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BioAssay: AID 492998

A Cell Based Secondary Assay to Explore Cytotoxicity in Vero E6 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosis

Project Overview: This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the non-replicating, drug-tolerant Mycobacterium tuberculosis bacteriocidal assay. ..more
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 Tested Compounds
 Tested Compounds
All(2294)
 
 
Active(554)
 
 
Inactive(1740)
 
 
 Tested Substances
 Tested Substances
All(2294)
 
 
Active(554)
 
 
Inactive(1740)
 
 
 Related BioAssays
 Related BioAssays
AID: 492998
Data Source: Southern Research Specialized Biocontainment Screening Center (TB_Hung_Vero_1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-12-17

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 554
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 AID
489018A Cell Based Secondary Assay to Explore Cytotoxicity in HepG2 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory depositor-specified cross reference: Primary screen hits in HepG2 cytotoxicity screen
489025A Cell Based Secondary Assay to Explore Cytotoxicity in THP-1 Cells of Compounds that Modulate Non-Replicating, Drug-tolerant Mycobacterium tuberculosisConfirmatory depositor-specified cross reference: Primary screen hits in THP1 cytotoxicity screen
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
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
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 Vero E6 cells viability as a secondary screen to the non-replicating, drug-tolerant Mycobacterium tuberculosis bacteriocidal assay.

In this assay, we treated Vero E6 cells with compounds selected as "hits" in the M. tuberculosis (AID 488890) assay for 72 hours over a 10 point 2-fold dilution series, ranging from 0.034 uM to 20 uM. Following 72 hours of treatment, relative viable cell number was determined using a Cell Titer Glo (Promega). Each plate contained 64 replicates of vehicle treated cells which served as negative controls.
Protocol
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. 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 and carrier control (DMSO) 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 muL 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 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 40-80 with "Inactive" compounds scoring 0.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: VERO 76
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)
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