Luminescence Microorganism-Based Dose Confirmation HTS to Identify Compounds Cytotoxic to SK(-)GAS Group A Streptococcus
Keywords: Streptokinase, inhibition, growth, bacterial, group A streptococcus, virulence, dose response ..more
BioActive Compounds: 2879
Keywords: Streptokinase, inhibition, growth, bacterial, group A streptococcus, virulence, dose response
The strain used for this assay is a control GAS strain, SK(-)GAS carrying the kanamycin resistant gene under the control of an SK-unrelated promoter (Perez-Casal, J Bacteriology, 173 (8), 2617-24, 1991). Compounds that inhibit the streptokinase-independent 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 in primary screen were tested in this assay in 6-point 3-fold dilution doses with starting concentrations at 15 uM 0.2%DMSO. Briefly, SK(-)GAS at OD600 equal to 0.007 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 IC50 determination were performed as described below. The results are reported as IC50 values. IC50 values obtained from this counter screen and those from retest using the SKKanGAS strain may be compared to illustrate selectivity of compounds to inhibit cell growth in the context of kanamycin resistance of these engineered strains.
Day 1 Streak out SK(-)GAS for colonies on THY/Strept (THY with streptomycin 100 μg/mL) plate; 37oC O/N
Day 2 Grow an overnight culture in THY/Strept from a single colony, 37oC O/N
Day 3 In the morning, measure OD600 of the overnight culture, and then dilute 1:20 into fresh THY/Strept in flask(s). Monitor OD600 until it reaches 0.6-0.8 (between 3-5 hrs).
Dispense assay plates at 30ul/well of THY/Kanamycin (40ug/ml Kan) with Combi
Load plates to incubators.
Dilute the 0.6-0.8OD culture down to OD 0.018 into cold (4oC) THY/Kanamycin
Seed cells onto the pinned plates at 20ul/well with Combi; let plates sit at RT for 30 minutes
Pin ~100 nL of compounds
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)