A High-Throughput Screening Assay to Identify Drugs that Can Treat Long QT Syndrome Caused by Trafficking-Deficient K(V)11.1 (hERG) Variants

Loss-of-function (LOF) variants in the K(V)11.1 potassium channel cause long QT syndrome (LQTS). Most variants disrupt intracellular channel transport (trafficking) to the cell membrane. Since some channel inhibitors improve trafficking of K(V)11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to the identification of drug candidates. The thallium (Tl+) flux assay technique, widely used for drug screening, was optimized using human embryonic kidney (HEK-293) cells expressing a trafficking-deficient K(V)11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z') scores comparing vehicle to E-4031, a drug that increases K(V)11.1 membrane trafficking. The optimized assay was validated by immunoblot, electrophysiology experiments, and a pilot drug screen. The combination of: 1) truncating the trafficking-deficient variant K(V)11.1-G601S (K(V)11.1-G601S-G965*X) with the addition of 2) K(V)11.1 channel activator (VU0405601) and 3) cesium (Cs+) to the Tl+ flux assay buffer resulted in an outstanding Z' of 0.83. To validate the optimized trafficking assay, we carried out a pilot screen that identified three drugs (ibutilide, azaperone, and azelastine) that increase K(V)11.1 trafficking. The new assay exhibited 100% sensitivity and specificity. Immunoblot and voltage-clamp experiments confirmed that all three drugs identified by the new assay improved membrane trafficking of two additional LQTS K(V)11.1 variants. We report two new ways to increase target-specific activity in trafficking assays-genetic modification and channel activation-that yielded a novel HTS assay for identifying drugs that improve membrane expression of pathogenic K(V)11.1 variants. SIGNIFICANCE STATEMENT This manuscript reports the development of a high-throughput assay (thallium flux) to identify drugs that can increase function in K(V)11.1 variants that are trafficking-deficient. Two key aspects that improved the resolving power of the assay and could be transferable to other ion channel trafficking-related assays include genetic modification and channel activation.