Facing small and biased data dilemma in drug discovery with enhanced federated learning approaches.

Artificial intelligence (AI) models usually require large amounts of high-quality training data, which is in striking contrast to the situation of small and biased data faced by current drug discovery pipelines. The concept of federated learning has been proposed to utilize distributed data from different sources without leaking sensitive information of the data. This emerging decentralized machine learning paradigm is expected to dramatically improve the success rate of AI-powered drug discovery. Here, we simulated the federated learning process with different property and activity datasets from different sources, among which overlapping molecules with high or low biases exist in the recorded values. Beyond the benefit of gaining more data, we also demonstrated that federated training has a regularization effect superior to centralized training on the pooled datasets with high biases. Moreover, different network architectures for clients and aggregation algorithms for coordinators have been compared on the performance of federated learning, where personalized federated learning shows promising results. Our work demonstrates the applicability of federated learning in predicting drug-related properties and highlights its promising role in addressing the small and biased data dilemma in drug discovery.

Inhibition of the hERG potassium channel by phenanthrene: a polycyclic aromatic hydrocarbon pollutant.

The lipophilic polycyclic aromatic hydrocarbon (PAH) phenanthrene is relatively abundant in polluted air and water and can access and accumulate in human tissue. Phenanthrene has been reported to interact with cardiac ion channels in several fish species. This study was undertaken to investigate the ability of phenanthrene to interact with hERG (human Ether-à-go-go-Related Gene) encoded Kv11.1 K+ channels, which play a central role in human ventricular repolarization. Pharmacological inhibition of hERG can be proarrhythmic. Whole-cell patch clamp recordings of hERG current (IhERG) were made from HEK293 cells expressing wild-type (WT) and mutant hERG channels. WT IhERG1a was inhibited by phenanthrene with an IC50 of 17.6 ± 1.7 µM, whilst IhERG1a/1b exhibited an IC50 of 1.8 ± 0.3 µM. WT IhERG block showed marked voltage and time dependence, indicative of dependence of inhibition on channel gating. The inhibitory effect of phenanthrene was markedly impaired by the attenuated inactivation N588K mutation. Remarkably, mutations of S6 domain aromatic amino acids (Y652, F656) in the canonical drug binding site did not impair the inhibitory action of phenanthrene; the Y652A mutation augmented IhERG block. In contrast, the F557L (S5) and M651A (S6) mutations impaired the ability of phenanthrene to inhibit IhERG, as did the S624A mutation below the selectivity filter region. Computational docking using a cryo-EM derived hERG structure supported the mutagenesis data. Thus, phenanthrene acts as an inhibitor of the hERG K+ channel by directly interacting with the channel, binding to a distinct site in the channel pore domain.

Assessment of proarrhythmogenic risk for chloroquine and hydroxychloroquine using the CiPA concept.

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

TSSF-hERG: A machine-learning-based hERG potassium channel-specific scoring function for chemical cardiotoxicity prediction.

The human ether-à-go-go-related gene (hERG) encodes the Kv11.1 voltage-gated potassium ion (K+) channel that conducts the rapidly activating delayed rectifier current (IKr) in cardiomyocytes to regulate the repolarization process. Some drugs, as blockers of hERG potassium channels, cannot be marketed due to prolonged QT intervals, as well known as cardiotoxicity. Predetermining the binding affinity values between drugs and hERG through in silico methods can greatly reduce the time and cost required for experimental verification. In this study, we collected 9,215 compounds with AutoDock Vina's docking structures as training set, and collected compounds from four references as test sets. A series of models for predicting the binding affinities of hERG blockers were built based on five machine learning algorithms and combinations of interaction features and ligand features. The model built by support vector regression (SVR) using the combination of all features achieved the best performance on both tenfold cross-validation and external verification, which was selected and named as TSSF-hERG (target-specific scoring function for hERG). TSSF-hERG is more accurate than the classic scoring function of AutoDock Vina and the machine-learning-based generic scoring function RF-Score, with a Pearson's correlation coefficient (Rp) of 0.765, a Spearman's rank correlation coefficient (Rs) of 0.757, a root-mean-square error (RMSE) of 0.585 in a tenfold cross-validation study. All results demonstrated that TSSF-hERG would be useful for improving the power of binding affinity prediction between hERG and compounds, which can be further used for prediction or virtual screening of the hERG-related cardiotoxicity of drug candidates.

Intracellular uptake of agents that block the hERG channel can confound the assessment of QT interval prolongation and arrhythmic risk.

