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BioAssay: AID 1918

High throughput discovery of novel modulators of ROMK K+ channel activity: Primary Screen

The Renal Outer Medullary Potassium channel (ROMK, Kir1.1) is expressed in the renal tubule where it critically regulates fluid and electrolyte homeostasis (Hebert, 2005). An emerging body of evidence suggests that ROMK could be a target for a novel class loop diuretic that lowers blood pressure while preserving plasma potassium levels (Ji, 2008). Furthermore, homozygous loss-of-function more ..
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 Tested Compounds
 Tested Compounds
All(125267)
 
 
Active(2463)
 
 
Inactive(122804)
 
 
 Tested Substances
 Tested Substances
All(125296)
 
 
Active(2464)
 
 
Inactive(122832)
 
 
AID: 1918
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (JD001_primary_5mM)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-09-03
Modify Date: 2009-12-04

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: Potassium inwardly-rectifying channel, subfamily J, member 1 [Homo sapiens]
Description ..   
Protein Family: Inward rectifier potassium channel

Gene:KCNJ1     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 2463
Related Experiments
AIDNameTypeComment
2436High-throughput Discovery of Novel Modulators of ROMK K+ Channel ActivitySummarydepositor-specified cross reference
2753High throughput discovery of novel modulators of ROMK K+ channel activity: Dose-Response AssayConfirmatorydepositor-specified cross reference
1916High throughput discovery of novel modulators of ROMK K+ channel activity: Analog Dose-ResponseConfirmatorysame project related to Summary assay
1917High throughput discovery of novel modulators of ROMK K+ channel activity: Retest of Primary HitsOthersame project related to Summary assay
1922High throughput discovery of novel modulators of ROMK K+ channel activity: Selectivity by Patch ClampOthersame project related to Summary assay
1924High throughput discovery of novel modulators of ROMK K+ channel activity: Ancillary ActivityOthersame project related to Summary assay
435017High throughput discovery of novel modulators of ROMK K+ channel activity: Analog Library TestingConfirmatorysame project related to Summary assay
Description:
Assay Provider: Jerod Denton
Assay Provider Affiliation: Vanderbilt University
Grant Title: High throughput discovery of novel modulators of ROMK K+ channel activity
Grant Number: R21 NS057041-01

The Renal Outer Medullary Potassium channel (ROMK, Kir1.1) is expressed in the renal tubule where it critically regulates fluid and electrolyte homeostasis (Hebert, 2005). An emerging body of evidence suggests that ROMK could be a target for a novel class loop diuretic that lowers blood pressure while preserving plasma potassium levels (Ji, 2008). Furthermore, homozygous loss-of-function mutations in the gene encoding ROMK (KCNJ1) cause antenatal Bartter syndrome, a severe salt and water wasting disease in infants (Simon,1996). ROMK is thus an important pharmacological target for the management of disease. Its actual therapeutic value and drugability, however, are unknown due to the lack of small-molecule probes targeting the channel. The discovery of ROMK modulators will provide important new tools for studying the structure, function and therapeutic potential of ROMK and other inward rectifying potassium channels.
Protocol
The purpose of this assay was to test the small molecule library provided by the Molecular Libraries Small Molecule Repository (MLSMR, distributed by DPI-Biofocus) for the ability to modulate thalluium flux in a ROMK-expressing cell line. All compounds were tested in single at 10uM final concentration.
High-Throughput Screening
Experiments were done as previously described (Lewis, 2009). Briefly, cells were plated in clear-bottomed, black-walled 384-well plates and cultured overnight in serum-free media containing Tet (for induction of ROMK expression) at 37 degrees C in the presence of 5% CO2. The following day, the cells were combined with dye and incubated for 20min at RT, followed by automated washing using the ELx405 plate washer (BioTek, Winooski, VT). The plate was loaded into the Functional Drug Screening System 6000 (FDSS6000; Hamamatsu, Tokyo, Japan) to obtain an initial baseline image (F0) (10 images at 1 Hz; excitation, 470 +/- 20 nm; emission, 540 +/- 30 nm). DMSO vehicle control, TPNQ or test compound was added to each plate for final concentrations of 0.1 %, 2 uM and 10 uM, respectively. Four columns in each plate were filled with vehicle control or TPNQ-containing buffer for determination of Z', while the remaining 20 columns were used for compound screening at 10uM final concentration. The compound and cells were allowed to incubate for 20 min at RT, then the plate was reloaded into the FDSS and a second baseline acquired. The FDSS's integrated pipettor added thallium stimulus (125 mM sodium gluconate, 12 mM thallium sulfate, 1 mM magnesium sulfate, 1.8 mM calcium gluconate, 5 mM glucose, 10 mM HEPES, pH 7.3, adjusted to 343 mOsm with sucrose) while imaging of the flux continued at 1 Hz for 2 min total sampling time.
Data Analysis and Statistics
The kinetic fluorescence values (F) from each well were divided by the initial frame of the read (F0) to give the static ratio (F/F0) which corrects for variability in cell number and dye loading. The second baseline reading was used to assess fluorescent compound artifacts, and those wells where the reading exceeded 70% of the mean fluorescence for the plate were excluded from analysis. The slope of the static ratio from 7 to 12 seconds was calculated and the value (Value) compared with the average distribution of the remaining compound wells assumed to Gaussian. Hits were selected as any compound whose value property was at least 3 standard deviations from the value_mean property *or* whose bscore property (Malo, 2006) was at least 3 standard deviations from the bscore_mean in an automated data analysis pipeline generated with Pipeline Pilot (Accelrys, San Diego, CA) and R statistics package (www.r-project.org). TPNQ and vehicle control wells were included on every plate and used to calculate the Z' (Zhang, 1999). For those compounds picked as 'hits', Outcome = Active and Score = 100, all remaining compound were assigned Outcome = Inactive and Score = 0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ValueThe raw fluorescence intensities were divided by the initial fluorescence, and the slope from 7 to 12 seconds after thallium addition was calculated and labeled 'Value'.Float
2Value_meanMean 'Value' on a per-plate basisFloat
3Value_stddevStandard deviation of 'Value' on a per-plate basisFloat
4BscoreBscore polish of 'Value'Float
5Bscore_meanMean 'Bscore' on a per-plate basisFloat
6Bscore_stddevStandard deviation of 'Bscore' on a per-plate basisFloat
7AB_meanMean of the vehicle control (assay buffer + 0.1% DMSO) on a per-plate basisFloat
8TPNQ_meanMean of the TPNQ control on a per-plate basisFloat
9AB_stddevStandard deviation of the vehicle control (assay buffer + 0.1% DMSO) on a per-plate basisFloat
10TPNQ_stddevStandard deviation of the TPNQ control on a per-plate basisFloat
11zprime_AB_TPNQZ factor calculation of vehicle and TPNQ control populations on a per-plate basisFloat
Additional Information
Grant Number: R21 NS057041-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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