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.

Characterization of Vixotrigine, a Broad-Spectrum Voltage-Gated Sodium Channel Blocker.

Voltage-gated sodium channels (Navs) are promising targets for analgesic and antiepileptic therapies. Although specificity between Nav subtypes may be desirable to target specific neural types, such as nociceptors in pain, many broadly acting Nav inhibitors are clinically beneficial in neuropathic pain and epilepsy. Here, we present the first systematic characterization of vixotrigine, a Nav blocker. Using recombinant systems, we find that vixotrigine potency is enhanced in a voltage- and use-dependent manner, consistent with a state-dependent block of Navs. Furthermore, we find that vixotrigine potently inhibits sodium currents produced by both peripheral and central nervous system Nav subtypes, with use-dependent IC50 values between 1.76 and 5.12 µM. Compared with carbamazepine, vixotrigine shows higher potency and more profound state-dependent inhibition but a similar broad spectrum of action distinct from Nav1.7- and Nav1.8-specific blockers. We find that vixotrigine rapidly inhibits Navs and prolongs recovery from the fast-inactivated state. In native rodent dorsal root ganglion sodium channels, we find that vixotrigine shifts steady-state inactivation curves. Based on these results, we conclude that vixotrigine is a broad-spectrum, state-dependent Nav blocker. SIGNIFICANCE STATEMENT: Vixotrigine blocks both peripheral and central voltage-gated sodium channel subtypes. Neurophysiological approaches in recombinant systems and sensory neurons suggest this block is state-dependent.

A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm.

INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

Applying the CiPA approach to evaluate cardiac proarrhythmia risk of some antimalarials used off-label in the first wave of COVID-19.

We applied a set of in silico and in vitro assays, compliant with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm, to assess the risk of chloroquine (CLQ) or hydroxychloroquine (OH-CLQ)-mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin (ERT) and azithromycin (AZI), drugs repurposed during the first wave of coronavirus disease 2019 (COVID-19). Each drug or drug combination was tested in patch clamp assays on seven cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay) using control (healthy) or high-risk cell populations, and in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. In each assay, concentration-response curves encompassing and exceeding therapeutic free plasma levels were generated. Both CLQ and OH-CLQ showed blocking activity against some potassium, sodium, and calcium currents. CLQ and OH-CLQ inhibited IKr (half-maximal inhibitory concentration [IC50 ]: 1 µM and 3-7 µM, respectively) and IK1 currents (IC50 : 5 and 44 µM, respectively). When combining OH-CLQ with AZI, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC50  > 300-1000 µM). Using Virtual Assay, both antimalarials affected several TdP indicators, CLQ being more potent than OH-CLQ. Effects were more pronounced in the high-risk cell population. In hiPSC-derived cardiomyocytes, all drugs showed early after-depolarizations, except AZI. Combining CLQ or OH-CLQ with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off-label use in COVID-19, CLQ and OH-CLQ use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.

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.

Increased cardiac risk in concomitant methadone and diazepam treatment: pharmacodynamic interactions in cardiac ion channels.

Methadone, a synthetic opioid for treatment of chronic pain and withdrawal from opioid dependence, has been linked to QT prolongation, potentially fatal torsades de pointes, and sudden cardiac death. Concomitant use of diazepam or other benzodiazepines in methadone maintenance treatment can increase the risk of sudden death. Therefore, we determined the effects of methadone and diazepam singly and in combination on cardiac action potentials (APs) and on the major ion channels responsible for cardiac repolarization. Using patch clamp recording in human stem cell-derived cardiomyocytes and stably transfected mammalian cells, we found that methadone produced concentration-dependent AP prolongation and ion channel block at low micromolar concentrations: hERG (IC50 = 1.7 µM), hNav1.5 (11.2 µM tonic block; 5.5 µM phasic block), and hCav1.2 (26.7 µM tonic block; 7.7 µM phasic block). Methadone was less potent in hKv4.3/hKChIP2.2 (IC50 = 39.0 µM) and hKvLQT1/hminK (53.3 µM). In contrast, diazepam blocked channels only at much higher concentrations and had no effect on AP duration at 1 µM. However, coadministration of 1-µM diazepam with methadone caused a statistically significant increase in AP duration and a 4-fold attenuation of hNav1.5 block (IC50 values were 44.2 µM and 26.6 µM, respectively, for tonic and phasic block), with no significant effect on methadone-induced block of hERG, hCav1.2, hKv4.3/hKChIP2.2, and hKvLQT1/hminK channels. Thus, although diazepam alone does not prolong the QT interval, the relief of methadone-induced Na channel block may leave hERG K channel block uncompensated, thereby increasing cardiac risk.

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.

