article reviews evidence for the impact of SQT1-3 missense potassium channel gene mutations on the electrophysiological properties of I Kr , I Ks and I K1 and of the links between these changes and arrhythmia susceptibility. Data from experimental and simulation studies and future directions for research in this field are considered. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

hERG is considered to be a primary anti-target in the drug development process, as the K + channel encoded by hERG plays an important role in cardiac re-polarization. It is desirable to address the hERG safety liability during early-stage development to avoid the expenses of validating leads that will eventually fail at a later stage. We have previously reported the development of highly potent quinazoline-based TLR7 and TLR9 antagonists for possible application against autoimmune disease. Initial experimental hERG assessment showed that most of the lead TLR7 and TLR9 antagonists suffer from hERG liability rendering them ineffective for further development. The present study herein describes a coordinated strategy to integrate the understanding from structure-based protein-ligand interaction to develop non- hERG binders with IC 50 >30 μM with retention of TLR7/9 antagonism through a single point change in the scaffold. This structure-guided strategy can serve as a prototype for abolishing hERG liability during lead optimization., (© 2023 Wiley-VCH GmbH.)

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 EC 50 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 EC 50 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 [ 3 H]-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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Given the direct association with malignant ventricular arrhythmias, cardiotoxicity is a major concern in drug design. In the past decades, computational models based on the quantitative structure-activity relationship have been proposed to screen out cardiotoxic compounds and have shown promising results. The combination of molecular fingerprint and the machine learning model shows stable performance for a wide spectrum of problems; however, not long after the advent of the graph neural network (GNN) deep learning model and its variant (e.g., graph transformer), it has become the principal way of quantitative structure-activity relationship-based modeling for its high flexibility in feature extraction and decision rule generation. Despite all these progresses, the expressiveness (the ability of a program to identify non-isomorphic graph structures) of the GNN model is bounded by the WL isomorphism test, and a suitable thresholding scheme that relates directly to the sensitivity and credibility of a model is still an open question. Methods: In this research, we further improved the expressiveness of the GNN model by introducing the substructure-aware bias by the graph subgraph transformer network model. Moreover, to propose the most appropriate thresholding scheme, a comprehensive comparison of the thresholding schemes was conducted. Results: Based on these improvements, the best model attains performance with 90.4% precision, 90.4% recall, and 90.5% F1-score with a dual-threshold scheme (active: < 1 μ M ; non-active: > 30 μ M ). The improved pipeline (graph subgraph transformer network model and thresholding scheme) also shows its advantages in terms of the activity cliff problem and model interpretability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Zhu, Izu, Chen-Izu, Ono, Altaf-Ul-Amin, Kanaya and Huang.)

Recent updates and modifications to the clinical ICH E14 and nonclinical ICH S7B guidelines, which both relate to the evaluation of drug-induced delayed repolarization risk, provide an opportunity for nonclinical in vivo electrocardiographic (ECG) data to directly influence clinical strategies, interpretation, regulatory decision-making and product labeling. This opportunity can be leveraged with more robust nonclinical in vivo QTc datasets based upon consensus standardized protocols and experimental best practices that reduce variability and optimize QTc signal detection, i.e., demonstrate assay sensitivity. The immediate opportunity for such nonclinical studies is when adequate clinical exposures (e.g., supratherapeutic) cannot be safely achieved, or other factors limit the robustness of the clinical QTc evaluation, e.g., the ICH E14 Q5.1 and Q6.1 scenarios. This position paper discusses the regulatory historical evolution and processes leading to this opportunity and details the expectations of future nonclinical in vivo QTc studies of new drug candidates. The conduct of in vivo QTc assays that are consistently designed, executed and analyzed will lead to confident interpretation, and increase their value for clinical QTc risk assessment. Lastly, this paper provides the rationale and basis for our companion article which describes technical details on in vivo QTc best practices and recommendations to achieve the goals of the new ICH E14/S7B Q&As, see Rossman et al., 2023 (this journal)., Competing Interests: Declaration of Competing Interest All authors are employed by pharmaceutical companies or contract research organizations., (Copyright © 2023 Amgen, Inc. Published by Elsevier Inc. All rights reserved.)

