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BioAssay: AID 489018

A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosis

Project Overview: This functional assay was developed for detection of compounds inhibiting HepG2 cells viability as a secondary screen to the non-replicating, drug-tolerant M. tuberculosis bacteriocidal assay (AID 488890). 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(2294)
 
 
Active(350)
 
 
Inactive(1944)
 
 
 Tested Substances
 Tested Substances
All(2294)
 
 
Active(350)
 
 
Inactive(1944)
 
 
 Related BioAssays
 Related BioAssays
AID: 489018
Data Source: Southern Research Specialized Biocontainment Screening Center (TB_Hung_HepG2_1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-11-18
Modify Date: 2010-11-30

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 350
Related Experiments
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AIDNameTypeProbeComment
488890Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tuberculosisConfirmatory depositor-specified cross reference: Primary/Confirmatory Inhibition Screen
488929Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tuberculosis - SummarySummary1 depositor-specified cross reference: Summary Assay ID
492998A Cell Based Secondary Assay to Explore Cytotoxicity in Vero E6 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory depositor-specified cross reference
687034Broad Institute Elucidation of the physiology of non-replicating, drug tolerant Mycobacterium tuberculosis: Probe to inhibit the Transition from Non-Replication to Replication Inhibitor Probe ProjectSummary depositor-specified cross reference
489025A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
492952A Cell Based Secondary Assay to Explore Compounds that Modulate Non-Replicating, Drug-tolerant Compounds in Replicating H37Rv TB of Mycobacterium tuberculosisConfirmatory same project related to Summary assay
651617Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651618Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651619Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651620Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651850Inhibition of Mycobacterium tuberculosis during logarithmic growth, as enumerated by colony forming units_2157-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651851Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteriaConfirmatory same project related to Summary assay
651856Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowthConfirmatory same project related to Summary assay
651857Inhibition of Mycobacterium tuberculosis during starvation growth, as enumerated by colony forming units_2157-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651946HeLa Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-04_Other_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651947HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Other_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651949HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Other_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
687033Broad Institute Elucidation of the physiology of non-replicating, drug tolerant Mycobacterium tuberculosis: Probe for both Non-Replicating & Replicating organisms Inhibitor Probe ProjectSummary same project related to Summary assay
743175Elucidation of physiology of non-replicating, drug-tolerant Mycobacterium tuberculosis Measured in Microorganism System Using Plate Reader - 2157-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
743183Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
743186TB:Log Assay, OD600 Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
489025A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory same project related to Summary assay
492952A Cell Based Secondary Assay to Explore Compounds that Modulate Non-Replicating, Drug-tolerant Compounds in Replicating H37Rv TB of Mycobacterium tuberculosisConfirmatory same project related to Summary assay
651617Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651618Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteria Measured in Microorganism System Using Plate Reader - 2157-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651619Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
651620Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowth Measured in Microorganism System Using Plate Reader - 2157-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651851Counterscreen for inhibitors of non replicating M. tb using log phase replicating mycobacteriaConfirmatory same project related to Summary assay
651856Secondary assay to identify inhibitors of non-replicating M. tb using luciferase expression without log phase outgrowthConfirmatory same project related to Summary assay
651946HeLa Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-04_Other_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651947HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Other_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651949HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Other_Dose_DryPowder_Activity_Set6Confirmatory same project related to Summary assay
Description:
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Deborah Hung, Massachusetts Institute of Technology
Award: 1 RO3 MH087444-01

Project Overview: This functional assay was developed for detection of compounds inhibiting HepG2 cells viability as a secondary screen to the non-replicating, drug-tolerant M. tuberculosis bacteriocidal assay (AID 488890). 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
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 41-80 with "Inactive" compounds scoring 0.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: HEPG2
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1CC50 ModifierString
2CC50*FloatμM
3CC50 Std Dev ModifierString
4CC50 Std DevFloat
5CC50 Hill SlopeFloat
6CC50 Normalized Chi2Float
7% Cell Viability @ 20 uM (20μM**)Float%
8% Cell Viability @ 10 uM (10μM**)Float%
9% Cell Viability @ 5 uM (5μM**)Float%
10% Cell Viability @ 2.5 uM (2.5μM**)Float%
11% Cell Viability @ 1.25 uM (1.25μM**)Float%
12% Cell Viability @ 0.625 uM (0.625μM**)Float%
13% Cell Viability @ 0.313 uM (0.313μM**)Float%
14% Cell Viability @ 0.156 uM (0.156μM**)Float%
15% Cell Viability @ 0.078 uM (0.078μM**)Float%
16% Cell Viability @ 0.039 uM (0.039μM**)Float%
17Max % ViabilityMaximum % Viability observed across all concentrations.Float%
18Max Viability ConcConcentration at which Maximum % Viability was observed.FloatμM
19Min % ViabilityMinimum % Viability observed across all concentrations.Float%
20Min Viability ConcConcentration at which Minimum % Viability was observed.FloatμM

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 RO3 MH087444-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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