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: 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., Beiyan Zou Ph.D., Shunyou Long M.S., Amy Scott, Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.

Name: Primary cell-based high-throughput screening assay for identification of compounds that potentiate KCNQ2 potassium channels

Description:


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 [9].

In the mid-90s, a compound known as D-23129, or retigabine, was initially developed to target GABAnergic signaling [10, 11]. Despite its poor effect on the GABA pathway, retigabine was shown to have potent anti-convulsant activity. In 1997, retigabine was also found to induce the opening of voltage-gated potassium channels at resting membrane potentials, and since then, through a variety of other studies, this has been established to be its main mechanism of action. Retigabine, acting on KCNQ2/3 potassium channels which code M-currents, is now being used for treatment of epilepsy. None of the anti-convulsants that are in clinical use today have a comparable mechanism of action, i.e., through ligand activation of voltage-gated potassium channels. Several potentiation compounds are synthesized according to the scaffold of retigabine. However, considering the poor potency of retigabine and several other undesirable characteristics including its broad action on KCNQ2, 3, 4, and 5 [12], it is not clear whether these related structures will fundamentally improve therapeutic efficacy. Hence, it argues for the performance of a non-biased screen for new structures with potentiation activity.

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.

Principle of the assay

The Tl+ ion, which is permeable through potassium channels, serves as a surrogate for K+ flux [13]. 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, extracellular Tl+ flux into cells through open KCNQ2 channels, and the KCNQ2 potassium channel, when bound to a dye, produces a fluorescent signal that is monitored in real-time by a fluorescence imaging plate reader [14, 15].The binding to Tl+ causes increase in fluorescence. If activity of KCNQ2 is potentiated by a test compound, the fluorescent signal increases.



Keywords:

KCNQ2, HTS assay, 384, primary, agonist, activator, potentiator, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.


References:
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. Rostock, A. et al. D-23129: a new anticonvulsant with a broad spectrum activity in animal models of epileptic seizures. Epilepsy Res. 1996. 23, 211-223. PMID: 8739124
11. Tober, C., Rostock, A., Rundfeldt, C. & Bartsch, R. D-23129: a potent anticonvulsant in the amygdala kindling model of complex partial seizures. Eur. J. Pharmacol. 1996. 303, 163-169. PMID: 8813562
12. Gribkoff, V.K. (2003) The therapeutic potential of neuronal KCNQ channel modulators. Expert Opin. Ther. Targets. 2003. 7, 737-748. PMID: 14640909.
13. 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.
14. 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.
15. 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.
16. 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.
17. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2), 167-175. PMID: 16465162.

Assay overview:

The purpose of this assay is to identify compounds that potentiate KCNQ2 potassium channels. This assay employs a CHO-K1 cell line that stably expresses the KCNQ2 potassium channel. The cells are treated with test compounds, followed by measurement of intracellular thallium, as monitored by a thallium-sensitive fluorescent dye, FluxOR. As designed, compound effects on the KCNQ2 channel were measured by thallium assay, using a commercial kit, the FluxOR detection kit. Those CHO-K1 cells stably expressing KCNQ2 channels were plated into 384-well plates. On the following day, cells were loaded with a thallium-sensitive dye, FluxOR, and then incubated with assay buffer, followed by the addition of compounds. Cells were incubated with 10 uM compound for 20 minutes, and detected upon the addition of stimulus solution (2.5 mM K2SO4 and 2.5 mM Tl2SO4).The fluorescence of FluxOR was measured on a Hamamatsu FDSS 6000 kinetic imaging plate reader. Compound effect was evaluated by the calculated FluxOR fluorescence ratio, normalized with negative controls. If the compound causes more than 3 times the standard deviation of the B-scores of the library compounds, the compound is then considered to be active as a potentiator of the KCNQ2 channel.


Protocol for the KCNQ2 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 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 ZTZ240 (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' factors [16].
16. Calculate B scores [17] 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 an active (Value=2) potentiator of the KCNQ2 channels. Otherwise, it is designated as inactive (value=1).
18. Score assignment: An active test compound is assigned a score between 0 and 100 by calculation of INT(100*LOG(B Score Potentiator Ratio), they are normalized to the smallest and largest LOG(B Score Potentiator Ratio), B Score Potentiator Ratio, as in the result definition.


List of reagents

1. KCNQ2-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. ZTZ-240 (Synthesized)
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 may include, but are not limited to: unintended chemicals or dust in or on 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

Grant Number: 1 R03 DA027716-01