|Primary cell-based high-throughput screening assay for identification of compounds that inhibit KCNQ1 potassium channels - BioAssay Summary
Assay Implementation: Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Haibo Yu Ph.D., Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
BioActive Compounds: 3878
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Meng Wu, Ph.D.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH090837-01
Grant Proposal PI: Meng Wu, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Haibo Yu Ph.D., Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
HTS execution: Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Kaiping Xu M.S., Meng Wu Ph.D.
Name: Primary cell-based high-throughput screening assay for identification of compounds that inhibit KCNQ1 potassium channels
Voltage-gated potassium channels [1,2] are tetrameric membrane proteins that selectively conduct K+ across cellular membranes, thus open, close, and inactivate in response to changes in transmembrane voltage . Individual subtypes of these potassium channels often have a unique expression pattern allowing cells to "fine-tune" membrane potentials and excitability according to their respective physiological functions . Dysfunctions of these electrical excitability controlling proteins, either congenital or acquired, are attributed to a variety of diseases [5,6], such as cardiac arrhythmias, diabetes, hypertension, and epilepsy. Specific modulation of individual potassium channel types therefore represents an enormous potential for the development of physiological tool compounds and new drugs [7-9].
KCNQ1 (Kv7.1, KvLQT) [10,11] is an alpha-subunit subtype of voltage-gated KCNQ potassium channel family, which is composed of five members of KCNQ1-KCNQ5. They share between 30% and 65% amino acid identity. A classical KCNQ alpha-subunit is composed of six transmembrane segments, including a voltage-sensor segment and a pore domain [12-15]. Unique from other members of KCNQ family , KCNQ1 has been generally absent from neuronal tissues, mainly expressed in heart, kidney, small intestine, pancreas, prostate and other non-excitable epithelial tissues. Also contrast to other members of KCNQ family which form both alpha-subunit homo- and heterotetrameric channels, KCNQ1 channels only form alpha-subunit homotetramers . They commonly co-assemble with beta-subunit KCNE proteins to give rise to functional variations in different tissues.
These molecular assemblies have afforded KCNQ1 with two important physiological functions: 1) repolarization of the cardiac tissue following an action potential and 2) water and salt transport in epithelial tissues. Mutations in this gene are associated with hereditary long QT syndrome, diabetics , Romano-Ward syndrome, Jervell and Lange-Nielsen syndrome  and familial atrial fibrillation , as well as impairment of cyclic AMP-stimulated intestinal secretion of chloride ions related to cystic fibrosis [21,22] and pathological forms of secretary diarrhea [23-25]. Furthermore, drug-induced acquired KCNQ1 and KCNQ1/KCNE dysfunctions also raise concerns of KCNQ1/KCNE as potential hERG-like drug safety issue in pharmaceutical development .
For their pharmacological responses, KCNQ1/KCNE heteromultimers function differently from KCNQ1 alone. Initial discovery of KCNQ1 modulators is focused on the KCNQ1 (and KCNQ1/KCNE1 IKs) inhibitors , from earlier Chromonal 293B, linopirdine and XE991, to new family of potent inhibitors, i.e. Merck-IKs (IC50 ~0.08 nM), JNJ 303(IC50 0.064 uM) and JNJ282 (IC50 0.001 uM).
Systemic compound screens for KCNQ1 and its heteromultimers have not been reported. Here the assay, Tl+-based fluorescence assay in 384 format by FDSS, therefore, was used for the identification of inhibitory compounds acting on KCNQ1 from a large MLSMR compound library.
Principle of the assay
The Tl+ ion, which is permeable through potassium channels, serves as a surrogate for K+ flux . The thallium-sensitive dye is loaded into cells, and, in the absence of Tl+, exhibits very low basal fluorescence. Upon the addition of Tl+ onto cells expressing potassium channels, in this case, KCNQ1 potassium channel, extracellular Tl+ flux into cells through open KCNQ1 channels, and when bound to the dye, produce a fluorescent signal that is monitored in real-time by a fluorescence imaging plate reader [29, 30].
KCNQ1, HTS assay, 384, primary, antagonist, inhibitor, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN.
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The purpose of this assay is to identify compounds that inhibit KCNQ1 potassium channels. This assay employs CHO-K1 cells line that stably expresses KCNQ1 potassium channel. The cells are treated with test compounds, followed by measurement of intracellular thallium, as monitored by a commercially available thallium-sensitive fluorescent dye, FluxOR. Compound effect was evaluated by the calculated FluxOR fluorescence ratio, normalized with negative controls. If the compound has a B score less than the mean plus 3 standard deviation of the B-scores of the library compounds, the compound is then considered to be active as an inhibitor of the KCNQ1 channels.
Protocol for the KCNQ1 project:
1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50 ug/ml streptomycin, and 500 ug/ml G418.
2. Cell plating: Add 50 ul/well of 120,000 cells/ml re-suspended in DMEM/F12 medium with 10% FBS
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 25 ul /well of 1x FluxOR solution to cells
5. Incubate 90 minutes at room temperature (RT) in the dark
6. Prepare 7.5X compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer(IC0), activator control R-L3 and inhibitor control XE991 (all with DMSO concentrations matched to that of test compounds)
7. Remove FluxOR dye solution and add 20 ul /well of assay buffer to cells
8. Add 4 ul of 7.5x compound stock into the cell plates via Cybi-Well system
9. Incubate all cell plates for 20 minutes at RT in the dark
10. Prepare 5x stimulus buffer containing 12.5 mM K2SO4 and 12.5 mM Tl2SO4
11. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
12. Measure fluorescence for 10 seconds at 1Hz to establish baseline
13. Depolarize cells with 6 ul/well of stimulus buffer and continue measuring fluorescence for 110 seconds
14. Calculate ratio readout as F(max-min)/F0
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors .
16. Calculate B scores  for test compounds using ratios calculated in Step 14.
17. Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (<=3*SD), AND the B score of initial fluorescence intensity is within 3 times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as active (Value=2) as an inhibitor of the KCNQ1 channels. Otherwise, it is designated as inactive (value=1).
18. Score assignment: An active test compound is assigned a score between 4 and 100 by calculation of INT(((LOG(Abs([Bscore Ratio]))-0.747)/0.828)*100), they are normalized to the smallest and largest LOG (Abs([Bscore Ratio]), Bscore Ratio, as in the result definition.
List of reagents
1. KCNQ1-CHO cell lines (provided by JHICC)
2. PBS: pH7.4 (Gibco, Cat#10010)
3. Medium: DMEM/F12 50/50 (Mediatech, Cat#15-090-CV)
4. Fetal Bovine Serum (Gemini, Cat# 100-106)
5. 200 mM L-Glutamine (Gibco, Cat#25030)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. 0.05% Trypsin-EDTA (Gibco, Cat#25300)
8. Geneticin: (Gibco, Cat#11811-031)
9. HEPES (Sigma, Cat#H4034)
10. R-L3 (Tocris Bioscience)
11. FluxOR detection kit (Invitrogen, Cat #F10017): FluxOR, assay buffer and stimulus buffer.
12. Triple-layer flask (VWR, Cat #62407-082)
13. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)