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BioAssay: AID 504351

Dose 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_Set3

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 multi-point, multi-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
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Active(4)
 
 
Inactive(36)
 
 
Inconclusive(16)
 
 
 Tested Substances
 Tested Substances
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Active(4)
 
 
Inactive(38)
 
 
Inconclusive(16)
 
 
AID: 504351
Data Source: Broad Institute (2014-03_Inhibitor_Dose_DryPowder_Activity_Set3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-02-24
Hold-until Date: 2011-03-12
Modify Date: 2011-03-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 4
Depositor Specified Assays
AIDNameTypeComment
1677Broad Institute MLPCN Streptokinase Expression Inhibition ProjectsummarySummary assay
Description:
Keywords:
Group A streptococcus, GAS, streptokinase, virulence, inhibition, viability

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 multi-point, multi-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). 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
Protocol:

Day 1 Streak out UMAA 2616 for colonies on THY/S (THY with streptomycin 100 ug/mL) plate; 37 degrees C O/N

Day 2 Grow an overnight culture in THY/S from a single colony, 37 degrees 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 degrees C) THY/S; stir at 4 degrees 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 degrees C for 6 hrs in Liconic incubator

Pellet cells at 3000 rpm

Transfer 10 uL/well supernatant to clear plates (Nunc 242757) for assay with Cybi-Well Vario (CyBio), store at -20oC.
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 2x10 uL/well supernatant (thawed and warmed up to RT) in clear SK assay plates with Combi

Read OD405 at 0 hrs

37oC for 2.5 hrs

Read OD405

37oC for 2.5 hrs

Read OD405
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) and positive control wells (PC; n=9) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

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 (10)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
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): AC50
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)

PUBCHEM_ACTIVITY_OUTCOME:
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 >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
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.

DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.

Note:
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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3PUBCHEM_ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
4PUBCHEM_ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
5PUBCHEM_ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
6pAC50_MEqual to -1*log10(AC50)String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_0.0075uM_(%)_TEST1 (0.0075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.0235uM_(%)_TEST1 (0.0235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.068uM_(%)_TEST1 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.21uM_(%)_TEST1 (0.21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.6uM_(%)_TEST1 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_1.8uM_(%)_TEST1 (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_5.6uM_(%)_TEST1 (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_16uM_(%)_TEST1 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25PUBCHEM_ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
26PUBCHEM_ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
27PUBCHEM_ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
28Activity_at_50uM_(%)_TEST1 (50μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29AC50_Qualifier_TEST2'>','=', or '<'String
30AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
31pAC50_M_TEST2Equal to -1*log10(AC50)String
32Hill_Slope_TEST2The slope at AC50Float
33S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
34Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
35Num_Points_TEST2The number of data points used to generate the plotInteger
36Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
37Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
38Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
39Activity_at_0.0075uM_(%)_TEST2 (0.0075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_0.0235uM_(%)_TEST2 (0.0235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_0.068uM_(%)_TEST2 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_0.21uM_(%)_TEST2 (0.21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_0.6uM_(%)_TEST2 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_1.8uM_(%)_TEST2 (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_5.6uM_(%)_TEST2 (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46Activity_at_16uM_(%)_TEST2 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47Activity_at_50uM_(%)_TEST2 (50μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
48PUBCHEM_ACTIVITY_OUTCOME_TEST3The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
49PUBCHEM_ACTIVITY_SCORE_TEST3The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
50PUBCHEM_ASSAYDATA_COMMENT_TEST3Comments relevant to the subset of data included in the indicated Test number.String
51AC50_Qualifier_TEST3'>','=', or '<'String
52AC50_uM_TEST3The concentration at which activity reaches 50% of the maximumFloatμM
53pAC50_M_TEST3Equal to -1*log10(AC50)String
54Hill_Slope_TEST3The slope at AC50Float
55S0_(%)_TEST3The fitted activity value at zero concentrationFloat%
56Sinf_(%)_TEST3The fitted activity value at infinite concentrationFloat%
57Num_Points_TEST3The number of data points used to generate the plotInteger
58Max_Activity_(%)_TEST3The maximum activity value observed, based on mean of replicates per concentrationFloat%
59Max_Activity_Conc_uM_TEST3The concentration at which the maximum activity is observedFloatμM
60Max_Concentration_uM_TEST3Maximum valid test concentrationFloatμM
61Activity_at_0.0075uM_(%)_TEST3 (0.0075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
62Activity_at_0.0235uM_(%)_TEST3 (0.0235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
63Activity_at_0.068uM_(%)_TEST3 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
64Activity_at_0.21uM_(%)_TEST3 (0.21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
65Activity_at_0.6uM_(%)_TEST3 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
66Activity_at_1.8uM_(%)_TEST3 (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
67Activity_at_5.6uM_(%)_TEST3 (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
68Activity_at_16uM_(%)_TEST3 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
69Activity_at_50uM_(%)_TEST3 (50μM**)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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