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BioAssay: AID 2388

Luminesence Cell-Based Secondary Assay to Identify Inhibitors of Calcineurin

Assay Overview: Method for determining if compound acts as Calcineurin inhibitor. S cerevisiae expressing beta-galactosidase driven by 4 tandem copies of the calcineurin-dependent response element (CDRE) is exposed to compounds followed by challenge with CaCl2 stressor. Potential inhibition of calcineurin function will be evaluated by loss of signal. ..more
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 Tested Compounds
 Tested Compounds
All(342)
 
 
Active(23)
 
 
Inactive(319)
 
 
 Tested Substances
 Tested Substances
All(344)
 
 
Active(23)
 
 
Inactive(321)
 
 
AID: 2388
Data Source: Broad Institute (2037-06_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-22
Modify Date: 2011-10-11

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 23
Depositor Specified Assays
AIDNameTypeComment
2007Summary of Broad Institute MLPCN Reversing Antifungal Drug Resistance Project.summarySummary
1979Fluorescence Cell-Based Primary HTS of C.albicans growth in the presence of Fluconazole and compoundscreeningPrimary HTS
Description:
Keywords: Candida albicans, drug resistance, Fluconazole, calcineurin, stress response

Assay Overview: Method for determining if compound acts as Calcineurin inhibitor. S cerevisiae expressing beta-galactosidase driven by 4 tandem copies of the calcineurin-dependent response element (CDRE) is exposed to compounds followed by challenge with CaCl2 stressor. Potential inhibition of calcineurin function will be evaluated by loss of signal.

Expected Outcome: Reduction of signal indicates calcineurin inhibition by compound.
Protocol
1) Grow reporter strain in SD-URE media overnight @ 23 degrees C.
2) Dilute cells to OD = 0.01 in SD-URE media.
3) Add 35 ul of culture to each well of 384 white plate.
4) Pin 100 nL compounds into plates, using 5uM FK506 as positive control.
5) Incubate plates for 75 minutes at 23C, with agitation.
6) Add 5 ul of SD-URA with 1.25M Calcium chloride to plates.
7) Incubate at 30 C for 90 minutes, with agitation.
8) Add 40 ul Gal-Screen reagent.
9) Incubate at 30C for 25 minutes, then read luminescence.
Comment
Dose Data Analysis

Neutral control (NC) wells and positive control (PC) wells were included on every plate.
Active inhibitor compounds result in decreased luminescent signal.

The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.

No plate pattern correction algorithm was applied.

IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.


PubChem Activity Score and Outcome

PUBCHEM_ACTIVITY_SCORE:

Inactive compounds = 0
Active compounds = -10*Log(IC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
IC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
or
The fit was not valid due to poor fit quality.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3EC50 Standard Errorthe standard error for the calculated EC50 valueFloatμM
4S0the fitted activity level at zero concentrationFloat%
5SInfthe fitted activity level at infinite concentrationFloat%
6Hill Slopethe slope at EC50Float
7Num. Pointsthe number of data points included in the plotInteger
8Max. Activitythe maximum activity value observedFloat%
9Activity at 0.135uM (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.285uM (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.560uM (0.56μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 1.10uM (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 2.10uM (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 2.35uM (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 4.60uM (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 9.00uM (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity at 18.00uM (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH086456-01

Data Table (Concise)
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