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BioAssay: AID 588529

Anti-Fungal Single Agent: CaCi2 Measured in Microorganism System Using Plate Reader - 2148-01_Inhibitor_Dose_CherryPick_Activity

Assay Overview: The basic assay strategy will consist of fluconazole-resistant C. albicans clinical isolate cultured in microtiter plates. Test compounds that inhibit subsequent growth will merit further evaluation. 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 more ..
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 Tested Compounds
 Tested Compounds
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Active(39)
 
 
 Tested Substances
 Tested Substances
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Active(39)
 
 
 Related BioAssays
 Related BioAssays
AID: 588529
Data Source: Broad Institute (2148-01_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-17
Hold-until Date: 2012-10-14
Modify Date: 2012-10-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 39
Related Experiments
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AIDNameTypeComment
588528Broad Institute Reversing Antifungal Drug Resistance: Single Agent Probe Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
1979Fluorescence Cell-Based Primary HTS of C.albicans growth in the presence of Fluconazole and compoundScreeningsame project related to Summary assay
588530CaCi17 Retest at Dose Measured in Microorganism System Using Plate Reader - 2148-02_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
588634Antifungal Single Agent - Mammalian Cell Toxicity Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2148-03_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
602184Anti-Fungal Single Agent: S.cerevisiae growth, Glycerol Measured in Microorganism System Using Plate Reader - 2148-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602185Anti-Fungal Single Agent: C.glabrata growth, Glucose Measured in Microorganism System Using Plate Reader - 2148-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602186Anti-Fungal Single Agent: C.glabrata growth, Glycerol Measured in Microorganism System Using Plate Reader - 2148-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602187Anti-Fungal Single Agent: S.cerevisiae growth, Glucose Measured in Microorganism System Using Plate Reader - 2148-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602188Antifungal Single Agent - Mammalian Cell Toxicity Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2148-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602189CaCi17 Retest at Dose Measured in Microorganism System Using Plate Reader - 2148-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602190Anti-Fungal Single Agen: CaCi2 Measured in Microorganism System Using Plate Reader - 2148-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602394Antifungal Single Agent - Mammalian Cell Toxicity Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2148-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623897CaCi17 Retest at Dose Measured in Microorganism System Using Plate Reader - 2148-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623899Anti-Fungal Single Agent: CaCi2 Measured in Microorganism System Using Plate Reader - 2148-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623903Anti-Fungal Single Agent: C.glabrata growth, Glycerol Measured in Microorganism System Using Plate Reader - 2148-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623904Anti-Fungal Single Agent: C.glabrata growth, Glucose Measured in Microorganism System Using Plate Reader - 2148-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623965Anti-Fungal Single Agent: C.glabrata growth, Glycerol Measured in Microorganism System Using Plate Reader - 2148-04_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
623967Anti-Fungal Single Agent: C.glabrata growth, Glucose Measured in Microorganism System Using Plate Reader - 2148-05_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
623969Anti-Fungal Single Agent: S.cerevisiae growth, Glycerol Measured in Microorganism System Using Plate Reader - 2148-06_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
623971Anti-Fungal Single Agent: S.cerevisiae growth, Glucose Measured in Microorganism System Using Plate Reader - 2148-07_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
623981Anti-Fungal Single Agen: CaCi2 Measured in Microorganism System Using Plate Reader - 2148-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
623982CaCi17 Retest at Dose Measured in Microorganism System Using Plate Reader - 2148-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
624006Anti-Fungal Single Agen: CaCi2 Measured in Microorganism System Using Plate Reader - 2148-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
624012Anti-Fungal Single Agent: S.cerevisiae growth, Glycerol Measured in Microorganism System Using Plate Reader - 2148-06_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
624013Anti-Fungal Single Agent: C.glabrata growth, Glucose Measured in Microorganism System Using Plate Reader - 2148-05_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
624014Anti-Fungal Single Agent: C.glabrata growth, Glycerol Measured in Microorganism System Using Plate Reader - 2148-04_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
624025Anti-Fungal Single Agent: S.cerevisiae growth, Glucose Measured in Microorganism System Using Plate Reader - 2148-07_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
Description:
Keywords: Candida albicans, glycolysis, respiration


Assay Overview: The basic assay strategy will consist of fluconazole-resistant C. albicans clinical isolate cultured in microtiter plates. Test compounds that inhibit subsequent growth will merit further evaluation. 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: Reduction of signal due to cell death.
Protocol
Protocol:
Positive Control:
Amphotericin B 10 uM in well
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-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 30 degrees C overnight.
Read OD 600 of fungal culture using standard plate reader. Dilute to starting OD of the fungal inoculum of 0.00015 A600.
384 well black sterile Corning plates (PN 3712)
Add Pen/Strep at 0.1 ml per 10 ml media.
Using Combi, dispense 40 uL/well of fungal suspension into all wells.
Pin 100 nL from compound plates into assay plates.
Incubate plates in humidified (90 % humidity) incubator at 37 degrees C without agitation for 48 hrs.
Add 5 uL Alamar Blue (1:40 dilution in PBS) to each well. Incubate 2 hours. Read fluorescence on standard plate reader.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) 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 (2)(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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Organism-based
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_0.011uM_(%) (0.011μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.035uM_(%) (0.035μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.1uM_(%) (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.3uM_(%) (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.9uM_(%) (0.9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_2.85uM_(%) (2.85μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_8uM_(%) (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_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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