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BioAssay: AID 504910

A 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)

Project Overview: This functional assay was developed for detection of compounds inhibiting HepG2 cells viability as a secondary screen to the M. tuberculosis bacteriocidal assay that compares growth in media containing glycerol versus growth in media without glycerol (AID 449762). The HepG2 line was selected for cytotoxicity studies since it is derived from a human liver fibroblast and would be more indicative of the type of cell that M. tuberculosis would infect and be applicable as a drug target. ..more
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 Tested Compounds
 Tested Compounds
All(69)
 
 
Active(8)
 
 
Inactive(61)
 
 
 Tested Substances
 Tested Substances
All(69)
 
 
Active(8)
 
 
Inactive(61)
 
 
 Related BioAssays
 Related BioAssays
AID: 504910
Data Source: Southern Research Specialized Biocontainment Screening Center (TB_Inh_HepG2_4)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-07-05
Hold-until Date: 2012-04-12
Modify Date: 2012-04-12

Data Table ( Complete ):           Active    All
BioActive Compounds: 8
Depositor Specified Assays
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AIDNameTypeComment
449762High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Mediaconfirmatoryprimary & confirmatory screen (w/Glycerol)
493198High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compoundsconfirmatoryconfirmatory screen (w/Glycerol)
504556High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (2)confirmatoryconfirmatory screen (w/Glycerol)
504646High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (3)confirmatoryconfirmatory screen (w/Glycerol)
504857High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (4)confirmatoryconfirmatory screen (w/Glycerol)
449764A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerolconfirmatorycounter screen (No Glycerol)
493181A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compoundsconfirmatorycounter screen (No Glycerol)
504564A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (2)confirmatorycounter screen (No Glycerol)
504645A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (3)confirmatorycounter screen (No Glycerol)
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)confirmatorycounter screen (No Glycerol)
435019A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Mycobacterium TuberculosisconfirmatoryVero Cytotox
504335A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis.confirmatoryVero Cytotox
504562A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (2)confirmatoryVero Cytotox
504684A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (3)confirmatoryVero Cytotox
504854A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (4)confirmatoryVero Cytotox
504642A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without GlycerolconfirmatoryHepG2 Cytotox
504682A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Mycobacterium tuberculosis in Media Containing Glycerol versus Media without Glycerol (2)confirmatoryHepG2 Cytotox
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)confirmatoryHepG2 Cytotox
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 GlycerolconfirmatoryTHP1 Cytotox
504683A 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 (2)confirmatoryTHP1 Cytotox
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)confirmatoryTHP1 Cytotox
449775Summary of Assays used to Identify Novel Compounds That Inhibit Mycobacterium Tuberculosis in 7H9 Media with GlycerolsummarySummary AID
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
588447High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (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
602435High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (9)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
602431A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (8)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
588445A Cell Based Secondary Assay To Explore Vero Cell Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (6)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
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:
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: William Bishai, Johns Hopkins University
Award: 1 R03 MH084877-01A1

Project Overview: This functional assay was developed for detection of compounds inhibiting HepG2 cells viability as a secondary screen to the M. tuberculosis bacteriocidal assay that compares growth in media containing glycerol versus growth in media without glycerol (AID 449762). The HepG2 line was selected for cytotoxicity studies since it is derived from a human liver fibroblast and would be more indicative of the type of cell that M. tuberculosis would infect and be applicable as a drug target.

In this assay, HepG2 cells were treated with compounds selected as "hits" in the M. tuberculosis assay for 72 hours over a 10 point 2-fold dilution series, ranging from 20 uM to 0.078 microM. tuberculosis 0.39 uM. Following 72 hour incubation period, relative viable cell number was determined using Cell Titer Glo (Promega). Each plate contained 64 replicates of vehicle treated cells which served as negative controls.
Protocol
Cell Culture: HepG2 cells were subcultured every 7 days in E-MEM with 10% fetal bovine serum, incubated at 37 degrees C in 5% carbon dioxide. Cells were passaged as needed, harvested from flasks using 0.25% trypsin-EDTA and maintained for no more than 20 passages.

Compound Dosing/Plating: Compounds or carrier control (DMSO) were diluted to 6X in complete growth medium and 5 microL was dispensed into 384-well black clear-bottom tissue culture treated plates.

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: Following the 72 hour incubation period, the assay plates were equilibrated to room temperature for 10 min and twenty-five muL of Cell Titer Glo reagent (Promega) was added to each well using a WellMate (Matrix, Hudson, NH) and the plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Perkin Elmer Envision microplate reader with an integration time of 0.1 s.

