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Inhibition of hERG Channel Trafficking: An Under‐Explored Mechanism for Drug‐Induced QT Prolongation

Functional blockage of the cardiac potassium channel encoded by the human ether-go-go-related gene (hERG or KCNH2) is responsible for the rapidly activating component of the delayed rectifier K current (Ikr), which can delay ventricular cell repolarization. As a result of this, the QT interval on an electrocardiogram is prolonged, and can potentially lead to lethal cardiac arrhythmias. This serious cardiotoxicity has resulted in the withdrawal of approved drugs from the market, and discontinuation of clinical trials on development candidates. Drugs that cause long QT syndrome (LQTS) generally exert a direct blockade of the hERG channel by binding to a common site defined by the key residues Tyr652 and Phe656 in the S6 pore domain of the hERG channel. As measured by the in vitro patch clamp technique, acute inhibition of the Ikr current and its recovery are essentially instantaneous, with blockade and relief occurring within minutes. However, a number of drugs, including the commonly prescribed azole antifungal ketoconazole and the antidepressant fluoxetine, a selective serotonin reuptake inhibitor (Figure 1), have recently been shown to induce LQTS by an indirect, chronic inhibitory mechanism that reduces hERG channel expression at the cell surface. It is now apparent that most of the genetic mutations in hERG that cause LQTS do so by this indirect mechanism, denoted as a class 2 effect. Ketoconazole acutely inhibits the Ikr current in hERG-expressing human embryonic kidney (hERG-HEK 293) cells with an IC50 value of 1.92 mm. As revealed by Western blot analysis, and by peak tail current measurement, ketoconazole also reduced cell surface expression of the mature, fully glycosylated 155 kD hERG channel after 48 h of incubation, with 50% inhibition observed at concentrations above 10 mm. A greater than 50% reduction in cell surface expression of the hERG channel persisted for up to 48 h after incubation with 30 mm of ketoconazole, and recovery was slow after drug washout, with about 74% of the channels restored after 48 h. The direct and indirect inhibition of the hERG channel by ketoconazole are independent of each other but the effect is additive. Similarly, the IC50 values determined for the acute inhibition of the Ikr current in hERG-HEK 293 cells by fluoxetine was 0.7 mm, while that for cell surface expression, as measured by Western blot image density, was 2.7 mm. Norfluoxetine, a metabolite of fluoxetine, was also active in these assays although two to threefold less potent. Following treatment with 30 mm fluoxetine, the reduction of cell surface expression of hERG and recovery took hours, with 100% restoration occurring at ~24 h after drug washout. Ketoconazole and fluoxetine are thus categorized as dual mode inhibitors of the hERG channel, a property common to many acute inhibitors. Preliminary results using an antibody-based chemiluminescent assay (HERG-Lite), which measures the cell surface expression of hERG in HEK 293 cells, showed that 20 known direct hERG inhibitors, representing 40% of those tested, also exhibited the indirect mechanism of inhibition that leads to reduced cell surface expression, including: diphenhydramine, terfenadine, tamoxifen, chlorpromazine and lovastatin. However, there are examples of hERG inhibitors that exert their action only by reducing cell surface expression of the protein, most prominently pentamidine, an antiprotozoal agent used for treating Pneumocystis carinii pneumonia, and probucol, a cholesterol-lowering agent (Figure 2). Significantly, pentamidine and probucol have also been shown to reduce Ikr tail current, and prolong the action potential duration in guinea pigs and neonatal rat ventricular myocytes via this indirect mechanism, Figure 1. Examples of dual mode inhibitors that directly block hERG channels and also reduce hERG channel expression at the cell surface.