Antifungal Drug Resistance - Single Agent Toxicity Measured in Microorganism System Using Plate Reader - 2037-07_Inhibitor_Dose_DryPowder_Activity_Set3
Assay Overview: Fluorescent counterscreen of C. albicans growth in the absence of fluconazole with compound, to determine whether compound is toxic in its own right or only in presence of fluconazole. A clinical isolate of fluconazole-resistant C. albicans will be cultured in 384-well format in the absence of a sub-lethal concentration of fluconazole. Test compounds will be pinned into individual wells and those compounds that inhibit subsequent yeast growth in the presence of fluconazole, but not in its absence, will be identified as hits meriting further evaluation. ..more
Keywords: Candida albicans, drug resistance, fluconazole, Hsp90, stress response
Assay Overview: Fluorescent counterscreen of C. albicans growth in the absence of fluconazole with compound, to determine whether compound is toxic in its own right or only in presence of fluconazole. A clinical isolate of fluconazole-resistant C. albicans will be cultured in 384-well format in the absence of a sub-lethal concentration of fluconazole. Test compounds will be pinned into individual wells and those compounds that inhibit subsequent yeast growth in the presence of fluconazole, but not in its absence, will be identified as hits meriting further evaluation.
Expected Outcome: loss of signal indicates broad toxicity rather than specificity of action in concert with fluconazole.
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 30 deg C overnight.
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 15 mM 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 37 deg C without agitation for 48 hrs.
Dilute Alamar Blue Reagent (Biosource International Cat# DAL1100) 1:40 in Ca/Mg-free PBS. Add 5 ul/well to plates with CombinL (final dilution factor 1:200). Incubate 2 hrs at room temperature. 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.
1) The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between
the derived 'Maximal_Activity' and the apparent most active data point.
2) There were no active compounds in this assay, and therefore no AC50 values could be determined.
Categorized Comment - additional comments and annotations
Assay Type: Toxicity
Assay Format: Organism-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)