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BioAssay: AID 1902

Luminescence Microorganism-Based Dose Confirmation HTS to Identify Inhibitors of Streptokinase Promotor Activity

Keywords: Streptokinase, inhibition, growth, bacterial, group A streptococcus, virulence, dose response ..more
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 Tested Compounds
 Tested Compounds
All(3225)
 
 
Active(2944)
 
 
Inactive(281)
 
 
 Tested Substances
 Tested Substances
All(3227)
 
 
Active(2946)
 
 
Inactive(281)
 
 
AID: 1902
Data Source: Broad Institute (2014-01_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-08-17

Data Table ( Complete ):           Active    All
Target
Sequence: streptokinase A precursor [Streptococcus pyogenes M1 GAS]
Description ..   
Protein Family: Staphylokinase/Streptokinase family

Gene:SKA     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 2944
Depositor Specified Assays
AIDNameTypeProbeComment
1662MLPCN Streptokinase Expression Inhibitionscreening Primary HTS
1677Broad Institute MLPCN Streptokinase Expression Inhibition Projectsummary3 Project Summary
2477Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to Streptococcus.confirmatory
2137Absorbance Microorganism-Based Dose Response Followup to Identify Inhibitors of Streptokinase Expressionconfirmatory
2138Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to Streptococcusconfirmatory
1914Absorbance Microorganism-Based Dose Response HTS to Identify Inhibitors of Streptokinase Expressionconfirmatory
1915Luminescence Microorganism-Based Dose Response HTS to Identify Compounds Cytotoxic to Streptococcusconfirmatory
2470Absorbance Microorganism-Based Dose Response Followup to Identify Inhibitors of Streptokinase Expression.confirmatory
Description:
Keywords: Streptokinase, inhibition, growth, bacterial, group A streptococcus, virulence, dose response

Assay Overview:
The strain used for this assay is the same GAS strain used in primary screen carrying the Kanamycin resistant gene under the control of the streptokinase promoter (SKKanGAS). Compounds that inhibit streptokinase promoter activity lead to a decrease of the kanamycin resistant gene product and cell death in the presence of kanamycin (Sigma K1637). Active compounds identified from primary screen were tested in this assay in 6-point 3-fold dilution doses with starting concentrations at 15 uM 0.2%DMSO. Briefly, SKKanGAS at OD600 equal to 0.015 were plated onto 384-well plates (Corning 3570) and incubated with test compounds in the presence of 40 ug/ml of kanamycin in Todd-Hewitt Broth medium for 6 hours before cell survival is assessed by BacTiterGlo reagent (Promega G8233). Detailed procedures are described in Protocol section.

Expected Outcome:
Normalization of the HTS data across runs, calibration to the positive control and EC50 determination were performed as described below. The results are reported as EC50 values.
Protocol
Assay Protocol:
Day 1 Streak out SKKanGAS for colonies on THY/S/S agar plate (Todd-Hewitt Broth with streptomycin 100 ug/mL, spectinomycin 100 ug/ml, 1.5% agar); 37oC O/N
Day 2 Grow an overnight culture in THY/S/S from a single colony, 37oC O/N
Day 3 Measure OD600 of the overnight culture (typically over OD600 1.2), and then dilute 1:20 into fresh THY/S/S in a flask. Monitor OD600 until it reaches 0.6-0.9.
Dispense assay plates with 50ul/well of cells at final OD600 of 0.015 in THY/Kanamycin (40ug/ml Kan) with Multidrop Combi (ThermoFisher)
Pin 100 nL of compounds into medium to reach a final average concentration of 7.5 uM
Incubate at 37oC for 6 hr at >85% humidity
Cool plates to RT for 30 minutes
Add 30 uL/well 80% BacTiterGlo diluted in PBS
RT 10 minutes
Read on Envision for luminescence with a 384-well aperture and regular sensitivity.
Comment
Dose Response Data Analysis:
32 Negative control (DMSO) and 32 positive control (Tetracycline, PubChem CID 9848033) wells were included on every plate.

All wells tested were background subtracted using the median of the negative controls wells on the same plate and scaled using the median value of the positive control wells on the same plate. DMSO plates were run before and after the compound plate and Genedata's runwise additive correction was used with those as calibration plates to correct for plate effects.

EC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (v6.0.1). EC50 values were extrapolated up to 1 log
over the highest tested concentration.

PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3EC50 Stderrthe standard error for the calculated EC50 valueFloatμM
4S0the fitted activity level at zero concentrationFloat%
5SInfthe fitted activity level at infinite concentrationFloat%
6Hill Slopethe slope at EC50Float
7# of Points Fitthe number of data points included in the plotInteger
8Max Activitythe maximum activity value observedFloat%
9Activity at 0.06uM (0.06μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.18uM (0.18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.56uM (0.56μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 1.60uM (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 5.00uM (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 15.00uM (15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03DA026214-01

Data Table (Concise)
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