Enhancing hERG Risk Assessment with Interpretable Classificatory and Regression Models.

The human Ether-à-go-go-Related Gene (hERG) is a transmembrane protein that regulates cardiac action potential, and its inhibition can induce a potentially deadly cardiac syndrome. In vitro tests help identify hERG blockers at early stages; however, the high cost motivates searching for alternative, cost-effective methods. The primary goal of this study was to enhance the Pred-hERG tool for predicting hERG blockage. To achieve this, we developed new QSAR models that incorporated additional data, updated existing classificatory and multiclassificatory models, and introduced new regression models. Notably, we integrated SHAP (SHapley Additive exPlanations) values to offer a visual interpretation of these models. Utilizing the latest data from ChEMBL v30, encompassing over 14,364 compounds with hERG data, our binary and multiclassification models outperformed both the previous iteration of Pred-hERG and all publicly available models. Notably, the new version of our tool introduces a regression model for predicting hERG activity (pIC50). The optimal model demonstrated an R2 of 0.61 and an RMSE of 0.48, surpassing the only available regression model in the literature. Pred-hERG 5.0 now offers users a swift, reliable, and user-friendly platform for the early assessment of chemically induced cardiotoxicity through hERG blockage. The tool provides versatile outcomes, including (i) classificatory predictions of hERG blockage with prediction reliability, (ii) multiclassificatory predictions of hERG blockage with reliability, (iii) regression predictions with estimated pIC50 values, and (iv) probability maps illustrating the contribution of chemical fragments for each prediction. Furthermore, we implemented explainable AI analysis (XAI) to visualize SHAP values, providing insights into the contribution of each feature to binary classification predictions. A consensus prediction calculated based on the predictions of the three developed models is also present to assist the user's decision-making process. Pred-hERG 5.0 has been designed to be user-friendly, making it accessible to users without computational or programming expertise. The tool is freely available at http://predherg.labmol.com.br.

Toward Quantitative Models in Safety Assessment: A Case Study to Show Impact of Dose-Response Inference on hERG Inhibition Models.

Due to challenges with historical data and the diversity of assay formats, in silico models for safety-related endpoints are often based on discretized data instead of the data on a natural continuous scale. Models for discretized endpoints have limitations in usage and interpretation that can impact compound design. Here, we present a consistent data inference approach, exemplified on two data sets of Ether-à-go-go-Related Gene (hERG) K+ inhibition data, for dose-response and screening experiments that are generally applicable for in vitro assays. hERG inhibition has been associated with severe cardiac effects and is one of the more prominent safety targets assessed in drug development, using a wide array of in vitro and in silico screening methods. In this study, the IC50 for hERG inhibition is estimated from diverse historical proprietary data. The IC50 derived from a two-point proprietary screening data set demonstrated high correlation (R = 0.98, MAE = 0.08) with IC50s derived from six-point dose-response curves. Similar IC50 estimation accuracy was obtained on a public thallium flux assay data set (R = 0.90, MAE = 0.2). The IC50 data were used to develop a robust quantitative model. The model's MAE (0.47) and R2 (0.46) were on par with literature statistics and approached assay reproducibility. Using a continuous model has high value for pharmaceutical projects, as it enables rank ordering of compounds and evaluation of compounds against project-specific inhibition thresholds. This data inference approach can be widely applicable to assays with quantitative readouts and has the potential to impact experimental design and improve model performance, interpretation, and acceptance across many standard safety endpoints.

Critical Assessment of Artificial Intelligence Methods for Prediction of hERG Channel Inhibition in the "Big Data" Era.

