Luminescence Microorganism-Based Dose Response Followup to Identify Compounds Cytotoxic to Streptococcus
This assay measures cell viability and serves as a counter screen 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 more ..
BioActive Compounds: 18
Group A streptococcus, GAS, streptokinase, virulence, inhibition, viability, EC50
This assay measures cell viability and serves as a counter screen 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 in a separate assay. Cell pellets were assayed for viability by BacTiterGlo reagent (Promega G8233). IC50 values were generated as described in the Data Analysis/Comments section. Selectivity is determined by comparing IC50 values of reduction of 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, 37'C 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) with Cybi-Well Vario (CyBio).
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
Each plate contained neutral (negative) control, positive controls for SK expression, and positive controls for toxicity.
Viability values were derived as follows:
1) Luminescence signals (RLU) 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 (Tetracycline, PubChem CID 9848033) on the same plate.
2) IC50 values were calculated using the Smart Fit strategy of Genedata Screener Condoseo (v6.0.1). 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)