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Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking.

ABSTRACT: The antiarrhythmic drug amiodarone delays cardiac repolarisation through inhibition of hERG-encoded potassium channels responsible for the rapid delayed rectifier potassium current (IKr). This study aimed to elucidate molecular determinants of amiodarone binding to the hERG channel. Whole-cell patch-clamp recordings were made at 37°C of ionic current (IhERG) carried by wild-type (WT) or mutant hERG channels expressed in HEK293 cells. Alanine mutagenesis and ligand docking were used to investigate the roles of pore cavity amino-acid residues in amiodarone binding. Amiodarone inhibited WT outward IhERG tails with a half-maximal inhibitory concentration (IC50) of ?45nM, whilst inward IhERG tails in a high K(+) external solution ([K(+)]e) of 94mM were blocked with an IC50 of 117.8nM. Amiodarone's inhibitory action was contingent upon channel gating. Alanine-mutagenesis identified multiple residues directly or indirectly involved in amiodarone binding. The IC50 for the S6 aromatic Y652A mutation was increased to ?20-fold that of WT IhERG, similar to the pore helical mutant S624A (?22-fold WT control). The IC50 for F656A mutant IhERG was ?17-fold its corresponding WT control. Computational docking using a MthK-based hERG model differentiated residues likely to interact directly with drug and those whose Ala mutation may affect drug block allosterically. The requirements for amiodarone block of aromatic residues F656 and Y652 within the hERG pore cavity are smaller than for other high affinity IhERG inhibitors, with relative importance to amiodarone binding of the residues investigated being S624A?Y652A>F656A>V659A>G648A>T623A.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC4959829 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking.

Zhang Yihong Y Colenso Charlotte K CK El Harchi Aziza A Cheng Hongwei H Witchel Harry J HJ Dempsey Chris E CE Hancox Jules C JC

Biochemical pharmacology 20160530

The antiarrhythmic drug amiodarone delays cardiac repolarisation through inhibition of hERG-encoded potassium channels responsible for the rapid delayed rectifier potassium current (IKr). This study aimed to elucidate molecular determinants of amiodarone binding to the hERG channel. Whole-cell patch-clamp recordings were made at 37°C of ionic current (IhERG) carried by wild-type (WT) or mutant hERG channels expressed in HEK293 cells. Alanine mutagenesis and ligand docking were used to investigat ...[more]

PMID: 27256139

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Project description:Background: In the absence of SARS-CoV-2 specific antiviral treatments, various repurposed pharmaceutical approaches are under investigation for the treatment of COVID-19. Antiviral drugs considered for this condition include atazanavir, remdesivir, lopinavir-ritonavir, and favipiravir. Whilst the combination of lopinavir and ritonavir has been previously linked to prolongation of the QTc interval on the ECG and risk of torsades de pointes arrhythmia, less is known in this regard about atazanavir, remdesivir, and favipiravir. Unwanted abnormalities of drug-induced QTc prolongation by diverse drugs are commonly mediated by a single cardiac anti-target, the hERG potassium channel. This computational modeling study was undertaken in order to explore the ability of these five drugs to interact with known determinants of drug binding to the hERG channel pore. Methods: Atazanavir, remdesivir, ritonavir, lopinavir and favipiravir were docked to in silico models of the pore domain of hERG, derived from cryo-EM structures of hERG and the closely related EAG channel. Results: Atazanavir was readily accommodated in the open hERG channel pore in proximity to the S6 Y652 and F656 residues, consistent with published experimental data implicating these aromatic residues in atazanavir binding to the channel. Lopinavir, ritonavir, and remdesivir were also accommodated in the open channel, making contacts in a model-dependent fashion with S6 aromatic residues and with residues at the base of the selectivity filter/pore helix. The ability of remdesivir (at 30 μM) to inhibit the channel was confirmed using patch-clamp recording. None of these four drugs could be accommodated in the closed channel structure. Favipiravir, a much smaller molecule, was able to fit within the closed channel and could adopt multiple binding poses in the open channel, but with few simultaneous interactions with key binding residues. Only favipiravir and remdesivir showed the potential to interact with lateral pockets below the selectivity filter of the channel. Conclusions: All the antiviral drugs studied here can, in principle, interact with components of the hERG potassium channel canonical binding site, but are likely to differ in their ability to access lateral binding pockets. Favipiravir's small size and relatively paucity of simultaneous interactions may confer reduced hERG liability compared to the other drugs. Experimental structure-function studies are now warranted to validate these observations.

Project description:BACKGROUND AND PURPOSE: Amiodarone (2-n-butyl-3-[3,5 diiodo-4-diethylaminoethoxybenzoyl]-benzofuran, B2-O-CH(2)CH(2)-N-diethyl) is an effective class III antiarrhythmic drug demonstrating potentially life-threatening organ toxicity. The principal aim of the study was to find amiodarone analogues that retained human ether-a-go-go-related protein (hERG) channel inhibition but with reduced cytotoxicity. EXPERIMENTAL APPROACH: We synthesized amiodarone analogues with or without a positively ionizable nitrogen in the phenolic side chain. The cytotoxic properties of the compounds were evaluated using HepG2 (a hepatocyte cell line) and A549 cells (a pneumocyte line). Interactions of all compounds with the hERG channel were measured using pharmacological and in silico methods. KEY RESULTS: Compared with amiodarone, which displayed only a weak cytotoxicity, the mono- and bis-desethylated metabolites, the further degraded alcohol (B2-O-CH(2)-CH(2)-OH), the corresponding acid (B2-O-CH(2)-COOH) and, finally, the newly synthesized B2-O-CH(2)-CH(2)-N-pyrrolidine were equally or more toxic. Conversely, structural analogues such as the B2-O-CH(2)-CH(2)-N-diisopropyl and the B2-O-CH(2)-CH(2)-N-piperidine were significantly less toxic than amiodarone. Cytotoxicity was associated with a drop in the mitochondrial membrane potential, suggesting mitochondrial involvement. Pharmacological and in silico investigations concerning the interactions of these compounds with the hERG channel revealed that compounds carrying a basic nitrogen in the side chain display a much higher affinity than those lacking such a group. Specifically, B2-O-CH(2)-CH(2)-N-piperidine and B2-O-CH(2)-CH(2)-N-pyrrolidine revealed a higher affinity towards hERG channels than amiodarone. CONCLUSIONS AND IMPLICATIONS: Amiodarone analogues with better hERG channel inhibition and cytotoxicity profiles than the parent compound have been identified, demonstrating that cytotoxicity and hERG channel interaction are mechanistically distinct and separable properties of the compounds.

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Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking.

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Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking.

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