AID	Name	Type	Comment
2753	High throughput discovery of novel modulators of ROMK K+ channel activity: Dose-Response Assay	confirmatory
2436	High-throughput Discovery of Novel Modulators of ROMK K+ Channel Activity	summary

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.

The purpose of this assay was to retest primary hit compounds resupplied by Biofocus-DPI in duplicate at 10uM using the primary assay conditions.

High-Throughput Screening
As described previously (Lewis, 2009), 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 assay buffer (0.44 mM NaH2PO4, 4.17 mM NaHCO3, 137.93 mM NaCl, 0.338 mM Na2HPO4, 20 mM HEPES, 0.25 mM K2SO4, adjusted to 343 mOsm with sucrose) 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 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 slope of the static ratio from 7 to 12 seconds was calculated and the value (Value) compared with the mean of the negative control population (assay buffer + 0.1% DMSO). An automated data analysis pipeline generated with Pipeline Pilot (Accelrys, San Diego, CA) and R statistics package (www.r-project.org) was used. TPNQ and assay buffer control wells were included on every plate and used to calculate the Z' (Zhang et al., 1999). Compounds that differed from the negative control by 3 standard deviations in both replicates were assigned Outcome = Active and Score = 100. Compounds where duplicate values differed where assigned Outcome = Active and Score = 50. All other compounds were assigned Outcome = Inactive and Score = 0.

Grant Number: R21 NS057041-01