AID	Name	Type	Comment
1677	Broad Institute MLPCN Streptokinase Expression Inhibition Project	summary	Summary assay

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). AC50 values were generated as described in the Data Analysis/Comments section. Selectivity is determined by comparing AC50 values of reduction of SK activity and AC50 values of reduction of viability.

Expected Outcome:
The project is looking for compounds that selectively reduce expression of SK activity without inhibiting cell growth.

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

Day 2 Grow an overnight culture in THY/S from a single colony, 37 deg C overnight

Day 3 In the morning, dilute 1:20 of the overnight 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 deg C) THY/S; stir at 4 deg C overnight


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 deg 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 -20 deg 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 2x10 uL/well supernatant (thawed and warmed up to RT) in clear SK assay plates with Combi

Read OD405 at 0 hrs

37 deg C for 2.5 hrs

Read OD405

37 deg C for 2.5 hrs

Read OD405

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.

Grant Number: 1R03DA026214-01