AID	Name	Type	Comment
2007	Summary of Broad Institute MLPCN Reversing Antifungal Drug Resistance Project.	summary	Summary assay

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 NIH 3T3 mammalian fibroblasts cultured in 384-well format in the presence of a sub-toxic concentration of fluconazole. Test compounds that do not inhibit subsequent growth in the presence of fluconazole will merit further evaluation for their selectivity for fungal inhibition over mammalian cell toxicity. This whole cell phenotypic screening approach will only capture compounds that are capable of entering and accumulating in cells to bioactive concentrations. 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: Wells in which there is toxicity will show a reduced fluorescence intensity due to a reduction in the amount of Alamar Blue dye metabolized by fewer viable cells.

Protocol:
Stock solutions
Geldanamycin (GA) 15mM stock sol'n in DMSO
Pen/Strep 100x in PBS

Assay Medium:

Optimem medium (Invitrogen Cat#31985-070, Lot# 548536)
2.5% (v/v) Fetal Bovine Serum (Hyclone Cat#30071.03, Lot# ARF26748)
1% (v/v) Pen/Strep solution (Invitrogen Cat#15140-122, Lot#529891)

Cell Inoculum
Test Strain: NIH-3T3 mammalian fibroblasts (ATCC #CRL1658)

Concentration in assay plate: Cells plated at 6,000/well in 20 uL assay medium and cultured overnight at 37C under 5% CO2 in 384-well, transparent bottom, black, tissue culture-treated, bar-coded assay plates

Procedure: After overnight culture, compounds are pinned into wells at 100nL/well using the CyBio pinning instrument. After compounds are pinned, an additional 20 uL of assay medium supplemented with fluconazole (16 ug/mL, Sequoia Research) is added to each well. The final nominal concentration in the well will be a dose range of 26 - 0.195 uM of test compound and 8 ug/mL fluconazole. The plates will be returned to the tissue culture incubator and culture continued for 48 hrs at 37C under 5% CO2. At the completion of this incubation, Alamar Blue solution diluted 1:40 will be added to each well (10uL/well) to achieve a final dilution of 1:200. Plates will be incubated for a further 2-3 hrs at 37C under 5% CO2 and then fluorescence intensity as a measure of relative viable cell number will be determined on an Envision plate reading fluorometer.

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).


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.


PUBCHEM_ACTIVITY_OUTCOME:
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:

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.

Grant Number: 1 R03 MH086456-01