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BioAssay: AID 504499

Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set9

Assay Overview: Fluorescent counterscreen of C. albicans growth in the absence of fluconazole with compound, to determine whether compound is toxic in its own right or only in presence of fluconazole. A clinical isolate of fluconazole-resistant C. albicans will be cultured in 384-well format in the absence of a sub-lethal concentration of fluconazole. Test compounds will be pinned into individual wells and those compounds that inhibit subsequent yeast growth in the presence of fluconazole, but not in its absence, will be identified as hits meriting further evaluation. ..more
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 Tested Compounds
 Tested Compounds
All(28)
 
 
Active(2)
 
 
Inactive(26)
 
 
 Tested Substances
 Tested Substances
All(28)
 
 
Active(2)
 
 
Inactive(26)
 
 
 Related BioAssays
 Related BioAssays
AID: 504499
Data Source: Broad Institute (2037-07_Inhibitor_Dose_DryPowder_Activity_Set9)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-21
Hold-until Date: 2012-03-21
Modify Date: 2012-03-21

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
2007Summary of Broad Institute MLPCN Reversing Antifungal Drug Resistance Project.Summary3 depositor-specified cross reference: Summary assay
1979Fluorescence Cell-Based Primary HTS of C.albicans growth in the presence of Fluconazole and compoundScreening same project related to Summary assay
2327Fluorescence Cell-Based Secondary Assay for toxicity in mammalian fibroblastsConfirmatory same project related to Summary assay
2387Fluorescence Cell-Based Secondary Assay to Measure Toxicity of Compounds Not in the Presence of FluconazoleConfirmatory same project related to Summary assay
2388Luminesence Cell-Based Secondary Assay to Identify Inhibitors of CalcineurinConfirmatory same project related to Summary assay
2400Luminesence Cell-Based Secondary Assay to Identify Inhibitors of Hsp90Confirmatory same project related to Summary assay
2423Fluorescence Cell-Based Secondary Assay to Identify Inhibitors of Resistant C. albicans Growth in the Presence of FluconazoleConfirmatory same project related to Summary assay
2467Fluorescence Cell-Based Retest of C. albicans Growth in the Presence of FluconazoleConfirmatory same project related to Summary assay
434923Fluorescence Cell-Based Secondary Assay for Toxicity in Mammalian FibroblastsConfirmatory same project related to Summary assay
434930Fluorescence Cell-Based Retest of C. albicans growth in the presence of fluconazole and compoundConfirmatory same project related to Summary assay
434935Fluorescence Cell-Based Secondary Assay of Resistant C. albicans Growth in the Presence of FluconazoleConfirmatory same project related to Summary assay
434938Luminescence Cell-Based Secondary Assay to Identify Inhibitors of Hsp90Confirmatory same project related to Summary assay
434945Fluorescence Cell-Based Secondary Assay to Identify Inhibitors of CalcineurinConfirmatory same project related to Summary assay
434946Fluorescence Cell-Based Secondary Assay to Measure Toxicity of Compounds Not in the Presence of Fluconazole.Confirmatory same project related to Summary assay
488802Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488807Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488809Antifungal Drug Resistance - Mammalian Cell Toxicity Measured in Cell-Based System Using Plate Reader - 2037-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488836Reversing Antifungal Drug Resistance Measured in Microorganism System Using Plate Reader - 2037-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488893Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
488905Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
488907Reversing Antifungal Drug Resistance Measured in Microorganism System Using Plate Reader - 2037-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
488909Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
488911Antifungal Drug Resistance - Mammalian Cell Toxicity Measured in Cell-Based System Using Plate Reader - 2037-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
488930Reversing Antifungal Drug Resistance Measured in Microorganism System Using Plate Reader - 2037-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
488932Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
488933Antifungal Drug Resistance - Mammalian Cell Toxicity Measured in Cell-Based System Using Plate Reader - 2037-04_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
488937Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
488943Reversing Antifungal Drug Resistance Measured in Microorganism System Using Plate Reader - 2037-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
488950Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
493064Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
493069Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
493070Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
493080Antifungal Retest at Dose Measured in Microorganism System Using Plate Reader - 2037-09_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
493081Antifungal Retest at Dose Measured in Microorganism System Using Plate Reader - 2037-09_Inhibitor_DoseNoFile_DryPowder_ActivityConfirmatory same project related to Summary assay
493082Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_DoseNoFile_DryPowder_ActivityConfirmatory same project related to Summary assay
493089Antifungal Retest at Dose Measured in Microorganism System Using Plate Reader - 2037-09_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
493099Antifungal Drug Resistance - Mammalian Cell Toxicity Measured in Cell-Based System Using Plate Reader - 2037-04_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
493134Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatory same project related to Summary assay
493147Antifungal Drug Resistance - Mammalian Cell Toxicity Measured in Cell-Based System Using Plate Reader - 2037-04_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
493149Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set9Confirmatory same project related to Summary assay
493150Antifungal Retest at Dose Measured in Microorganism System Using Plate Reader - 2037-09_Inhibitor_Dose_DryPowder_Activity_Set5Confirmatory same project related to Summary assay
493157Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set8Confirmatory same project related to Summary assay
504390Antifungal Drug Resistance - Hsp90 Inhibitors Measured in Microorganism System Using Plate Reader - 2037-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504502Antifungal Retest at Dose Measured in Microorganism System Using Plate Reader - 2037-09_Inhibitor_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
504504Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set10Confirmatory same project related to Summary assay
504525Antifungal Drug Resistance - Mammalian Cell Toxicity Measured in Cell-Based System Using Plate Reader - 2037-04_Inhibitor_Dose_DryPowder_Activity_Set7Confirmatory same project related to Summary assay
Description:
Keywords: Candida albicans, drug resistance, fluconazole, Hsp90, stress response

