MLPCN Streptokinase Expression Inhibition
The goal of the project is to identify and develop novel antibiotic small molecules through inhibiting streptokinase (SK) expression in group A streptococcus (GAS). Based on the critical role of SK in GAS pathogenicity, we are attempting to identify chemical compounds that specifically reduce the expression of bacterial SK, without interfering with bacterial viability. Specifically, a probe is a more ..
BioActive Compounds: 3959
Depositor Specified Assays
Broad Institute MLPCN Streptokinase Expression Inhibition Project
Project ID: 2014
Keywords: Streptokinase, inhibition, growth, bacterial, group A streptococcus, virulence
Primary Collaborators: Hongmin Sun, University of Missouri-Columbia, email@example.com
The goal of the project is to identify and develop novel antibiotic small molecules through inhibiting streptokinase (SK) expression in group A streptococcus (GAS). Based on the critical role of SK in GAS pathogenicity, we are attempting to identify chemical compounds that specifically reduce the expression of bacterial SK, without interfering with bacterial viability. Specifically, a probe is a small molecule that shows no less than one hundred fold selection for the primary strain (SKKanGAS, see below) vs a control strain SK(-)GAS (see below) in growth-based assays and shows significant inhibition of SK secretion in abiochemical assay that measure SK activity. The more potent and specific probe will be used to further explore the role of SK in GAS pathogenicity.
Assay: The strain used for this project is a GAS strain carrying the kanamycin resistant gene under the control of the streptokinase promoter (SKKanGAS). Compounds that inhibit streptokinase promoter activity lead to a decrease of the kanamycin resistant gene product and cell death in the presence of kanamycin (Sigma K1637). Briefly, SKKanGAS at OD600 equal to 0.015 were plated onto 384-well plates (Corning 3570) and incubated with test compounds (7.5 uM, 0.2%DMSO) in the presence of 40 ug/ml of kanamycin in Todd-Hewitt Broth medium for 6 hours before cell survival is assessed by BacTiterGlo reagent (Promega G8233). Selectivity is determined by comparing growth inhibition between SKKanGAS and another GAS strain, SK(-)GAS where the kanamycin resistant gene is under the control of an SK-independent promoter.
Outcome: A decrease in the luminescent signal will identify compounds that either inhibit the SK promoter, or inhibit the cell growth independent of inhibiting the SK promoter. Normalization of the HTS data across runs and calibration to the positive control was performed as described below. The results are reported as % inhibition. Specificity for SK promoter inhibition will be determined in a secondary assay where a GAS strain carrying kanamycin resistant gene which is not controlled by the SK promoter will be used.
Day 1 Streak out SKKanGAS for colonies on THY/S/S agar plate (Todd-Hewitt Broth with streptomycin 100 ug/mL, spectinomycin 100 ug/ml, 1.5% agar); 37oC O/N
Day 2 Grow an overnight culture in THY/S/S from a single colony, 37oC O/N
Day 3 Measure OD600 of the overnight culture (typically over OD600 1.2), and then dilute 1:20 into fresh THY/S/S in a flask. Monitor OD600 until it reaches 0.6-0.9.
Dispense assay plates with 50ul/well of cells at final OD600 of 0.015 in THY/Kanamycin (40ug/ml Kan) with Multidrop Combi (ThermoFisher)
Pin 100 nL of compounds into medium to reach a final average concentration of 7.5 uM
Incubate at 37oC for 6 hr at >85% humidity
Cool plates to RT for 30 minutes
Add 30 uL/well 80% BacTiterGlo diluted in PBS
RT 10 minutes
Read on Envision for luminescence with a 384-well aperture and regular sensitivity.
This assay belongs to specific project (2014, MLPCN Streptokinase Expression Inhibition ) at the Broad Institute of MIT and Harvard
HTS Data Analysis:
Negative control wells (DMSO) were included on every plate.
Two plates containing the positive control at various doses were included in every assay run. In order to provide a consistent calibration for all runs, we defined the positive control value to be the value observed when the positive control was present at 1.2974uM.
The PubChem_Activity_Score was derived using the follow procedure:
1. A background-subtracted value was calculated for each well by subtracting the median value of the negative control wells on each plate from the value of each well on that plate.
2. Systematic plate effects were corrected for each run. A plate effect correction matrix was derived by calculating the median value of all non-positive-control wells for each well location (e.g. C07) across all the plates in a run. This correction matrix was then smoothed using an inverse-distance weighted median filter and subtracted from all wells on the plates in the same run resulting in a background-subtracted, plate-effect-corrected value for each well.
3. An activity score was derived for each well by dividing the background-subtracted, plate-effect-corrected value for each well by the median of the background-subtracted, plate-effect-corrected value of the positive control wells in the same run and multiplying the resulting fraction by 100.
4. The final PubChem_Activity_Score represents the mean of all valid replicate activity scores obtained.
The PubChem_Activity_Outcome class was assigned as described below:
Activity_Outcome = 1 (inactive)
PubChem_Activity_Score <50 and all replicate activity scores <50.
Activity_Outcome = 2 (active)
Activity_Outcome = 3 (inconclusive)
PubChem_Activity_Score < 50 with at least one replicate activity score >=50.
Activity_Outcome = 4
Data Table (Concise)