Two synthetic phenylethylamines, N-methyl-1-(naphthalen-2-yl)propan-2-amine (MNA) and 1-phenyl-2-pyrrolidinylpentane (prolintane), are being abused by people seeking hallucinogens for pleasure. These new psychotropic substances may provoke problems because there is no existing information about their toxicity and pharmacological behaviors. Therefore, we evaluated the safety of nerves and cardiovascular systems by determining toxicity after MNA and prolintane drugs administrations to mice and rat. Consequently, side effects such as increased spontaneous motion and body temperature were observed in oral administration of MNA. In addition, both substances reduced motor coordination levels. The IHC tests were conducted to see whether the immune response also shows abnormalities in brain tissue compared to the control group. It has been confirmed that the length of allograft inflammatory factor 1(IBA-1), an immune antibody known as microglia marker, has been shortened. We identified that a problem with the contact between synapses and neurons might be possibly produced. In the assessment of the cardiac toxicity harmfulness, no substances have been confirmed to be toxic to myocardial cells, but at certain concentrations, they have caused the QT prolongation, an indicator of ventricular arrhythmia. In addition, the hERG potassium channel, the biomarker of the QT prolongation, has been checked for inhibition. The results revealed that the possibility of QT prolongation through the hERG channel could not be excluded, and the two substances can be considered toxic that may cause ventricular arrhythmia. In sum, this study demonstrated that the possibility of toxicity in MNA and prolintane compounds might bring many harmful effects on nerves and hearts.

Mutation in the hERG gene leading to partial or complete blockade of the rapid delayed rectifier current causes Long QT Type 2 (LQT2) phenotype, the second most common form of Long QT Syndrome. However, the exact involvement of the His-Purkinje System (HPS) remains elusive. We utilized a finite element model of the rabbit ventricles integrated with a HPS to elucidate the role of HPS during LQT2-mediated arrhythmia. Following the induction of persistent reentry from an ectopic stimulus, we isolated the HPS at different time points. Moreover, we varied the coupling resistance and the number of myocytes at the Purkinje-Myocardial Junctions (PMJs) to ascertain how the junctional parameters altered reentry dynamics. Reentry was terminated with the earliest termination time for reentry coinciding with the earliest time the HPS was isolated. This observation provides evidence of direct involvement of the HPS during LQT2-mediated ventricular arrhythmia. Increasing the coupling resistance or the number of myocytes at the PMJs reduced the percentage of successful retrograde propagation during reentry. Thus, the HPS alters reentry dynamics. Our multi-scale computer modeling outcomes offer important new understandings of probable arrhythmia mechanisms under LQT2 circumstances.

The repolarizing current (I kr ) produced by the hERG potassium channel forms a major component of the cardiac action potential and blocking this current by small molecule drugs can lead to life-threatening cardiotoxicity. Understanding the mechanisms of drug-mediated hERG inhibition is essential to develop a second generation of safe drugs, with minimal cardiotoxic effects. Although various computational tools and drug design guidelines have been developed to avoid binding of drugs to the hERG pore domain, there are many other aspects that are still open for investigation. This includes the use computational modelling to study the implications of hERG mutations on hERG structure and trafficking, the interactions of hERG with hERG chaperone proteins and with membrane-soluble molecules, the mechanisms of drugs that inhibit hERG trafficking and drugs that rescue hERG mutations. The plethora of available experimental data regarding all these aspects can guide the construction of much needed robust computational structural models to study these mechanisms for the rational design of safe drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Microelectrode array studies (MEAs) and voltage sensing optical technique on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were combined with ionic current measurements in mammalian cell lines , In-silico simulations of cardiac action potentials (APs) and statistic regression analysis. Proarrhythmic effects of 12 Evodia preparations, containing different amounts of the hERG inhibitors dehydroevodiamine (DHE) and hortiamine were analysed. Extracts produced different prolongation of the AP, occurrence of early after depolarisations and triangulation of the AP in hiPSC-CMs depending on the contents of the hERG inhibitors. DHE and hortiamine dose-dependently prolonged the field potential duration in hiPSC-CMs studied with MEAs. In-silico simulations of ventricular AP support a scenario where proarrhythmic effects of Evodia extracts are predominantly caused by the content of the selective hERG inhibitors. Statistic regression analysis revealed a high torsadogenic risk for both compounds that was comparable to drugs assigned to the high-risk category in a CiPA study., Competing Interests: The authors have no competing interests to declare., (© 2023 Published by Elsevier B.V.)

