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BioAssay: AID 2753

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

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
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Active(114)
 
 
Inactive(127)
 
 
 Tested Substances
 Tested Substances
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Active(114)
 
 
Inactive(127)
 
 
AID: 2753
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (ROMK_DR)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-04-06
Modify Date: 2010-08-09

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 114
Related Experiments
AIDNameTypeComment
1917High throughput discovery of novel modulators of ROMK K+ channel activity: Retest of Primary HitsOtherdepositor-specified cross reference
1918High throughput discovery of novel modulators of ROMK K+ channel activity: Primary ScreenScreeningdepositor-specified cross reference
1922High throughput discovery of novel modulators of ROMK K+ channel activity: Selectivity by Patch ClampOtherdepositor-specified cross reference
2436High-throughput Discovery of Novel Modulators of ROMK K+ Channel ActivitySummarydepositor-specified cross reference
1916High throughput discovery of novel modulators of ROMK K+ channel activity: Analog Dose-ResponseConfirmatorysame project related to Summary assay
1924High throughput discovery of novel modulators of ROMK K+ channel activity: Ancillary ActivityOthersame 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 to give 'Value'. 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 5mM potassium buffer control wells were included on every plate and used to calculate the Z' (Zhang et al., 1999). Pubchem 'Outcome' and 'Activity' were assigned as follows: 1) samples with 2 or more replicates with 'Fit' were 'Active' and '100'; 2) samples with 2 or more replicates with 'Trend' were 'Active' and '50'; 3)samples with 1 'Fit' and 1 'Trend' were 'Active' and '50'; and 4)all other samples were 'Inactive' and '0'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1VUIDVanderbilt internal identifierString
2FitCategory for replicate 1Qualitative assessment of the quality of the curve. Options are "fit," "trend," and "no fit" for replicate 1String
3FitCategory for replicate 2Qualitative assessment of the quality of the curve. Options are "fit," "trend," and "no fit" for replicate 2String
4FitCategory for replicate 3Qualitative assessment of the quality of the curve. Options are "fit," "trend," and "no fit" for replicate 3String
5top for replicate 1If the fit converged, then this is the calculated value for the top parameter for replicate 1Float
6top for replicate 2If the fit converged, then this is the calculated value for the top parameter for replicate 2Float
7top for replicate 3If the fit converged, then this is the calculated value for the top parameter for replicate 3Float
8bottom for replicate 1If the fit converged, then this is the calculated value for the bottom parameter for replicate 1Float
9bottom for replicate 2If the fit converged, then this is the calculated value for the bottom parameter for replicate 2Float
10bottom for replicate 3If the fit converged, then this is the calculated value for the bottom parameter for replicate 3Float
11slope for replicate 1If the fit converged, then this is the calculated value for the slope parameter for replicate 1Float
12slope for replicate 2If the fit converged, then this is the calculated value for the slope parameter for replicate 2Float
13slope for replicate 3If the fit converged, then this is the calculated value for the slope parameter for replicate 3Float
14logEC50 for replicate 1If the fit converged, then this is the calculated value for theLogEC50 parameter for replicate 1Float
15logEC50 for replicate 2If the fit converged, then this is the calculated value for theLogEC50 parameter for replicate 2Float
16logEC50 for replicate 3If the fit converged, then this is the calculated value for theLogEC50 parameter for replicate 3Float
17EC50 for replicate 1EC50 value in micromolar for replicate 1FloatμM
18EC50 for replicate 2EC50 value in micromolar for replicate 2FloatμM
19EC50 for replicate 3*EC50 value in micromolar for replicate 3FloatμM
20top_stderr for replicate 1Standard error for the calculated top value for replicate 1Float
21top_stderr for replicate 2Standard error for the calculated top value for replicate 2Float
22top_stderr for replicate 3Standard error for the calculated top value for replicate 3Float
23bottom_stderr for replicate 1Standard error for the calculated bottom value for replicate 1Float
24bottom_stderr for replicate 2Standard error for the calculated bottom value for replicate 2Float
25bottom_stderr for replicate 3Standard error for the calculated bottom value for replicate 3Float
26slope_stderr for replicate 1Standard error for the calculated slope value for replicate 1Float
27slope_stderr for replicate 2Standard error for the calculated slope value for replicate 2Float
28slope_stderr for replicate 3Standard error for the calculated slope value for replicate 3Float
29logEC50_stderr for replicate 1Standard error for the calculated LogEC50 value for replicate 1Float