BACKGROUND: Oliceridine is a biased ligand at the µ-opioid receptor recently approved for the treatment of acute pain. In a thorough QT study, corrected QT (QTc) prolongation displayed peaks at 2.5 and 60 minutes after a supratherapeutic dose. The mean plasma concentration peaked at 5 minutes, declining rapidly thereafter. OBJECTIVE: The purpose of this study was to examine the basis for the delayed effect of oliceridine to prolong the QTc interval. METHODS: Repolarization parameters and tissue accumulation of oliceridine were evaluated in rabbit left ventricular wedge preparations over a period of 5 hours. The effects of oliceridine on ion channel currents were evaluated in human embryonic kidney and Chinese hamster ovary cells. Quinidine was used as a control. RESULTS: Oliceridine and quinidine produced a progressive prolongation of the QTc interval and action potential duration over a period of 5 hours, paralleling slow progressive tissue uptake of the drugs. Oliceridine caused modest prolongation of these parameters, whereas quinidine produced a prominent prolongation of action potential duration and QTc interval as well as development of early afterdepolarization (after 2 hours), resulting in a high torsades de pointes score. The 50% inhibitory concentration values for the oliceridine inhibition of the rapidly activating delayed rectifier current (human ether a-go-go current) and late sodium channel current were 2.2 and 3.45 µM when assessed after traditional acute exposure but much lower after 3 hours of drug exposure. CONCLUSION: Our findings suggest that a gradual increase of intracellular access of drugs to the hERG channels as a result of their intracellular uptake and accumulation can significantly delay effects on repolarization, thus confounding the assessment of QT interval prolongation and arrhythmic risk when studied acutely. The multi-ion channel effects of oliceridine, late sodium channel current inhibition in particular, point to a low risk of devloping torsades de pointes.

Proarrhythmic effects induced by benzethonium chloride and domiphen bromide in vitro and in vivo.

Benzethonium chloride (BZT) and domiphen bromide (DMP) are widely used as antimicrobials in drugs, vaccines and industry. However, no cardiac safety data has been developed on both compounds. Previously we reported BZT and DMP as high-affinity human ether-a-go-go related gene (HERG) channel inhibitors with unknown proarrhythmic risk. Here, we investigate the cardiotoxicity of BZT and DMP in vitro and in vivo, aiming to improve the safety-in-use of both antimicrobials. In the present study, human iPSC derived cardiomyocytes (hiPSC-CMs) were generated and rabbit models were used to examine the proarrhythmic potential of BZT and DMP. Our results found that BZT and DMP induced time- and dose-dependent decrease in the contractile parameters of hiPSC-CMs, prolonged FPDc (≥ 0.1 µM), caused tachycardia/fibrillation-like oscillation (0.3-1 µM), ultimately progressing to irreversible arrest of beating (≥ 1 µM). The IC50 values of BZT and DMP derived from normalized beat rate were 0.13 µM and 0.10 µM on hiPSC-CMs at 76 days. Moreover, in vivo rabbit ECG data demonstrated that 12.85 mg/kg BZT and 3.85 mg/kg DMP evoked QTc prolongation, noncomplex arrhythmias and ventricular tachycardias. Our findings support the cardiac safety of 0.01 µM BZT/DMP in vitro and the intravenous infusion of 3.85 mg/kg BZT and 1.28 mg/kg DMP in vivo, whereas higher concentrations of both compounds cause mild to moderate cardiotoxicity that should not be neglected during medical and industrial applications.

Mechanisms of gefitinib-induced QT prolongation.

Gefitinib, a tyrosine kinase inhibitor, was the first targeted therapy for non-small cell lung cancer (NSCLC). Gefitinib could block human Ether-à-go-go-Related Gene (hERG) channel, an important target in drug-induced long QT syndrome. However, it is unclear whether gefitinib could induce QT interval prolongation. Here, whole-cell patch-clamp technique was used for evaluating the effect of gefitinib on rapidly-activating delayed rectifier K+ current (IKr), slowly-activating delayed rectifier K+ current (IKs), transient outward potassium current (Ito), inward rectifier K+ current (IK1) and on action potentials in guinea pig ventricular myocytes. The Langendorff heart perfusion technique was used to determine drug effect on the ECG. Gefitinib depressed IKr by binding to open and closed hERG channels in a concentration-dependent way (IC50: 1.91 µM). The inhibitory effect of gefitinib on wildtype hERG channels was reduced at the hERG mutants Y652A, S636A, F656V and S631A (IC50: 8.51, 13.97, 18.86, 32.99 µM), indicating that gefitinib is a pore inhibitor of hERG channels. In addition, gefitinib accelerated hERG channel inactivation and decreased channel steady-state inactivation. Gefitinib also decreased IKs with IC50 of 23.8 µM. Moreover, gefitinib increased action potential duration (APD) in guinea pig ventricular myocytes and the corrected QT interval (QTc) in isolated perfused guinea pig hearts in a concentration-dependent way (1-30 µM). These findings indicate that gefitinib could prolong QTc interval by potently blocking hERG channel, modulating kinetic properties of hERG channel. Partial block of KCNQ1/KCNE1 could also contribute to delayed repolarization and prolonged QT interval. Thus, caution should be taken when gefitinib is used for NSCLC treatment.