Data Analysis: Sixty-four control wells containing cells treated with DMSO vehicle 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.
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" and CC50 values were not determined.

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 average 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
1Average CC50 ModifierString
2Average CC50*FloatμM
3CC50 Modifier Rep 1String
4CC50 Rep 1FloatμM
5CC50 Std Dev Rep 1Float
6CC50 Hill Slope Rep 1Float
7CC50 NormChi2 Rep 1Float
8CC50 Modifier Rep 2String
9CC50 Rep 2FloatμM
10CC50 Std Dev Rep 2Float
11CC50 Hill Slope Rep 2Float
12CC50 NormChi2 Rep 2Float
13CC50 Modifier Rep 3String
14CC50 Rep 3FloatμM
15CC50 Std Dev Rep 3Float
16CC50 Hill Slope Rep 3Float
17CC50 NormChi2 Rep 3Float
18Cell Viability @ 50 uM Rep 1 (50μM**)Float%
19Cell Viability @ 25 uM Rep 1 (25μM**)Float%
20Cell Viability @ 12.5 uM Rep 1 (12.5μM**)Float%
21Cell Viability @ 6.25 uM Rep 1 (6.25μM**)Float%
22Cell Viability @ 3.125 uM Rep 1 (3.125μM**)Float%
23Cell Viability @ 1.563 uM Rep 1 (1.563μM**)Float%
24Cell Viability @ 0.782 uM Rep 1 (0.781μM**)Float%
25Cell Viability @ 0.391 uM Rep 1 (0.391μM**)Float%
26Cell Viability @ 0.195 uM Rep 1 (0.195μM**)Float%
27Cell Viability @ 0.098 uM Rep 1 (0.098μM**)Float%
28Cell Viability @ 0.049 uM Rep 1 (0.049μM**)Float%
29Cell Viability @ 50 uM Rep 2 (50μM**)Float%
30Cell Viability @ 25 uM Rep 2 (25μM**)Float%
31Cell Viability @ 12.5 uM Rep 2 (12.5μM**)Float%
32Cell Viability @ 6.25 uM Rep 2 (6.25μM**)Float%
33Cell Viability @ 3.125 uM Rep 2 (3.125μM**)Float%
34Cell Viability @ 1.563 uM Rep 2 (1.563μM**)Float%
35Cell Viability @ 0.781 uM Rep 2 (0.781μM**)Float%
36Cell Viability @ 0.391 uM Rep 2 (0.391μM**)Float%
37Cell Viability @ 0.195 uM Rep 2 (0.195μM**)Float%
38Cell Viability @ 0.098 uM Rep 2 (0.098μM**)Float%
39Cell Viability @ 50 uM Rep 3 (50μM**)Float%
40Cell Viability @ 25 uM Rep 3 (25μM**)Float%
41Cell Viability @ 12.5 uM Rep 3 (12.5μM**)Float%
42Cell Viability @ 6.25 uM Rep 3 (6.25μM**)Float%
43Cell Viability @ 3.125 uM Rep 3 (3.125μM**)Float%
44Cell Viability @ 1.563 uM Rep 3 (1.563μM**)Float%
45Cell Viability @ 0.781 uM Rep 3 (0.781μM**)Float%
46Cell Viability @ 0.391 uM Rep 3 (0.391μM**)Float%
47Cell Viability @ 0.195 uM Rep 3 (0.195μM**)Float%
48Cell Viability @ 0.098 uM Rep 3 (0.098μM**)Float%
49Cell Viability @ 50 uM Rep 4 (50μM**)Float%
50Cell Viability @ 25 uM Rep 4 (25μM**)Float%
51Cell Viability @ 12.5 uM Rep 4 (12.5μM**)Float%
52Cell Viability @ 6.25 uM Rep 4 (6.25μM**)Float%
53Cell Viability @ 3.125 uM Rep 4 (3.125μM**)Float%
54Cell Viability @ 1.563 uM Rep 4 (1.563μM**)Float%
55Cell Viability @ 0.782 uM Rep 4 (0.781μM**)Float%
56Cell Viability @ 0.391 uM Rep 4 (0.391μM**)Float%
57Cell Viability @ 0.195 uM Rep 4 (0.195μM**)Float%
58Cell Viability @ 0.098 uM Rep 4 (0.098μM**)Float%
59Cell Viability @ 0.049 uM Rep 4 (0.049μM**)Float%
60VerificationString

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

Data Table (Concise)
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