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.

The hERG channel activator, RPR260243, enhances protective IKr current early in the refractory period reducing arrhythmogenicity in zebrafish hearts.

Human ether-à-go-go related gene (hERG) K+ channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach.NEW & NOTEWORTHY hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.

Revealing Molecular Determinants of hERG Blocker and Activator Binding.

The Kv11.1 potassium channel, encoded by the human ether-a-go-go-related gene (hERG), plays an essential role in the cardiac action potential. hERG blockade by small molecules can induce "torsade de pointes" arrhythmias and sudden death; as such, it is an important off-target to avoid during drug discovery. Recently, a cryo-EM structure of the open channel state of hERG was reported, opening the door to in silico docking analyses and interpretation of hERG structure-activity relationships, with a view to avoiding blocking activity. Despite this, docking directly to this cryo-EM structure has been reported to yield binding modes that are unable to explain known mutagenesis data. In this work, we use molecular dynamics simulations to sample a range of channel conformations and run ensemble docking campaigns at the known hERG binding site below the selectivity filter, composed of the central cavity and the four deep hydrophobic pockets. We identify a hERG conformational state allowing discrimination of blockers vs nonblockers from docking; furthermore, the binding pocket agrees with mutagenesis data, and blocker binding modes fit the hERG blocker pharmacophore. We then use the same protocol to identify a binding pocket in the hERG channel pore for hERG activators, again agreeing with the reported mutagenesis. Our approach may be useful in drug discovery campaigns to prioritize candidate compounds based on hERG liability via virtual docking screens.

Toward a Structural View of hERG Activation by the Small-Molecule Activator ICA-105574.

Outward current conducted by human ether-à-go-go-related gene type 1 (hERG1) K+ channels is important for action potential repolarization in the human ventricle. Rapid, voltage-dependent inactivation greatly reduces outward currents conducted by hERG1 channels and involves conformational changes in the ion selectivity filter (SF). Recently, compounds have been found that activate hERG1 channel function by modulating gating mechanisms such as reducing inactivation. Such activating compounds could represent a novel approach to prevent arrhythmias associated with prolonged ventricular repolarization associated with inherited or acquired long QT syndrome. ICA-105574 (ICA), a 3-nitro-n-(4-phenoxyphenyl) benzamide derivative activates hERG1 by strongly attenuating pore-type inactivation. We previously mapped the putative binding site for ICA to a hydrophobic pocket located between two adjacent subunits. Here, we used the recently reported cryoelectron microscopy structures of hERG1 to elucidate the structural mechanisms by which ICA influences the stability of the SF. By combining molecular dynamics simulations, voltage-clamp electrophysiology, and the synthesis of novel ICA derivatives, we provide atomistic insights into SF dynamics and propose a structural link between the SF and S6 segments. Further, our study highlights the importance of the nitro moiety, at the meta position of the benzamide ring, for the activity of ICA and reveals that the (bio)isosteric substitution of this side chain can switch the activity to weak inhibitors. Our findings indicate that ICA increases the stability of the SF to attenuate channel inactivation, and this action requires a fine-tuned compound geometry.

A pharmacological master key mechanism that unlocks the selectivity filter gate in K+ channels.

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.

Lipid roles in hERG function and interactions with drugs.

Human-ether-a-go-go-related channel (hERG) is a voltage gated potassium channel (Kv11.1) abundantly expressed in heart and brain tissues. In addition to playing an important role in mediation of repolarizing K+ currents (IKr) in Action Potential (AP), hERG is notorious for its propensity to interact with various medications. The drug-induced block of K+ currents across hERG channel are strongly associated with dysrhythmic conditions collectively known as drug-induced long-QT-syndrome. The recent availability of the high-resolution Cryo-EM structures for the hERG channel has provided unique opportunity to resolve structural mechanisms involved into the process of voltage-gating of hERG channels, map various roles played by components of ventricular and neuronal membranes and then to connect it to cellular pathways through which diverse chemical compounds might be affecting function of the channel. Specifically, lipids and lipid derivatives such as polyunsaturated fatty acids (PUFAs), ceramides and steroids have been shown to directly interact with the lipid facing amino acids in various Kv channels including hERG. In this review, possible lipophilic pathways of hERG activators and blockers, together with the existence of fenestration windows and effects of PUFAs, ceramides and steroids are explored throughout different sections. Finally, the interplay between long QT inducing drugs and phospholipidosis is briefly discussed.

CiPA challenges and opportunities from a non-clinical, clinical and regulatory perspectives. An overview of the safety pharmacology scientific discussion.

