A Pharmacologically Validated, High-Capacity, Functional Thallium Flux Assay for the Human Ether-à-go-go Related Gene Potassium Channel

Abstract

The voltage-gated potassium channel, human Ether-à-go-go related gene (hERG), represents the molecular component of IKr, one of the potassium currents involved in cardiac action potential repolarization. Inhibition of IKr increases the duration of the ventricular action potential, reflected as a prolongation of the QT interval in the electrocardiogram, and increases the risk for potentially fatal ventricular arrhythmias. Because hERG is an appropriate surrogate for IKr, hERG assays that can identify potential safety liabilities of compounds during lead identification and optimization have been implemented. Although the gold standard for hERG evaluation is electrophysiology, this technique, even with the medium capacity, automated instruments that are currently available, does not meet the throughput demands for supporting typical medicinal chemistry efforts in the pharmaceutical environment. Assays that could provide reliable molecular pharmacology data, while operating in high capacity mode, are therefore desirable. In the present study, we describe a high-capacity, 384- and 1,536-well plate, functional thallium flux assay for the hERG channel that fulfills these criteria. This assay was optimized and validated using different structural classes of hERG inhibitors. An excellent correlation was found between the potency of these agents in the thallium flux assay and in electrophysiological recordings of channel activity using the QPatch automated patch platform. Extension of this study to include 991 medicinal chemistry compounds from different internal drug development programs indicated that the thallium flux assay was a good predictor of in vitro hERG activity. These data suggest that the hERG thallium flux assay can play an important role in supporting drug development efforts.

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

“…S7B strategy has been gradually questioned as an inaccurate surrogate for TdP risk in the last decade. Thus the comprehensive in vitro proarrhythmia assay (CiPA), a combo-cardiac safety assay, has been proposed to evaluate proarrhythmic risk for candidate drugs [8] . Other six human cardiac ion channels [I Na (I NaF , I NaL ), I to , I CaL , I Ks and I K1 ] present in ventricular myocardium are suggested to join hERG as first CiPA assays [8] .…”

“…Thus the comprehensive in vitro proarrhythmia assay (CiPA), a combo-cardiac safety assay, has been proposed to evaluate proarrhythmic risk for candidate drugs [8] . Other six human cardiac ion channels [I Na (I NaF , I NaL ), I to , I CaL , I Ks and I K1 ] present in ventricular myocardium are suggested to join hERG as first CiPA assays [8] . To comply with this, authors would suggest that the hERG liability compounds should be further tested in other cardiac ion channel panels to predict their potential proarrythmic potential.…”

“…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] .…”

“…Ion flux assays with surrogate ions of Rubidium (Rb + ) or Thallium (Tl + ) have generated effective values for detecting potassium channel activity in high-throughput formats. However, ion flux assays are performed under less physiologically relevant conditions [7,8] [8] . Recent advance in automated electrophysiology technology has provided platforms such as IonWorks Quattro that can generate data with high information content and good quality at higher throughput [9][10][11] .…”

Investigation of Cardiovascular Effects of Tetrahydro-β-carboline sstr3 antagonists

“…A thallium flux assay was used to assess the effect of added serum to the response of the hERG channel. 25 Table 6 shows the effect of added dog serum to the ability of MK-4256 to block the response of the hERG channel. Despite being highly protein bound (dog Fu = 0.8%), MK-4256 exhibited no serum shift.…”