Pyrethroid neurotoxicity studies with bifenthrin indicate a mixed Type I/II mode of action.

BACKGROUND: Bifenthrin is usually considered a Type I pyrethroid, because it lacks an α-CN group present in Type II pyrethroids, but some previous studies suggest a mixed Type I/II mode-of-action. Results are presented for bifenthrin in a rat developmental neurotoxicity (DNT) study along with effects on Na currents in human VGSC subtypes. Molecular modeling comparisons were also made for bifenthrin and other pyrethroids. RESULTS: In a rat DNT study, bifenthrin produced tremors and clonic convulsions in dams and pups and slightly reduced acoustic startle response amplitude, and increased Tmax, at PND20 in females. Similar blood levels of bifenthrin were measured in dams and pups at each dose level i.e. no concentration in pups. In human VGSC experiments, using the Nav1.8 subtype, bifenthrin's effects on inactivation were slight, as for Type II pyrethroids, but without large prolongation of the tail current (deactivation) seen with Type II. Molecular modeling of bifenthrin indicates that the o-Me group may occupy a similar space to the α-CN group of cypermethrin and fenpropathrin. CONCLUSION: In a DNT study and on human Nav1.8 tail currents bifenthrin showed Type I and II effects, similar to some published studies. Overall, bifenthrin acts as a mixed Type I/II pyrethroid. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Alfuzosin delays cardiac repolarization by a novel mechanism.

The United States Food and Drug Administration (FDA) uses alfuzosin as an example of a drug having QT risk in humans that was not detected in nonclinical studies. FDA approval required a thorough clinical QT study (TCQS) that was weakly positive at high doses. The FDA has used the clinical/nonclinical discordance as a basis for mandatory TCQS, and this requirement has serious consequences for drug development. For this reason, we re-examined whether nonclinical signals of QT risk for alfuzosin were truly absent. Alfuzosin significantly prolonged action potential duration (APD)(60) in rabbit Purkinje fibers (p < 0.05) and QT in isolated rabbit hearts (p < 0.05) at the clinically relevant concentration of 300 nM. In man, the QT interval corrected with Fridericia's formula increased 7.7 ms, which exceeds the 5.0-ms threshold for a positive TCQS. Effects on hK(v)11.1, hK(v)4.3, and hK(v)7.1/hKCNE1 potassium currents and calcium current were not involved. At 300 nM, approximately 30x C(max), alfuzosin significantly increased whole-cell peak sodium (hNa(v)1.5) current (p < 0.05), increased the probability of late hNa(v)1.5 single-channel openings, and significantly shortened the slow time constant for recovery from inactivation. Alfuzosin also increased hNa(v)1.5 burst duration and number of openings per burst between 2- and 3-fold. Alfuzosin is a rare example of a non-antiarrhythmic drug that delays cardiac repolarization not by blocking hK(v)11.1 potassium current, but by increasing sodium current. Nonclinical studies clearly show that alfuzosin increases plateau potential and prolongs APD and QT, consistent with QT prolongation in man. The results challenge the FDA grounds for the absolute primacy of TCQS based on the claim of a false-negative, nonclinical study on alfuzosin.

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.

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors.

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and ß-subunit composition. The α6ß2ß3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3ß2ß3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration-approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3ß2ß3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6ß2ß3 nicotinic receptors.

Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors.

The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and ß-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3ß4, α3ß4α5, α4ß2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity.

HERG channel trafficking.

Mutations in the cardiac potassium channel hERG/IKr cause inherited long QT syndrome with increased susceptibility to ventricular arrhythmias. Several mutations in hERG produce trafficking-deficient channels that are retained in the endoplasmic reticulum (ER). Surface expression of certain mutations (i.e. hERG G601S) can be restored by specific channel blockers. Although hERG currents have been studied extensively, little is known about proteins in the processing pathway. Using biochemical and electrophysiological assays we show that the cytosolic chaperones Hsp70 and Hsp90 interact transiently with wild-type hERG. Inhibition of Hsp90 prevents maturation and reduces hERG/IKr currents. Trafficking-deficient mutants remain tightly associated with chaperones in the ER until trafficking is restored, e.g. by channel blockers. hERG/chaperone complexes represent novel targets for therapeutic compounds with cardiac liability such as arsenic, which is used in the treatment of leukaemias. Arsenic interferes with the formation of hERG/chaperone complexes and inhibits hERG maturation causing ECG abnormalities. We conclude that Hsp9O and Hsp70 are crucial for productive folding of wild-type hERG. Therapeutic compounds that inhibit chaperone function produce a novel form of acquired long QT syndrome not by direct channel block but by reduced surface expression due to an acquired trafficking defect of hERG.

