Name: Primary cell-based high-throughput screening assay for identification of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
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: Elena Makhina Ph.D., University of Pittsburgh
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA026212-01
Grant Proposal PI: Elena Makhina Ph.D., University of Pittsburgh
Assay Implementation: Meng Wu Ph.D., Amy Scott, Shunyou Long M.S., Joseph Babcock, Beiyan Zou Ph.D., 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 inhibit/block inward-rectifying potassium ion channel Kir2.1


Description:

Potassium (K+) channels are well recognized as a target in the treatment of cardiovascular, neurological, renal and metabolic disorders [1, 2]. However, the number of ion channel drugs is strikingly small, compared to the number of ion channel genes. Most of the known K+ channel modulators lack potency and specificity. The reason for slow development of K+ channel drugs lies largely in the low capacity and high cost of electrophysiology-based screening techniques.

The overall goal of the project is to discover inhibitors of the Kir2.1 channel with EC50 < 1 uM. Identified compounds will serve as both a basis for development of clinical K+ channel drugs, and as a basic study of K+ channel physiology with novel mechanisms of action.

The Kir2.1 channel (KCNJ2) is a member of inwardly-rectifying potassium channels, expressing two transmembrane-spanning segments, flanking a conserved pore region. Inward rectifier K+ channels pass prominent inward currents, and mediate the cardiac inwardly-rectifying potassium current (IK1) [3], while outward currents are largely blocked. The rectification process is mediated through blocking of the channel pore by intracellular Mg2+ and polyamines, and is specific for individual Kir subtypes [4, 5]. Specific modulators of the Kir2.1 channel serve as leads for development of anti-arrhythmic drugs, including drugs for treatment of short- and long-QT syndromes, as well as for the treatment of Andersen syndrome. Several Kir2.1 inhibitor candidates have been discovered [6] [7], i.e. Chlorpromazine, Celastrol, and Gambogic acid. However, a highly specific, high potency small molecule is still in need, not only for the mechanistic probing of the ion channel activities, but also to generate leads for therapeutics for related diseases.

Principle of the assay
The Tl+ ion, which is permeable through potassium channels, serves as a surrogate for K+ flux [8]. 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, inwardly-rectifying potassium ion channels (Kir2.1)--extracellular Tl+ flux into cells through open Kir2.1 channels, and when bound to a dye, produces a fluorescent signal that is monitored in real-time by a fluorescence imaging plate reader [9, 10]. If the activity of Kir2.1 is blocked/inhibited by a test compound, the fluorescent signal remains low/does not increase.


Keywords:

Kir2.1, inward rectifying HTS assay, 384, primary, antagonist, inhibitor, blocker, FDSS, Thallium, fluorescence, Kinetic, FluxOR, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.

References:

1. Kaczorowski, G.J., et al., Ion Channels as Drug Targets: The Next GPCRs. J. Gen. Physiol., 2008. 131(5): p. 399-405. PMID: 18411331
2. Garcia, M.L. and G.J. Kaczorowski, Potassium Channels as Targets for Therapeutic Intervention. Sci. STKE, 2005. 2005(302): p. pe46-. PMID: 16174819
3. Lopatin, A.N. and C.G. Nichols, Inward Rectifiers in the Heart: An Update on IK1. Journal of Molecular and Cellular Cardiology, 2001. 33(4): p. 625-638. PMID: 11273717
4. Ficker, E., et al., Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science, 1994. 266(5187): p. 1068-1072. PMID: 7973666
5. Dhamoon, A.S., et al., Unique Kir2.x Properties Determine Regional and Species Differences in the Cardiac Inward Rectifier K+ Current. Circ Res, 2004. 94(10): p. 1332-1339. PMID: 15087421
6. Sun, H., et al., Chronic Inhibition of Cardiac Kir2.1 and hERG Potassium Channels by Celastrol with Dual Effects on Both Ion Conductivity and Protein Trafficking. J. Biol. Chem., 2006. 281(9): p. 5877-5884. PMID: 16407206
7. Zaks-Makhina, E., et al., Novel Neuroprotective K+ Channel Inhibitor Identified by High-Throughput Screening in Yeast. Mol Pharmacol, 2004. 65(1): p. 214-219. PMID: 14722253
8. Delpire, E., et al., Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2. Proceedings of the National Academy of Sciences, 2009. 106(13): p. 5383-5388. PMID: 19279215
9. 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): p. 671-677. PMID: 15634793
10. 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): p. 1213-1224. PMID: 18171729
11. 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): p. 67-73. PMID: 10838414
12. Malo, N., et al., Statistical practice in high-throughput screening data analysis. Nat Biotech, 2006. 24(2): p. 167-175. PMID: 16465162

Assay overview:

Primary cell-based high-throughput screening assay for identification of compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1

The purpose of this assay is to identify test compounds that inhibit/block inward rectifying potassium ion channel Kir2.1. This assay employs a HEK293 cell line that stably expresses Kir2.1 channels. 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 Kir2.1 channel were measured by thallium assay, using the FluxOR detection kit, a commercial kit. Those HEK293 cells stably expressing Kir2.1 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. Compounds were added to the assay buffer. Cells were incubated with 10 uM compound for 20 minutes, and detected upon the addition of stimulus solution (5 mM K2SO4 and 1.4 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 less than 3 times the standard deviation of the B-scores of the library compounds, the compound is then considered to be active as an inhibitor/blocker of the Kir2.1 channels.


Protocol for the Kir2.1 project:

1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50ug/ml streptomycin, and 500ug/ml G418.
2. Cell plating: Add 50 ul/well of 300,000 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), and IC100 of Chlorpromaizne (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 25 mM K2SO4 and 7 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. Add 6 ul/well of stimulus buffer onto cells 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' prime factors [11].
16. Calculate B scores [12] for test compounds using ratios calculated in Step 14.
17. Outcome assignment: If the B score of the test compound is less than minus 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 2 times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as active as an inhibitor/blocker of the Kir2.1 channels. Otherwise, it is designated as inactive.
18. Score assignment: An active test compound is assigned a score between 0 and 100 by calculation of 100*(0- Integer ([B Score Inhibitor Ratio]))/20, B Score Inhibitor Ratio, as in the result definition. Among the active compounds in the assay, the activity
score range is 95-20. All inactive test compounds are assigned to the score 0.

List of reagents

1. Kir2.1 HEK293 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. G418 (Geneticin): (Gibco, Cat#11811-031)
9. HEPES (Sigma, Cat#H4034)
10. Chlorpromazine hydrochloride (Sigma, C8138)
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)6663 and Lot #8163495)
14. 10x HBSS (Gibco, Cat#14065)

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.

Grant Number: 1 R03 DA027716-01