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BioAssay: AID 1914

Absorbance Microorganism-Based Dose Response HTS to Identify Inhibitors of Streptokinase Expression

The goal of this assay is to identify compounds that specifically reduce streptokinase expression without inhibiting cell growth. Active compounds from the primary screen were tested in 6-point, 3-fold dilution doses with starting concentration at 15 uM as follows. GAS UMAA2166 at OD600 equal to 0.015 were plated onto 384-well plates (Corning 3570) and incubated with test compounds in more ..
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 Tested Compounds
 Tested Compounds
All(3264)
 
 
Active(773)
 
 
Inactive(1638)
 
 
Inconclusive(854)
 
 
 Tested Substances
 Tested Substances
All(3266)
 
 
Active(774)
 
 
Inactive(1638)
 
 
Inconclusive(854)
 
 
AID: 1914
Data Source: Broad Institute (2014-03_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-09-02
Modify Date: 2009-09-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 773
Related Experiments
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AIDNameTypeProbeComment
1662MLPCN Streptokinase Expression InhibitionScreening depositor-specified cross reference: Primary HTS
1677Broad Institute MLPCN Streptokinase Expression Inhibition ProjectSummary3 depositor-specified cross reference: Project Summary
1900Luminescence Microorganism-Based Dose Confirmation HTS to Identify Compounds Cytotoxic to SK(-)GAS Group A StreptococcusConfirmatory depositor-specified cross reference: Counter Screen
1902Luminescence Microorganism-Based Dose Confirmation HTS to Identify Inhibitors of Streptokinase Promotor ActivityConfirmatory depositor-specified cross reference: Retest at Dose
2137Absorbance Microorganism-Based Dose Response Followup to Identify Inhibitors of Streptokinase ExpressionConfirmatory depositor-specified cross reference
2138Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to StreptococcusConfirmatory depositor-specified cross reference
2470Absorbance Microorganism-Based Dose Response Followup to Identify Inhibitors of Streptokinase Expression.Confirmatory depositor-specified cross reference
2477Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to Streptococcus.Confirmatory depositor-specified cross reference
1915Luminescence Microorganism-Based Dose Response HTS to Identify Compounds Cytotoxic to StreptococcusConfirmatory same project related to Summary assay
504351Dose responses of compound inhibitions on streptokinase expression and effects on viability in Group A streptococci Measured in Microorganism System Using Plate Reader - 2014-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504396Dose responses of compound inhibitions on streptokinase expression and effects on viability in Group A streptococci Measured in Microorganism System Using Plate Reader - 2014-03_Inhibitor_Dose_DryPowder_Viability_Set3Confirmatory same project related to Summary assay
Description:
Keywords:
Group A streptococcus, GAS, streptokinase, expression, virulence, inhibition, dose response, EC50

Assay Overview:
The goal of this assay is to identify compounds that specifically reduce streptokinase expression without inhibiting cell growth. Active compounds from the primary screen were tested in 6-point, 3-fold dilution doses with starting concentration at 15 uM as follows. GAS UMAA2166 at OD600 equal to 0.015 were plated onto 384-well plates (Corning 3570) and incubated with test compounds in Todd-Hewitt Broth medium supplemented with 100 ug/ml of streptomycin for 6 hours before cells were pelleted. Supernatant of cells was removed and tested for SK activity by measuring activation of plasminogen in an absorbance-based assay as described in the Protocol section. Cell pellets were assayed for viability by BacTiterGlo reagent (Promega G8233). Normalized SK activity values were generated by dividing values of SK activity by the values of viability reading, and used to generate IC50 values as described in the Data Analysis/Comments section. Results for viability alone are reported in a separate assay. Selectivity is determined by comparing IC50 values of reduction of normalized SK activity and IC50 values of reduction of viability.

Expected Outcome:
The project is looking for compounds that selectively reduce expression of SK activity without inhibiting cell growth.
Protocol
Day 1 Streak out UMAA 2616 for colonies on THY/S (THY with streptomycin 100 μg/mL) plate; 37'C O/N

Day 2 Grow an overnight culture in THY/S from a single colony, 37oC O/N

Day 3 In the morning, dilute 1:20 of the O/N culture into fresh THY/S in flask. Monitor OD600 until it reaches 0.6-0.8 (between 3-5 hrs).

Dilute the 0.8OD Culture down to OD 0.038 into cold (4'C) THY/S; stir at 4'C O/N

Day 4 Dispense assay plates (Corning 3570) at 30ul/well of THY/S with Combi (Thermo)

Pin Compounds (100 nL) using Cybi-Well (CyBio)

Seed cells (warmed up to RT) onto the pinned plates at 20ul/well with Combi

37'C for 6 hrs in Liconic incubator

Pellet cells at 3000 rpm

Transfer 2 x 10 uL/well supernatant to clear plates (Nunc 242757) for assay with Cybi-Well Vario (CyBio), store at -20'C.
Cool the culture plates to RT for 30 minutes
Add 50 uL/well of 1/2x BacTiterGlo (Promega G8233)
RT 10 minutes, Read for luminescence

SK activity is measured as follows:
Add 50 uL/well of SK assay solution (5 uL of human plasma, 1.2 uL of 4.2mg/ml S2403, 43.8 uL of PBS) to the 10 uL/well supernatant (thawed and warmed up to RT) in clear SK assay plates with Combi

Read OD405 at 0 hrs

37'C for 2.5 hrs

Read OD405

37'C for 2.5 hrs

Read OD405
Comment
A. Normalized SK expression values were derived by
1) subtracting intial OD405 values at time 0 hrs from final OD405 values at time 2.5 hrs, to determine the change in absorbance (deltaOD405);
2) dividing this subtracted value by the luminescence (RLU) reading to normalize for cell number variation, resulting in a normalized SK expression value: deltaOD405/RLU
3) normalized SK expression values were background subtracted using the median of the 32 negative controls wells on the same plate and scaled using the median value of the 32 positive control wells located on two separate control plates in the same run.
B. IC50 values were calculated based on the normalized, scaled SK expression values using the Smart Fit strategy of Genedata Screener Condoseo (v6.0.1). IC50 values were extrapolated up to 1 log over the highest tested concentration.

PUBCHEM_ACTIVITY_SCORE:

Inactive compounds = 0
Active compounds = -10*Log(IC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
IC50 <= 1 log over the highest tested concentration
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Organism-based
From ChEMBL:
Assay Type: Functional
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 Standard Errorthe 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)
Data Table ( Complete ):     View Active Data    View All Data
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