hERG (human Ether-a-go-go-Related Gene) is a gene encoding alpha subunit of potassium channel. This channel is responsible for the repolarizing current in cardiac cycle. The inhibition of the channel or mutations may cause the adverse condition called long QT syndrome. In the past few years, several drugs have been withdrawn from the market due to their interaction with certain ion channels that may cause life-threatening arrhythmias and sudden cardiac death. Invitrogen’s Predictor™ hERG Fluorescence Polarization Assay provides valuable information about possible binding of test compounds to hERG channel followed by QT prolongation on echocardiogram (ECG). Although manual patch-clamp testing is the gold standard for this purpose, biochemical hERG assay allows checking multiple compounds at the earliest stages of drug discovery. Screening compounds for hazardous side effects on hERG channel early in the drug development process is likely to cut the drug development costs.
We use Predictor™ hERG Fluorescence Polarisation Assay (Invitrogen, Cat# PV5365). The assay employs a membrane fraction containing hERG channel protein (Predictor™ hERG Membrane) and a high-affinity red fluorescent hERG channel ligand, or “tracer” (Predictor™ hERG Tracer Red), in a fluorescence polarization (FP)-based format. In the case a tested compound is hERG channel protein binder, it competes for binding with the tracer and, as a result, decreases polarization of plane-polarized light. The decrease of fluorescence polarisation is measured using Tecan ULTRA microplate reader. Typically, three dilutions of the tested compounds are assessed: 1 µM, 5 µM and 20 µM. All concentration points are tested in duplicates. The set of controls (Buffer Blank, Assay blank, Free Tracer Control, Positive control, Reference control) is assayed in 6 repeats each in each experimental batch. The positive control (compound E-4031) is used at 30 µM concentration. The results are expressed as % of inhibition, compared to the positive control. IC50 can be determined upon customer’s request in case a significant hERG channel protein inhibition is observed.
A minimal weighted amount of dry compound, 1 mg, or 200 µL of 10mM stock DMSO solution, is required for this assay.