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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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 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
 Related BioAssays
 Related BioAssays
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 ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
624280Broad Institute Inhibition of T.cruzi replication in culture Inhibitor Probe ProjectsummarySummary 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).
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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