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BioAssay: AID 1716

Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Counterscreen with HEK cells

Cation-chloride cotransporters such as K-Cl cotransport and Na-K-2Cl cotransport play major roles in a variety of physiological settings, including the modulation of GABAergic synaptic transmission. For instance, KCC2, a neuronal-specific K-Cl cotransporter is up-regulated in the brain during postnatal development, and is responsible for lowering the intracellular Cl- concentration in neurons, more ..
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 Tested Compounds
 Tested Compounds
All(3695)
 
 
Active(1712)
 
 
Inactive(1549)
 
 
Inconclusive(434)
 
 
 Tested Substances
 Tested Substances
All(3695)
 
 
Active(1712)
 
 
Inactive(1549)
 
 
Inconclusive(434)
 
 
 Related BioAssays
 Related BioAssays
AID: 1716
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (ED001_AID1)
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-05-05

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 1712
Related Experiments
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AIDNameTypeProbeComment
1456Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Primary ScreenOther depositor-specified cross reference
1799Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Antagonist Probe SummarySummary1 depositor-specified cross reference
1713Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Secondary Assay with KCC2 cellsOther same project related to Summary assay
1714Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Secondary Assay 3 with KCC2 cellsOther same project related to Summary assay
1715Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Secondary Assay 2 with KCC2 cellsOther same project related to Summary assay
1717Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Retesting of KCC2 cells with OuabainOther same project related to Summary assay
1718Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Counterscreen 2 with HEK cellsOther same project related to Summary assay
1723Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Dose-Dependent Assay 2 with KCC2Confirmatory same project related to Summary assay
1734Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Rubidium FluxConfirmatory same project related to Summary assay
1735Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Dose-dependent Counterscreen 2 with HEK cellsConfirmatory same project related to Summary assay
1736Identification of Novel Modulators of Cl- dependent Transport Process via HTS; Dose-dependent Assay with KCC2Confirmatory same project related to Summary assay
1737Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Dose-dependent Assay 3 with KCC2Confirmatory same project related to Summary assay
1738Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Dose-dependent Counterscreen 3 with HEK cellsConfirmatory same project related to Summary assay
1753Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Dose-Dependent Counterscreen with HEK cellsConfirmatory same project related to Summary assay
1793Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Antagonist Ancillary ProfileOther same project related to Summary assay
Description:
Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters
Assay Provider: Eric Delpire
Assay Provider Affliation: Vanderbilt University
Grant Title: Identification of Novel Modulators of Cl- dependent Transport Process via HTS
Grant Number: R21NS053658-01

Cation-chloride cotransporters such as K-Cl cotransport and Na-K-2Cl cotransport play major roles in a variety of physiological settings, including the modulation of GABAergic synaptic transmission. For instance, KCC2, a neuronal-specific K-Cl cotransporter is up-regulated in the brain during postnatal development, and is responsible for lowering the intracellular Cl- concentration in neurons, thus promoting GABA inhibition. Reduction in KCC2 expression results in brain hyperexcitability, as demonstrated by animal models. Furthermore, KCC2 expression is decreased in brain tissue isolated from epileptic patients.

There are very few pharmacological agents that affect K-Cl cotransporters. First, there are no specific inhibitors of K-Cl cotransporters. Furosemide is mostly used to inhibit K-Cl cotransporter function, but the diuretic is not very potent and is not specific as it inhibits the Na-K-2Cl cotransporter (diuretic effect), many Cl- channels including the GABAA receptor. Finding new inhibitors will provide important tools for the study of KCC2 in modulating inhibitory neurotransmission. Second, there are also no compounds known to activate K-Cl cotransporter, except for N-ethylmaleimide, which affects many cellular processes as an unspecific alkylating agent. Finding a specific agent that increase KCC2 function would potentially have therapeutic value, as increased KCC2 function reduces susceptibility to epileptic seizures.

The purpose of this assay was to test compounds identified as 'hits' from the Molecular Libraries Small Molecule Repository (MLSMR) at a single concentration in the presence of ouabain against HEK cells that do not contain the cation-chloride cotransporter, KCC2. Compounds active in this assay were disregarded as potential KCC2 probe molecules.
Protocol
METHOD:
1. Human embryonic kidney cells were plated at 20,000 cells/well in Dulbecco's modified medium (DMEM), 41.6% F12 (Gibco catalog 11320-033) in 384 well plate, black, clear bottom, poly-D-lysine coated (Greiner catalog 781946).
2. Cells were incubated overnight at 37 degrees C in 5%CO2.
3. Cells were loaded with 0.5 micromolar FluoZin2-AM dye (Invitrogen catalog number F24189) in assay buffer (Hanks Buffered Salt Solution, 20 mM HEPES, 0.2 mM ouabain) for 48 minutes.
4. Dye was removed and the plate imaged using the Hamamatsu FDSS kinetic plate reader equipped with 480 nanometer excitation and 540 nanometer emission filters.
5. Compounds in DMSO were added to a final concentration of 10 micromolar (0.1% DMSO final concentration).
6. Thallium buffer stimulus (125 mM sodium bicarbonate, 12 mM thallium sulfate, 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 10 mM HEPES, pH 7.3.) was added and images collected at 1 Hz.
7. Assay buffer containing DMSO (0.1% final concentration) was used as the negative control and 2 mM bumetanide was used as the positive control on each plate.

DATA PROCESSING:
1. The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated and labeled 'Value1' for replicate 1 and 'Value2' for replicate 2.
2. Comparison of each compound well with the mean + 3 standard deviations of the DMSO negative control population on a per plate basis resulted in wells designated as 'Outcome' = 'Active' and 'Score' = '100' based on a statistical significance of greater than 99.7 % confidence. Wells that did not significantly vary were labeled 'Outcome' = 'Inactive' and 'Score' = '0'. Wells where duplicate testing resulted in discrepant values were designated 'Outcome' = 'Inconclusive' and 'Score' = '50'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Value1The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated and labeled 'Value1' for the first replicate.Float
2Value2The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated and labeled 'Value2' for the second replicate.Float
3Value1_meanMean 'Value1' on a per-plate basisFloat
4Value2_meanMean 'Value2' on a per-plate basisFloat
5Value1_stddevStandard deviation of 'Value1' on a per-plate basisFloat
6Value2_stddevStandard deviation of 'Value2' on a per-plate basisFloat
7Bumet_mean1Mean of the bumetanide positive control for the first replicate on a per-plate basisFloat
8Bumet_mean2Mean of the bumetanide positive control for the second replicate on a per-plate basisFloat
9Bumet_stddev1Standard deviation of the bumetanide positive control for the first replicate on a per-plate basisFloat
10Bumet_stddev2Standard deviation of the bumetanide positive control for the second replicate on a per-plate basisFloat
11DMSO_mean1Mean of the DMSO vehicle control for the first replicate on a per-plate basisFloat
12DMSO_mean2Mean of the DMSO vehicle control for the second replicate on a per-plate basisFloat
13DMSO_stddev1Standard deviation of the DMSO vehicle control for the first replicate on a per-plate basisFloat
14DMSO_stddev2Standard deviation of the DMSO vehicle control for the second replicate on a per-plate basisFloat
Additional Information
Grant Number: R21NS053658-01

Data Table (Concise)
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