Bookmark and Share
BioAssay: AID 1738

Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Dose-dependent Counterscreen 3 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 ..
_
   
 Tested Compounds
 Tested Compounds
All(45)
 
 
Active(4)
 
 
Inactive(37)
 
 
Inconclusive(4)
 
 
 Tested Substances
 Tested Substances
All(45)
 
 
Active(4)
 
 
Inactive(37)
 
 
Inconclusive(4)
 
 
 Related BioAssays
 Related BioAssays
AID: 1738
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (ED001_CRC6)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-05-08

Data Table ( Complete ):           Active    All
BioActive Compounds: 4
Depositor Specified Assays
AIDNameTypeComment
1456Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Primary Screenother
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 of interest in dose-response against HEK cells in the presence of sodium-free buffer.
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 (140 mM N-methylglucamine.Cl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 5 mM HEPES) 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 were tested in triplicate as 11-point 3-fold dilutions starting at 30 uM.
6. Thallium buffer stimulus (125 mM N-methyl-glucamine , 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 (0.3% 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 'Value' for each test well.
2. The values for each dose-response curve for each compound were treated as a group and fit to a variable-slope sigmoidal curve using Pipeline Pilot and the R-statistics package 'drc' module.
3. Plate-based control statistics were calculated using Pipeline Pilot.
4. Replicate group measurements for each compound were treated independently rather than averaged over the replicates.
5. Individual curves were classified into a 'fit category' as either 'fit,' 'trend,' or 'no fit.' 'No fit' was used to describe groups where the curve fit did not converge. 'Fit' was used to describe groups where three or more actual data points lay on both sides of the calculated EC50. In cases where two or fewer data points were on either side of the EC50, the group was called a 'trend.' This was done to acknowledge the increased uncertainty in curves where the calculated EC50 is less reliable.
6. The overall activity outcome is an aggregate of all successful fits. If a plurality of the replicates for a compound resulted in a fit or a trend, that compound was considered active.
7. The activity score was assigned as 100 when 2 or 3 out of the 3 replicates were considered 'fit.' The score was assigned as 50 when 2 or 3 of 3 replicates were considered 'trend' or if one 'fit,' one 'trend,' and one 'no fit' existed for a set of replicates. A score of 0 was assigned when fewer than 2 of 3 replicates gave either 'fit' or 'trend.'
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1bottom 1If the fit converged, then this is the calculated value for the bottom parameter for the first replicateFloat
2bottom 2If the fit converged, then this is the calculated value for the bottom parameter for the second replicateFloat
3bottom 3If the fit converged, then this is the calculated value for the bottom parameter for the third replicateFloat
4bottom_pr 1p value for the calculated bottom fit parameter for the first replicateFloat
5bottom_pr 2p value for the calculated bottom fit parameter for the second replicateFloat
6bottom_pr 3p value for the calculated bottom fit parameter for the third replicateFloat
7bottom_stderr 1The standard error calculated for the bottom parameter for the first replicateFloat
8bottom_stderr 2The standard error calculated for the bottom parameter for the second replicateFloat
9bottom_stderr 3The standard error calculated for the bottom parameter for the third replicateFloat
10BUMET_mean 1Mean value for the bumetanide controls on the plate for the first replicateFloat
11BUMET_mean 2Mean value for the bumetanide controls on the plate for the second replicateFloat
12BUMET_mean 3Mean value for the bumetanide controls on the plate for the third replicateFloat
13BUMET_stddev 1Standard deviation of the mean for the bumetanide controls on the plate for the first replicateFloat
14BUMET_stddev 2Standard deviation of the mean for the bumetanide controls on the plate for the second replicateFloat
15BUMET_stddev 3Standard deviation of the mean for the bumetanide controls on the plate for the third replicateFloat
16DMSO_mean 1Mean value for the DMSO controls on the plate for the first replicateFloat
17DMSO_mean 2Mean value for the DMSO controls on the plate for the second replicateFloat
18DMSO_mean 3Mean value for the DMSO controls on the plate for the third replicateFloat
19DMSO_stddev 1Standard deviation of the mean for the DMSO controls on the plate for the first replicateFloat
20DMSO_stddev 2Standard deviation of the mean for the DMSO controls on the plate for the second replicateFloat
21DMSO_stddev 3Standard deviation of the mean for the DMSO controls on the plate for the third replicateFloat
22EC50 1*EC50 value in micromolar for the first replicateFloatμM
23EC50 2EC50 value in micromolar for the second replicateFloatμM
24EC50 3EC50 value in micromolar for the second replicateFloatμM
25EC50_stderr 1Standard error for the calculated EC50 value for the first replicateFloat
26EC50_stderr 2Standard error for the calculated EC50 value for the second replicateFloat
27EC50_stderr 3Standard error for the calculated EC50 value for the second replicateFloat
28FitCategory 1Qualitative assessment of the quality of the curve. Options are "fit," "trend," and "no fit" for the first replicate.String
29FitCategory 2Qualitative assessment of the quality of the curve. Options are "fit," "trend," and "no fit" for the second replicate.String
30FitCategory 3Qualitative assessment of the quality of the curve. Options are "fit," "trend," and "no fit" for the third replicate.String
31logEC50 1If the fit converged, then this is the calculated value for the logEC50 parameter for the first replicateFloat
32logEC50 2If the fit converged, then this is the calculated value for the logEC50 parameter for the second replicateFloat
33logEC50 3If the fit converged, then this is the calculated value for the logEC50 parameter for the third replicateFloat
