| Assay Method Information | |
| | hERG Inhibition |
| Description: | Studies were conducted at Cyprotex Discovery in Cheshire, UK according to the contractor's protocol. The studies were performed on an IonWorks HT instrument (Molecular Devices Corporation), which automatically performs electrophysiology measurements in 48 single cells simultaneously in a specialised 384-well plate (PatchPlate ). The cells used were Chinese hamster ovary (CHO) cells stably transfected with hERG (cell-line obtained from Cytomyx, UK). A single-cell suspension was prepared in extracellular solution (Dulbecco's phosphate buffered saline with calcium and magnesium pH 7-7.2) and aliquots added automatically to each well of a PatchPlate . The cells were then positioned over a small hole at the bottom of each well by applying a vacuum beneath the plate to form an electrical seal. The vacuum was applied through a single compartment common to all wells which was filled with intracellular solution (buffered to pH 7.2 with HEPES). The resistance of each seal was measured via a common ground-electrode in the intracellular compartment and individual electrodes placed into each of the upper wells.Electrical access to the cell was then achieved by circulating a perforating agent, amphotericin, underneath the PatchPlate and then measuring the pre-compound hERG current. An electrode was positioned in the extracellular compartment and a holding potential of −80 mV applied for 15 seconds. The hERG channels were then activated by applying a depolarising step to +40 mV for 5 seconds and then clamped at −50 mV for 4 seconds to elicit the hERG tail current, before returning to −80 mV for 0.3 seconds. Test compound was then added automatically to the upper wells of the PatchPlate from a 96-well microtitre plate containing a range of concentrations of compound TBAP-01. Quinidine, an established hERG inhibitor, was included as an experimental control. TBAP-01 was dissolved in DMSO and assayed at final concentrations ranging from 100 μM to 32 nM in 0.25% DMSO. Buffer containing 0.25% DMSO was included as a negative control. The test compound was left in contact with the cells for 300 seconds before recording currents using the same voltage-step protocol as in the pre-compound scan. Each concentration was tested in 4 replicate wells. |
| Affinity data for this assay | |
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