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P11031Aequorin Plasmids A Novel Tool for Generating Cell Lines for Luminescence Assays
Presenter Sandra Flores, PerkinElmer, Belgium
Additional Authors: Beatrice Goxe, Vincent Dupriez, Patricia Buchlin, Fanny Huberty, Stephane Parent, Janet Park
The potential for cardiotoxic side-effects associated with new chemical entities continues to challenge the development of small molecule based therapies. In many cases, these intolerable side-effects are precipitated by acquired (or drug-induced) long QT syndrome (LQT) which can lead to the characteristic ventricular arrhythmia known as Torsade de Pointes and ultimately sudden death.
A mechanistic link between LQT syndrome and loss of function in the human ether-a-go-go related gene (hERG) potassium channel has been appreciated for over a decade. Although patch-clamp electrophysiology remains the gold standard for determining the interaction of compounds with the function of the hERG channel, the cost of these assays remains high. However, because the bulk of the compounds that block the hERG channel do so by interaction with two rings of aromatic residues (Tyr652 and Phe656) near the inner cavity of the channel (Mitcheson et al, 2000; Fernadez et al, 2004), radioligand displacement assays have proven to be a cost-effective initial funnel for hERG channel liability at early stages of compound development. Speed and cost still suffer however as radioligand displacement assays remain heterogeneous and depend on the procurement and disposal costs of radioligands. We describe here the tools and methodology required to build a HT assay for rapid determination of hERG channel affinity based on fluorescent polarization principles. A number of known high-affinity blockers for the hERG channel provides a wide choice of scaffolds for the fluorescent tracer molecule. The limiting factor in the development of this assay is the expression of high levels of hERG channel protein in a recombinant cell line. Here we describe our approach to generate, identify and subclone a cell line that expresses sufficiently high levels of hERG channel protein. By using membrane preparations derived from this cell line, we have examined a series of candidate fluorescent hERG-blocking compounds and enabled a fluorescent polarization assay. This assay has identified the IC50 values of known hERG channel blockers within a 3-fold range of the published values determined by patch-clamp recordings. Additional optimization of the typical parameters (buffers, incubation time, assay plate) has provided a HT solution suitable for rapid determination of hERG channel affinity.