34logEC50_pr 1p value for the calculated logEC50 for the first replicateFloat
35logEC50_pr 2p value for the calculated logEC50 for the second replicateFloat
36logEC50_pr 3p value for the calculated logEC50 for the second replicateFloat
37logEC50_stderr 1The standard error calculated for the logEC50 parameter for the first replicateFloat
38logEC50_stderr 2The standard error calculated for the logEC50 parameter for the second replicateFloat
39logEC50_stderr 3The standard error calculated for the logEC50 parameter for the third replicateFloat
40Rsquared 1R-squared value for the fitted versus raw values for the first replicateFloat
41Rsquared 2R-squared value for the fitted versus raw values for the second replicateFloat
42Rsquared 3R-squared value for the fitted versus raw values for the third replicateFloat
43slope 1If the fit converged, then this is the calculated value for the slope parameter for the first replicateFloat
44slope 2If the fit converged, then this is the calculated value for the slope parameter for the second replicateFloat
45slope 3If the fit converged, then this is the calculated value for the slope parameter for the third replicateFloat
46slope_pr 1p value for the calculated slope parameter for the first replicateFloat
47slope_pr 2p value for the calculated slope parameter for the second replicateFloat
48slope_pr 3p value for the calculated slope parameter for the third replicateFloat
49slope_stderr 1The standard error calculated for the slope parameter for the first replicateFloat
50slope_stderr 2The standard error calculated for the slope parameter for the second replicateFloat
51slope_stderr 3The standard error calculated for the slope parameter for the third replicateFloat
52top 1If the fit converged, then this is the calculated value for the top parameter for the first replicateFloat
53top 2If the fit converged, then this is the calculated value for the top parameter for the second replicateFloat
54top 3If the fit converged, then this is the calculated value for the top parameter for the third replicateFloat
55top_pr 1p value for the calculated top fit parameter for the first replicateFloat
56top_pr 2p value for the calculated top fit parameter for the second replicateFloat
57top_pr 3p value for the calculated top fit parameter for the third replicateFloat
58top_stderr 1The standard error calculated for the top parameter for the first replicateFloat
59top_stderr 2The standard error calculated for the top parameter for the second replicateFloat
60top_stderr 3The standard error calculated for the top parameter for the third replicateFloat
61Value at 0.123 micromolar 1 (0.123μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
62Value at 0.123 micromolar 2 (0.123μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
63Value at 0.123 micromolar 3 (0.123μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
64Value at 0.37 micromolar 1 (0.37μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
65Value at 0.37 micromolar 2 (0.37μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
66Value at 0.37 micromolar 3 (0.37μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
67Value at 1.11 micromolar 1 (1.11μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
68Value at 1.11 micromolar 2 (1.11μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
69Value at 1.11 micromolar 3 (1.11μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
70Value at 1.37e-002 micromolar 1 (0.0137μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
71Value at 1.37e-002 micromolar 2 (0.0137μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
72Value at 1.37e-002 micromolar 3 (0.0137μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
73Value at 1.52e-003 micromolar 1 (0.00152μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
74Value at 1.52e-003 micromolar 2 (0.00152μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
75Value at 1.52e-003 micromolar 3 (0.00152μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
76Value at 10 micromolar 1 (10μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
77Value at 10 micromolar 2 (10μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
78Value at 10 micromolar 3 (10μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
79Value at 3.33 micromolar 1 (3.33μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
80Value at 3.33 micromolar 2 (3.33μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
81Value at 3.33 micromolar 3 (3.33μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
82Value at 30 micromolar 1 (30μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
83Value at 30 micromolar 2 (30μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
84Value at 30 micromolar 3 (30μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
85Value at 4.12e-002 micromolar 1 (0.0412μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
86Value at 4.12e-002 micromolar 2 (0.0412μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
87Value at 4.12e-002 micromolar 3 (0.0412μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
88Value at 4.57e-003 micromolar 1 (0.00457μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
89Value at 4.57e-003 micromolar 2 (0.00457μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
90Value at 4.57e-003 micromolar 3 (0.00457μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
91Value at 5.08e-004 micromolar 1 (0.000508μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the first replicate.Float
92Value at 5.08e-004 micromolar 2 (0.000508μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the second replicate.Float
93Value at 5.08e-004 micromolar 3 (0.000508μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated for the third replicate.Float
94zprime_DMSO_BUMET 1The calculated z' value between the DMSO and bumetanide controls on the plate for the first replicateFloat
95zprime_DMSO_BUMET 2The calculated z' value between the DMSO and bumetanide controls on the plate for the second replicateFloat
96zprime_DMSO_BUMET 3The calculated z' value between the DMSO and bumetanide controls on the plate for the third replicateFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R21NS053658-01

Data Table (Concise)
PageFrom: