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BioAssay: AID 493007

Validation assay for identification of compounds that potentiate KCNQ1 potassium channels

Assay Implementation: Zhihong Lin Ph.D., Xiaofang Huang M.S., Shunyou Long M.S., Owen McManus, Ph.D., Meng Wu Ph.D. ..more
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 Tested Compounds
 Tested Compounds
All(2435)
 
 
Active(1024)
 
 
Inactive(1411)
 
 
 Tested Substances
 Tested Substances
All(2435)
 
 
Active(1024)
 
 
Inactive(1411)
 
 
AID: 493007
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ1_Act_Vali)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-12-28

Data Table ( Complete ):           Active    All
Target
Sequence: potassium voltage-gated channel subfamily KQT member 1 isoform 1 [Homo sapiens]
Description ..   
Protein Family: KCNQ voltage-gated potassium channel

Gene:KCNQ1     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 1024
Depositor Specified Assays
AIDNameTypeComment
2648Primary cell-based high-throughput screening assay for identification of compounds that potentiate/activate KCNQ1 potassium channelsscreening
2699Summary of assays for compounds that potentiate/activate KCNQ1 potassium channelssummary
602121SAR analysis for compounds that activate KCNQ1 potassium channels in the KCNQ1 expressing cells on automated patch clamp 2confirmatory
493186Specificity screen assay against KCNQ1/E1 for identification of compounds that potentiate KCNQ1 potassium channelsscreening
588673SAR analysis for compounds that activate KCNQ1 potassium channels in the KCNQ1 expressing cells on automated patch clampconfirmatory
602306SAR analysis for compounds that activate KCNQ1 potassium channels in the KCNQ1 expressing cells on automated patch clamp IIIconfirmatory
493184Secondary automated electrophysiology assay of compounds that potentiate KCNQ1 potassium channelsscreening
Description:
Data Source: Johns Hopkins Ion Channel Center (JHICC, JHICC_KCNQ1_Act_Vali)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed, Duplicate

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., Owen McManus, Ph.D., Meng Wu Ph.D.

Name: Validation assay for identification of compounds that potentiate KCNQ1 potassium channels

Description:

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 [3]. 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 [4]. 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 [16], 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 [10]. 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 [18], Romano-Ward syndrome, Jervell and Lange-Nielsen syndrome [19] and familial atrial fibrillation [20], 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 [17].

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 [17]. In contrast to KCNQ1 channel blockers, only until recently have KCNQ1 channel activators/ potentiators been generating a lot of interests partially due to KCNQ1/KCNE activators might be useful agents to counteract the loss of delayed rectifier function in LQT syndromes, as well as counter target of other KCNQ family members for potential drugs for the treatment of epilepsy and neuropathic pain. Overall there are a very limited number of KCNQ1 activators/ potentiators, a further limited number of KCNQ1/E1 heteromultimer-specific modulators, and no reported KCNQ1/E2 or KCNQ1/E3 heteromultimer-specific modulators. This has hindered a more systematic study to understand the roles of on beta-subunits. Therefore it justifies the necessity of primary high throughput screen of KCNQ1 with the MLSMR library of >300,000-500,000 compounds covering large chemical space. Here the assay, Tl+-based fluorescence assay in 384 format by FDSS, therefore, was used for the identification of activating/potentiating compounds acting on KCNQ1 from the large MLSMR compound library.


Principle of the assay

The Tl+ ion, which is permeable through potassium channels, serves as a surrogate for K+ flux [26]. 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 [27, 28].The binding to Tl+ causes increase in fluorescence. If the activity of KCNQ1 is potentiated by a test compound, the fluorescent signal increase is enhanced.

Assay overview:

To validate the hit compounds that potentiate KCNQ1 potassium channels from the primary screen, a CHO-K1 cell line that stably expresses KCNQ1 potassium channel is employed. 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, from the duplicates.

Keywords:

KCNQ1, HTS assay, 384, primary, agonist, activator, potentiator, allosteric, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, MLSMR, Molecular Libraries Probe Production Centers Network, MLPCN.


