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BioAssay: AID 1979

Fluorescence Cell-Based Primary HTS of C.albicans growth in the presence of Fluconazole and compound

Assay Overview: The basic assay strategy will consist of fluconazole-resistant C. albicans clinical isolate cultured in 1536-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 more ..
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 Tested Compounds
 Tested Compounds
All(302455)
 
 
Active(1736)
 
 
Inactive(295112)
 
 
Inconclusive(5614)
 
 
 Tested Substances
 Tested Substances
All(302509)
 
 
Active(1737)
 
 
Inactive(295158)
 
 
Inconclusive(5614)
 
 
AID: 1979
Data Source: Broad Institute (2037-01_INHIBITORS_SINGLE-POINT_MLPCN-HTS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-10-14

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1736
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
2467Fluorescence Cell-Based Retest of C. albicans Growth in the Presence of Fluconazoleconfirmatory
434923Fluorescence Cell-Based Secondary Assay for Toxicity in Mammalian Fibroblastsconfirmatory
434930Fluorescence Cell-Based Retest of C. albicans growth in the presence of fluconazole and compoundconfirmatory
2327Fluorescence Cell-Based Secondary Assay for toxicity in mammalian fibroblastsconfirmatory
2388Luminesence Cell-Based Secondary Assay to Identify Inhibitors of Calcineurinconfirmatory
434946Fluorescence Cell-Based Secondary Assay to Measure Toxicity of Compounds Not in the Presence of Fluconazole.confirmatory
434935Fluorescence Cell-Based Secondary Assay of Resistant C. albicans Growth in the Presence of Fluconazoleconfirmatory
434938Luminescence Cell-Based Secondary Assay to Identify Inhibitors of Hsp90confirmatory
2387Fluorescence Cell-Based Secondary Assay to Measure Toxicity of Compounds Not in the Presence of Fluconazoleconfirmatory
2007Summary of Broad Institute MLPCN Reversing Antifungal Drug Resistance Project.summary3
2400Luminesence Cell-Based Secondary Assay to Identify Inhibitors of Hsp90confirmatory
588528Broad Institute Reversing Antifungal Drug Resistance: Single Agent Probe Inhibitor Probe Projectsummary
2423Fluorescence Cell-Based Secondary Assay to Identify Inhibitors of Resistant C. albicans Growth in the Presence of Fluconazoleconfirmatory
434945Fluorescence Cell-Based Secondary Assay to Identify Inhibitors of Calcineurinconfirmatory
Description:
Broad Institute: Reversing Antifungal Drug Resistance

Project ID: 2037

Keywords: Candida albicans, drug resistance, fluconazole, Hsp90, Calcineurin, stress response

Primary Collaborators: Susan Lindquist, Whitehead Institute for Biomedical Research, sll@wi.mit.edu

Assay Overview: The basic assay strategy will consist of fluconazole-resistant C. albicans clinical isolate cultured in 1536-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.

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 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.
Protocol
Stock solutions
Geldanamycin (GA) 15mM stock solution 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)

Fungal Inoculum
Test Strain: C. albicans CaCi-2 (Redding et al., 1994, Clin Infect Dis 24:235-247)
Inoculate 500 ul of strain from cryo-preserved 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 to starting OD of the fungal inoculum of 0.00015 A600.

1536-well Assay-ready plates (Aurora #29844) prepared in duplicate with 7.5 nL of compound pre-dispensed by Echo (Labcyte)

Prepare Geldanamycin positive control solution:
1 ml 15mM stock solution Geldanamycin
4 ml RPMI Growth Medium
20 ul Fluconazole stock solution
50 ul Pen/Strep
Dispense 400 nL into positive control wells using Combi NL (Thermo.)

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 CombiNL, dispense 8 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 1.6 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.
Comment
HTS Data Analysis:
128 negative control wells (DMSO) and 128 positive control wells (Geldanamycin, 700 uM) were included on every plate.

The PubChem_Activity_Score was derived using the follow procedure:
1. A background-subtracted value was calculated for each well by subtracting the median value of the negative control wells on each plate from the value of each well on that plate.

2. An activity score was derived for each well by dividing the background-subtracted value for each well by the median of the background-subtracted value of the positive control wells in the same run and multiplying the resulting fraction by 100.

3. Runwise patterns were smoothed using an additive algorithm in Genedata software

4. The final PubChem_Activity_Score represents the mean of all valid replicate activity scores obtained.

The PubChem_Activity_Outcome class was assigned as described below:

Activity_Outcome = 1 (inactive)
PubChem_Activity_Score for more than half the replicate activity scores < 75.

Activity_Outcome = 2 (active)
PubChem_Activity_Score for more than half the replicate activity scores > 75

Activity_Outcome = 3 (inconclusive)
PubChem_Activity_Score where one half of the replicates activity scores > 75 and one half < 75
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two
samples. Computed as the absolute value of the cosine between the "replicate vector"
(ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility.
NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid
Float
2BROAD_SCREENING_RUNIDSThis is a comma separated list of unique IDs given to each screening run at the Broad Institute.String
3REPLICATE_A_ACTIVITY_SCOREThe calculated activity score of the indicated replicate.Float%
4REPLICATE_B_ACTIVITY_SCOREThe calculated activity score of the indicated replicate.Float%
5REPLICATE_C_ACTIVITY_SCOREThe calculated activity score of the indicated replicate.Float%
6REPLICATE_D_ACTIVITY_SCOREThe calculated activity score of the indicated replicate.Float%
7DATE_REPORTEDDate data reported internallyString
Additional Information
Grant Number: 1 R03 MH086456-01

Data Table (Concise)
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