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BioAssay: AID 1511

Primary cell-based high-throughput screening assay for identification of compounds that protect hERG from block by proarrhythmic agents

Assay Implementation: Beiyan Zou Ph.D., Shunyou Long M.S., Amy Scott M.S., Haibo Yu Ph.D., Meng Wu Ph.D., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
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 Tested Compounds
 Tested Compounds
All(305614)
 
 
Active(1552)
 
 
Inactive(304062)
 
 
 Tested Substances
 Tested Substances
All(305679)
 
 
Active(1552)
 
 
Inactive(304127)
 
 
AID: 1511
Data Source: Johns Hopkins Ion Channel Center (JHICC_hERG)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-01-15
Modify Date: 2010-03-27

Data Table ( Complete ):           Active    All
Target
Sequence: putative potassium channel subunit
Description ..   
Protein Family: CAP_ED

Gene:KCNH2     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 1552
Depositor Specified Assays
Show more
AIDNameTypeComment
1680Summary of efforts in the identification of compounds that protect hERG from block by proarrhythmic agentssummarySummary assay for hERG project.
1835Confirmatory screen for compounds that protect hERG from block by proarrhythmic agentsscreeningConfirmatory assay in duplicates for 2405 compounds and 749 are active.
1839Counter screen for compounds that protect hERG from block by proarrhythmic agentsscreeningCounter screen assay against the parental cell line for 2405 compounds, among which 216 are active.
1942KCNQ2 Counter screen for compounds that protect hERG from block by proarrhythmic agentsscreeningCounter screen assay for KCNQ2 potentiators with 2405 compounds tested, and 578 are active.
1946KCNQ2 Counter screen for inhibitors that protect hERG from block by proarrhythmic agentsscreeningCounter screen assay for KCNQ2 inhibitors with 2405 compounds tested, and 180 are active.
2103Secondary screen for compounds that protect hERG from block by proarrhythmic agentsscreeningSecondary screen of 29 compounds using automated patch clamp, but none is active.
2121Confirmation Dose Response screen for compounds that protect hERG from block by proarrhythmic agentsconfirmatoryDosage response assay for 222 compounds where 113 are active.
2321Testing for compounds that protect hERG from block by proarrhythmic agents using manual patch clampscreeningManual patch clamp assay of 9 test compounds, but none is active.
2605Screening compounds that protect hERG from block by proarrhythmic agents using manual patch clampscreeningManual patch clamp assay of 5 active test compounds.
2638Dosage response for compounds that protect hERG from block by proarrhythmic agents using manual patch clampconfirmatoryManual patch clamp dosage measurements for 3 compounds, 1 is active.
Description:
Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Assay Provider: Dr. Sabina Kupershmidt, Vanderbilt University Medical Center
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R03MH084820-01
Grant Proposal PI: Dr. Sabina Kupershmidt, Vanderbilt University Medical Center
Assay Implementation: Beiyan Zou Ph.D., Shunyou Long M.S., Amy Scott M.S., Haibo Yu Ph.D., Meng Wu Ph.D., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.

Name: Identification of compounds that protect hERG from block by proarrhythmic agents

Description:

The acquired/drug-induced Long QT Syndrome (aLQTS) is both a threat to public health and a major stumbling block for drug development [1-3]. It is most often caused through unintended block of the cardiac repolarizing potassium channel, IKr, encoded by the Human Ether-a-go-go Related Gene (hERG) [4]. hERG is susceptible to inhibition by even transient exposure to a diverse array of common therapeutic compounds, including antihistamines, antidepressants, antibiotics, antiemetics, and antiarrhythmics which in turn provokes the life-threatening cardiac arrhythmia torsades de pointes in predisposed individuals. Indeed, several successful drugs (e.g., cisapride, seldane) had to be withdrawn from the market due to arrhythmia development caused by this mechanism [5-8]. Previous studies have shown that Ikr can be protected from block by known hERG antagonists through the activity of the K+ channel regulator 1 (KCR1) protein, which was characterized as an enzyme that is likely to function in the cellular glycosylation pathway[9, 10]. This finding led to a hypothesis that identification of a small molecule that mimics the activity of KCR1 and diminishes Ikr block by known hERG inhibitors may lead to the development of a co-drug to protect against the development of the aLQTS. Compounds that exhibit such activity would be expected to include neutral antagonists that bind to the same site as hERG blockers but do not favor the "blocked conformation" of the pore, hERG agonists that competitively bind to the same site as hERG inhibitors, or compounds that prevent the access of a hERG inhibitor to its cognate binding site at the intracellular aspect of the pore region. High-throughput methods are used to:

1. Screen a library of 305,722 compounds for agents that increase the IC70 of the well-characterized hERG inhibitor, dofetilide, using a high-throughput Tl+ flux assay [11-13]. The Tl+ ion serves as a surrogate for K+ flux, and, when bound to a dye, produces a fluorescent signal that is monitored in real time by a fluorescence imaging plate reader. A robust and reproducible assay in 384-well format has been developed to show a dose-dependent signal in response to dofetilide and tolerance to the compound vehicle, DMSO. Completion of a preliminary screen using 3,520 compounds demonstrated that the assay is ready to identify chemical compounds that alter the response of Ikr to dofetilide [14-17].

