TY - JOUR
T1 - A High-Throughput Screening Assay to Identify Drugs that Can Treat Long QT Syndrome Caused by Trafficking-Deficient KV11.1 (hERG) VariantsS
AU - Egly, Christian L.
AU - Blackwell, Daniel J.
AU - Schmeckpeper, Jeffrey
AU - Delisle, Brian P.
AU - Weaver, C. David
AU - Knollmann, Björn C.
N1 - Publisher Copyright:
© 2022 by The American Society.
PY - 2022/4/1
Y1 - 2022/4/1
N2 - Loss-of-function (LOF) variants in the KV11.1 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 KV11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to the identification of drug candidates. The thallium (Tl1) flux assay technique, widely used for drug screening, was optimized using human embryonic kidney (HEK-293) cells expressing a trafficking-deficient KV11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z') 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. The combination of: 1) truncating the trafficking-deficient variant KV11.1-G601S (KV11.1-G601S-G965*X) with the addition of 2) KV11.1 channel activator (VU0405601) and 3) cesium (Cs1) to the Tl1 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 KV11.1 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 KV11.1 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 KV11.1 variants.
AB - Loss-of-function (LOF) variants in the KV11.1 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 KV11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to the identification of drug candidates. The thallium (Tl1) flux assay technique, widely used for drug screening, was optimized using human embryonic kidney (HEK-293) cells expressing a trafficking-deficient KV11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z') 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. The combination of: 1) truncating the trafficking-deficient variant KV11.1-G601S (KV11.1-G601S-G965*X) with the addition of 2) KV11.1 channel activator (VU0405601) and 3) cesium (Cs1) to the Tl1 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 KV11.1 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 KV11.1 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 KV11.1 variants.
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U2 - 10.1124/molpharm.121.000421
DO - 10.1124/molpharm.121.000421
M3 - Article
C2 - 35125346
AN - SCOPUS:85127092925
SN - 0026-895X
VL - 101
SP - 236
EP - 245
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 4
ER -