The rise of novel artificial intelligence (AI) methods necessitates their benchmarking against classical machine learning for a typical drug-discovery project. Inhibition of the potassium ion channel, whose alpha subunit is encoded by the human ether-à-go-go-related gene (hERG), leads to a prolonged QT interval of the cardiac action potential and is a significant safety pharmacology target for the development of new medicines. Several computational approaches have been employed to develop prediction models for the assessment of hERG liabilities of small molecules including recent work using deep learning methods. Here, we perform a comprehensive comparison of hERG effect prediction models based on classical approaches (random forests and gradient boosting) and modern AI methods [deep neural networks (DNNs) and recurrent neural networks (RNNs)]. The training set (∼9000 compounds) was compiled by integrating the hERG bioactivity data from the ChEMBL database with experimental data generated from an in-house, high-throughput thallium flux assay. We utilized different molecular descriptors including the latent descriptors, which are real-value continuous vectors derived from chemical autoencoders trained on a large chemical space (>1.5 million compounds). The models were prospectively validated on ∼840 in-house compounds screened in the same thallium flux assay. The best results were obtained with the XGBoost method and RDKit descriptors. The comparison of models based only on latent descriptors revealed that the DNNs performed significantly better than the classical methods. The RNNs that operate on SMILES provided the highest model sensitivity. The best models were merged into a consensus model that offered superior performance compared to reference models from academic and commercial domains. Furthermore, we shed light on the potential of AI methods to exploit the big data in chemistry and generate novel chemical representations useful in predictive modeling and tailoring a new chemical space.

Toward in vivo-relevant hERG safety assessment and mitigation strategies based on relationships between non-equilibrium blocker binding, three-dimensional channel-blocker interactions, dynamic occupancy, dynamic exposure, and cellular arrhythmia.

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.

Assessment of spatial heterogeneity of ventricular repolarization after multi-channel blocker drugs in healthy subjects.

BACKGROUND AND OBJECTIVES: In contrast to potassium channel blockers, drugs affecting multiple channels seem to reduce torsadogenic risks. However, their effect on spatial heterogeneity of ventricular repolarization (SHVR) is still matter of investigation. Aim of this work is to assess the effect of four drugs blocking the human ether-à-go-go-related gene (hERG) potassium channel, alone or in combination with other ionic channel blocks, on SHVR, as estimated by the V-index on short triplicate 10 s ECG. METHODS: The V-index is an estimate of the standard deviation of the repolarization times of the myocytes across the entire myocardium, obtained from multi-lead surface electrocardiograms. Twenty-two healthy subjects received a pure hERG potassium channel blocker (dofetilide) and 3 other drugs with additional varying degrees of sodium and calcium (L-type) channel block (quinidine, ranolazine, and verapamil), as well as placebo. A one-way repeated-measures Friedman test was performed to compare the V-index over time. RESULTS: Computer simulations and Bland-Altman analysis supported the reliability of the estimates of V-index on triplicate 10 s ECG. Ranolazine, verapamil and placebo did not affect the V-index. On the contrary, after quinidine and dofetilide administration, an increase of V-index from predose to its peak value was observed (ΔΔV-index values were 19 ms and 27 ms, respectively, p < 0.05). CONCLUSIONS: High torsadogenic drugs (dofetilide and quinidine) affected significantly the SHVR, as quantified by the V-index. The metric has therefore a potential in assessing drug arrhythmogenicity.

An assessment of KIR channel function in human cerebral arteries.

In the rodent cerebral circulation, inward rectifying K+ (KIR) channels set resting tone and the distance over which electrical phenomena spread along the arterial wall. The present study sought to translate these observations into human cerebral arteries obtained from resected brain tissue. Computational modeling and a conduction assay first defined the impact of KIR channels on electrical communication; patch-clamp electrophysiology, quantitative PCR, and immunohistochemistry then characterized KIR2.x channel expression/activity. In keeping with rodent observations, computer modeling highlighted that KIR blockade should constrict cerebral arteries and attenuate electrical communication if functionally expressed. Surprisingly, Ba2+ (a KIR channel inhibitor) had no effect on human cerebral arterial tone or intercellular conduction. In alignment with these observations, immunohistochemistry and patch-clamp electrophysiology revealed minimal KIR channel expression/activity in both smooth muscle and endothelial cells. This absence may be reflective of chronic stress as dysphormic neurons, leukocyte infiltrate, and glial fibrillary acidic protein expression was notable in the epileptic cortex. In closing, KIR2.x channel expression is limited in human cerebral arteries from patients with epilepsy and thus has little impact on resting tone or the spread of vasomotor responses. NEW & NOTEWORTHY KIR2.x channels are expressed in rodent cerebral arterial smooth muscle and endothelial cells. As they are critical to setting membrane potential and the distance signals conduct, we sought to translate this work into humans. Surprisingly, KIR2.x channel activity/expression was limited in human cerebral arteries, a paucity tied to chronic brain stress in the epileptic cortex. Without substantive expression, KIR2.x channels were unable to govern arterial tone or conduction.