Antiepileptic Drug Tiagabine Does Not Directly Target Key Cardiac Ion Channels Kv11.1, Nav1.5 and Cav1.2.

Tiagabine is an antiepileptic drug used for the treatment of partial seizures in humans. Recently, this drug has been found useful in several non-epileptic conditions, including anxiety, chronic pain and sleep disorders. Since tachycardia-an impairment of cardiac rhythm due to cardiac ion channel dysfunction-is one of the most commonly reported non-neurological adverse effects of this drug, in the present paper we have undertaken pharmacological and numerical studies to assess a potential cardiovascular risk associated with the use of tiagabine. A chemical interaction of tiagabine with a model of human voltage-gated ion channels (VGICs) is described using the molecular docking method. The obtained in silico results imply that the adverse effects reported so far in the clinical cardiological of tiagabine could not be directly attributed to its interactions with VGICs. This is also confirmed by the results from the isolated organ studies (i.e., calcium entry blocking properties test) and in vivo (electrocardiogram study) assays of the present research. It was found that tachycardia and other tiagabine-induced cardiac complications are not due to a direct effect of this drug on ventricular depolarization and repolarization.

Arrhythmogenic and antiarrhythmic actions of late sustained sodium current in the adult human heart.

Late sodium current (late INa) inhibition has been proposed to suppress the incidence of arrhythmias generated by pathological states or induced by drugs. However, the role of late INa in the human heart is still poorly understood. We therefore investigated the role of this conductance in arrhythmias using adult primary cardiomyocytes and tissues from donor hearts. Potentiation of late INa with ATX-II (anemonia sulcata toxin II) and E-4031 (selective blocker of the hERG channel) slowed the kinetics of action potential repolarization, impaired Ca2+ homeostasis, increased contractility, and increased the manifestation of arrhythmia markers. These effects could be reversed by late INa inhibitors, ranolazine and GS-967. We also report that atrial tissues from donor hearts affected by atrial fibrillation exhibit arrhythmia markers in the absence of drug treatment and inhibition of late INa with GS-967 leads to a significant reduction in arrhythmic behaviour. These findings reveal a critical role for the late INa in cardiac arrhythmias and suggest that inhibition of this conductance could provide an effective therapeutic strategy. Finally, this study highlights the utility of human ex-vivo heart models for advancing cardiac translational sciences.

Rutaecarpine targets hERG channels and participates in regulating electrophysiological properties leading to ventricular arrhythmia.

Drug-mediated or medical condition-mediated disruption of hERG function accounts for the main cause of acquired long-QT syndrome (acLQTs), which predisposes affected individuals to ventricular arrhythmias (VA) and sudden death. Many Chinese herbal medicines, especially alkaloids, have risks of arrhythmia in clinical application. The characterized mechanisms behind this adverse effect are frequently associated with inhibition of cardiac hERG channels. The present study aimed to assess the potent effect of Rutaecarpine (Rut) on hERG channels. hERG-HEK293 cell was applied for evaluating the effect of Rut on hERG channels and the underlying mechanism. hERG current (IhERG ) was measured by patch-clamp technique. Protein levels were analysed by Western blot, and the phosphorylation of Sp1 was determined by immunoprecipitation. Optical mapping and programmed electrical stimulation were used to evaluate cardiac electrophysiological activities, such as APD, QT/QTc, occurrence of arrhythmia, phase singularities (PSs), and dominant frequency (DF). Our results demonstrated that Rut reduced the IhERG by binding to F656 and Y652 amino acid residues of hERG channel instantaneously, subsequently accelerating the channel inactivation, and being trapped in the channel. The level of hERG channels was reduced by incubating with Rut for 24 hours, and Sp1 in nucleus was inhibited simultaneously. Mechanismly, Rut reduced threonine (Thr)/ tyrosine (Tyr) phosphorylation of Sp1 through PI3K/Akt pathway to regulate hERG channels expression. Cell-based model unables to fully reveal the pathological process of arrhythmia. In vivo study, we found that Rut prolonged QT/QTc intervals and increased induction rate of ventricular fibrillation (VF) in guinea pig heart after being dosed Rut for 2 weeks. The critical reasons led to increased incidence of arrhythmias eventually were prolonged APD90 and APD50 and the increase of DF, numbers of PSs, incidence of early after-depolarizations (EADs). Collectively, the results of this study suggest that Rut could reduce the IhERG by binding to hERG channels through F656 and Y652 instantaneously. While, the PI3K/Akt/Sp1 axis may play an essential role in the regulation of hERG channels, from the perspective of the long-term effects of Rut (incubating for 24 hours). Importantly, the changes of electrophysiological properties by Rut were the main cause of VA.