References:
1. Gutman, G. A., Chandy, K. G., Grissmer, S., et al. International Union of Pharmacology. LIII. Nomenclature and Molecular Relationships of Voltage-Gated Potassium Channels. Pharmacol Rev 57(4), 473-508 (2005) PMID: 16382104
2. Dai, S., Hall, D. D., and Hell, J. W. Supramolecular Assemblies and Localized Regulation of Voltage-Gated Ion Channels. Physiol. Rev. 89(2), 411-452 (2009) PMID: 19342611
3. Borjesson, S., and Elinder, F. Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels. Cell Biochemistry and Biophysics 52(3), 149-174 (2008) PMID: 18989792
4. Pischalnikova, A., and Sokolova, O. The Domain and Conformational Organization in Potassium Voltage-Gated Ion Channels. Journal of Neuroimmune Pharmacology 4(1), 71-82 (2009) PMID: 18836841
5. Peroz, D., Rodriguez, N., Choveau, F., et al. Kv7.1 (KCNQ1) properties and channelopathies. The Journal of Physiology 586(7), 1785-1789 (2008) PMID: 18174212
6. Cannon, S. C. Physiologic Principles Underlying Ion Channelopathies. Neurotherapeutics 4(2), 174-183 (2007) PMID: 17395127
7. Ahern, C. A., and Kobertz, W. R. Chemical Tools for K+ Channel Biology. Biochemistry 48(3), 517-526 (2008) PMID: 19113860
8. Wulff, H., and Zhorov, B. S. K+ Channel Modulators for the Treatment of Neurological Disorders and Autoimmune Diseases. Chemical Reviews 108(5), 1744-1773 (2008) PMID: 18476673
9. Wickenden, A. D. K+ channels as therapeutic drug targets. Pharmacology & Therapeutics 94(1-2), 157-182 (2002) PMID: 12191600
10. Jespersen, T., Grunnet, M., and Olesen, S.-P. The KCNQ1 Potassium Channel: From Gene to Physiological Function. Physiology 20(6), 408-416 (2005) PMID: 16287990
11. Mackie, A. R., and Byron, K. L. Cardiovascular KCNQ (Kv7) Potassium Channels: Physiological Regulators and New Targets for Therapeutic Intervention. Mol Pharmacol 74(5), 1171-1179 (2008) PMID: 18684841
12. Maljevic, S., Wuttke, T. V., and Lerche, H. Nervous system KV7 disorders: breakdown of a subthreshold brake. The Journal of Physiology 586(7), 1791-1801 (2008) PMID: 18238816
13. Robbins, J. KCNQ potassium channels: physiology, pathophysiology, and pharmacology. Pharmacology & Therapeutics 90(1), 1-19 (2001) PMID: 11448722
14. Hernandez, C. C., Zaika, O., Tolstykh, G. P., et al. Regulation of neural KCNQ channels: signalling pathways, structural motifs and functional implications. The Journal of Physiology 586(7), 1811-1821 (2008) PMID: 18238808
15. Delmas, P., and Brown, D. A. Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nat Rev Neurosci 6(11), 850-862 (2005) PMID: 16261179
16. Brown, D. A., and Passmore, G. M. Neural KCNQ (Kv7) channels. British Journal of Pharmacology 156(8), 1185-1195 (2009) PMID: 19298256
17. Towart, R., Linders, J. T. M., Hermans, A. N., et al. Blockade of the IKs potassium channel: An overlooked cardiovascular liability in drug safety screening? Journal of Pharmacological and Toxicological Methods 60(1), 1-10 (2009) PMID: 19439185
18. Jonsson, A., Isomaa, B., Tuomi, T., et al. A variant in the KCNQ1 gene predicts future type 2 diabetes and mediates impaired insulin secretion. Diabetes, 58(10) 2409-13 (2009) PMID: 19584308
19. Schmitt, N., Schwarz, M., Peretz, A., et al. A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly. EMBO J 19(3), 332-340 (2000) PMID: 10654932
20. OMIM. http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=607542 (2009)
21. Namkung, W., Song, Y., Mills, A. D., et al. In Situ Measurement of Airway Surface Liquid [K+] Using a Ratioable K+-sensitive Fluorescent Dye. J. Biol. Chem. 284(23), 15916-15926 (2009) PMID: 19364771
22. Moser, S., Harron, S., Crack, J., et al. Multiple KCNQ Potassium Channel Subtypes Mediate Basal Anion Secretion from the Human Airway Epithelial Cell Line Calu-3. Journal of Membrane Biology 221(3), 153-163 (2008) PMID: 18264812
23. Schroeder, B. C., Waldegger, S., Fehr, S., et al. A constitutively open potassium channel formed by KCNQ1 and KCNE3. Nature 403(6766), 196-199 (2000) PMID: 10646604
24. Greenwood, I., Yeung, S., Hettiarachi, S., et al. KCNQ-encoded channels regulate Na+ transport across H441 lung epithelial cells. Pflugers Archiv European Journal of Physiology 457(4), 785-794 (2009) PMID: 18663467
25. Matos, J. E., Sausbier, M., Beranek, G., et al. Role of cholinergic-activated K Ca 1.1 (BK), K Ca 3.1 (SK4) and K V 7.1 (KCNQ1) channels in mouse colonic Cl - secretion. Acta Physiologica 189(3), 251-258 (2007) PMID: 17305705
26. Delpire, E., et al., Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2. Proc Natl Acad Sci U S A., 2009. 106(13),5383-5388. PMID: 19279215.
27. Weaver, C.D., et al., A Thallium-Sensitive, Fluorescence-Based Assay for Detecting and Characterizing Potassium Channel Modulators in Mammalian Cells. J Biomol Screen, 2004. 9(8), 671-677. PMID: 15634793.
28. Niswender, C.M., et al., A Novel Assay of Gi/o-Linked G Protein-Coupled Receptor Coupling to Potassium Channels Provides New Insights into the Pharmacology of the Group III Metabotropic Glutamate Receptors. Mol Pharmacol, 2008. 73(4), 1213-1224. PMID: 18171729.
29. Zhang, J.-H., T.D.Y. Chung, and K.R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen, 1999. 4(2),67-73. PMID: 10838414.
30. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), 167-175. PMID: 16465162.
Protocol
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 [29].
16. Calculate the percentage of tested compounds with the following formula: Percentage (%)=100* (Ratio(cmpd)- AvgRatio(Buffer))/(AvgRatio(R-L3)-AvgRatio(Buffer)); Percentage(%): percentage change of compound readout over those of negative controls (Buffer), Ratio(cmpd): Ratio of the test compound. AvgRatio(Buffer): Ratio average of the negative controls with Buffer, Ratio(R-L3): Ratio of R-L3.
17. Outcome assignment: If the compound (the average of the duplicates of the Percentage (%, avPercent) as readout) causes more than those of negative controls (Buffer) plus 3SD of negative controls (Buffer), the compound is considered to be active (Value=2). Otherwise, it is designated as inactive (Value=1).
18. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(((Log(ABS(avPercent))-1.16)*60.13, avPercent, as in the result definition. The inactive test compounds are assigned a score of 0.

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)
Comment
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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1avPercent (10μM**)Average percent of the duplicates of each test compound readout (Percentage (%)) at a concentration of 10muM.Float

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH090837-01

Data Table (Concise)
Classification
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