Antifungal Drug Resistance - Hsp90 Inhibitors Measured in Microorganism System Using Plate Reader - 2037-05_Inhibitor_Dose_DryPowder_Activity_Set2
Assay Overview: Method for determining if compound acts as Hsp90 inhibitor. Saccharomyces strain expressing B-galactosidase driven by glucocorticoid response element is exposed to compounds. Loss of signal indicates interference with hsp90 function. ..more
BioActive Compounds: 6
Keywords: Candida albicans, drug resistance, fluconazole, Hsp90, stress response
Assay Overview: Method for determining if compound acts as Hsp90 inhibitor. Saccharomyces strain expressing B-galactosidase driven by glucocorticoid response element is exposed to compounds. Loss of signal indicates interference with hsp90 function.
Expected Outcome: Reduction of signal indicates hsp90 inhibition by compound.
1) Grow reporter strain in SD-ADE media overnight @ 37 degrees C.
2) Dilute cells to OD = 0.04 in SD-ADE media.
3) Add 20 ul of culture to each well of 384 white plate.
4) Pin 100 nL compounds into plates, using 5uM Radicicol as positive control.
5) Add 20 ul of 20uM DOC (steroid) in SD-ADE media to plates.
6) Incubate at 30 C for 75 minutes, with agitation.
7) Add 40 ul Gal-Screen reagent.
8) Incubate at 30C for 25 minutes, then read luminescence.
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).
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)
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.
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.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)