Assay Overview: Fluorescent counterscreen of C. albicans growth in the absence of fluconazole with compound, to determine whether compound is toxic in its own right or only in presence of fluconazole. A clinical isolate of fluconazole-resistant C. albicans will be cultured in 384-well format in the absence of a sub-lethal concentration of fluconazole. Test compounds will be pinned into individual wells and those compounds that inhibit subsequent yeast growth in the presence of fluconazole, but not in its absence, will be identified as hits meriting further evaluation.

Expected Outcome: loss of signal indicates broad toxicity rather than specificity of action in concert with fluconazole.
Protocol
Protocol:
Stock solutions
Geldanamycin (GA) 15mM stock sol'n in DMSO
Pen/Strep 100x in PBS

Synthetic Defined Growth Medium
RPMI 1640 medium, (powder without sodium bicarbonate; Invitrogen 31800-089)
Uridine 8 mg/ml in water (Sigma)
Glucose 40% (w/v) in water (Sigma)
MOPS Buffer (Sigma)
Pen/Strep

Fungal Inoculum
Test Strain: C. albicans CaCi-2
Inoculate 500 ul of strain from cryopreserved stock into 250 ml shaker flask containing 30 ml YPD medium. Shake at 30C O/N.
Spin down culture, pour off broth, wash with RPMI medium. Spin down, pour off broth, resuspend in RPMI medium.
Read OD 600 of fungal culture using standard plate reader. Dilute to starting OD of the fungal inoculum of 0.00015 A600.

Prepare Geldanamycin positive control solution:
1 ml 15mM stock solution Geldanamycin
4 ml RPMI Growth Medium
50 ul Pen/Strep
Dispense 400 nL into positive control wells using Combi NL (Thermo.)

Dispense 20 uL/well of RPMI synthetic defined medium into 384-well black plates.
Pin 100 nL from compound plates into assay plates.
Dispense 20 uL/well of fungal suspension into all wells.
Incubate plates in humidified (90 % humidity) incubator at 37C without agitation for 48 hrs.
Dilute Alamar Blue Reagent (Biosource International Cat# DAL1100) 1:40 in Ca/Mg-free PBS. Add 5 ul/well to plates with CombinL (final dilution factor 1:200). Incubate 2 hrs at RT. Read Relative Fluorescence Intensity (RFU) of wells on standard plate reader as measure of relative fungal growth.
Plate reader set-up- Envision, Perkin Elmer: Ex 544nm, Em 590nm, Bandwidth 12nm, Top read.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=168) and positive control wells (PC; n=18) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).

NORMALIZATION:
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 neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.1uM_(%) (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.195uM_(%) (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_26uM_(%) (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH086456-01

Data Table (Concise)
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