Development of the Predictor hERG Fluorescence Polarization Assay Using a Membrane Protein Enrichment Approach

Abstract

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.

Novel cell-free high-throughput screening method for pharmacological tools targeting K + channels

“…Our ability to predict which small molecules will block hERG is poor and therefore all compounds under consideration for drug development are tested in a hERG activity assay. Several assays exist: cell-based functional assays, such as thallium and/or rubidium flux, nonfunctional assays, such as ligand binding and/or fluorescence polarization, and electrophysiology (22,23,31,32). The gold standard is electrophysiology, but it can be slow and expensive when the need exists for evaluating many compounds.…”

Preclinical Drug Safety and Cardiac Ion Channel Screening

“…For example, a homogeneous, fluorescence polarization-based assay to characterize the affinity of small molecules for the hERG channel was recently reported [40]. This assay has demonstrated tight correlation with data obtained from patch-clamp assays.…”

Identification of Selective ERRγ Inverse Agonists

“…Fluorescence intensity (Excitation at 530 nm, Emission at 590 nm) was measured using a multi-mode microplate reader Synergy Neo (Biotek, Winooski, VT, USA). E-4031 was used as the reference positive standard (IC 50 = 10-90 nM) [20].…”