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BioAssay: AID 2423

Fluorescence Cell-Based Secondary Assay to Identify Inhibitors of Resistant C. albicans Growth in the Presence of Fluconazole

Assay Overview: The basic assay strategy will consist of highly Fuconazole-resistant C. albicans clinical isolate cultured in 384-well format in the presence of a sub-toxic concentration of Fluconazole. Test compounds that inhibit subsequent growth in the presence of Fluconazole will merit further evaluation for their synergy with Fluconazole. This whole cell phenotypic screening approach will more ..
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 Tested Compounds
 Tested Compounds
All(1836)
 
 
Active(1103)
 
 
Inactive(344)
 
 
Inconclusive(389)
 
 
 Tested Substances
 Tested Substances
All(1838)
 
 
Active(1105)
 
 
Inactive(344)
 
 
Inconclusive(389)
 
 
AID: 2423
Data Source: Broad Institute (2037-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-25
Hold-until Date: 2010-03-19
Modify Date: 2011-10-11

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1103
Related Experiments
Show more
AIDNameTypeProbeComment
1979Fluorescence Cell-Based Primary HTS of C.albicans growth in the presence of Fluconazole and compoundScreening depositor-specified cross reference: Primary HTS
2007Summary of Broad Institute MLPCN Reversing Antifungal Drug Resistance Project.Summary3 depositor-specified cross reference: Summary
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
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
504499Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set9Confirmatory 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:
Broad Institute: Reversing Antifungal Drug Resistance

Project ID: 2037

Keywords: Candida albicans, drug resistance, Fluconazole, Hsp90, Calcineurin, stress response

Primary Collaborators: Susan Lindquist, Whitehead Institute for Biomedical Research, sll@wi.mit.edu

Assay Overview: The basic assay strategy will consist of highly Fuconazole-resistant C. albicans clinical isolate cultured in 384-well format in the presence of a sub-toxic concentration of Fluconazole. Test compounds that inhibit subsequent growth in the presence of Fluconazole will merit further evaluation for their synergy with Fluconazole. This whole cell phenotypic screening approach will only capture compounds that retain activity in biological media and are capable of entering and accumulating in fungi to bioactive concentrations. Grossly cytotoxic compounds will be removed through subsequent counterscreens. Compound activity will be measured by the metabolism of Alamar Blue, a cell stain that is metabolized to a fluorescent product by living cells but that remains non-fluorescent in wells with growth-inhibited organisms.

Probe attributes:
a. Compounds that inhibit yeast growth in the presence, but not in the absence of 8 ug/ ml Fluconazole.
b. Compounds that show a 10-fold specificity between the primary Candida test strain and mammalian cells.
c. Compounds that exhibit 10X greater inhibition against fungus than the hsp90 chaperon and /or calcineurin.
d. IC50 < 1uM

Expected Outcome: Active wells will show a reduced fluorescence intensity due to a reduction in the amount of Alamar Blue dye metabolized by fewer viable microorganisms.
Protocol
Stock solutions
Geldanamycin (GA) 15mM stock solution in DMSO
Fluconazole (FLC, Sequoia Research Products Ltd) 2 mg/ml stock solution in PBS
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)

Fungal Inoculum
Test Strain: C. albicans CaCi-8 (Redding et al., 1994, Clin Infect Dis, 18: 240-242)
Inoculate 500 ul of strain from cryopreserved stock into 250 ml shaker flask containing 30 ml growth medium. Shake at 30C O/N.
Read OD 600 of fungal culture using standard plate reader. Dilute with Synthetic Defined Growth Medium to starting OD of the fungal inoculum of 0.00015 A600.

384-well plates (Corning).

Add Fluconazole stock solution (2 mg/mL in PBS) to Synthetic Defined Growth Medium to achieve 8 ug/ml. Add Pen/Strep at 0.1 ml per 10 ml media.
Using Thermo Combi, dispense 20 uL/well of Synthetic Defined Growth Medium into all wells.
Pin 100 nL from compound plates into assay plates using CyBiWell pinning instrument.
Add Fluconazole stock solution (2 mg/mL in PBS) to fungal inoculum to achieve 8 ug/ml. Add Pen/Strep at 0.1 ml per 10 ml media.
Using Thermo Combi, dispense 20 uL/well of Synthetic Defined Growth Medium plus culture 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 6.4 ul/well to plates with Combi (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
Dose Data Analysis

Neutral control (NC) wells and positive control (PC) wells were included on every plate.
Active inhibitor compounds result in decreased fluorescent 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 0.120uM (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.235uM (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.500uM (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 1.000uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 1.95uM (1.95μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 3.80uM (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 7.50uM (7.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 8.00uM (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity at 15.0uM (15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity at 16.0uM (16μM**)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)
Data Table ( Complete ):     View Active Data    View All Data
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