Revealing kinetics and state-dependent binding properties of IKur-targeting drugs that maximize atrial fibrillation selectivity.

The KV1.5 potassium channel, which underlies the ultra-rapid delayed-rectifier current (IKur) and is predominantly expressed in atria vs. ventricles, has emerged as a promising target to treat atrial fibrillation (AF). However, while numerous KV1.5-selective compounds have been screened, characterized, and tested in various animal models of AF, evidence of antiarrhythmic efficacy in humans is still lacking. Moreover, current guidelines for pre-clinical assessment of candidate drugs heavily rely on steady-state concentration-response curves or IC50 values, which can overlook adverse cardiotoxic effects. We sought to investigate the effects of kinetics and state-dependent binding of IKur-targeting drugs on atrial electrophysiology in silico and reveal the ideal properties of IKur blockers that maximize anti-AF efficacy and minimize pro-arrhythmic risk. To this aim, we developed a new Markov model of IKur that describes KV1.5 gating based on experimental voltage-clamp data in atrial myocytes from patient right-atrial samples in normal sinus rhythm. We extended the IKur formulation to account for state-specificity and kinetics of KV1.5-drug interactions and incorporated it into our human atrial cell model. We simulated 1- and 3-Hz pacing protocols in drug-free conditions and with a [drug] equal to the IC50 value. The effects of binding and unbinding kinetics were determined by examining permutations of the forward (kon) and reverse (koff) binding rates to the closed, open, and inactivated states of the KV1.5 channel. We identified a subset of ideal drugs exhibiting anti-AF electrophysiological parameter changes at fast pacing rates (effective refractory period prolongation), while having little effect on normal sinus rhythm (limited action potential prolongation). Our results highlight that accurately accounting for channel interactions with drugs, including kinetics and state-dependent binding, is critical for developing safer and more effective pharmacological anti-AF options.

The J to T-peak interval as a biomarker in drug safety studies: A method of accuracy assessment applied to two algorithms.

OBJECTIVES: To evaluate performance of J-to-T-peak (JTP) measurements of 12-lead ECGs, in a five-arm study using drugs with various levels of electrolyte channel block. METHODS: The novel evaluation method distinguishes between different aspects of measurement. "Random noise" is the variability among repeated measurements made without changing the conditions. "Context noise" is the variability of changes in context of the measurement, e.g. T-wave morphology, autonomic nervous system state. RESULTS: The average random noise of our RR-corrected JTPc measurements in standard deviations was 3.0 ms and not dependent on the drug. The average context noise was 4.0 ms for ranolazine, verapamil, and placebo, and 8.8 ms for dofetilide and quinidine. Measurement consistency is corroborated by linear fit confidence intervals of baseline- and placebo-corrected JTPc versus drug concentration. CONCLUSIONS: Systematic differences were found in JTPc drug response between the Mortara method and published data. Residual signal component in the context noise may influence future study design.

Evaluation of Batch Variations in Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes from 2 Major Suppliers.