30logEC50_stderr for replicate 2Standard error for the calculated LogEC50 value for replicate 2Float
31logEC50_stderr for replicate 3Standard error for the calculated LogEC50 value for replicate 3Float
32EC50_stderr for replicate 1Standard error for the calculated EC50 value for replicate 1Float
33EC50_stderr for replicate 2Standard error for the calculated EC50 value for replicate 2Float
34EC50_stderr for replicate 3Standard error for the calculated EC50 value for replicate 3Float
35top_pr for replicate 1p value for the calculated tope value for replicate 1Float
36top_pr for replicate 2p value for the calculated tope value for replicate 2Float
37top_pr for replicate 3p value for the calculated tope value for replicate 3Float
38bottom_pr for replicate 1p value for the calculated bottom value for replicate 1Float
39bottom_pr for replicate 2p value for the calculated bottom value for replicate 2Float
40bottom_pr for replicate 3p value for the calculated bottom value for replicate 3Float
41slope_pr for replicate 1p value for the calculated slope for replicate 1Float
42slope_pr for replicate 2p value for the calculated slope for replicate 2Float
43slope_pr for replicate 3p value for the calculated slope for replicate 3Float
44logEC50_pr for replicate 1p value for the calculated logEC50 for replicate 1Float
45logEC50_pr for replicate 2p value for the calculated logEC50 for replicate 2Float
46logEC50_pr for replicate 3p value for the calculated logEC50 for replicate 3Float
475mM_mean for replicate 1Mean of 5mM potassium positive control on a per-plate-basis for replicate 1Float
485mM_mean for replicate 2Mean of 5mM potassium positive control on a per-plate-basis for replicate 2Float
495mM_mean for replicate 3Mean of 5mM potassium positive control on a per-plate-basis for replicate 3Float
505mM_stddev for replicate 1Standard deviation of mean for 5mM potassium positive control on a per-plate-basis for replicate 1Float
515mM_stddev for replicate 2Standard deviation of mean for 5mM potassium positive control on a per-plate-basis for replicate 2Float
525mM_stddev for replicate 3Standard deviation of mean for 5mM potassium positive control on a per-plate-basis for replicate 3Float
53TPNQ_mean for replicate 1Mean of TPNQ negative control on a per-plate-basis for replicate 1Float
54TPNQ_mean for replicate 2Mean of TPNQ negative control on a per-plate-basis for replicate 2Float
55TPNQ_mean for replicate 3Mean of TPNQ negative control on a per-plate-basis for replicate 3Float
56TPNQ_stddev for replicate 1Standard deviation of mean for TPNQ negative control on a per-plate-basis for replicate 1Float
57TPNQ_stddev for replicate 2Standard deviation of mean for TPNQ negative control on a per-plate-basis for replicate 2Float
58TPNQ_stddev for replicate 3Standard deviation of mean for TPNQ negative control on a per-plate-basis for replicate 3Float
59zprime_5mM_TPNQ for replicate 1The calculated z' value between the TPNQ and 5mM Potassium controls on the plate for replicate 1Float
60zprime_5mM_TPNQ for replicate 2The calculated z' value between the TPNQ and 5mM Potassium controls on the plate for replicate 2Float
61zprime_5mM_TPNQ for replicate 3The calculated z' value between the TPNQ and 5mM Potassium controls on the plate for replicate 3Float
62Value at 2.54e-003 micromolar for replicate 1 (0.00254μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
63Value at 2.54e-003 micromolar for replicate 2 (0.00254μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
64Value at 2.54e-003 micromolar for replicate 3 (0.00254μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
65Value at 7.62e-003 micromolar for replicate 1 (0.00762μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
66Value at 7.62e-003 micromolar for replicate 2 (0.00762μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
67Value at 7.62e-003 micromolar for replicate 3 (0.00762μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
68Value at 2.29e-002 micromolar for replicate 1 (0.0229μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
69Value at 2.29e-002 micromolar for replicate 2 (0.0229μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
70Value at 2.29e-002 micromolar for replicate 3 (0.0229μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
71Value at 6.86e-002 micromolar for replicate 1 (0.0686μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
72Value at 6.86e-002 micromolar for replicate 2 (0.0686μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
73Value at 6.86e-002 micromolar for replicate 3 (0.0686μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
74Value at 0.206 micromolar for replicate 1 (0.206μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
75Value at 0.206 micromolar for replicate 2 (0.206μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
76Value at 0.206 micromolar for replicate 3 (0.206μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
77Value at 0.617 micromolar for replicate 1 (0.617μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
78Value at 0.617 micromolar for replicate 2 (0.617μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
79Value at 0.617 micromolar for replicate 3 (0.617μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
