Luminescence Microorganism-Based Dose Confirmation HTS to Identify Inhibitors of Streptokinase Promotor Activity
Keywords: Streptokinase, inhibition, growth, bacterial, group A streptococcus, virulence, dose response ..more
BioActive Compounds: 2944
Keywords: Streptokinase, inhibition, growth, bacterial, group A streptococcus, virulence, dose response
The strain used for this assay is the same GAS strain used in primary screen 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). Active compounds identified from primary screen were tested in this assay in 6-point 3-fold dilution doses with starting concentrations at 15 uM 0.2%DMSO. Briefly, SKKanGAS at OD600 equal to 0.015 were plated onto 384-well plates (Corning 3570) and incubated with test compounds 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). Detailed procedures are described in Protocol section.
Normalization of the HTS data across runs, calibration to the positive control and EC50 determination were performed as described below. The results are reported as EC50 values.
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.
Dose Response Data Analysis:
32 Negative control (DMSO) and 32 positive control (Tetracycline, PubChem CID 9848033) wells were included on every plate.
All wells tested 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. DMSO plates were run before and after the compound plate and Genedata's runwise additive correction was used with those as calibration plates to correct for plate effects.
EC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (v6.0.1). EC50 values were extrapolated up to 1 log
over the highest tested concentration.
Inactive compounds = 0
Active compounds = -10*Log(EC50)
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)