Drug-induced proarrhythmia is a major safety issue in drug development. Developing sensitive in vitro assays that can predict drug-induced cardiotoxicity in humans has been a challenge of toxicology research for decades. Recently, induced pluripotent stem cell-derived human cardiomyocytes (iPSC-hCMs) have become a promising model because they largely replicate the electrophysiological behavior of human ventricular cardiomyocytes. Patient-specific iPSC-hCMs have been proposed for personalized cardiac drug selection and adverse drug response prediction; however, many procedures are involved in cardiomyocytes differentiation and purification process, which may result in large line-to-line and batch-to-batch variations. Here, we examined the purity, cardiac ion channel gene expression profile, and electrophysiological response of 3 batches of iPSC-hCMs from each of 2 major cell suppliers. We found that iPSC-hCMs from both vendors had similar purities. Most of the cardiac ion channel genes were expressed uniformly among different batches of iCells, while larger variations were found in Cor.4U cells, particularly in the expression of CACNA1C, KCND2, and KCNA5 genes, which could underlie the differences in baseline beating rate (BR) and field potential duration (FPD) measurements. Although, in general, the electrophysiological responses of different batches of cells to Na+, Ca2+, Ikr, and Iks channel blockers were similar, with Ikr blocker-induced proarrhythmia, the sensitivities were depended on baseline BR and FPD values: cells that beat slower had longer FPD and greater sensitivity to drug-induced proarrhythmia. Careful evaluation of the performance of iPSC-hCMs and methods of data analysis is warranted for shaping regulatory standards in qualifying iPSC-hCMs for drug safety testing.

Fampridine and real-life walking in multiple sclerosis: Low predictive value of clinical test for habitual short-term changes.

BACKGROUND: Fampridine improves walking speed in patients with multiple sclerosis (MS) in performance-based tests. The impact on habitual mobility and its correlation with clinical tests has not been analysed. OBJECTIVE: To investigate the association between clinical response criteria and habitual mobility in MS patients starting a fampridine treatment. METHODS: During a four-week baseline-to-treatment study, we assessed in 28 patients (median EDSS 4.75, range 4-6.5) walking tests as the Timed-25-Foot-Walk (T25FW) and mobility questionnaires at day 0, 14 (start of treatment) and 28. Habitual steps and distance per day, total activity and walking speed was measured by accelerometry over four weeks. Beside improvement in real-life mobility, we investigated if such measures differed between non-responders and responders defined by a 20% improvement in clinical tests. RESULTS: All clinical test, patient reported outcomes and total activity improved significantly (p<0.05). 46% improved (any change >0) in three of four real-life measures. Change of the T25FW predicted only an increase of distance per day. Subjective rating of patients performed better by predicting distance and walking speed changes correctly. CONCLUSION: Fampridine might improve walking in daily life of MS, but clinical tests are weak predictors. Accelerometry opens a new perspective on mobility measurment, but the current data do not show a consistent effect on non-performance based accelerometry outcomes.

Multi-parametric assessment of cardiomyocyte excitation-contraction coupling using impedance and field potential recording: A tool for cardiac safety assessment.

INTRODUCTION: The ICH S7B guidelines recommend that all new chemical entities should be subjected to hERG repolarization screening due to its association with life-threatening "Torsades de Pointes" (TdP) arrhythmia. However, it has become evident that not all hERG channel inhibitors result in TdP and not all compounds that induce QT prolongation and TdP necessarily inhibit hERG. In order to address the limitations of the S7B/E14 guidelines, the FDA through a public/private partnership initiated the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative to examine the possible modification and refinement of the ICH E14/S7B guidelines. One of the main components of the CiPA initiative is to utilize a predictive assay system together with human cardiomyocytes for risk assessment of arrhythmia. METHOD: In this manuscript we utilize the xCELLigence® CardioECR system which simultaneously measures excitation-contraction coupling together with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to assess the effect of 8 reference compounds across 3 different independent sites. These 8 compounds were part of Phase I CiPA validation study. RESULTS: Our data demonstrate that hERG channel blockers, such as E4031 and moxifloxacin, prolonged field potential duration (FPD) at low concentration and induced arrhythmic beating activity as measured by field potential (FP) recording and impedance (IMP) recordings at higher concentrations. On the contrary, nifedipine, an inhibitor of calcium channel, didn't disrupt the periodicity of cell beating and weakened cell contractile activity and shortened FPD. Multichannel inhibitors, such as flecainide, quinidine and mexiletine, not only increased FPD and induced arrhythmia but also significantly reduced the amplitude of FP spike. JNJ303, an IKs inhibitor, only affected FPD. Comparison of the compound effect on FPD across the 3 different sites is consistent in terms of trend of the effect with observed 3-10 fold differences in minimal effective concentration at which a minimum of 10% response is detected. In addition, pentamidine, a hERG trafficking inhibitor which induced irregular beating activity over a more prolonged duration of time was readily flagged in this assay system. Taken together, this multi-parameter assay using hiPSC-CMs in conjunction with simultaneous measurement of ion channel activity and contractility can be a reliable approach for risk assessment of proarrhythmic compounds.

