AID	Name	Type	Comment
1456	Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Primary Screen	other
1799	Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Antagonist Probe Summary	summary

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 ouabain and bumetanide.

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 oubain, 2mM bumetanide) 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 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 (0.3% 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.'

Grant Number: R21NS053658-01