Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 236-245 |
| Number of pages | 10 |
| Journal | Molecular Pharmacology |
| Volume | 101 |
| Issue number | 4 |
| DOIs | |
| State | Published - Apr 1 2022 |
Bibliographical note
Publisher Copyright:© 2022 by The American Society.
Funding
This work was supported in part by National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI) [Grant R35-HL144980] (B.C.K.), [Grant T32-5T32GM007569-44] (B.C.K., C.L.E.), Shared Instrumentation [Grant 1S10OD021734] (Panoptic), and NHLBI [Grant F32-HL140874] (D.J.B.); the Leducq Foundation [Grant 18CVD05] (B.C.K.); the American Heart Association [Grant 19SFRN34830019] (B.C.K.); and a PhRMA Foundation Postdoctoral Award (C.L.E.). Thallium flux experiments were performed in the Vanderbilt High-Throughput Screening (HTS) Core Facility with assistance provided by Corbin Whitwell, David Westover, and Debbie Mi. The HTS Core receives support from the Vanderbilt Institute of Chemical Biology and the Vanderbilt Ingram Cancer Center (P30 CA68485). Special thanks to Eric Delpire for help with molecular biology and RACE protocols. HEK-293 cells expressing N470D and G601S were kindly donated from Craig January and Brett Kroncke respectively.
| Funders | Funder number |
|---|---|
| Vanderbilt Institute of Chemical Biology | |
| National Institutes of Health (NIH) | |
| National Heart, Lung, and Blood Institute (NHLBI) | 1S10OD021734, F32-HL140874, R35-HL144980 |
| National Heart, Lung, and Blood Institute (NHLBI) | |
| National Institute of General Medical Sciences | T32GM007569 |
| National Institute of General Medical Sciences | |
| American the American Heart Association | 19SFRN34830019 |
| American the American Heart Association | |
| Pharmaceutical Research and Manufacturers of America Foundation | |
| Vanderbilt Ingram Cancer Center | P30 CA68485 |
| Vanderbilt Ingram Cancer Center | |
| Fondation Leducq | 18CVD05 |
| Fondation Leducq |
ASJC Scopus subject areas
- Molecular Medicine
- Pharmacology