Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel.

CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

A general procedure to select calibration drugs for lab-specific validation and calibration of proarrhythmia risk prediction models: An illustrative example using the CiPA model.

INTRODUCTION: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet. METHODS: The proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction. RESULTS: The selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation). DISCUSSION: This work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm.

Assessment of an In Silico Mechanistic Model for Proarrhythmia Risk Prediction Under the CiPA Initiative.

The International Council on Harmonization (ICH) S7B and E14 regulatory guidelines are sensitive but not specific for predicting which drugs are pro-arrhythmic. In response, the Comprehensive In Vitro Proarrhythmia Assay (CiPA) was proposed that integrates multi-ion channel pharmacology data in vitro into a human cardiomyocyte model in silico for proarrhythmia risk assessment. Previously, we reported the model optimization and proarrhythmia metric selection based on CiPA training drugs. In this study, we report the application of the prespecified model and metric to independent CiPA validation drugs. Over two validation datasets, the CiPA model performance meets all pre-specified measures for ranking and classifying validation drugs, and outperforms alternatives, despite some in vitro data differences between the two datasets due to different experimental conditions and quality control procedures. This suggests that the current CiPA model/metric may be fit for regulatory use, and standardization of experimental protocols and quality control criteria could increase the model prediction accuracy even further.

Vanoxerine: cellular mechanism of a new antiarrhythmic.

INTRODUCTION: There remains an unmet need for safe and effective antiarrhythmic drugs, especially for the treatment of atrial fibrillation. Vanoxerine is a drug that is free of adverse cardiac events in normal volunteers, yet is a potent blocker of the hERG (hK(v)11.1) cardiac potassium channel. Consequently,we hypothesized that vanoxerine might also be a potent blocker of cardiac calcium (Ca) and sodium (Na) currents, and would not affect transmural dispersion of repolarization. METHODS: The whole cell patch clamp technique was used to measure currents from cloned ion channels overexpressed in stable cell lines and single ventricular myocytes. We measured intracellular action potentials from canine ventricular wedges and Purkinje fibers using sharp microelectrode technique. RESULTS: We found that vanoxerine was a potent hK(v)11.1 blocker, and at submicromolar concentrations, it blocked Ca and Na currents in a strongly frequency-dependent manner. In the canine ventricular wedge preparation vanoxerine did not significantly affect transmural action potential waveforms, QT interval or transmural dispersion of repolarization. CONCLUSIONS: Vanoxerine (1) is a potent blocker of cardiac hERG, Na and Ca channels; (2) block is strongly frequency-dependent especially for Na and Ca channels; and (3) transmural dispersion of ventricular repolarization is unaffected. The multichannel block and repolarization uniformity resemble the effects of amiodarone, the exemplar atrial fibrillation drug. Vanoxerine is a completely different chemical and has none of amiodarone's toxic effects. Vanoxerine has characteristics of a potentially effective and safe antiarrhythmic.

An ion channel library for drug discovery and safety screening on automated platforms.

Ion channels represent the third largest class of targets in drug discovery after G-protein coupled receptors and kinases. In spite of this ranking, ion channels continue to be under exploited as drug targets compared with the other two groups for several reasons. First, with 400 ion channel genes and an even greater number of functional channels due to mixing and matching of individual subunits, a systematic collection of ion channel-expressing cell lines for drug discovery and safety screening has not been available. Second, the lack of high-throughput functional assays for ion channels has limited their use as drug targets. Now that automated electrophysiology has come of age and provided the technology to assay ion channels at medium to high throughput, we have addressed the need for a library of ion channel cell lines by constructing the Ion Channel Panel (ChanTest Corp., Cleveland, OH). From 400 ion channel genes, a collection of 82 of the most relevant human ion channels for drug discovery, safety, and human disease has been assembled.Each channel has been stably overexpressed in human embryonic kidney 293 or Chinese hamster ovary cells. Cell lines have been selected and validated on automated electrophysiology systems to facilitate cost-effective screening for safe and selective compounds at earlier stages in the drug development process. The screening and validation processes as well as the relative advantages of different screening platforms are discussed.

HERG-Lite: a novel comprehensive high-throughput screen for drug-induced hERG risk.

INTRODUCTION: Direct block of I(Kr) by non-antiarrhythmic drugs (NARDs) is a major cause of QT prolongation and torsades de pointes (TdP), and has made the hERG potassium channel a major target of drug safety programs in cardiotoxicity. Block of hERG currents is not the only way that drugs can adversely impact the repolarizing current I(Kr), however. We have shown recently that two drugs in clinical use do not block hERG but produce long QT syndrome (LQTS) and TdP by inhibiting trafficking of hERG to the cell surface. To address the need for an inexpensive, rapid, and comprehensive assay to predict both types of hERG risk early in the drug development process, we have developed a novel antibody-based chemiluminescent assay called HERG-Lite. METHODS: HERG-Lite monitors the expression of hERG at the cell surface in two different stable mammalian cell lines. One cell line acts as a biosensor for drugs that inhibit hERG trafficking, while the other predicts hERG blockers based on their ability to act as pharmacological chaperones. In this study, we have validated the HERG-Lite assay using a panel of 100 drugs: 50 hERG blockers and 50 nonblockers. RESULTS: HERG-Lite correctly predicted hERG risk for all 100 test compounds with no false positives or negatives. All 50 hERG blockers were detected as drugs with hERG risk in the HERG-Lite assay, and fell into two classes: B (for blocker) and C (for complex; block and trafficking inhibition). DISCUSSION: HERG-Lite is the most comprehensive assay available for predicting drug-induced hERG risk. It accurately predicts both channel blockers and trafficking inhibitors in a rapid, cost-effective manner and is a valuable non-clinical assay for drug safety testing.

Deferoxamine promotes survival and prevents electrocardiographic abnormalities in the gerbil model of iron-overload cardiomyopathy.

We investigated the time course of electrocardiographic (ECG) changes in the Mongolian gerbil model of iron overload and the effects of the iron chelator deferoxamine (DFO) on these changes. Iron overload was produced with weekly subcutaneous injections of low doses (200 mg/kg/wk) or high doses (800 mg/kg/wk) of iron-dextran. DFO was administered subcutaneously at a dose of 200 mg/kg/day to high-dose animals. Our results show that (1) survival of iron-overloaded gerbils is dose-dependent, with median survival times of 68 and 14 weeks for low- and high-dose animals, respectively; (2) both low and high doses produce prolongation of the PR interval and bradycardia in early stages and prolongation of the QT interval, premature ventricular contractions, variable degrees of atrioventricular block, changes in the ST segment, and T-wave inversion at later stages coinciding with the development of heart failure; (3) DFO prevented death during 20 weeks of high-dose iron-dextran; (4) DFO prevented ECG changes, although delayed prolongation of PR intervals and QRS complexes occurred; and (5) despite marked prolongation of survival and prevention of ECG changes, DFO had modest effects on total cardiac iron content. We speculate that DFO chelates a small iron pool located within the cytoplasm of iron-overloaded cardiomyocytes.