The Safety Pharmacology Society organized a scientific session at its annual conference in 2017 to discuss the challenges and opportunities of the Comprehensive In-Vitro Proarrhythmia Assay (CiPA) paradigm. Our intention was to raise awareness of this initiative with its members and also to gauge the extent to which safety pharmacologists have incorporated the CiPA testing strategy within the pharmaceutical industry. CiPA offers many potential opportunities including 1) a focus on proarrhythmic risk (as opposed to QTc prolongation), 2) providing scientific rationale to support the continued development of compounds that may have a poor selectivity over hERG whilst also blocking other inward currents and 3) reducing the extent of ECG monitoring in clinical trials with a greater influence of the non-clinical studies. Such opportunities may speed drug development and reduce costs. However, there are also challenges for CiPA implementation. For example, the mixed ion channel paradigm does not easily lend itself to a prospective drug discovery strategy although testing for such effects can be achieved with assays with good throughput. However, it should also be recognized that compounds with a mixed ion channel profile might also have properties that are undesirable to treat non-life threatening indications. All components of CiPA (nonclinical and clinical) require validation, particularly as a composite package to impact drug development and evaluation. One of the significant discussion points was that the existing regulatory guidance supports the use of components of CiPA through follow-up studies. A survey of the conference audience showed that the level of awareness of CiPA is quite high and that companies are already conducting some testing against a wider panel of cardiac ion channels beyond hERG. However, the adoption of other technologies (stem cell derived cardiac myocytes and in silico modeling) is less well developed. Taken together, the session demonstrated the potential advantages of CiPA, but also some significant challenges.

Regulation of Kv4.3 and hERG potassium channels by KChIP2 isoforms and DPP6 and response to the dual K+ channel activator NS3623.

Transient outward potassium current (Ito) contributes to early repolarization of many mammalian cardiac action potentials, including human, whilst the rapid delayed rectifier K+ current (IKr) contributes to later repolarization. Fast Ito channels can be produced from the Shal family KCNDE gene product Kv4.3s, although accessory subunits including KChIP2.x and DPP6 are also needed to produce a near physiological Ito. In this study, the effect of KChIP2.1 & KChIP2.2 (also known as KChIP2b and KChIP2c respectively), alone or in conjunction with the accessory subunit DPP6, on both Kv4.3 and hERG were evaluated. A dual Ito and IKr activator, NS3623, has been recently proposed to be beneficial in heart failure and the action of NS3623 on the two channels was also investigated. Whole-cell patch-clamp experiments were performed at 33 ±â€¯1 °C on HEK293 cells expressing Kv4.3 or hERG in the absence or presence of these accessory subunits. Kv4.3 current magnitude was augmented by co-expression with either KChIP2.2 or KChIP2.1 and KChIP2/DPP6 with KChIP2.1 producing a greater effect than KChIP2.2. Adding DPP6 removed the difference in Kv4.3 augmentation between KChIP2.1 and KChIP2.2. The inactivation rate and recovery from inactivation were also altered by KChIP2 isoform co-expression. In contrast, hERG (Kv11.1) current was not altered by co-expression with KChIP2.1, KChIP2.2 or DPP6. NS3623 increased Kv4.3 amplitude to a similar extent with and without accessory subunit co-expression, however KChIP2 isoforms modulated the compound's effect on inactivation time course. The agonist effect of NS3623 on hERG channels was not affected by KChIP2.1, KChIP2.2 or DPP6 co-expression.

Discovery of a new mexiletine-derived agonist of the hERG K+ channel.

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in the rapid component (IKr) of cardiac action potential repolarization phase. A large number of structurally different compounds block hERG and cause a high risk of arrhythmias. Among the drugs that block hERG channel, a few compounds have been identified as hERG channel activators. Such compounds may be useful, at least in theory, for the treatment of long term QT syndrome. Here we describe a new activator of hERG channel, named MC450. This compound is a symmetric urea, derived from (R)-mexiletine. Using patch-clamp recordings, we found that MC450 increased the activation current of hERG channel, with an EC50 of 41±4µM. Moreover MC450 caused a depolarizing shift in the voltage dependence of inactivation from -64.1±1.2mV (control), to -35.9±1.4mV, whereas it had no effect on the voltage dependence of activation. Furthermore, MC450 slowed current inactivation and the effect of MC450 was attenuated by the inactivation-impaired double mutant G628C/S631C.

ß-Arrestin-Mediated Regulation of the Human Ether-a-go-go-Related Gene Potassium Channel.

The rapidly activating delayed rectifier K+ channel (IKr) is encoded by the human ether-a-go-go-related gene (hERG), which is important for the repolarization of the cardiac action potential. Mutations in hERG or drugs can impair the function or decrease the expression level of hERG channels, leading to long QT syndrome. Thus, it is important to understand hERG channel trafficking and its regulation. For this purpose, G protein-coupled receptors (GPCRs), which regulate a vast array of cellular processes, represent a useful route. The development of designer GPCRs known as designer receptors exclusively activated by designer drugs (DREADDs) has made it possible to dissect specific GPCR signaling pathways in various cellular systems. In the present study, by expressing an arrestin-biased M3 muscarinic receptor-based DREADD (M3D-arr) in stable hERG-expressing human embryonic kidney (HEK) cells, we demonstrate that ß-arrestin signaling plays a role in hERG regulation. By exclusively activating M3D-arr using the otherwise inert compound, clozapine-N-oxide, we found that M3D-arr activation increased mature hERG expression and current. Within this paradigm, M3D-arr recruited ß-arrestin-1 to the plasma membrane, and promoted phosphoinositide 3-kinase-dependent activation of protein kinase B (Akt). The activated Akt acted through phosphatidylinositol 3-phosphate 5-kinase and Rab11 to facilitate hERG recycling to the plasma membrane. Potential ß-arrestin signaling-mediated increases in hERG and IKr were also observed in hERG-HEK cells as well as in neonatal rat ventricular myocytes treated with the muscarinic agonist carbachol. These findings provide novel insight into hERG trafficking and regulation.