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
Related BioAssays
Related BioAssays
Target
BioActive Compounds: 23
Depositor Specified Assays
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.
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.
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.
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
Show more |
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | EC50 Qualifier | '>', '=', or '<' | String | |||
| 2 | EC50* | the concentration whereupon perceived activity reaches 50% of the maximum | | Float | μM | |
| 3 | EC50 Standard Error | the standard error for the calculated EC50 value | | Float | μM | |
| 4 | S0 | the fitted activity level at zero concentration | | Float | % | |
| 5 | SInf | the fitted activity level at infinite concentration | | Float | % | |
| 6 | Hill Slope | the slope at EC50 | | Float | ||
| 7 | Num. Points | the number of data points included in the plot | | Integer | ||
| 8 | Max. Activity | the maximum activity value observed | | Float | % | |
| 9 | Activity at 0.135uM (0.135μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 10 | Activity at 0.285uM (0.285μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 11 | Activity at 0.560uM (0.56μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 12 | Activity at 1.10uM (1.1μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 13 | Activity at 2.10uM (2.1μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 14 | Activity at 2.35uM (2.35μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 15 | Activity at 4.60uM (4.6μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 16 | Activity at 9.00uM (9μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 17 | Activity at 18.00uM (18μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % |
* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH086456-01
Data Table (Concise)
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