Many studies have shown the high efficacy of histamine H 3 receptor ligands in preventing weight gain. In addition to evaluating the efficacy of future drug candidates, it is very important to assess their safety profile, which is established through numerous tests and preclinical studies. The purpose of the present study was to evaluate the safety of histamine H 3 /sigma-2 receptor ligands by assessing their effects on locomotor activity and motor coordination, as well as on the cardiac function, blood pressure, and plasma activity of certain cellular enzymes. The ligands tested at a dose of 10 mg/kg b.w. did not cause changes in locomotor activity (except for KSK-74) and did not affect motor coordination. Significant reductions in blood pressure were observed after the administration of compounds KSK-63, KSK-73, and KSK-74, which seems logically related to the increased effect of histamine. Although the results of in vitro studies suggest that the tested ligands can block the human ether-a-go-go-related gene (hERG) potassium channels, they did not affect cardiac parameters in vivo. It should be noted that repeated administration of the tested compounds prevented an increase in the activity of alanine aminotransferase (AlaT) and gamma-glutamyl transpeptidases (gGT) observed in the control animals fed a palatable diet. The obtained results show that the ligands selected for this research are not only effective in preventing weight gain but also demonstrate safety in relation to the evaluated parameters, allowing the compounds to proceed to the next stages of research.

In accord with International Conference on Harmonization S7B guidelines, an in vitro human ether-a-go-go-related gene (hERG) assay is one component of an integrated risk assessment for delayed ventricular repolarization. Function of hERG could be affected by direct (acute) mechanisms, or by indirect (chronic) mechanisms. Some approved oligonucleotide therapeutics had submitted hERG data to regulatory agents, which were all collected with the same protocol used for small-molecule testing (incubation time <20 min; acute), however, oligonucleotides have unique mechanisms and time courses of action (indirect). To reframe the hERG testing strategy for silencing RNA (siRNA), an investigation was performed to assess the time course for siRNA-mediated inhibition of hERG function and gene expression. Commercially available siRNAs of hERG were evaluated in a stable hERG-expressed cell line by whole-cell voltage clamp using automated electrophysiology and polymerase chain reaction. In the acute hERG study, no effects were observed after treatment with 100 nM siRNA for 20 min. The chronic effects of 100 nM siRNAs on hERG function were evaluated and recorded over 8-48 h following transfection. At 8 h there was no significant effect, whereas 77% reduction was observed at 48 h. Measurement of hERG mRNA levels demonstrated a 79% and 93% decrease of hERG mRNA at 8 and 48 h, respectively, consistent with inhibition of hERG transcription. The results indicate that an anti-hERG siRNA requires a long exposure time (48 h) in the hERG assay to produce a maximal reduction in hERG current; short exposures (20 min-8 h) had no effect. These findings imply that off-target profiling of novel oligonucleotides could benefit from using hERG protocol with long incubation times to de-risk potential off-target (indirect) effects on the hERG channel. This hERG assay modification may be important to consider if the findings are used to support an integrated nonclinical-clinical risk assessment for QTc (the duration of the QT interval adjusted for heart rate) prolongation.

The evaluation of the inhibitory activities of drugs on multiple cardiac ion channels is required for the accurate assessment of proarrhythmic risks. Moreover, the in silico prediction of such inhibitory activities of drugs on cardiac channels can improve the efficiency of the drug-development process. Here, we performed molecular docking simulations to predict the complex structures of 25 reference drugs that were proposed by the Comprehensive in vitro Proarrhythmia Assay consortium using two cardiac ion channels, the human ether-a-go-go-related gene (hERG) potassium channel and human Na V 1.5 (hNa V 1.5) sodium channel, with experimentally available structures. The absolute binding free energy (Δ G bind ) values of the predicted structures were calculated by a molecular dynamics-based method and compared with the experimental half-maximal inhibitory concentration (IC 50 ) data. Furthermore, the regression analysis between the calculated values and negative of the common logarithm of the experimental IC 50 ) revealed that the calculated values of four and ten drugs deviated significantly from the regression lines of the hERG and hNa 50 ) revealed that the calculated values of four and ten drugs deviated significantly from the regression lines of the hERG and hNa V 1.5 channels, respectively. We reconsidered the docking poses and protonation states of the drugs based on the experimental data and recalculated their Δ G bind values (coefficients of determination=0.791 and 0.613 for the hERG and hNa G bind values of 24 and 19 drugs correlated with their experimental pIC 50 values (coefficients of determination=0.791 and 0.613 for the hERG and hNa V 1.5 channels, respectively). Thus, the regression analysis between the calculated Δ G bind and experimental IC 50 data ensured the realization of an increased number of reliable complex structures., Competing Interests: The authors declare no conflict of interest., (2023 THE BIOPHYSICAL SOCIETY OF JAPAN.)

