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BioAssay: AID 651885

Inhibition of intracellular CAI/72 strain of Trypanosoma cruzi in bovine embryo skeletal muscle cells: high content imaging Measured in Cell-Based System Using Imaging - 2138-07_Inhibitor_SinglePoint_DryPowder_Activity

Assay Overview: The assay detects the effects of compounds on intracellular T. cruzi amastigotes. Bovine Embryo Skeletal Muscle (BESM) cells are infected with CA-I/72 clinical strain of T.cruzi in a clear bottom 96-well plate. Extracellular T. cruzi is washed away and varying concentrations of compound are added to the well and incubated for 72 hrs. Cells are fixed and stained with a nuclear dye Hoechst and the number of T. cruzi vs. mammalian host cell nuclei are imaged, categorized by size, and counted using the IN Cell Analyzer 2000. ..more
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 Tested Compounds
 Tested Compounds
All(63)
 
 
Active(28)
 
 
Inactive(23)
 
 
Inconclusive(12)
 
 
 Tested Substances
 Tested Substances
All(64)
 
 
Active(28)
 
 
Inactive(23)
 
 
Inconclusive(13)
 
 
 Related BioAssays
 Related BioAssays
AID: 651885
Data Source: Broad Institute (2138-07_Inhibitor_SinglePoint_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-12-07
Hold-until Date: 2013-07-15
Modify Date: 2013-07-16

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 28
Related Experiments
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AIDNameTypeProbeComment
624280Broad Institute Inhibition of T.cruzi replication in culture Inhibitor Probe ProjectSummary1 depositor-specified cross reference: summary assay
624255Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_SinglePoint_HTS_ActivityScreening same project related to Summary assay
651739Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_SinglePoint_CherryPick_ActivityOther same project related to Summary assay
651740Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_SinglePoint_CherryPick_Activity_Set2Other same project related to Summary assay
651742NIH/3T3 (mouse embryonic fibroblast) toxicity Measured in Cell-Based System Using Plate Reader - 2138-02_Inhibitor_SinglePoint_CherryPick_ActivityOther same project related to Summary assay
651744NIH/3T3 (mouse embryonic fibroblast) toxicity Measured in Cell-Based System Using Plate Reader - 2138-02_Inhibitor_SinglePoint_CherryPick_Activity_Set2Other same project related to Summary assay
651817NIH/3T3 (mouse embryonic fibroblast ) toxicity Measured in Cell-Based System Using Plate Reader - 2017-02_Inhibitor_Dose_CherryPick_Activity_Set2Confirmatory same project related to Summary assay
651818Intracellular Trypanosomes Measured in Cell-Based/Microorganism System Using Plate Reader - 2017-01_Inhibitor_Dose_CherryPick_Activity_Set3Confirmatory same project related to Summary assay
651844NIH/3T3 (mouse embryonic fibroblast) toxicity Measured in Cell-Based System Using Plate Reader - 2138-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651845Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651869Cidal vs. static assay: inhibition of T. cruzi (CAI/72) after prolonged treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-08_Inhibitor_SinglePoint_DryPowder_Activity_Set2Other same project related to Summary assay
651877Cidal vs. static assay: inhibition of T. cruzi (CAI/72) after prolonged treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-08_Inhibitor_SinglePoint_DryPowder_ActivityOther same project related to Summary assay
651881Inhibition of T. cruzi Tulahuen strain amastigotes in L-6 muscle cells Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2138-03_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651888Inhibition of Plasmodium falcipirum NF54 Measured in Cell-Based System Using Plate Reader - 2138-11_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651889Inhibition of Plasmodium falcipirum NF54 Measured in Cell-Based System Using Plate Reader - 2138-11_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651890Inhibition of T. cruzi Tulahuen strain amastigotes in L-6 muscle cells Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2138-03_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651891Inhibition of L. donovani axenic amastigotes Measured in Cell-Based System Using Plate Reader - 2138-04_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651892Inhibition of L. donovani axenic amastigotes Measured in Cell-Based System Using Plate Reader - 2138-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651893Inhibition of Trypanosoma brucei rhodesiense STIB900 Measured in Cell-Based System Using Plate Reader - 2138-05_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651894Inhibition of Trypanosoma brucei rhodesiense STIB900 Measured in Cell-Based System Using Plate Reader - 2138-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651895L-6 rat myocyte cell toxicity Measured in Cell-Based System Using Plate Reader - 2138-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
651896L-6 rat myocyte cell toxicity Measured in Cell-Based System Using Plate Reader - 2138-06_Inhibitor_Dose_CherryPick_ActivityConfirmatory same project related to Summary assay
651897Inhibition of Trypanosoma cruzi Cruzain Measured in Biochemical System Using Plate Reader - 2138-09_Inhibitor_SinglePoint_DryPowder_ActivityConfirmatory same project related to Summary assay
651903Intracellular Trypanosomes Measured in Cell-Based/Microorganism System Using Plate Reader - 2017-01_Inhibitor_SinglePoint_HTS_Activity_Set2Screening same project related to Summary assay
652275Inhibition of T.cruzi proliferation in culture Measured in Cell-Based System Using Plate Reader - 2138-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
652278Cidal vs. static assay: inhibition of T. cruzi (CAI/72) in J774 macrophages after prolonged compound treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-13_Inhibitor_SinglePoint_DryPowder_Activity_Set2Other same project related to Summary assay
652279Cidal vs. static assay: inhibition of T. cruzi (CAI/72) in J774 macrophages after prolonged compound treatment and treatment withdrawal Measured in Cell-Based System Using Imaging - 2138-13_Inhibitor_SinglePoint_DryPowder_ActivityOther same project related to Summary assay
652280NIH/3T3 (mouse embryonic fibroblast) toxicity Measured in Cell-Based System Using Plate Reader - 2138-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
686919Inhibition of Trypanosoma cruzi Cruzain Measured in Biochemical System Using Plate Reader - 2138-09_Inhibitor_Dose_DryPowder_ActivityOther same project related to Summary assay
Description:
Keywords: Trypanosoma cruzi, amastigotes, high content screening, Chagas Disease


