Fluorescence Cell-Based Secondary Assay to Measure Toxicity of Compounds Not in the Presence of Fluconazole
Assay Overview: The basic assay strategy will consist of NIH 3T3 mammalian fibroblasts cultured in 384-well format in the presence of a sub-toxic concentration of Fluconazole. Test compounds that do not inhibit subsequent growth in the presence of Fluconazole will merit further evaluation for their non-toxicity. This whole cell phenotypic screening approach will only capture compounds that are more ..
BioActive Compounds: 94
Keywords: Candida albicans, drug resistance, Fluconazole, Hsp90, Calcineurin, stress response
Primary Collaborators: Susan Lindquist, Whitehead Institute for Biomedical Research, email@example.com
Assay Overview: The basic assay strategy will consist of NIH 3T3 mammalian fibroblasts cultured in 384-well format in the presence of a sub-toxic concentration of Fluconazole. Test compounds that do not inhibit subsequent growth in the presence of Fluconazole will merit further evaluation for their non-toxicity. This whole cell phenotypic screening approach will only capture compounds that are capable of entering and accumulating in cells to bioactive concentrations. 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.
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: Wells in which there is toxicity will show a reduced fluorescence intensity due to a reduction in the amount of Alamar Blue dye metabolized by fewer viable cells.
Geldanamycin (GA) 15mM stock solution 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)
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 6.4 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.
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)