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BioAssay: AID 2467

Fluorescence Cell-Based Retest of C. albicans Growth in the Presence of Fluconazole

Assay Overview: The basic assay strategy will consist of Fluconazole-resistant C. albicans clinical isolate cultured in 384-well dose-response 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 more ..
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 Tested Compounds
 Tested Compounds
All(1836)
 
 
Active(1097)
 
 
Inactive(614)
 
 
Inconclusive(125)
 
 
 Tested Substances
 Tested Substances
All(1838)
 
 
Active(1099)
 
 
Inactive(614)
 
 
Inconclusive(125)
 
 
 Related BioAssays
 Related BioAssays
AID: 2467
Data Source: Broad Institute (2037-01_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-03

Data Table ( Complete ):           Active    All
BioActive Compounds: 1097
Depositor Specified Assays
AIDNameTypeComment
1979Fluorescence Cell-Based Primary HTS of C.albicans growth in the presence of Fluconazole and compoundscreeningPrimary HTS
2007Summary of Broad Institute MLPCN Reversing Antifungal Drug Resistance Project.summaryProject Summary
Description:
Broad Institute MLPCN Antifungal Project

Project ID: 2037

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

Assay Overview: The basic assay strategy will consist of Fluconazole-resistant C. albicans clinical isolate cultured in 384-well dose-response 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.

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-2 (Redding et al., 1994, Clin Infect Dis 24:235-247)
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 to starting OD of the fungal inoculum of 0.00015 A600.

1536-well Assay-ready plates (Aurora #29844) prepared in duplicate with 7.5 nL of compound pre-dispensed by Echo (Labcyte.)

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

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 Combi NL, dispense 8 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 1.6 ul/well to plates with Combi NL (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
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon activity reaches 50% of the maximum.FloatμM
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 0.0300uM (0.03μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.0600uM (0.06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.120uM (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 0.235uM (0.235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 0.500uM (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 1.000uM (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 1.95uM (1.95μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 3.80uM (3.8μM**)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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