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BioAssay: AID 1717

Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Retesting of KCC2 cells with Ouabain

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(1781)
 
 
Inactive(1232)
 
 
Inconclusive(682)
 
 
 Tested Substances
 Tested Substances
All(3695)
 
 
Active(1781)
 
 
Inactive(1232)
 
 
Inconclusive(682)
 
 
 Related BioAssays
 Related BioAssays
AID: 1717
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (ED001_AID5)
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-05-05

Data Table ( Complete ):           Active    All
BioActive Compounds: 1781
Depositor Specified Assays
AIDNameTypeComment
1456Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Primary Screenother
1793Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Antagonist Ancillary Profileother
1799Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Antagonist Probe Summarysummary
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 expressing the cation-chloride cotransporter, KCC2 (same as primary screening conditions).
Protocol
METHOD:
1. Human embryonic kidney cells expressing KCC2 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 oubain) 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. Each compound well was compared with the mean + 3 standard deviations of the DMSO negative control population on a per plate basis. Compounds designated as 'Outcome' = 'Active' and 'Score' = '100' were statistical significant with greater than 99.7 % confidence than the negative control for the majority of the replicate testings. Wells that did not significantly vary in the majority of the replicates were labeled 'Outcome' = 'Inactive' and 'Score' = '0'. All other wells where multiple testings 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
3Value3The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated and labeled 'Value3' for the third replicate.Float
4Value4The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated and labeled 'Value4' for the fourth replicate.Float
5Value5The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated and labeled 'Value5' for the fifth replicate.Float
6Value6The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated and labeled 'Value6' for the sixth replicate.Float
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_mean3Mean of the bumetanide positive control for the third replicate on a per-plate basisFloat
10Bumet_mean4Mean of the bumetanide positive control for the fourth replicate on a per-plate basisFloat
11Bumet_mean5Mean of the bumetanide positive control for the fifth replicate on a per-plate basisFloat
12Bumet_mean6Mean of the bumetanide positive control for the sixth replicate on a per-plate basisFloat
13Bumet_stddev1Standard deviation of the bumetanide positive control for the first replicate on a per-plate basisFloat
14Bumet_stddev2Standard deviation of the bumetanide positive control for the second replicate on a per-plate basisFloat
15Bumet_stddev3Standard deviation of the bumetanide positive control for the third replicate on a per-plate basisFloat
16Bumet_stddev4Standard deviation of the bumetanide positive control for the fourth replicate on a per-plate basisFloat
17Bumet_stddev5Standard deviation of the bumetanide positive control for the fifth replicate on a per-plate basisFloat
18Bumet_stddev6Standard deviation of the bumetanide positive control for the sixth replicate on a per-plate basisFloat
19DMSO_mean1Mean of the DMSO vehicle control for the first replicate on a per-plate basisFloat
20DMSO_mean2Mean of the DMSO vehicle control for the second replicate on a per-plate basisFloat
21DMSO_mean3Mean of the DMSO vehicle control for the third replicate on a per-plate basisFloat
22DMSO_mean4Mean of the DMSO vehicle control for the fourth replicate on a per-plate basisFloat
23DMSO_mean5Mean of the DMSO vehicle control for the fifth replicate on a per-plate basisFloat
24DMSO_mean6Mean of the DMSO vehicle control for the sixth replicate on a per-plate basisFloat
25DMSO_stddev1Standard deviation of the DMSO vehicle control for the first replicate on a per-plate basisFloat
26DMSO_stddev2Standard deviation of the DMSO vehicle control for the second replicate on a per-plate basisFloat
27DMSO_stddev3Standard deviation of the DMSO vehicle control for the third replicate on a per-plate basisFloat
28DMSO_stddev4Standard deviation of the DMSO vehicle control for the fourth replicate on a per-plate basisFloat
29DMSO_stddev5Standard deviation of the DMSO vehicle control for the fifth replicate on a per-plate basisFloat
30DMSO_stddev6Standard deviation of the DMSO vehicle control for the sixth replicate on a per-plate basisFloat
31zprime_DMSO_BUMET_1Z factor calculation of DMSO and bumetanide control populations for the first replicate. (http://en.wikipedia.org/wiki/Z-factor)Float
32zprime_DMSO_BUMET_2Z factor calculation of DMSO and bumetanide control populations for the second replicate. (http://en.wikipedia.org/wiki/Z-factor)Float
33zprime_DMSO_BUMET_3Z factor calculation of DMSO and bumetanide control populations for the third replicate. Float
34zprime_DMSO_BUMET_4Z factor calculation of DMSO and bumetanide control populations for the fourth replicate. Float
35zprime_DMSO_BUMET_5Z factor calculation of DMSO and bumetanide control populations for the fifth replicate. Float
36zprime_DMSO_BUMET_6Z factor calculation of DMSO and bumetanide control populations for the sixth replicate. Float
Additional Information
Grant Number: R21NS053658-01

Data Table (Concise)
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