Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set3
Assay Overview: The basic assay strategy will consist of highly fluconazole-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 ..
BioActive Compounds: 12
Keywords: Candida albicans, drug resistance, fluconazole, Hsp90, Calcineurin, stress response
Broad Institute: Reversing Antifungal Drug Resistance
Project ID: 2037
Primary Collaborators: Susan Lindquist, Whitehead Institute for Biomedical Research, firstname.lastname@example.org
Assay Overview: The basic assay strategy will consist of highly fluconazole-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. This assay measures compound in activity in a more resistant clinical isolate (CaCi-8) compared to the primary assay strain (CaCi-2).
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 chaperone 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.
Geldanamycin (GA) 15mM stock sol'n 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-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.
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) 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 (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).
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.
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) 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
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 not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)
PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)