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BioAssay: AID 651897

Inhibition of Trypanosoma cruzi Cruzain Measured in Biochemical System Using Plate Reader - 2138-09_Inhibitor_SinglePoint_DryPowder_Activity

Cruzain or cruzipain is a cystein protease that has been identified as a potential target for drug development against Chagas disease. This assay was designed to test if compounds identified in a phenotypic screen target cruzain. Compounds were tested against purified recombinant cruzain. Cruzain (4 nM) was incubated with test compound in for 5 min at room temperature, then a buffer containing more ..
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AID: 651897
Data Source: Broad Institute (2138-09_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
Target
BioActive Compound: 1
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
651885Inhibition 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_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
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: Cruzain, Cruzipain, cysteine protease


Assay Overview:

Cruzain or cruzipain is a cystein protease that has been identified as a potential target for drug development against Chagas disease. This assay was designed to test if compounds identified in a phenotypic screen target cruzain. Compounds were tested against purified recombinant cruzain. Cruzain (4 nM) was incubated with test compound in for 5 min at room temperature, then a buffer containing Z-Phe-Arg-AMC was added to enzyme-compound mixture to give 10 microM substrate. The rate of free AMC release was measured at excitation and emission wavelengths of 355 and 460 nm, respectively, with a microtiter plate spectrofluorimeter for 3 min. Percentage inhibition of test compound was calculated relative to the DMSO control (0% inhibition control). IC50 values were determined.

Expected Outcome: If compounds inhibit cruzain protease activity, a decrease in free AMC release would be observed.
Protocol
Cruzain inhibition assay: Methods for routine screening of the inhibition of cruzain are published[Ang KK, Ratman J, Gut J, Legac J, Hansell E, Mackey Z, Skrypczynska KM, Debnath A, Recombinant cruzain was expressed and purified. For IC50 determinations, compounds were serially diluted in DMSO in the range of 25 microM - 0.001 microM for cruzain. Protease inhibition assays for cruzain were carried out in 384 well plate format. Cruzain (4 nM) was incubated with test compound in 100 mM sodium acetate, pH 5.5, containing 5 mM DTT and 0.01% Triton X-100 (buffer A), for 5 min at room temperature (the higher concentration of TbCatB was required due to its lower intrinsic activity against the fluorogenic substrate). Then buffer A containing Z-Phe-Arg-AMC (Bachem) was added to enzyme-compound mixture to give 10 microM substrate in a final assay volume of 200 microL. The rate of free AMC release was measured at excitation and emission wavelengths of 355 and 460 nm, respectively, with a microtiter plate spectrofluorimeter (SpectraMax M5, Molecular Devices) for 3 min. Percentage inhibition of test compound was calculated relative to the DMSO control (0% inhibition control). IC50 values were determined with Prism 4 software (GraphPad, San Diego, CA) fitted by nonlinear regression to the variable-slope sigmoidal dose-response formula y=100/[1 + 10;(log IC50 - X);H, where H is the Hill coefficient (slope factor).
Comment
The reported IC50 of Cruzain by K11777 is 4 nM. To determine the activity score, the IC50 of K1177 was divided by compound nanomolar IC50 and multiplied by 10,000.
The compound was considered active if 100% inhibition was achieved by the highest dose tested (250uM).
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50(uM)*FloatμM

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

Data Table (Concise)
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