Flavonoids and hERG channels: Friends or foes?

The cardiac action potential is regulated by several ion channels. Drugs capable to block these channels, in particular the human ether-à-go-go-related gene (hERG) channel, also known as KV11.1 channel, may lead to a potentially lethal ventricular tachyarrhythmia called "Torsades de Pointes". Thus, evaluation of the hERG channel off-target activity of novel chemical entities is nowadays required to safeguard patients as well as to avoid attrition in drug development. Flavonoids, a large class of natural compounds abundantly present in food, beverages, herbal medicines, and dietary food supplements, generally escape this assessment, though consumed in consistent amounts. Continuously growing evidence indicates that these compounds may interact with the hERG channel and block it. The present review, by examining numerous studies, summarizes the state-of-the-art in this field, describing the most significant examples of direct and indirect inhibition of the hERG channel current operated by flavonoids. A description of the molecular interactions between a few of these natural molecules and the Rattus norvegicus channel protein, achieved by an in silico approach, is also presented.

COVID-19 Drugs Chloroquine and Hydroxychloroquine, but Not Azithromycin and Remdesivir, Block hERG Potassium Channels.

Drug-induced long QT syndrome (LQTS) is an established cardiac side effect of a wide range of medications and represents a significant concern for drug safety. The rapidly and slowly activating delayed rectifier K+ currents, mediated by channels encoded by the human ether-a-go-go-related gene (hERG) and KCNQ1 + KCNE1, respectively, are two main currents responsible for ventricular repolarization. The common cause for drugs to induce LQTS is through impairing the hERG channel. For the recent emergence of COVID-19, caused by severe acute respiratory syndrome coronavirus 2, several drugs have been investigated as potential therapies; however, there are concerns about their QT prolongation risk. Here, we studied the effects of chloroquine, hydroxychloroquine, azithromycin, and remdesivir on hERG channels. Our results showed that although chloroquine acutely blocked hERG current (IhERG), with an IC50 of 3.0 µM, hydroxychloroquine acutely blocked IhERG 8-fold less potently, with an IC50 of 23.4 µM. Azithromycin and remdesivir did not acutely affect IhERG When these drugs were added at 10 µM to the cell culture medium for 24 hours, remdesivir increased IhERG by 2-fold, which was associated with an increased mature hERG channel expression. In addition, these four drugs did not acutely or chronically affect KCNQ1 + KCNE1 channels. Our data provide insight into COVID-19 drug-associated LQTS and cardiac safety concerns. SIGNIFICANCE STATEMENT: This work demonstrates that, among off-label potential COVID-19 treatment drugs chloroquine, hydroxychloroquine, azithromycin, and remdesivir, the former two drugs block hERG potassium channels, whereas the latter two drugs do not. All four drugs do not affect KCNQ1 + KCNE1. As hERG and KCNQ1 + KCNE1 are two main K+ channels responsible for ventricular repolarization, and most drugs that induce long QT syndrome (LQTS) do so by impairing hERG channels, these data provide insight into COVID-19 drug-associated LQTS and cardiac safety concerns.

Discovery of Atabecestat (JNJ-54861911): A Thiazine-Based ß-Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor Advanced to the Phase 2b/3 EARLY Clinical Trial.

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

Discovery of a novel series of indolinylpyrimidine-based GPR119 agonists: Elimination of ether-a-go-go-related gene liability using a hydrogen bond acceptor-focused approach.