ABT-333 (dasabuvir) is an antiviral agent used in hepatitis C treatment. The molecule, similarly to some inhibitors of hERG channels, responsible for the delayed rectifier potassium current (I Kr ), contains the methanesulfonamide group. Reduced I Kr current leads to long QT syndrome and early afterdepolarizations (EADs), therefore potentially causing life-threatening arrhythmias and sudden cardiac death. Our goal was to investigate the acute effects of ABT-333 in enzymatically isolated canine left ventricular myocardial cells. Action potentials (APs) and ion currents were recorded with a sharp microelectrode technique and whole-cell patch clamp, respectively. Application of 1 μM ABT-333 prolonged the AP in a reversible manner. The maximal rates of phases 0 and 1 were irreversibly decreased. Higher ABT-333 concentrations caused larger AP prolongation, elevation of the early plateau potential, and reduction of maximal rates of phases 0, 1, and 3. EADs occurred in some cells in 3-30 μM ABT-333 concentrations. The 10 μM ABT-333-sensitive current, recorded with AP voltage clamp, contained a late outward component corresponding to I Kr and an early outward one corresponding to transient outward potassium current (I to ). ABT-333 reduced hERG-channel-mediated ion current in a concentration-dependent, partially reversible manner with a half-inhibitory concentration of 3.2 μM. As the therapeutic plasma concentration of ABT-333 can reach the low μM range, ABT-333 application carries a risk of cardiac side effects especially in case of coadministration with strong inhibitors of CYP2C8.

Drug-induced cardiotoxicity represents one of the most critical safety concerns in the early stages of drug development. The blockade of the human ether-à-go-go-related potassium channel (hERG) is the most frequent cause of cardiotoxicity, as it is associated to long QT syndrome which can lead to fatal arrhythmias. Therefore, assessing hERG liability of new drugs candidates is crucial to avoid undesired cardiotoxic effects. In this scenario, computational approaches have emerged as useful tools for the development of predictive models able to identify potential hERG blockers. In the last years, several efforts have been addressed to generate ligand-based (LB) models due to the lack of experimental structural information about hERG channel. However, these methods rely on the structural features of the molecules used to generate the model and often fail in correctly predicting new chemical scaffolds. Recently, the 3D structure of hERG channel has been experimentally solved enabling the use of structure-based (SB) strategies which may overcome the limitations of the LB approaches. In this study, we compared the performances achieved by both LB and SB classifiers for hERG-related cardiotoxicity developed by using Random Forest algorithm and employing a training set containing 12789 hERG binders. The SB models were trained on a set of scoring functions computed by docking and rescoring calculations, while the LB classifiers were built on a set of physicochemical descriptors and fingerprints. Furthermore, models combining the LB and SB features were developed as well. All the generated models were internally validated by ten-fold cross-validation on the TS and further verified on an external test set. The former revealed that the best performance was achieved by the LB model, while the model combining the LB and the SB attributes displayed the best results when applied on the external test set highlighting the usefulness of the integration of LB and SB features in correctly predicting unseen molecules. Overall, our predictive models showed satisfactory performances providing new useful tools to filter out potential cardiotoxic drug candidates in the early phase of drug discovery., Competing Interests: FL and AB were employed by the EXSCALATE, Dompé Farmaceutici SpA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vittorio, Lunghini, Pedretti, Vistoli and Beccari.)

The hERG channel is one of the essential ion channels composing the cardiac action potential and the toxicity assay for new drug. Recently, the comprehensive in vitro proarrhythmia assay (CiPA) was adopted for cardiac toxicity evaluation. One of the hurdles for this protocol is identifying the kinetic effect of the new drug on the hERG channel. This procedure included the model-based parameter identification from the experiments. There are many mathematical methods to infer the parameters; however, there are two main difficulties in fitting parameters. The first is that, depending on the data and model, parametric inference can be highly time-consuming. The second is that the fitting can fail due to local minima problems. The simplest and most effective way to solve these issues is to provide an appropriate initial value. In this study, we propose a deep learning-based method for improving model fitting by providing appropriate initial values, even the right answer. We generated the dataset by changing the model parameters and trained our deep learning-based model. To improve the accuracy, we used the spectrogram with time, frequency, and amplitude. We obtained the experimental dataset from https://github.com/CardiacModelling/hERGRapidCharacterisation. Then, we trained the deep-learning model using the data generated with the hERG model and tested the validity of the deep-learning model with the experimental data. We successfully identified the initial value, significantly improved the fitting speed, and avoided fitting failure. This method is useful when the model is fixed and reflects the real data, and it can be applied to any in silico model for various purposes, such as new drug development, toxicity identification, environmental effect, etc. This method will significantly reduce the time and effort to analyze the data., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Song, Kim and Leem.)