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 ..
BioActive Compounds: 51
Group A streptococcus, GAS, streptokinase, expression, virulence, inhibition, dose response, EC50
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.
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 ##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
37'C for 2.5 hrs
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.
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).
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%
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
* Activity Concentration.
Data Table (Concise)