We previously identified a novel series of indolinylpyrimidine derivatives exemplified by 2 in Figure 1, which is an indoline based derivative, as potent GPR119 agonists. Despite the attractive potency of 2, this compound inhibited the human ether-a-go-go-related gene (hERG) K+ channel. We elucidated crucial roles of the methylsulfonyl group of 2 in its interaction with the hERG channel and the GPR119 receptor, presumably as a hydrogen bond acceptor (HBA). To remove the undesirable hERG inhibitory activity, a strategy was implemented to arrange an HBA on a less conformationally flexible framework at the indoline 5-position instead of the methylsulfonyl group. This successfully led to the discovery of a piperidinone ring as a desirable motif at the indoline 5-position, which could minimize hERG liability as shown by 24b. Further optimization focused on the reduction of lipophilicity in terms of more favorable drug-like properties. Consequently, the introduction of a hydroxy group at the 3-position of the piperidinone ring effectively reduced lipophilicity without compromising GPR119 potency, resulting in the identification of (3S)-3-hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]- 2,3-dihydro-1H-indol-5-yl}piperidin-2-one ((S)-29) as a novel, potent, and orally bioavailable GPR119 agonist with a well-balanced profile. The pharmacological effects of this compound were also confirmed after single and chronic oral administration in diabetic animal models.

Cryo-EM Structure of K+-Bound hERG Channel Complexed with the Blocker Astemizole.

The hERG channel is a voltage-gated potassium channel involved in cardiac repolarization. Off-target hERG inhibition by drugs has become a critical issue in the pharmaceutical industry. The three-dimensional structure of the hERG channel was recently reported at 3.8-Å resolution using cryogenic electron microscopy (cryo-EM). However, the drug inhibition mechanism remains unclear because of the scarce structural information regarding the drug- and potassium-bound hERG channels. In this study, we obtained the cryo-EM density map of potassium-bound hERG channel complexed with astemizole, a well-known hERG inhibitor that increases risk of potentially fatal arrhythmia, at 3.5-Å resolution. The structure suggested that astemizole inhibits potassium conduction by binding directly below the selectivity filter. Furthermore, we propose a possible binding model of astemizole to the hERG channel and provide insights into the unusual sensitivity of hERG to several drugs.

A system-level approach to investigate alloxan-induced toxicity in microtubule-binding protein to lead type 2 diabetes mellitus.

Microtubule-associated protein tau (MAPT) is a key protein, which is mainly identified as an essential factor for microtubule dynamics and neuronal outgrowth. Though tau has several functions, regulation of insulin signaling is one among them to control type 2 diabetes. Abnormal expression of tau protein leads to hyperphosphorylation and is known as tauopathies. The presence of alloxan occurs in refined wheat flour, especially in various baking products such as parotta, a well-known South Indian dish. In this study, the reduced form of alloxan called dialuric acid can enter the beta cells of islets of Langerhans and binds MAPT to induce toxicity by hyperphosphorylating the tau protein, which ultimately causes destruction to pancreatic beta cells, and it leads to diabetes mellitus. Here, the toxic effects of dialuric acid targeting MAPT through in silico computational predictions have been investigated. The 3D structure of MAPT protein was constructed through I-Tasser, and it has been refined and validated by GalaxyRefine and PROCHECK. The structure of ligand was retrieved from PubChem. Molecular docking was accomplished by AutoDock 4.2 software, and the results indicate the strong binding affinity between dialuric acid and MAPT protein, and it showed a binding free energy (∆G) of - 3.72 kcal/mol. Dialuric acid binds with the active region SER 232 of MAPT whereby it hyperphosphorylates the protein to become toxic. Also, ADMET results strongly suggest that the compound dialuric acid possesses toxic property, and similarly, Ames test confirmed that it was found to be mutagenic. Thus, our results strongly revealed that dialuric acid was found to be toxic which could be able to damage the beta cells of the pancreas and abates insulin signaling, and finally, it leads to DM.

Targeting the Kv11.1 (hERG) channel with allosteric modulators. Synthesis and biological evaluation of three novel series of LUF7346 derivatives.