80Value at 1.85 micromolar for replicate 1 (1.85μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
81Value at 1.85 micromolar for replicate 2 (1.85μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
82Value at 1.85 micromolar for replicate 3 (1.85μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
83Value at 5.56 micromolar for replicate 1 (5.56μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
84Value at 5.56 micromolar for replicate 2 (5.56μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
85Value at 5.56 micromolar for replicate 3 (5.56μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
86Value at 16.7 micromolar for replicate 1 (16.7μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
87Value at 16.7 micromolar for replicate 2 (16.7μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
88Value at 16.7 micromolar for replicate 3 (16.7μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
89Value at 50 micromolar for replicate 1 (50μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
90Value at 50 micromolar for replicate 2 (50μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
91Value at 50 micromolar for replicate 3 (50μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
92Value at 150 micromolar for replicate 1 (150μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
93Value at 150 micromolar for replicate 2 (150μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
94Value at 150 micromolar for replicate 3 (150μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
95Value at 30 micromolar for replicate 1 (30μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
96Value at 30 micromolar for replicate 2 (30μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
97Value at 30 micromolar for replicate 3 (30μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
98Value at 10 micromolar for replicate 1 (10μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
99Value at 10 micromolar for replicate 2 (10μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
100Value at 10 micromolar for replicate 3 (10μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
101Value at 3.33 micromolar for replicate 1 (3.33μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
102Value at 3.33 micromolar for replicate 2 (3.33μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
103Value at 3.33 micromolar for replicate 3 (3.33μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
104Value at 1.11 micromolar for replicate 1 (1.11μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
105Value at 1.11 micromolar for replicate 2 (1.11μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
106Value at 1.11 micromolar for replicate 3 (1.11μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
107Value at 0.37 micromolar for replicate 1 (0.37μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
108Value at 0.37 micromolar for replicate 2 (0.37μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
109Value at 0.37 micromolar for replicate 3 (0.37μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
110Value at 0.123 micromolar for replicate 1 (0.123μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
111Value at 0.123 micromolar for replicate 2 (0.123μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
112Value at 0.123 micromolar for replicate 3 (0.123μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
113Value at 4.12e-002 micromolar for replicate 1 (0.0412μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
114Value at 4.12e-002 micromolar for replicate 2 (0.0412μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
115Value at 4.12e-002 micromolar for replicate 3 (0.0412μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
116Value at 1.37e-002 micromolar for replicate 1 (0.0137μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
117Value at 1.37e-002 micromolar for replicate 2 (0.0137μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
118Value at 1.37e-002 micromolar for replicate 3 (0.0137μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
119Value at 4.57e-003 micromolar for replicate 1 (0.00457μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
120Value at 4.57e-003 micromolar for replicate 2 (0.00457μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
121Value at 4.57e-003 micromolar for replicate 3 (0.00457μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
122Value at 1.52e-003 micromolar for replicate 1 (0.00152μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
123Value at 1.52e-003 micromolar for replicate 2 (0.00152μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
124Value at 1.52e-003 micromolar for replicate 3 (0.00152μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float
125Value at 5.08e-004 micromolar for replicate 1 (0.000508μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 1Float
126Value at 5.08e-004 micromolar for replicate 2 (0.000508μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 2Float
127Value at 5.08e-004 micromolar for replicate 3 (0.000508μM**)The raw fluorescence intensities were divided by the initial fluorescence, and the slope after thallium addition was calculated for replicate 3Float

* 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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