A High-throughput Assay to Identify Drugs that can Treat Long QT Syndrome Caused by Trafficking-deficient KV11.1 (hERG) Variants

Background and Purpose: Loss-of-function (LOF) variants in the K_V11.1 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_V11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to identify drug candidates. Methods: 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_V11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z9) scores comparing vehicle to E-4031, a drug that increases Kv11.1 membrane trafficking. The optimized assay was validated by immunoblot, electrophysiology experiments, and a pilot drug screen. Results: The combination of: 1) truncating the trafficking-deficient variant K_V11.1-G601S (K_V11.1-G601S-G965*X), and addition of 2) K_V11.1 channel activator (VU0405601) and 3) cesium (Cs⁺) to the Tl⁺ flux assay buffer resulted in an outstanding Z9 of 0.83. To validate the optimized trafficking assay, we carried out a pilot screen that identified three drugs (ibutilide, azaperone, and azelastine) as drugs that increase K_V11.1 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_V11.1 variants. Conclusion: We report two new ways to increase target-specific activity in trafficking assays - genetic modification and channel activation - which yielded a novel HTS assay for identifying drugs that improve membrane expression of pathogenic K_V11.1 variants. Significance Statement This paper involves the development of an HTS method to rapidly discover new drugs that increase membrane trafficking of LQTS K_V11.1 variants. 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.