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.

Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X).

OBJECTIVE: The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, I(Kr). The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and I(Kr) currents was investigated in this study. METHODS: hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native I(Kr) currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. RESULTS: Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC(50) values of 1.0 and 13.2 muM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of -20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 muM BIM I reduced native I(Kr) currents by 69.2% and lead to action potential prolongation. CONCLUSION: In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and I(Kr) currents.

Inhibition of human ether-a-go-go-related gene potassium channels by alpha 1-adrenoceptor antagonists prazosin, doxazosin, and terazosin.

Human ether-a-go-go-related gene (HERG) potassium channels are expressed in multiple tissues including the heart and adenocarcinomas. In cardiomyocytes, HERG encodes the alpha-subunit underlying the rapid component of the delayed rectifier potassium current, I(Kr), and pharmacological reduction of HERG currents may cause acquired long QT syndrome. In addition, HERG currents have been shown to be involved in the regulation of cell proliferation and apoptosis. Selective alpha 1-adrenoceptor antagonists are commonly used in the treatment of hypertension and benign prostatic hyperplasia. Recently, doxazosin has been associated with an increased risk of heart failure. Moreover, quinazoline-derived alpha 1-inhibitors induce apoptosis in cardiomyocytes and prostate tumor cells independently of alpha1-adrenoceptor blockade. To assess the action of the effects of prazosin, doxazosin, and terazosin on HERG currents, we investigated their acute electrophysiological effects on cloned HERG potassium channels heterologously expressed in Xenopus oocytes and HEK 293 cells.Prazosin, doxazosin, and terazosin blocked HERG currents in Xenopus oocytes with IC(50) values of 10.1, 18.2, and 113.2 microM respectively, whereas the IC(50) values for HERG channel inhibition in human HEK 293 cells were 1.57 microM, 585.1 nM, and 17.7 microM. Detailed biophysical studies revealed that inhibition by the prototype alpha 1-blocker prazosin occurred in closed, open, and inactivated channels. Analysis of the voltage-dependence of block displayed a reduction of inhibition at positive membrane potentials. Frequency-dependence was not observed. Prazosin caused a negative shift in the voltage-dependence of both activation (-3.8 mV) and inactivation (-9.4 mV). The S6 mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) HERG current blockade, indicating that prazosin binds to a common drug receptor within the pore-S6 region. In conclusion, this study demonstrates that HERG potassium channels are blocked by prazosin, doxazosin, and terazosin. These data may provide a hypothetical molecular explanation for the apoptotic effect of quinazoline-derived alpha1-adrenoceptor antagonists.

Evaluating state dependence and subtype selectivity of calcium channel modulators in automated electrophysiology assays.

Voltage-gated Ca2+ channels play essential roles in control of neurosecretion and muscle contraction. The pharmacological significance of Cav channels stem from their identification as the molecular targets of calcium blockers used in the treatment of cardiovascular diseases, such as hypertension, angina, and arrhythmia, and neurologic diseases, such as pain and seizure. It has been proposed that state-dependent Cav inhibitors, that is, those that preferentially bind to channels in open or inactivated states, may improve the therapeutic window over relatively state-independent Cav inhibitors. High-throughput fluorescent-based functional assays have been useful in screening chemical libraries to identify Cav inhibitors. However, hit confirmation, mechanism of action, and subtype selectivity are better suited to automated patch clamp assays that have sufficient capacity to handle the volume of compounds identified during screening, even of modest sized libraries (≤500,000 compounds), and the flexible voltage control that allows evaluation of state-dependent drug blocks. IonWorks Barracuda (IWB), the newest generation of IonWorks instruments, provides the opportunity to accelerate the Cav drug discovery studies in an automated patch clamp platform in 384-well format capable of medium throughput screening and profiling studies. We have validated hCav1.2, hCav2.1, hCav2.2, and hCav3.2 channels assays on the IWB platform (population patch clamp mode) and demonstrated that the biophysical characteristics of the channels (activation, inactivation, and steady-state inactivation) obtained with the IWB system are consistent with known subtype-specific characteristics. Using standard reference compounds (nifedipine, BAY K8644, verapamil, mibefradil, and pimozide), we demonstrated subtype-selective and state- and use-dependent characteristics of drug-channel interactions. Here we describe the design and validation of novel robust high-throughput Cav channel assays on the IWB platform. The assays can be used to screen focused compound libraries for state-dependent Cav channel antagonists, to prioritize compounds for potency or to counterscreen for Cav subtype selectivity.

MICE models: superior to the HERG model in predicting Torsade de Pointes.

Drug-induced block of the cardiac hERG (human Ether-à-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Nav1.5 and Cav1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Cav1.2.