We synthesized and evaluated three novel series of substituted benzophenones for their allosteric modulation of the human Kv11.1 (hERG) channel. We compared their effects with reference compound LUF7346 previously shown to shorten the action potential of cardiomyocytes derived from human stem cells. Most compounds behaved as negative allosteric modulators (NAMs) of [3H]dofetilide binding to the channel. Compound 9i was the most potent amongst all ligands, remarkably reducing the affinity of dofetilide in competitive displacement assays. One of the other derivatives (6k) tested in a second radioligand binding set-up, displayed unusual displacement characteristics with a pseudo-Hill coefficient significantly distinct from unity, further indicative of its allosteric effects on the channel. Some compounds were evaluated in a more physiologically relevant context in beating cardiomyocytes derived from human induced pluripotent stem cells. Surprisingly, the compounds tested showed effects quite different from the reference NAM LUF7346. For instance, compound 5e prolonged, rather than shortened, the field potential duration, while it did not influence this parameter when the field potential was already prolonged by dofetilide. In subsequent patch clamp studies on HEK293 cells expressing the hERG channel the compounds behaved as channel blockers. In conclusion, we successfully synthesized and identified new allosteric modulators of the hERG channel. Unexpectedly, their effects differed from the reference compound in functional assays on hERG-HEK293 cells and human cardiomyocytes, to the extent that the compounds behaved as stand-alone channel blockers.

Toxicological Screening of Four Bioactive Citroflavonoids: In Vitro, In Vivo, and In Silico Approaches.

Many studies describe different pharmacological effects of flavonoids on experimental animals and humans. Nevertheless, few ones are confirming the safety of these compounds for therapeutic purposes. This study aimed to investigate the preclinical safety of naringenin, naringin, hesperidin, and quercetin by in vivo, in vitro, and in silico approaches. For this, an MTT-based cytotoxicity assay in VERO and MDCK cell lines was performed. In addition, acute toxicity was evaluated on Wistar rats by OECD Guidelines for the Testing of Chemicals (Test No. 423: Acute Oral Toxicity-Class Method). Furthermore, we used the ACD/Tox Suite to predict toxicological parameters such as hERG channel blockade, CYP450 inhibition, and acute toxicity in animals. The results showed that quercetin was slightly more cytotoxic on cell lines (IC50 of 219.44 ± 7.22 mM and 465.41 ± 7.44 mM, respectively) than the other citroflavonoids. All flavonoids exhibited an LD50 value > 2000 mg/kg, which classifies them as low-risk substances as OECD guidelines established. Similarly, predicted LD50 was LD50 > 300 to 2000 mg/kg for all flavonoids as acute toxicity assay estimated. Data suggests that all these flavonoids did not show significant toxicological effects, and they were classified as low-risk, useful substances for drug development.

Characterization of the Synergistic Inhibition of IK(erg) and IK(DR) by Ribociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor.

Ribociclib (RIB, LE011, Kisqali®), an orally administered inhibitor of cyclin-dependent kinase-4/6 (CDK-4/6) complex, is clinically effective for the treatment of several malignancies, including advanced breast cancer. However, information regarding the effects of RIB on membrane ion currents is limited. In this study, the addition of RIB to pituitary tumor (GH3) cells decreased the peak amplitude of erg-mediated K+ current (IK(erg)), which was accompanied by a slowed deactivation rate of the current. The IC50 value for RIB-perturbed inhibition of deactivating IK(erg) in these cells was 2.7 µM. In continued presence of µM RIB, neither the subsequent addition of 17ß-estradiol (30 µM), phorbol 12-myristate 13-acetate (10 µM), or transforming growth factor-ß (1 µM) counteracted the inhibition of deactivating IK(erg). Its presence affected the decrease in the degree of voltage-dependent hysteresis for IK(erg) elicitation by long-duration triangular ramp voltage commands. The presence of RIB differentially inhibited the peak or sustained component of delayed rectifier K+ current (IK(DR)) with an effective IC50 of 28.7 or 11.4 µM, respectively, while it concentration-dependently decreased the amplitude of M-type K+ current with IC50 of 13.3 µM. Upon 10-s long membrane depolarization, RIB elicited a decrease in the IK(DR) amplitude, which was concomitant with an accelerated inactivation time course. However, the inability of RIB (10 µM) to modify the magnitude of the hyperpolarization-activated cation current was disclosed. The mean current-voltage relationship of IK(erg) present in HL-1 atrial cardiomyocytes was inhibited in the presence of RIB (10 µM). Collectively, the hyperpolarization-activated cation current was observed. RIB-mediated perturbations in ionic currents presented herein are upstream of its suppressive action on cytosolic CDK-4/6 activities and partly participates in its modulatory effects on the functional activities of pituitary tumor cells (e.g., GH3 cells) or cardiac myocytes (e.g., HL-1 cells).

Functional Impact of BeKm-1, a High-Affinity hERG Blocker, on Cardiomyocytes Derived from Human-Induced Pluripotent Stem Cells.

IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.