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

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 Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition.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.

SUBMITTER: Titus SA

PROVIDER: S-EPMC2766802 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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

Titus Steven A SA Beacham Daniel D Shahane Sampada A SA Southall Noel N Xia Menghang M Huang Ruili R Hooten Elizabeth E Zhao Yong Y Shou Louie L Austin Christopher P CP Zheng Wei W

Analytical biochemistry 20090705 1

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 inhibiti ...[more]

PMID: 19583963

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Project description:The human ether-à-go-go-1 (h-eag1) K(+) channel is expressed in a variety of cell lines derived from human malignant tumors and in clinical samples of several different cancers, but is otherwise absent in normal tissues. It was found to be necessary for cell cycle progression and tumorigenesis. Specific inhibition of h-eag1 expression leads to inhibition of tumor cell proliferation. We report here that h-eag1 expression is controlled by the p53-miR-34-E2F1 pathway through a negative feed-forward mechanism. We first established E2F1 as a transactivator of h-eag1 gene through characterizing its promoter region. We then revealed that miR-34, a known transcriptional target of p53, is an important negative regulator of h-eag1 through dual mechanisms by directly repressing h-eag1 at the post-transcriptional level and indirectly silencing h-eag1 at the transcriptional level via repressing E2F1. There is a strong inverse relationship between the expression levels of miR-34 and h-eag1 protein. H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity. Therefore, p53 negatively regulates h-eag1 expression by a negative feed-forward mechanism through the p53-miR-34-E2F1 pathway. Inactivation of p53 activity, as is the case in many cancers, can thus cause oncogenic overexpression of h-eag1 by relieving the negative feed-forward regulation. These findings not only help us understand the molecular mechanisms for oncogenic overexpression of h-eag1 in tumorigenesis but also uncover the cell-cycle regulation through the p53-miR-34-E2F1-h-eag1 pathway. Moreover, these findings place h-eag1 in the p53-miR-34-E2F1-h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1. Our study therefore fills the gap between p53 pathway and its cellular function mediated by h-eag1.

Dataset Information

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

Publications

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

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