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BioAssay: AID 1916

High throughput discovery of novel modulators of ROMK K+ channel activity: Analog Dose-Response

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 ..
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 Tested Compounds
 Tested Compounds
All(20)
 
 
Active(11)
 
 
Inactive(7)
 
 
Unspecified(2)
 
 
 Tested Substances
 Tested Substances
All(20)
 
 
Active(11)
 
 
Inactive(7)
 
 
Unspecified(2)
 
 
AID: 1916
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (JD001_inhib_analog_crc)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-09-03
Hold-until Date: 2010-02-23
Modify Date: 2010-02-23

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: Potassium inwardly-rectifying channel, subfamily J, member 1 [Homo sapiens]
Description ..   
Protein Family: Inward rectifier potassium channel

Gene:KCNJ1     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 11
Related Experiments
AIDNameTypeComment
2436High-throughput Discovery of Novel Modulators of ROMK K+ Channel ActivitySummarydepositor-specified cross reference
1917High throughput discovery of novel modulators of ROMK K+ channel activity: Retest of Primary HitsOthersame project related to Summary assay
1918High throughput discovery of novel modulators of ROMK K+ channel activity: Primary ScreenScreeningsame project related to Summary assay
1922High throughput discovery of novel modulators of ROMK K+ channel activity: Selectivity by Patch ClampOthersame project related to Summary assay
1924High throughput discovery of novel modulators of ROMK K+ channel activity: Ancillary ActivityOthersame project related to Summary assay
2753High throughput discovery of novel modulators of ROMK K+ channel activity: Dose-Response AssayConfirmatorysame project related to Summary assay
435017High throughput discovery of novel modulators of ROMK K+ channel activity: Analog Library TestingConfirmatorysame project related to Summary assay
Description:
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.
Protocol
The purpose of this assay to test synthesized compounds in thallium flux for their ability to dose-dependently inhibit ROMK function.
Experimental methods were as previously described (Lewis, 2009). Briefly, cells were plated in black-walled, clear-bottom plates and treated with Tet overnight to induce the expression of ROMK in serum-free media. The cells were loaded with FluoZin2 dye, incubated for 20 min at RT and washed 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). The plate was imaged on the Hamamatsu FDSS 6000 system to obtain F0, followed by compound addition. Compounds were serially diluted 3-fold to generate 11-point concentration curves. After 20 min at RT, thallium stimulus buffer (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) was added while simultaneously imaging for a total of 2 min acquisition time.
Data Handling:
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 for each compound concentration. 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). The data were normalized to % TPNQ activity and the concentration response curves fit using Prism 5.0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1LMLIDInternal ReferenceString
2VUIDInternal Reference 2String
3R1 C1 (0.0169μM**)Value for replicate 1 at concentration 1FloatμM
4R1 C2 (0.0508μM**)Value for replicate 1 at concentration 2FloatμM
5R1 C3 (0.152μM**)Value for replicate 1 at concentration 3FloatμM
6R1 C4 (0.457μM**)Value for replicate 1 at concentration 4FloatμM
7R1 C5 (1.37μM**)Value for replicate 1 at concentration 5FloatμM
8R1 C6 (4.12μM**)Value for replicate 1 at concentration 6FloatμM
9R1 C7 (12.3μM**)Value for replicate 1 at concentration 7FloatμM
10R1 C8 (37μM**)Value for replicate 1 at concentration 8FloatμM
11R1 C9 (111μM**)Value for replicate 1 at concentration 9FloatμM
12R1 C10 (333μM**)Value for replicate 1 at concentration 10FloatμM
13R1 C11 (1000μM**)Value for replicate 1 at concentration 11FloatμM
14R2 C1 (0.0169μM**)Value for replicate 2 at concentration 1FloatμM
15R2 C2 (0.0508μM**)Value for replicate 2 at concentration 2FloatμM
16R2 C3 (0.152μM**)Value for replicate 2 at concentration 3FloatμM
17R2 C4 (0.457μM**)Value for replicate 2 at concentration 4FloatμM
18R2 C5 (1.37μM**)Value for replicate 2 at concentration 5FloatμM
19R2 C6 (4.12μM**)Value for replicate 2 at concentration 6FloatμM
20R2 C7 (12.3μM**)Value for replicate 2 at concentration 7FloatμM
21R2 C8 (37μM**)Value for replicate 2 at concentration 8FloatμM
22R2 C9 (111μM**)Value for replicate 2 at concentration 9FloatμM
23R2 C10 (333μM**)Value for replicate 2 at concentration 10FloatμM
24R2 C11 (1000μM**)Value for replicate 2 at concentration 11FloatμM
25R3 C1 (0.0169μM**)Value for replicate 3 at concentration 1FloatμM
26R3 C2 (0.0508μM**)Value for replicate 3 at concentration 2FloatμM
27R3 C3 (0.152μM**)Value for replicate 3 at concentration 3FloatμM
28R3 C4 (0.457μM**)Value for replicate 3 at concentration 4FloatμM
29R3 C5 (1.37μM**)Value for replicate 3 at concentration 5FloatμM
30R3 C6 (4.12μM**)Value for replicate 3 at concentration 6FloatμM
31R3 C7 (12.3μM**)Value for replicate 3 at concentration 7FloatμM
32R3 C8 (37μM**)Value for replicate 3 at concentration 8FloatμM
33R3 C9 (111μM**)Value for replicate 3 at concentration 9FloatμM
34R3 C10 (333μM**)Value for replicate 3 at concentration 10FloatμM
35R3 C11 (1000μM**)Value for replicate 3 at concentration 11FloatμM
36R4 C1 (0.0169μM**)Value for replicate 4 at concentration 1FloatμM
37R4 C2 (0.0508μM**)Value for replicate 4 at concentration 2FloatμM
38R4 C3 (0.152μM**)Value for replicate 4 at concentration 3FloatμM
39R4 C4 (0.457μM**)Value for replicate 4 at concentration 4FloatμM
40R4 C5 (1.37μM**)Value for replicate 4 at concentration 5FloatμM
41R4 C6 (4.12μM**)Value for replicate 4 at concentration 6FloatμM
42R4 C7 (12.3μM**)Value for replicate 4 at concentration 7FloatμM
43R4 C8 (37μM**)Value for replicate 4 at concentration 8FloatμM
44R4 C9 (111μM**)Value for replicate 4 at concentration 9FloatμM
45R4 C10 (333μM**)Value for replicate 4 at concentration 10FloatμM
46R4 C11 (1000μM**)Value for replicate 4 at concentration 11FloatμM
47Fit ResultFit type as determined by PrismString
48Best-fit Value LogIC50Best fit value for the LogIC50Float
49Std. Error LogIC50Standard Error for the LogIC50Float
50CI LogIC5095% Confidence Intervals for the LogIC50String
51Best-fit Value HillSlopeBest-fit value for Hill SlopeFloat
52Std. Error HillSlopeStandard Error for the Hill SlopeFloat
53CI HillSlope95% Confidence Intervals for the Hill SlopeString
54Best-Fit Value IC50 (M)Best-fit value for the IC50 in MolarFloat
55CI IC50 (M)95% Confidence Intervals for the IC50 value in MolarString
56Best-Fit Value IC50 (uM)*Best-fit value for the IC50 in micromolarFloatμM
57Degrees of FreedomDegrees of FreedomInteger
58R2R2Float
59Absolute Sum of SquaresAbsolute Sum of SquaresString
60Number of PointsNumber of Points analyzedInteger

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R21 NS057041-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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