Confirmatory screen for compounds that inhibit KCNQ2 potassium channels
Assay Implementation: Haibo Yu Ph.D., Kaiping Xu, Shunyou Long M.S, Joseph Babcock,David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D. ..more
BioActive Compounds: 569
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center (JHICC_KCNQ2_Inh_Vali)
BioAssay Type: Confirmatory, Confirmatory 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: Min Li, Ph.D.
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA027716-01
Grant Proposal PI: Min Li, Ph.D., Johns Hopkins University School of Medicine
Assay Implementation: Haibo Yu Ph.D., Kaiping Xu, Shunyou Long M.S, Joseph Babcock,David Meyers Ph.D., Meng Wu Ph.D., Owen McManus Ph.D.
Voltage-gated potassium (K) channels are critical for neuronal function in excitable tissues such as brain and heart. They are also found in non-excitable tissues important for other functions such as hormone secretion, oxygen-sensing and immune responses. There are more than 100 genes in human genome encoding different but homologous potassium channels. Voltage-gated K+ channels, exemplified by the Shaker K+ channel, share considerable sequence similarity. Isolation and characterization of bioactive chemical probes could form important pharmacological foundation, providing a great deal of insights into the structure and function.
The KCNQ (or also called Kv7) channel family includes five members, KCNQ1 to KCNQ5; KCNQ are members of Kv channel superfamily[1-6]. Different from other Kv channel classes, KCNQ channels commonly display a sub-threshold opening (i.e., at a voltage near resting potential) and are regulated by GPCR signaling, e.g., muscarinic receptors [7, 8]. KCNQ2-5 channels are primarily expressed in the brain, while KCNQ1 is abundantly expressed in cardiac tissue. Among congenital long QT patients, a disease which affects 1 in 2,500 live births, the most prevalent mutations are within the KCNQ1 allele. Mutations in KCNQ2 and KCNQ3 are known to cause benign familial neonatal convulsion (BFNC), a rare form of epilepsy .
Systemic compound screens for M-current have not been reported. Because of the rapid progress of molecular cloning and functional characterization, KCNQ2 has been validated as the key molecular target of M-current. It is therefore feasible to design non-biased high-throughput screens specifically targeting to KCNQ2 channels. This justifies use of the Tl+-based fluorescence assay in 384-well format by FDSS for the identification of modulatory compounds acting on KCNQ2 from a large compound library.
The purpose of this assay is to validate compounds identified as inhibitors in primary screen assay (PubChem AID 2156). This assay employs the same experimental conditions as presented in the primary screen assay. Compounds were tested in duplicates and their effects were evaluated by the calculated fluorescence ratio percentage, normalized with negative control. If the compound causes 3SD (of negative controls) or more activity decrease (from mean of negative controls) in both duplicates, and is active in the primary screen, the compound is considered to be active.
KCNQ2, HTS assay, 384, primary, antagonist, inhibitor, blocker, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
1. Charlier, C. et al. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat. Genet. 1998.18, 53-55, PMID: 9425900
2. Gutman, G.A. et al. International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels. Pharmacol. Rev. 2003. 55, 583-586, PMID: 14657415
3. Kubisch, C. et al. KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness. Cell. 1999. 96, 437-446, PMID: 10025409
4. Schroeder, B.C., Hechenberger, M., Weinreich, F., Kubisch, C. & Jentsch, T.J. KCNQ5, a novel potassium channel broadly expressed in brain, mediates M-type currents. J. Biol. Chem. 2000, 275, 24089-24095,PMID: 10816588.
5. Singh, N.A. et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat. Genet. 1998.18, 25-29, PMID: 9425895.
6. Wang, Q. et al. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat. Genet. 1996, 12, 17-23, PMID: 8528244.
7. Brown, D.A. et al. Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neurone. Nature. 1980. 283, 673-676. PMID: 6965523.
8. Marrion, N.V. Control of M-current. Annu. Rev. Physiol.1997.59, 483-504, PMID: 9074774.
9. Maljevic, S. et al. Nervous system KV7 disorders: breakdown of a subthreshold brake. J Physiol. 2008. 586, 1791-1801. PMID: 18238816.
10. 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.
11. 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.
12. 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.
13. 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.
14. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), 167-175. PMID: 16465162.
Protocol for the KCNQ2 screen:
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 darkness.
6. Prepare 7.5X compound plates and control plates on the Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (IC0), ICmax of 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 darkness.
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'.
16. Calculate the percentage of tested compounds with the following formula:
Percentage(%): percentage change of compound readout over those of negative controls (vehicle control), The effect of inhibition is reflected by the negative Percentage (%).
Ratio(cmpd): Ratio of the test compound.
AvgRatio(NC): Ratio average of the negative controls with stimulus buffer.
Ratio(Blank): Ratio of the blank control without stimulus buffer.
17. Outcome assignment:
If the compound causes 3SD (of negative controls) or more activity decrease (from mean of negative controls) in both duplicates, and is active in the primary screen, the compound is considered to be active. (Value=2) Otherwise, it is designated as inactive (Value=1).
18. Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned a score between 0 and 100 by calculation of INT (100*(Percentage(%)-Min(%))/(Max(%)-Min(%))), where: Percentage (%) is the percentage average of the duplicates of the test compound over the negative controls, as in Result Definitions; Min(%) is the percentage(%) of the compound with the minimal inhibition effect among the active compounds; Max(%) is the percentage(%) of the compound with the maximal inhibition effect among the active compounds.
List of reagents
1. KCNQ2-CHO cell line (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)
11.FluxOR detection kit (Invitrogen, Cat #F10017): FluxOR, assay buffer and
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 may include, but are not limited to: unintended chemicals or dust in or under the wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, compounds that induce K/Tl flux independent of KCNQ2, compounds that directly interact with the Tl sensitive dye molecule, 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)