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 Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition.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

Profiling Diverse Compounds by Flux- and Electrophysiology-Based Primary Screens for Inhibition of Human Ether-à-go-go Related Gene Potassium Channels

“…That question has been addressed in a number of publications using IC 50 values calculated from small sets of compounds typically with defined hERG-blocking properties (e.g., refs. 11,29 ). The data presented here provide more relevant argument for the importance of electrophysiological assays using statistical data from a much larger collection of naïve compounds.…”

“…[19][20][21] The primary approach has involved the use of surrogate ions such as rubidium (Rb þ ) or thallium (Tl þ ), which may be detected by atomic absorption spectrometry 22,23 or fluorescent dyes. 8,10,11 With the advent of automated patch-clamps, 6,[24][25][26][27] it is now possible to consider whether the more direct measurements of channel electrophysiological activity obtained from the automated patch-clamp justifies its higher implementation cost in high-throughput screens. It is therefore important to obtain more specific and quantitative information to assess the cost and benefit comparatives between assays employing electrophysiological methods versus surrogate ion flux measurements.…”

“…The former is usually carried out by taking advantage of fluorescent dyes that selectively bind to the targeted ions. 8,10,11 The latter is often based on the membrane potential changes following ion fluxes. 12,13 Using surrogate ionic fluxes, several large HTS campaigns have been recently described in published reports 14,15 and in a public database (PubChem AIDs 1456, 1511, 1672, 1918, 2156, and 2239).…”

“…The described assay conditions are comparable to what has been reported by others, as judged by IC 50 values. 11,29 The resultant hits from these screens under different selective criteria were analyzed and compared to assess these two assays. This approach differs from previously published analyses of hERG assays in which comparison of IC 50 values of mostly known hERG blockers was performed to identify the preferred approach for safety evaluation and compound optimization.…”

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay

“…However those models have not yet produced satisfactory predictions given limited compounds and differential data quality caused by variations in detection methodologies. Several high-throughput screening approaches have been established for identification of hERG modulators, including ion flux assay (Rb + ) and fluorescence-based assay (Tl + ), etc [8,[35][36][37][38] . Although these assays could identify hERG modulators effectively and showed better correlation with electrophysiological assays, the interferences from the parental cells may interact with compounds, and thus cause higher false-positive or falsenegative hit rate [7] .…”