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 ..
BioActive Compounds: 1097
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.
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)
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.
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
Inactive compounds = 0
Active compounds = -10*Log(IC50)
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%
The fit was not valid due to poor fit quality.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)