High throughput discovery of novel modulators of ROMK K+ channel activity: Primary Screen
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 more ..
BioActive Compounds: 2463
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 test the small molecule library provided by the Molecular Libraries Small Molecule Repository (MLSMR, distributed by DPI-Biofocus) for the ability to modulate thalluium flux in a ROMK-expressing cell line. All compounds were tested in single at 10uM final concentration.
Experiments were done as previously described (Lewis, 2009). Briefly, 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 vehicle control 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 plate was reloaded into the FDSS and a second baseline acquired. 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 second baseline reading was used to assess fluorescent compound artifacts, and those wells where the reading exceeded 70% of the mean fluorescence for the plate were excluded from analysis. The slope of the static ratio from 7 to 12 seconds was calculated and the value (Value) compared with the average distribution of the remaining compound wells assumed to Gaussian. Hits were selected as any compound whose value property was at least 3 standard deviations from the value_mean property *or* whose bscore property (Malo, 2006) was at least 3 standard deviations from the bscore_mean in an automated data analysis pipeline generated with Pipeline Pilot (Accelrys, San Diego, CA) and R statistics package (www.r-project.org). TPNQ and vehicle control wells were included on every plate and used to calculate the Z' (Zhang, 1999). For those compounds picked as 'hits', Outcome = Active and Score = 100, all remaining compound were assigned Outcome = Inactive and Score = 0.
Data Table (Concise)