Assay Overview: Method for determining if compound acts as Hsp90 inhibitor. Saccharomyces strain expressing B-galactosidase driven by glucocorticoid response element is exposed to compounds. Loss of signal indicates interference with hsp90 function. ..more
Target
| Sequence: heat shock protein 90 [Candida albicans] Protein Family: HATPase_c Related Protein 3D Structures |
BioActive Compounds: 158
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 2007 | Summary of Broad Institute MLPCN Reversing Antifungal Drug Resistance Project. | summary | Summary |
| 1979 | Fluorescence Cell-Based Primary HTS of C.albicans growth in the presence of Fluconazole and compound | screening | Primary HTS |
| 434938 | Luminescence Cell-Based Secondary Assay to Identify Inhibitors of Hsp90 | confirmatory |
Description:
Keywords: Candida albicans, drug resistance, Fluconazole, Hsp90, stress response
Assay Overview: Method for determining if compound acts as Hsp90 inhibitor. Saccharomyces strain expressing B-galactosidase driven by glucocorticoid response element is exposed to compounds. Loss of signal indicates interference with hsp90 function.
Expected Outcome: Reduction of signal indicates hsp90 inhibition by compound.
Assay Overview: Method for determining if compound acts as Hsp90 inhibitor. Saccharomyces strain expressing B-galactosidase driven by glucocorticoid response element is exposed to compounds. Loss of signal indicates interference with hsp90 function.
Expected Outcome: Reduction of signal indicates hsp90 inhibition by compound.
Protocol
1) Grow reporter strain in SD-ADE media overnight @ 37 degrees C.
2) Dilute cells to OD = 0.04 in SD-ADE media.
3) Add 20 ul of culture to each well of 384 white plate.
4) Pin 100 nL compounds into plates, using 5uM Radicicol as positive control.
5) Add 20 ul of 20uM DOC (steroid) in SD-ADE media to plates.
6) Incubate at 30 C for 75 minutes, with agitation.
7) Add 40 ul Gal-Screen reagent.
8) Incubate at 30C for 25 minutes, then read luminescence.
2) Dilute cells to OD = 0.04 in SD-ADE media.
3) Add 20 ul of culture to each well of 384 white plate.
4) Pin 100 nL compounds into plates, using 5uM Radicicol as positive control.
5) Add 20 ul of 20uM DOC (steroid) in SD-ADE media to plates.
6) Incubate at 30 C for 75 minutes, with agitation.
7) Add 40 ul Gal-Screen reagent.
8) 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 0.12uM (0.12μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 10 | Activity at 0.235uM (0.235μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 11 | Activity at 0.50uM (0.5μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 12 | Activity at 1.00uM (1μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 13 | Activity at 1.95uM (1.95μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 14 | Activity at 3.80uM (3.8μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 15 | Activity at 7.50uM (7.5μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 16 | Activity at 8.00uM (8μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 17 | Activity at 15.00uM (15μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 18 | Activity at 16.00uM (16μM**) | 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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