In vivo-based yeast HTS counterscreen to detect compounds rescuing yeast growth/survival of Saccharomyces cerevisiae SKN7-mediated toxicity Measured in Whole Organism System Using Plate Reader - 2120-02_Inhibitor_Dose_CherryPick_Activity
In this counterscreen , the aim is to elimnate compounds with non-specific effects on yeast growth such as suppression of galactose induction. Briefly, a strain expressing a toxicity inducing protein unrelated to HSP40 (SKN7, a yeast transcriptional regulator) under the control of the galactose promoter will be used to test for unspecific effects of the hits on yeast growth. The capacity of small more ..
BioActive Compounds: 351
Depositor Specified Assays
Keywords: Heat shock proteins, malaria, transcription
Assay Overview (Counterscreen, Growth rescue of yeast SKN7-mediated toxicity):
In this counterscreen , the aim is to elimnate compounds with non-specific effects on yeast growth such as suppression of galactose induction. Briefly, a strain expressing a toxicity inducing protein unrelated to HSP40 (SKN7, a yeast transcriptional regulator) under the control of the galactose promoter will be used to test for unspecific effects of the hits on yeast growth. The capacity of small molecules to block SKN7-mediated yeast toxicity and induce yeast growth will be measured through luminescence mediated by the presence of intracellular ATP in yeast and luciferin as an indicator of cell viability.
Identification of probe(s) inhibiting yeast SKN7-mediated yeast toxicity. Compounds with an AbsAC40 (Absolute active concentration at an arbitrary value of 40) below 20 uM will be considered hits and will be discarded for further studies.
SKN7 Growth Rescue Assay Protocol:
Day 1 (overnight growth culture):
1 - Thaw vial containing Saccaromyces cerevisiae W303a delta pdr1 delta pdr3 p416-SKN7 yeast glycerol stock (Provided by Dr. Susan Lindquist's lab) in 20 ml SRaff-Ura media (recipe below) and grow yeast overnight at 23degreeC with shaking.
Day 2 (Yeast Plating)
2- Check OD600 to ensure culture remains in log phase (OD600 0.2-0.8) until ready to use for plating in assay format.
3 - After taking the OD at 600nm, dispense 6 ul of SKN7 yeast at OD (600 nm) of 0.015 in SGal-Ura using multidrop dispenser (Thermo Scientific) with small tubing cassette (Thermo Scientific) in assay ready 1536-well black plates (Aurora, cat# 00019180BK) with compound already present in the well (7.5 nl) 8 concentrations, 3 fold dilution (starting concentration 10 mM ).
4 - Incubate plates at 30degreeC for 3 days in humidified environment.
Day 3 (Adding Bactiter-Glo reagent and Reading on Envision)
5. Add 2 ul Bactiter-Glo (Promega, G8233) using multidrop dispenser (small tubing cassette)(Thermo Scientific). Read immediately luminescence (ultrasensitive setting) on Envision.
Overnight growing media (SRaff-Ura):
1.7 g Yeast Nitrogen Base (YNB) media without amino acids, without ammonium sulfate (Sigma, Cat#Y1251)0.77g CSM-Ura drop-out powder (Sunrise Science Products, Catalog #1004-100)1g L-Glutamic acid, monosodium salt (Sigma # G1626)20g raffinose (Sigma, Cat# R0250)1L H20
Screening media (SGal-Ura):1.7 g Yeast Nitrogen Base (YNB) without amino acids, without ammonium sulfate (Sigma, Cat#Y1251)0.77g CSM-Ura drop-out powder (Sunrise Science Products, catalog #1004-100)1g L-Glutamic acid, monosodium salt (Sigma # G1626)20g D(+) galactose (Sigma, Cat#G5388)1L H20
Positive control media (SGlu-Ura):1.7 g Yeast Nitrogen Base YNB without amino acids, without ammonium sulfate (Sigma, Cat#Y1251)0.77g CSM-Ura drop-out powder (Sunrise Science Products, catalog #1004-100)1g L-Glutamic acid, monosodium salt (Sigma # G1626)20g glucose (Sigma, G7528)1L H20
Stir for 30-60 mins at RTFilter into 0.2 uM filter flaskKeep media at room temperature until use within 2 months.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=288) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing 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 Max_Concentration.
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
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): absACnn, the concentration at which the curve crosses threshold 40.0
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 >= 30% but <= 70%, 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)