2. Determine if the isolated hits also affect the IC70 of a range of drugs with divergent chemical structures (moxifloxacin, clozapine[18], droperidol [5-6,19-21]) and clinical applications in the Tl+ flux assay.

These findings will be validated through standard patch-clamp assays. Structure Activity Relationship (SAR) studies will be initiated around the most interesting chemical scaffolds.

The development of a small molecule chemical agent that could be safely co-administered with clinically useful pharmaceuticals to protect susceptible patients from hERG inhibitors and the subsequent development of arrhythmias would improve public health and greatly facilitate the drug discovery process.


Principle of the assay

Tl+ is permeable through potassium channels. The thallium-sensitive dye is loaded into cells and exhibits very low basal fluorescence in the absence of Tl+. Upon depolarization of cells expressing potassium channels, in this case, human ether-a-go-go-related gene (hERG) channels, extracellular Tl+ flux into cells through activated hERG channels. The binding to Tl+ causes increase in fluorescence. If activity of hERG is potentiated by a test compound, the fluorescent signal increases; however, when the hERG channel is blocked by a test compound, low or no increase of the fluorescent signal is observed.

1. Defective drugs. adrugrecall.com. 2005. (Available at URL: http://www.adrugrecall.com/seldane/seldane.html )
2. Alexandrou AJ, Duncan RS, Sullivan A, Hancox JC, Leishman DJ, Witchel HJ, Leaney JL. Mechanism of hERG K+ channel blockade by the fluoroquinolone antibiotic moxifloxacin. British Journal of Pharmacology. 2006. 147:905-916, PMID: 16474415.
3. Carmeliet E. Voltage- and time-dependent block of the delayed K+ current in cardiac myocytes by dofetilide. The Journal of pharmacology and experimental therapeutics. 1992. 262 (2): 809-17, PMID: 1501123
4. Davie C, Pierre-Valentin J, Pollard C, Standen N, Mitcheson J, Alexander P, Thong B. Comparative pharmacology of guinea pig cardiac myocyte and cloned hERG (I(Kr)) channel. Journal of cardiovascular electrophysiology. 2004.15 (11): 1302 - 1309, PMID: 15574182.
5. Drolet B, Zhang S, Deschenes D, Rail J, Nadeau S, Zhou Z, January CT, Turgeon J. Droperidol lengthens cardiac repolarization due to block of the rapid component of the delayed rectifier potassium current. Journal of
cardiovascular electrophysiology. 1999. 10 (12): 1597-604, PMID: 10636190.
6. FDA. FDA Strengthens Warnings for Droperidol. http://www.fda.gov/bbs/topics/answers/2001/ans01123.html 2007.
7. Ficker E, Jarolimek W, Brown AM. Molecular determinants of inactivation and dofetilide block in ether a-go-go (EAG) channels and EAG-related K(+) channels. Molecular pharmacology. 2001. 60 (6): 1343-1348, PMID: 11723241.
8. Generali J. Drugs with Black Box Warnings - Comprehensive List. http://www.formularyproductions.com/master/showpage.php?dir=blackbox&whichpage=9 2007.
9. Kupershmidt S, Yang IC-H, Hayashi K, Wei J, Chanthaphaychith S, Petersen CI, Johns DC, George ALJ, Roden DM, Balser JR. IKr drug response is modulated by KCR1 in transfected cardiac and noncardiac cell lines The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2003. 17 (15): 2263-5, PMID: 14525949
10. Nakajima T, Hayashi K, Viswanathan PC, Kim M-Y, Anghelescu M, Barksdale KA, Shuai W, Balser JR, Kupershmidt S. HERG is protected from pharmacological block by {alpha}-1,2-Glucosyltransferase function. J. Biol. Chem. 2007. 282 (8): 5506-5513, PMID: 17189275
11. Sui Y, Wu Z. Alternative Statistical Parameter for High-Throughput Screening Assay Quality Assessment. Journal of biomolecular screening :the official journal of the Society for Biomolecular Screening. 2007. 12(2): 229-34, PMID: 17218666.
12. Weaver CD, Harden D, Dworetzky SI, Robertson B, Knox RJ. A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells. Journal of biomolecular screening. 2004. 9 (8): 671-7, PMID: 15634793.
13. Zhang J-H, Chung TDY, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of biomolecular screening: the official journal of the Society for Biomolecular Screening. 1999. 4 (2):67-76, PMID: 10838414
14. Kiehn J, Lacerda AE, Wible B, Brown AM. Molecular physiology and pharmacology of HERG. Single-channel currents and block by dofetilide. Circulation. 1996.94 (10): 2572-9, PMID: 8921803
15. Kiehn J, Wible B, Lacerda AE, Brown AM. Mapping the block of a cloned human inward rectifier potassium channel by Dofetilide. Molecular pharmacology.1996. 50 (2): 380-7, PMID: 8700146
16. Lees-Miller JP, Duan Y, Teng GQ, Duff HJ. Molecular determinant of high-affinity dofetilide binding to HERG1 expressed in Xenopus oocytes: involvement of S6 sites. Molecular pharmacology. 2000. 57 (2): 367-74., PMID: 10648647.
17. Sanguinetti MC, Jiang C, Curran ME, Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell. 1995. 81: 299-307,PMID: 7736582.
18. Lee S-Y, Kim Y-J, Kim K-T, Choe H, Jo S-H. Blockade of HERG human K+ channels and IKr of guinea-pig cardiomyocytes by the antipsychotic drug clozapine. British Journal of Pharmacology. 2006. 148: 499-509, PMID: 16633353
19. Lischke V, Behne M, Doelken P, Schledt U, Probst S, Vettermann J. Droperidol causes a dose-dependent prolongation of the QT interval. Anesthesia and analgesia. 1994. 79 (5): 983-6, PMID: 7978420.
20. Snyders DJ, Chaudhary AA. High affinity open channel block by Dofetilide of HERG expressed in a human cell line. Molecular pharmacology. 1996. 49 (6): 949-55, PMID: 8649354.
21. Scuderi PE. Droperidol: Many Questions, Few Answers. Anesthesiology. 2003. 98 (2): 289-290,PMID: 12552182
Protocol
Assay overview:

