Elsevier

Analytical Biochemistry

Volume 394, Issue 1, 1 November 2009, Pages 30-38
Analytical Biochemistry

A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel

https://doi.org/10.1016/j.ab.2009.07.003Get rights and content

Abstract

Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.

Section snippets

Materials

BacMam hERG, FluxOR thallium assay kit, and cell culture reagents (Dulbecco’s modified Eagle’s medium [DMEM], Opti-MEM, penicillin/streptomycin, and TrypLE) were purchased from Invitrogen (Carlsbad, CA, USA). U-2 OS, CKO-K1, HEK293, and HeLa cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). The LOPAC 1280 library collection was purchased from Sigma–Aldrich (St. Louis, MO, USA). hERG

Assay principle

Thallium ions are used as surrogate ions in this assay to measure activity of the hERG potassium channel. This is possible because of the selective permeability of all potassium ion channels for thallium and the strong driving force for thallium entry into the cells when the channels are opened. In brief, FluxOR dye is loaded into the cells prior to the experiment (Fig. 1A). FluxOR dye contains aminomethyl (AM) ester groups that render the molecule membrane permeant from the extracellular

Discussion

Unlike other ion channels that interact only with ligands of specific structural classes, the hERG potassium ion channel can be blocked or modulated by a broad spectrum of structurally diverse compounds. Thus, the ligand binding assay has clear limitations on the assessment of compound activity at hERG channels because competition with a labeled ligand is limited to structurally similar compounds. This means that binding/displacement assays that pick up compounds occupying the same binding site

Acknowledgments

This research was supported by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research and the Intramural Research Programs of the National Human Genome Research Institute. The authors thank Paul Shinn for assistance with compound management. The authors also thank Shouming Du of Hamamatsu for technical support with the FDSS 7000 kinetic plate reader; Michael O’Grady, Matt Robers, and George Hanson of Invitrogen/Life Technologies and Blake Anson of Cellular Dynamics for

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