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BioAssay: AID 504504

Antifungal Drug Resistance - Resistant Strain Measured in Microorganism System Using Plate Reader - 2037-02_Inhibitor_Dose_DryPowder_Activity_Set10

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 ..
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 Tested Compounds
 Tested Compounds
All(28)
 
 
Active(13)
 
 
Inactive(13)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(28)
 
 
Active(13)
 
 
Inactive(13)
 
 
Inconclusive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 504504
Data Source: Broad Institute (2037-02_Inhibitor_Dose_DryPowder_Activity_Set10)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-21
Hold-until Date: 2012-03-21
Modify Date: 2012-03-21

Data Table ( Complete ):           Active    All
BioActive Compounds: 13
Depositor Specified Assays
AIDNameTypeComment
2007Summary of Broad Institute MLPCN Reversing Antifungal Drug Resistance Project.summarySummary assay
Description:
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, sll@wi.mit.edu

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 activity in a more resistant clinical isolate (CaCi-8) compared to the primary assay strain (CaCi-2).

Probe attributes:
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: 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
Protocol:
Stock solutions
Geldanamycin (GA) 15mM stock sol'n in DMSO
Fluconazole (FLC, Sequoia Research Products Ltd) 2 mg/ml stock sol'n 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-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.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=168) and positive control wells (PC; n=18) 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 (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).

NORMALIZATION:
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.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) 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
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
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 deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.1uM_(%) (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.195uM_(%) (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_26uM_(%) (26μM**)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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