A temperature-dependent in silico model of the human ether-à-go-go-related (hERG) gene channel.

INTRODUCTION: Current regulatory guidelines for assessing the risk of QT prolongation include in vitro assays assessing drug effects on the human ether-à-go-go-related (hERG; also known as Kv11.1) channel expressed in cell lines. These assays are typically conducted at room temperature to promote the ease and stability of recording hERG currents. However, the new Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm proposes to use an in silico model of the human ventricular myocyte to assess risk, requiring as input hERG channel pharmacology data obtained at physiological temperatures. To accommodate current industry safety pharmacology practices for measuring hERG channel activity, an in silico model of hERG channel that allows for the extrapolation of hERG assay data across different temperatures is desired. Because temperature may have an effect on both channel gating and drug binding rate, such models may need to have two components: a base model dealing with temperature-dependent gating changes without drug, and a pharmacodynamic component simulating temperature-dependent drug binding kinetics. As a first step, a base mode that can capture temperature effects on hERG channel gating without drug is needed. METHODS AND RESULTS: To meet this need for a temperature-dependent base model, a Markov model of the hERG channel with state transition rates explicitly dependent on temperature was developed and calibrated using data from a variety of published experiments conducted over a range of temperatures. The model was able to reproduce observed temperature-dependent changes in key channel gating properties and also to predict the results obtained in independent sets of new experiments. DISCUSSION: This new temperature-sensitive model of hERG gating represents an attempt to improve the predictivity of safety pharmacology testing by enabling the translation of room temperature hERG assay data to more physiological conditions. With further development, this model can be incorporated into the CiPA paradigm and also be used as a tool for developing insights into the thermodynamics of hERG channel gating mechanisms and the temperature-dependence of hERG channel block by drugs.

Implications of Dynamic Occupancy, Binding Kinetics, and Channel Gating Kinetics for hERG Blocker Safety Assessment and Mitigation.

Blockade of the hERG potassium channel prolongs the ventricular action potential (AP) and QT interval, and triggers early after depolarizations (EADs) and torsade de pointes (TdP) arrhythmia. Opinions differ as to the causal relationship between hERG blockade and TdP, the relative weighting of other contributing factors, definitive metrics of preclinical proarrhythmicity, and the true safety margin in humans. Here, we have used in silico techniques to characterize the effects of channel gating and binding kinetics on hERG occupancy, and of blockade on the human ventricular AP. Gating effects differ for compounds that are sterically compatible with closed channels (becoming trapped in deactivated channels) versus those that are incompatible with the closed/closing state, and expelled during deactivation. Occupancies of trappable blockers build to equilibrium levels, whereas those of non-trappable blockers build and decay during each AP cycle. Occupancies of ~83% (non-trappable) versus ~63% (trappable) of open/inactive channels caused EADs in our AP simulations. Overall, we conclude that hERG occupancy at therapeutic exposure levels may be tolerated for nontrappable, but not trappable blockers capable of building to the proarrhythmic occupancy level. Furthermore, the widely used Redfern safety index may be biased toward trappable blockers, overestimating the exposure-IC50 separation in nontrappable cases.

Cystathionine ß-synthase-derived hydrogen sulfide is involved in human malignant hyperthermia.

