hERG Potassium Channel Blockade Research Articles

Molecular Insights into hERG Potassium Channel Blockade by Lubeluzole

Background/Aims: Lubeluzole is a benzothiazole derivative that has shown neuroprotective properties in preclinical models of ischemic stroke. However, clinical research on lubeluzole is now at a standstill, since lubeluzole seems to be associated with the acquired long QT syndrome and ventricular arrhythmias. Since the cardiac cellular effects of lubeluzole have not been described thus far, an explanation for the lubeluzole-induced QT interval prolongation is lacking. Methods: We tested the affinity of lubeluzole, its enantiomer, and the racemate for hERG channel using the patch-clamp technique. We synthesized and tested two simplified model compounds corresponding to two moieties included in the lubeluzole structure. The obtained experimental results were rationalized by docking simulation on the recently reported cryo-electron microscopy (cryo-EM) structure of hERG. Group efficiency analysis was performed in order to individuate the fragment most contributing to binding. Results: We found that lubeluzole and its R enantiomer are highly potent inhibitors of human ether-ago-go-related gene (hERG) channel with an IC50 value of 12.9 ± 0.7 nM and 11.3 ± 0.8 nM, respectively. In the presence of lubeluzole, steady-state activation and inactivation of hERG channel were shifted to more negative potentials and inactivation kinetics was accelerated. Mutations of aromatic residues (Y652A and F656A) in the channel inner cavity significantly reduced the inhibitory effect of lubeluzole. Molecular docking simulations performed on the near atomic resolution cryo-electron microscopy structures of hERG supported the role of Y652 and F656 as the main contributors to high affinity binding. Group efficiency analysis indicated that both 1,3-benzothiazol-2-amine and 3-aryloxy-2-propanolamine moieties contribute to drug binding with the former giving higher contribution. Conclusions: This study suggests the possibility to modulate lubeluzole hERG blockade by introducing suitable substituents onto one or both constituting portions of the parent compound in order to either reduce potency (i. e. torsadogenic potential) or potentiate affinity (useful for class III antiarrhythmic and anticancer agent development).

Inhibition of hERG potassium channel by the antiarrhythmic agent mexiletine and its metabolite m-hydroxymexiletine.

Mexiletine is a sodium channel blocker, primarily used in the treatment of ventricular arrhythmias. Moreover, recent studies have demonstrated its therapeutic value to treat myotonic syndromes and to relieve neuropathic pain. The present study aims at investigating the direct blockade of hERG potassium channel by mexiletine and its metabolite m-hydroxymexiletine (MHM). Our data show that mexiletine inhibits hERG in a time- and voltage-dependent manner, with an IC50 of 3.7 ± 0.7 μmol/L. Analysis of the initial onset of current inhibition during a depolarizing test pulse indicates mexiletine binds preferentially to the open state of the hERG channel. Looking for a possible mexiletine alternative, we show that m-hydroxymexiletine (MHM), a minor mexiletine metabolite recently reported to be as active as the parent compound in an arrhythmia animal model, is a weaker hERG channel blocker, compared to mexiletine (IC50 = 22.4 ± 1.2 μmol/L). The hERG aromatic residues located in the S6 helix (Tyr652 and Phe656) are crucial in the binding of mexiletine and the different affinities of mexiletine and MHM with hERG channel are interpreted by modeling their corresponding binding interactions through ab initio calculations. The simulations demonstrate that the introduction of a hydroxyl group on the meta-position of the aromatic portion of mexiletine weakens the interaction of the drug xylyloxy moiety with Tyr652. These results provide further insights into the molecular basis of drug/hERG interactions and, in agreement with previously reported results on clofilium and ibutilide analogs, support the possibility of reducing hERG potency and related toxicity by modifying the aromatic pattern of substitution of clinically relevant compounds.

Ligand Structural Aspects of hERG Channel Blockade

Sudden death as a side effect of action of non-antiarrhythmic drugs is a major pharmacological safety concern facing the pharmaceutical industry and the health regulatory authorities. A number of drugs have been withdrawn from the market in recent years due to cardiovascular toxicity associated with undesirable blockade of hERG potassium channel. Pharmaceuticals of widely varying structure have been shown to interact with hERG. Defining the molecular features that confer hERG inhibitory activity has therefore become a focus of considerable computational and statistical modeling efforts. Some of the approaches are aimed primarily at filtering out potential hERG blockers in the context of virtual libraries, while others involve understanding structure-activity relationships governing hERG-drug interactions. The ability of models to produce structural hypotheses that can be tested by the project teams has become the key prerequisite driving their organization-wide adoption.

Slow recovery of hERG potassium channel blockade and voltage dependent unblock may contribute to the proarrhythmic properties of compounds.

Inhibition of the hERG channel can lead to cardiac dysrhythmias, but not all hERG blockers are proarrhythmic. We investigated the possibility that dissociation of a blocker from the channel could affect its proarrhythmic potential, using 4 compounds, 2 known proarrhythmics, terfenadine and astermizole and 2 blockers that have no history, or only a few reports of proarrhythmia, verapamil and fluoxetine. HEK 293 cells, stably transfected with HEK cDNA were held at −80 mV, pulsed to 20 mV, then stepped back to −50 mV for repetitive pulses over 6 min. as drug, in a concentration known to cause 50–90% inhibition was washed in. The voltage protocol was immediately repeated during a similar period of drug washout. Current recovery was complete with verapamil and fluoxetine, but full recovery was not achieved after treatment and washout of terfenadine or astemizole, even after 20 min. of washout with control solution, suggesting drug trapping within the channel. To test the voltage dependence of recovery from block, I-V relationships were measured; in control, in the presence of drug and after drug washout. Current recovery from fluoxetine and verapamil was complete at every voltage. Current recovery from terfenadine and astemizole showed voltage dependence with the greatest block at positive voltages. It is possible that the combination of drug trapping within the channel, and a greater block at positive voltages contributes to the proarrhythmic potential of terfenadine and astemizole.