Rb+ Flux through hERG Channels Affects the Potency of Channel Blocking Drugs: Correlation with Data Obtained Using a High-Throughput Rb+ Efflux Assay

Abstract

The nonradioactive Rb+ efflux assay has become a reliable and efficient high-throughput hERG screening method, but it is limited by its low sensitivity for potent hERG blockers. Using the patch clamp technique, the authors found that the low sensitivity is due in part to the use of Rb+ as the permeating cation in the assay. The affinities of the drugs measured by patch clamp technique in the presence of Rb+ were 3- to 10-fold lower than when measured by the same method in the presence of K+ ions. The apparent affinity of the drugs decreased even further when monitored bytheRb+ efflux assay. It was also observed that Rb+ had minimal effects on the activation properties of channels while there was a significant change in the half-inactivation potential. This voltage shift reduces hERG channel inactivation at efflux assay potentials, and will reduce the affinity of hERG-blocking drugs that bind to inactivated states of the channel. In combination with the effects of elevated extracellular ion concentrations, it is likely that Rb+ modulation of hERG channel inactivation is largely responsible for the reduced drug potencies observed in the Rb+ efflux assay.

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

“…This has been proposed to contribute to a right shift of IC 50 values for known compounds inhibiting hERG potassium channels. 40 In this case, using a higher concentration of testing compounds may yield a better overlap of hits identified by two different assays. Analysis of the Tl þ assay hits identified at 10 mM testing compound concentration ( Table 1 and Supplementary Fig.…”

“…Second, based on studies using known inhibitors of hERG potassium channels, it is clear that different compounds display varying degrees of right shift of potency, as large as 100-fold, in surrogate ion-based flux assays. 40 Therefore, the structural features of testing compounds may differentially affect the inhibitory potency in each assay. This factor may be especially more pronounced for hERG than for other channels because hERG is inhibited by a variety of structural classes.…”

Utility of hERG Assays as Surrogate Markers of Delayed Cardiac Repolarization and QT Safety

“…TOXICOLOGIC PATHOLOGY Another indirect approach employed to detect hERG channel-drug interactions is the ion flux assay. This assay measures reduced levels of radiolabeled Rb + efflux (reflecting diminished outward K + current through hERG channels) from hERG-transfected cells (Rezazadeh et al, 2004). Voltage-sensitive dyes have also been used to measure druginduced membrane depolarization of hERG transfected cells in which hERG/I Kr is the primary potassium current responsible for setting the resting membrane potential (Netzer et al, 2003).…”

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] .…”