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BioAssay: AID 504684

A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (3)

This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the beta-lactam sensitizing M. tuberculosis bacteriocidal assay. ..more
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 Tested Compounds
 Tested Compounds
All(63)
 
 
Active(40)
 
 
Inactive(23)
 
 
 Tested Substances
 Tested Substances
All(63)
 
 
Active(40)
 
 
Inactive(23)
 
 
 Related BioAssays
 Related BioAssays
AID: 504684
Data Source: Southern Research Specialized Biocontainment Screening Center (TB_Inh_Vero4)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-04-19
Hold-until Date: 2012-03-26
Modify Date: 2012-03-27

Data Table ( Complete ):           Active    All
BioActive Compounds: 40
Depositor Specified Assays
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AIDNameTypeComment
449672Growth inhibition of human HT-29 cells after 72 hrs by luminescent assay
449764A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerolconfirmatory
435019A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Mycobacterium Tuberculosisconfirmatory
493198High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compoundsconfirmatory
493181A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compoundsconfirmatory
449775Summary of Assays used to Identify Novel Compounds That Inhibit Mycobacterium Tuberculosis in 7H9 Media with Glycerolsummary
504335A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis.confirmatory
504562A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (2)confirmatory
504642A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerolconfirmatory
504640A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerolconfirmatory
504852A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (3)confirmatory
504853A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (3)confirmatory
504854A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (4)confirmatory
504857High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (4)confirmatory
504860A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (4)confirmatory
504897High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (6)confirmatory
504898A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (5)confirmatory
504901A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (6)confirmatory
504903High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (5)confirmatory
504909A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (5)confirmatory
504910A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (4)confirmatory
504911A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (4)confirmatory
588437A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (7)confirmatory
588441A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (5)confirmatory
588443A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (5)confirmatory
588445A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (6)confirmatory
588447High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (8)confirmatory
602431A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (8)confirmatory
602432A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (6)confirmatory
602433A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (6)confirmatory
602435High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (9)confirmatory
602437A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (8)confirmatory
Description:
This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the beta-lactam sensitizing M. tuberculosis bacteriocidal assay.

In this assay, we treated Vero E6 cells with compounds selected as hits in the M. tuberculosis assay for 72 hours over a 10 point 2-fold dilution series, ranging from 0.195 uM to 100 uM. Following 72 hours of treatment, relative viable cell number was determined using Cell Titer Glo from Promega. Each plate contained 64 replicates of vehicle treated cells which served as negative controls.
Protocol
Cell Culture: Vero E6 cells were subcultured every 7 days in E-MEM with 10% fetal bovine serum and 2 mM glutamine (complete growth medium), incubated at 37 degrees C in 5% carbon dioxide, and maintained for no more than 20 passages.

Compound Dosing/Plating: Carrier control / compounds were diluted in complete growth medium to prepare a 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume).

Cell Plating: Twenty uL of complete growth medium containing 3000 cells were dispensed per well. Plates were incubated at 37 C, 5% CO2 for 72h prior to endpoint detection.

Endpoint/Detection: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature for 10 min. Twenty-five uL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Envision microplate reader in luminescence mode with an integration time of 0.1 s.

Data Analysis: Sixty-four control wells containing cells treated with DMSO vehicle and were included on each assay plate. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med background)/(Med Cell Ctrl - Med background). The normalized % viability was plotted against the tested concentrations. The CC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0, respectively and allowing extrapolation to identify weakly active compounds.
Comment
Outcome: Compounds that showed <70% cell viability for at least one concentration were defined as "Active". If the % viability at all doses was >70%, the compound was defined as "Inactive".

Scoring: SRBCSC uses a three-tier scoring system where scores of 0-40 apply to primary screen data, 41-80 indicates confirmatory screen data, and 81-100 is reserved for confirmatory data on resynthesized compounds. Inactive compounds at any screening level receive a score of 0. In this confirmatory screen, "Active" compounds were scored based on CC50 results on a tier of 81-100 with "Inactive" compounds scoring 0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1CC50 ModifierString
2CC50*Cytotoxic concentrationFloatμM
3CC50 Std Dev ModifierString
4CC50 Std DevFloat
5CC50 Hill SlopeFloat
6CC50 Normalized Chi2Float
7% Cell Viability @ 100 uM (100μM**)Float%
8% Cell Viability @ 50 uM (50μM**)Float%
9% Cell Viability @ 25 uM (25μM**)Float%
10% Cell Viability @ 12.5 uM (12.5μM**)Float%
11% Cell Viability @ 6.25 uM (6.25μM**)Float%
12% Cell Viability @ 3.125 uM (3.125μM**)Float%
13% Cell Viability @ 1.563 uM (1.563μM**)Float%
14% Cell Viability @ 0.781 uM (0.781μM**)Float%
15% Cell Viability @ 0.391 uM (0.391μM**)Float%
16% Cell Viability @ 0.195 uM (0.195μM**)Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH084877-01A1

Data Table (Concise)
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