Hydrogen sulfide is an endogenous gasotransmitter and its mechanism of action involves activation of ATP-sensitive K(+) channels and phosphodiesterase inhibition. As both mechanisms are potentially involved in malignant hyperthermia (MH), in the present study we addressed the involvement of the L-cysteine/hydrogen sulfide pathway in MH. Skeletal muscle biopsies obtained from 25 MH-susceptible (MHS) and 56 MH-negative (MHN) individuals have been used to perform the in vitro contracture test (IVCT). Quantitative real-time PCR (qPCR) and Western blotting studies have also been performed. Hydrogen sulfide levels are measured in both tissue samples and plasma. In MHS biopsies an increase in cystathionine ß-synthase (CBS) occurs, as both mRNA and protein expression compared with MHN biopsies. Hydrogen sulfide biosynthesis is increased in MHS biopsies (0.128±0.12 compared with 0.943±0.13 nmol/mg of protein per min for MHN and MHS biopsies, respectively; P<0.01). Addition of sodium hydrosulfide (NaHS) to MHS samples evokes a response similar, in the IVCT, to that elicited by either caffeine or halothane. Incubation of MHN biopsies with NaHS, before caffeine or halothane challenge, switches an MHN to an MHS response. In conclusion we demonstrate the involvement of the L-cysteine/hydrogen sulfide pathway in MH, giving new insight into MH molecular mechanisms. This finding has potential implications for clinical care and could help to define less invasive diagnostic procedures.

Effects of the Concomitant Activation of ON and OFF Retinal Ganglion Cells on the Visual Thalamus: Evidence for an Enhanced Recruitment of GABAergic Cells.

A fundamental question in vision neuroscience is how parallel processing of Retinal Ganglion Cell (RGC) signals is integrated at the level of the visual thalamus. It is well-known that parallel ON-OFF pathways generate output signals from the retina that are conveyed to the dorsal lateral geniculate nucleus (dLGN). However, it is unclear how these signals distribute onto thalamic cells and how these two pathways interact. Here, by electrophysiological recordings and c-Fos expression analysis, we characterized the effects of pharmacological manipulations of the retinal circuit aimed at inducing either a selective activation of a single pathway, OFF RGCs [intravitreal L-(+)-2-Amino-4-phosphonobutyric, L-AP4] or an unregulated activity of all classes of RGCs (intravitreal 4-Aminopyridine, 4-AP). In in vivo experiments, the analysis of c-Fos expression in the dLGN showed that these two manipulations recruited active cells from the same area, the lateral edge of the dLGN. Despite this similarity, the unregulated co-activation of both ON and OFF pathways by 4-AP yielded a much stronger recruitment of GABAergic interneurons in the dLGN when compared to L-AP4 pure OFF activation. The increased activation of an inhibitory thalamic network by a high level of unregulated discharge of ON and OFF RGCs might suggest that cross-inhibitory pathways between opposing visual channels are presumably replicated at multiple levels in the visual pathway, thus increasing the filtering ability for non-informative or noisy visual signals.

Assessment of temperature-induced hERG channel blockade variation by drugs.

Drug-induced QT prolongation has been reported in humans and animals. This potentially lethal effect can be induced by drugs interacting with a cardiac potassium channel, namely hERG (human ether-a go-go-related gene) leading to arrhythmia or torsade de pointes (TdP). Hence, in vitro evaluation of therapeutics for their effects on the rapid delayed rectifier current (IKr) mediated by the K(+) ion channel encoded by hERG is a valuable tool for identifying potential arrhythmic side effects during drug safety testing. Our objective was to evaluate the temperature-induced hERG channel blockade variation by human and veterinary drugs using the IonFlux 16 system. A panel of eight drugs was tested for IKr inhibition at both ambient (23 °C) and physiological (37 °C) temperatures at various concentrations using IonFlux 16, an automated patch clamp system. Our results established that both amiodarone (IC(50) = 0.56 µM at 23 °C and 0.30 µM at 37 °C) and ß-estradiol (IC(50) = 24.72 µM at 23 °C and 8.17 µM at 37 °C) showed a dose-dependent IKr blockade with a higher blockade at 37 °C. Whereas, blockade of IKr by both ivermectin (IC(50) = 12.52 µM at 23 °C and 24.41 µM at 37 °C) and frusemide (IC(50) = 12.58 µM at 23 °C and 25.55 µM at 37 °C) showed a dose-dependent IKr blockade with a lower blockade at 37 °C. Gentamicin, enrofloxacin, xylazine and albendazole did not block IKr at both the assessed temperatures. Collectively, these results demonstrate that the effect of temperature variation should be taken into consideration during the evaluation of test drugs for their hERG channel blockade potential.