The purpose of this assay is to identify test compounds that are allosteric modulators (reduce or remove the inhibition of dofetilide) of human ether-a-go-go-related gene (hERG) channels. This assay employs a CHO cell line that stably expresses hERG 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 hERG channel were measured in the presence of IC70 of dofetilide by thallium assay, using a commercial kit, the FluxOR detection kit. The CHO cells stably expressing hERG channels were plated into 384-well plates. The following day, cells were loaded with a thallium-sensitive dye, FluxOR,and then incubated with assay buffer. Compounds with IC70 of dofetilide were added to assay buffer. Cells were incubated with 10 uM compound for 20 minutes, and then depolarized by incubation with 5 mM K2SO4 and 1.4 mM Tl2SO4. Fluorescence of FluxOR was measured on 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 5*SD, or more activity increase, the compound is considered to be active.


Protocol for the HERG 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 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 containing IC70 of dofetilide; controls are assay buffer, IC70, and ICmax of dofetilide (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. Depolarize cells with 6 ul/well of stimulus buffer and continue measuring fluorescence for 110 seconds
14. Calculate F(max-min)/F0
15. Calculate the average and standard deviation for negative and positive controls in each plate
16. Calculate the percentage with the following formula:
Percentage = Inhibition (%) - (Avg+5*SD)

Where
Percentage: percentage change of compound readout over those of negative controls with dofetilide with a concentration of its IC70 and 5 times of its standard deviation.

Inhibition (%): percentage of the compound readout normalized to those of the blank control without stimulus buffer (0) and negative control without inhibitors (100%).

Avg: average of inhibition (%) of those of negative controls with dofetilide with a concentration of its IC70.

SD: standard deviation of inhibition (%) of those of negative controls with dofetilide with a concentration of its IC70.
17. Score assignment: If Percentage <= 0, Score = 0; If Percentage >0, Score = Int(Percentage).

Where Percentage is defined above.

List of reagents

1. hERG 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. 0.25% Trypsin-EDTA (Gibco, Cat#25300)
8. Geneticin: (Gibco, Cat#11811-031)
9. HEPES (Sigma, Cat#H4034)
10. Dofetilide (Fisher, NC9753685)
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
1Percentage (10μM**) Normalized percent activation of the primary screen at a compound
concentration of 10 micromolar over the negative control with dofetilide at concentration of its IC70.
Float
2JHICC_EXT_DATASOURCE_REGID (10μM**) Compound ID of JHICCString

** Test Concentration.
Additional Information
Grant Number: 1R03MH084820-01

Data Table (Concise)
Classification
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