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BioAssay: AID 2138

Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to Streptococcus

This assay measures cell viability and serves as a counter screen 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 more ..
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 Tested Compounds
 Tested Compounds
All(62)
 
 
Active(18)
 
 
Inactive(44)
 
 
 Tested Substances
 Tested Substances
All(63)
 
 
Active(18)
 
 
Inactive(45)
 
 
 Related BioAssays
 Related BioAssays
AID: 2138
Data Source: Broad Institute (2014-03_INHIBITORS_DOSE-TITRATION_MLPCN-DRYPOWDER_TOXICITY)
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 ):           Active    All
BioActive Compounds: 18
Depositor Specified Assays
AIDNameTypeProbeComment
1662MLPCN Streptokinase Expression Inhibitionscreening Primary HTS
1677Broad Institute MLPCN Streptokinase Expression Inhibition Projectsummary3 Project Summary
1902Luminescence Microorganism-Based Dose Confirmation HTS to Identify Inhibitors of Streptokinase Promotor Activityconfirmatory Retest at Dose
1900Luminescence Microorganism-Based Dose Confirmation HTS to Identify Compounds Cytotoxic to SK(-)GAS Group A Streptococcusconfirmatory Counter Screen
1914Absorbance Microorganism-Based Dose Response HTS to Identify Inhibitors of Streptokinase Expressionconfirmatory
1915Luminescence Microorganism-Based Dose Response HTS to Identify Compounds Cytotoxic to Streptococcusconfirmatory
Description:
Keywords:
Group A streptococcus, GAS, streptokinase, virulence, inhibition, viability, EC50

Assay Overview:
This assay measures cell viability and serves as a counter screen 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 in a separate assay. Cell pellets were assayed for viability by BacTiterGlo reagent (Promega G8233). IC50 values were generated as described in the Data Analysis/Comments section. Selectivity is determined by comparing IC50 values of reduction of 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, 37'C 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) with Cybi-Well Vario (CyBio).
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
Comment
Each plate contained neutral (negative) control, positive controls for SK expression, and positive controls for toxicity.

Viability values were derived as follows:
1) Luminescence signals (RLU) 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 (Tetracycline, PubChem CID 9848033) on the same plate.
2) IC50 values were calculated 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(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.
Result Definitions
Show more
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_run2outcome run 2Float
10score_run2activity run 2Float
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 plotInteger
18Max. Activity_Run2the maximum activity value observedFloat%
19outcome_run3outcome run 3Float
20score_run3activity at run 3Float
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 plotInteger
28Max. Activity_Run3the maximum activity value observedFloat%
29outcome_run4outcome run 4Float
30score_run4activity for run 4Float
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 plotInteger
38Max. Activity_Run4the maximum activity value observedFloat%
39DATE_REPORTEDdate reported internallyString

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

Data Table (Concise)
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