Solubility assessment and on-line exposure confirmation in a patch-clamp assay for hERG (human ether-a-go-go-related gene) potassium channel inhibition.

INTRODUCTION: The hERG (human ether-a-go-go-related gene) potassium channel (KV11.1) is an important anti-target in drug discovery as its inhibition by small molecules has considerable promiscuity and is linked to an increased risk of the potentially fatal ventricular arrhythmia torsade de pointes. Therefore, great efforts are taken in the pharmaceutical industry to early on screen out compounds that block the channel. Early screening activities most often include compounds with sub-optimal physicochemical properties such as limited solubility. Therefore, careful monitoring of achieved compound concentration importantly supports the validity of experimental data. METHODS: A novel principle of exposure confirmation in a constant flow patch-clamp assay for hERG interaction is presented. Quantification is based on-real time UV absorption spectroscopy of the perfusion solution using long light path fiber optic flow cells. Calibration is performed using solutions which are confirmed by turbidometry to be free of precipitates. RESULTS: Turbidometry is shown to be sensitive enough to ensure valid calibration of the UV spectroscopic measurement. For a typical drug-like small molecule (verapamil) it is shown that even 30 nM can be accurately quantified using a 100 cm fiber optic flow cell. DISCUSSION: The combination of turbidometry and long light path fiber optic UV spectroscopy offers accurate, almost real-time exposure determination in a wide range of concentrations with little effort, affordable instrumentation, and no delay for data reporting. For research compounds with challenging physicochemical properties this method provides valuable data to support the validity of the measurements.

Stoichiometry of altered hERG1 channel gating by small molecule activators.

Voltage-gated K(+) channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K(+) selective conductance. In some K(+) channels, the selectivity filter can undergo a conformational change to reduce K(+) flux by a mechanism called C-type inactivation. In human ether-a-go-go-related gene 1 (hERG1) K(+) channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome.

[Comparative assessment of nephroprotective properties of potassium and calcium channel modulators in experimental renal injury].

The experiments in white laboratory rats have shown that a single intragasrtric administration of the new fluorine-containing potassium channel opener flocalin in a dose of 5 mg/kg in the initial stage of sublimate nephropathy increased the glomerular filtration rate, reduced creatininemia, increased urinary creatinine excretion, and decreased proteinuria. Under similar conditions, the administration of the calcium channel blocker diltiazem in a dose of 5 mg/kg (intragasrtric) showed a less pronounced antiproteinuric effect as compared to that of flocalin. A comparative assessment of the influence of flocalin and diltiazem on the basic renal function markers demonstrated predominant nephroprotective effect of flocalin in the treatment of acute toxic nephropathy.

Shaker IR T449 mutants separate C- from U-type inactivation.

Previous studies demonstrated that slow inactivation of the Shaker potassium channel can be made ~100-fold faster or slower by point mutations at a site in the outer pore (T449). However, the discovery that two forms of slow inactivation coexist in Shaker raises the question of which inactivation process is affected by mutation. Equivalent mutations in K(V)2.1, a channel exhibiting only U-type inactivation, have minimal effects on inactivation, suggesting that mutation of Shaker T449 acts on C-type inactivation alone, a widely held yet untested hypothesis. This study reexamines mutations at Shaker T449, confirming that T449A speeds inactivation and T449Y/V slow it. T449Y and T449V exhibit U-type inactivation that is enhanced by high extracellular potassium, in contrast to C-type inactivation in T449A which is inhibited by high potassium. Automated parameter estimation for a 12-state Markov model suggests that U-type inactivation occurs mainly from closed states upon weak depolarization, but primarily from the open state at positive voltages. The model also suggests that WT channels, which in this study exhibit mostly C-type inactivation, recover from inactivation through closed-inactivated states, producing voltage-dependent recovery. This suggests that both C-type and U-type inactivation involve both open-inactivated and closed-inactivated states.