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

Abstract

Compound effects on cloned human Ether-à-go-go related gene (hERG) potassium channels have been used to assess the potential cardiac safety liabilities of drug development candidate compounds. In addition to radioactive ligand displacement tests, two other common approaches are surrogate ion-based flux assays and electrophysiological recordings. The former has much higher throughput, whereas the latter measures directly the effects on ionic currents. Careful characterization in earlier reports has been performed to compare the relative effectiveness of these approaches for known hERG blockers, which often yielded good overall correlation. However, cases were reported showing significant and reproducible differences in potency and/or sensitivity by the two methods. This raises a question concerning the rationale and criteria on which an assay should be selected for evaluating unknown compounds. To provide a general basis for considering assays to profile large compound libraries for hERG activity, we have conducted parallel flux and electrophysiological analyses of 2,000 diverse compounds, representative of the 300,000 compound collection of NIH Molecular Library Small Molecular Repository (MLSMR). Our results indicate that at the conventional testing concentration 1.0 mM, the overlap between the two assays ranges from 32% to 50% depending on the hit selection criteria. There was a noticeable rate of false negatives by the thallium-based assay relative to electrophysiological recording, which may be greatly reduced under modified comparative conditions. As these statistical results identify a preferred method for cardiac safety profiling of unknown compounds, they suggest an efficient method combining flux and electrophysiological assays to rapidly profile hERG liabilities of large collection of naive compounds.

Antimalarial Efficacy of Hydroxyethylapoquinine (SN-119) and Its Derivatives

“…hERG channels were stably expressed in Chinese hamster ovary (CHO) cells which were held at Ϫ70 mV, and hERG currents were evoked by two voltage pulses (27). During the first pulse, cells were depolarized to ϩ40 mV for 2 s and hyperpolarized to Ϫ30 mV for 2 s. This was repeated after a 3-s hold at Ϫ70 mV.…”

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

“…We used a high-throughput fluorescence-based Tl ϩ influx assay (16,18), to test our hypothesis that a small chemical agent could modulate the sensitivity of the hERG channel to a known inhibitor. To this end we screened a library of compounds for agents that increased the IC 70 of dofetilide, a well characterized hERG inhibitor (21,22) in heterologous cells that stably expressed hERG currents.…”

“…Thallium-based Fluorescence Assay for High-throughput Screen-Assay development and high throughput screening were carried out according to the procedures detailed in AID 1511 (pubchem.ncbi.nlm.nih.gov) and published earlier (18).…”

hERGCentral: A Large Database to Store, Retrieve, and Analyze Compound-Human Ether-à-go-go Related Gene Channel Interactions to Facilitate Cardiotoxicity Assessment in Drug Development

“…The Molecular Libraries Small Molecule Repository (MLSMR) library was screened, which consisted of 318,950 compounds in 384-well plate format as single compound concentration at 5 mM in dimethyl sulfoxide (DMSO) (additional detail describing the library can be obtained from http://mlsmr.glpg.com). The automated patch clamp assay on hERG channel has been reported earlier by Zou et al 4 Briefly, compound effects were tested using dual compound additions at 1 mM and subsequently at 10 mM. The Chinese hamster ovary (CHO) cells stably expressing hERG channels were freshly dislodged from flasks and dispensed into a 384-well PPC plate.…”