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BioAssay: AID 2137

Absorbance Microorganism-Based Dose Response Followup 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(62)
 
 
Active(51)
 
 
Inactive(2)
 
 
Inconclusive(9)
 
 
 Tested Substances
 Tested Substances
All(63)
 
 
Active(52)
 
 
Inactive(2)
 
 
Inconclusive(9)
 
 
AID: 2137
Data Source: Broad Institute (2014-03_INHIBITORS_DOSE-TITRATION_MLPCN-DRYPOWDER)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-11-12
Hold-until Date: 2010-06-04
Modify Date: 2010-06-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 51
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
1914Absorbance Microorganism-Based Dose Response HTS to Identify Inhibitors of Streptokinase ExpressionConfirmatory depositor-specified cross reference
1915Luminescence Microorganism-Based Dose Response HTS to Identify Compounds Cytotoxic to StreptococcusConfirmatory depositor-specified cross reference
2138Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to StreptococcusConfirmatory same project related to Summary assay
2470Absorbance Microorganism-Based Dose Response Followup to Identify Inhibitors of Streptokinase Expression.Confirmatory same project related to Summary assay
2477Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to Streptococcus.Confirmatory 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 ##g/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
Data Analysis:

Each plate contained neutral (negative) control, positive controls for SK expression, and positive controls for toxicity.

A. Normalized SK expression values were derived by
1) subtracting initial 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 negative controls wells on the same plate and scaled using the median value of the positive control wells on the same plate.
B. IC50 values were calculated based on the normalized, scaled SK expression values using the Smart Fit strategy of Genedata Screener Condoseo (v7.0.3). IC50 values were extrapolated up to 1 log over the highest tested concentration.

PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)
NOTE: EC50 is reported in uM, so for a reported EC50 1.165 the formula above would read - -10*Log(1.165x10-6).

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

Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activtity >30% but <70%
or
The fit was not valid due to poor fit quality.

Each compound was retested at least once. These retests were analyzed as independent tests; therefore each test generated a separate set of curve data, including PUBCHEM_ACTIVITY_SCORE, PUBCHEM_ACTIVITY_OUTCOME, and EC50.
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
7Num. Pointsthe number of data points included in the plotInteger
8Max. Activitythe maximum activity value observedFloat%
9outcome_run2activity for run 2Integer
10score_run2score for run 2Integer
11EC50 Qualifier_Run2'>', '=', or '<'String
12EC50_Run2the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
13EC50 Standard Error_Run2the standard error for the calculated EC50 valueFloatμM
14S0_Run2the fitted activity level at zero concentrationFloat%
15SInf_Run2the fitted activity level at infinite concentrationFloat%
16Hill Slope_Run2the slope at EC50Float
17Num. Points_Run2the number of data points included in the plotFloat
18Max. Activity_Run2the maximum activity value observedthe maximum activity value observedFloat%
19outcome_run3activity for run 3Integer
20score_run3score for run 3Integer
21EC50 Qualifier_Run3'>', '=', or '<'String
22EC50_Run3the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
23EC50 Standard Error_Run3the standard error for the calculated EC50 valueFloatμM
24S0_Run3the fitted activity level at zero concentrationFloat%
25SInf_Run3the fitted activity level at infinite concentrationFloat%
26Hill Slope_Run3the slope at EC50Float
27Num. Points_Run3the number of data points included in the plotFloat
28Max. Activity_Run3the maximum activity value observedFloat%
29outcome_run4activity for run 4Integer
30score_run4score for run 4Integer
31EC50 Qualifier_Run4'>', '=', or '<'String
32EC50_Run4the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
33EC50 Standard Error_Run4the standard error for the calculated EC50 valueFloatμM
34S0_Run4the fitted activity level at zero concentrationFloat%
35SInf_Run4the fitted activity level at infinite concentrationFloat%
36Hill Slope_Run4the slope at EC50Float
37Num. Points_Run4the number of data points included in the plotFloat
38Max. Activity_Run4the maximum activity value observedFloat%
39Date_ReportedString

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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