Assay Overview: The assay detects the effects of compounds on intracellular T. cruzi amastigotes. Bovine Embryo Skeletal Muscle (BESM) cells are infected with CA-I/72 clinical strain of T.cruzi in a clear bottom 96-well plate. Extracellular T. cruzi is washed away and varying concentrations of compound are added to the well and incubated for 72 hrs. Cells are fixed and stained with a nuclear dye Hoechst and the number of T. cruzi vs. mammalian host cell nuclei are imaged, categorized by size, and counted using the IN Cell Analyzer 2000.

Expected Outcome: Compounds significantly suppressing luminescence, and therefore b-galactosidases expression will be identified as hits in the screen. Compounds that inhibit luminescence activity may kill T. cruzi, inhibit T. cruzi invasion or inhibit development of the parasite within the host cell. Compounds that are toxic to the host cell will be excluded in secondary assays.
Protocol
Microtiter-based 72 hour screen against T. cruzi amastigotes in host BESM cells

The screen employs the IN Cell Analyzer 2000 (GE Healthcare Life Sciences), a flexible, modular, cellular and subcellular imaging system for fast, automated imaging of fixed and live cells built around an automated epifluorescence-based microscope and image acquisition software. The screening is performed in sterile and fluorescence transmissible 96 wells-cell culture plates. Each well is seeded with 1000 BESM cells (Bovine Embryo Skeletal Muscle Cells) allowed to attach for several hours. The culture medium (RPMI 1640 with 5 % FBS) is then replaced with fresh medium containing 1000 T. cruzi trypomastigotes (Wild type CA-I/72 T. cruzi was isolated from an Argentinean chronic chagasic patient ) per well. Sixteen hours post-infection, cells are washed once to remove extracellular parasites, and maintained in medium containing serial dilutions of compounds for 72h at 370C. Cells are then washed once with PBS, fixed for 2 hours with 4% paraformaldehyde, and washed 1 time to remove the fixative. Then 50 microl of mounting medium containing a DNA fluorescent dye (i.e. DAPI or Hoechst) are added per well. The plates are kept in the dark at 4oC until scanned in the IN Cell Analyzer with a 10X objective, and the images are collected with appropriate fluorescent excitation/emission filters

At least 100 infected cells are recorded per well. The ratio of T. cruzi amastigotes per infected cell is calculated in untreated controls and compared to those obtained for the different compounds and conditions tested. A significant reduction in the E/C ratio indicates inhibition of parasite growth rate. This information provides a quantitative measure of growth inhibition during the first 72h post-infection.
Comment
In addition to IC50 values, IC90 and IC99 are provided for some active compounds. Also included but not used for calculation are Host cell toxicity measurements.

Compounds that did not achieve sufficient activity were listed with IC50 equal to the greatest tested concentration where applicable and should be interpreted as having an IC50 > 20000nM.

Compounds that had IC50 less than 3 times the control compound, Benznidazole, were labeled active. Those samples that had one test active and an other inactive were labeled inconclusive.

Pubchem Activity Score was calculated by dividing the IC50 of the control compound, Benznidazole, by the compound's average IC50 and multiplied by 100. Larger numbers are more active and lower numbers are less active.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50 (Test 1) (nM) FloatnM
2IC50 (Test 2) (nM)FloatnM
3Toxicity IC50 (Test 1) (nM)FloatnM
4Toxicity IC50 (Test 2) (nM)FloatnM
5IC50 (Test 3) (nM)FloatnM
6IC90 (Test 3) (nM)FloatnM
7IC99 (Test 3) (nM)FloatnM
8IC50 (Test 4) (nM)FloatnM
9IC90 (Test 4) (nM)FloatnM
10IC99 (Test 4) (nM)FloatnM
11Toxicity IC50 (Test 3) (nM)FloatnM
12Toxicity IC50 (Test 4) (nM)FloatnM
13Average IC50 OperatorString
14Average IC50 (uM)*FloatμM

* Activity Concentration.
Additional Information
Grant Number: 1 R03 MH085673-01

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