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BioAssay: AID 504656

Inhibitors of Cruzain: Trypanosoma Cruzi Growth Inhibition Assay

Cruzain is a cysteine protease from the tropical parasite Trypanosoma cruzi. This assay protocol is for testing followup compounds for T. Cruzi growth inhibition. ..more
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 504656
Data Source: NCGC (CRUZ561)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2011-04-13
Hold-until Date: 2012-03-31
Modify Date: 2012-03-31

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
Show more
AIDNameTypeComment
1476qHTS Assay for Promiscuous and Specific Inhibitors of Cruzain (without detergent)Confirmatorydepositor-specified cross reference: cruzain qHTS without detergent
1478qHTS Assay for Promiscuous and Specific Inhibitors of Cruzain (with detergent)Confirmatorydepositor-specified cross reference: cruzain qHTS with detergent
2158Confirmation qHTS Assay for Inhibitors of CruzainConfirmatorydepositor-specified cross reference: Confirmation qHTS Assay for Inhibitors of Cruzain
2161qHTS Assay for Inhibitors of Papain: Counterscreen for Cruzain AssayConfirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of Papain: Counterscreen for Cruzain Assay
2249Probe Development Summary of Promiscuous Inhibitors (Artifacts) of CruzainSummarydepositor-specified cross reference: Probe Development Summary of Promiscuous Inhibitors (Artifacts) of Cruzain
2383Probe Development Summary of Inhibitors of CruzainSummarydepositor-specified cross reference
2413Probe Summary: Docking False Negative Inhibitors of CruzainSummarysame project related to Summary assay
2737Confirmation Competitive Binding Assay for Inhibitors of CruzainConfirmatorysame project related to Summary assay
493215Inhibitors of Cruzain: Benzimidazole Series - Round 1Confirmatorysame project related to Summary assay
493215Inhibitors of Cruzain: Benzimidazole Series - Round 1Confirmatorysame project related to Summary assay
504751Inhibitors of Cruzain: Aqueous Profiling AssayOthersame project related to Summary assay
504752Inhibitors of Cruzain: PBS Stability Profiling AssayOthersame project related to Summary assay
504754Inhibitors of Cruzain: Metabolic Stability TheoryOthersame project related to Summary assay
504758Inhibitors of Cruzain: Caco-2 Permeability Profiling AssayOthersame project related to Summary assay
504759Inhibitors of Cruzain: Efflux Ratio ProfilingOthersame project related to Summary assay
504760Inhibitors of Cruzain: Mouse Plasma Stability ProfilingOthersame project related to Summary assay
Description:
Cruzain is a cysteine protease from the tropical parasite Trypanosoma cruzi. This assay protocol is for testing followup compounds for T. Cruzi growth inhibition.

See following references for additional details:

Jadhav A, et al. J Med Chem. 2010 Jan 14;53(1):37-51.
Mott BT, et al. J Med Chem. 2010 Jan 14;53(1):52-60.
Ferreira, et al. J Med Chem. 2010 Jul 8;53(13):4891-905.
Bettiol E, et al. PLoS Negl Trop Dis. 2009;3(2):e384.

Assay Provider: Shoichet, Brian; University of California, San Francisco and Ana Rodriguez, New York University School of Medicine.
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
Protocol
NIH/3T3 cells and parasites were harvested, washed once and resuspended in DMEM supplemented with 2% FBS and Pen-Strep-Glut. DMEM did not contain phenol red to avoid interference with the assay absorbance readings at 590nM. Different numbers of NIH/3T3 cells were seeded in 96-well plates. After 3 h, compounds were added at the indicated concentrations and mixed by pipetting. BZN tablets (Rochagan, Roche) dissolved in DMSO and 4uM Amphotericin B solution (Sigma-Aldrich) were used as positive controls. Different numbers of T. cruzi parasites were added in a final volume of 200 ul/well. After 4 days, 50ul of PBS containing 0.5% of the detergent NP40 and 100uM Chlorophenol Red-beta-D-galactoside (CPRG) (Fluka) were added. Plates were incubated at 37 degrees C for 4 h and absorbance was read at 590 nm using a Tecan Spectra Mini plate reader. To calculate the Z' factor, we used the formula described by Zhang et al.: Z' = 1-[(3sigma_c++3sigma_c-) / |uc+-uc-|] where sigma_c+ = standard deviation (SD) of positive control, sigma_c- = SD of negative control, uc+ = mean of positive control, uc- = mean of negative control. Subsequently, the best ratio was used for all growth inhibition assays (50,000 cells and parasites, multiplicity of infection (MOI) 1:1). To determine IC50 values, beta-gal activity (Abs590) was plotted against compound concentration for each compound. The IC50 was determined as the concentration at which the activity (absorbance) was half that in the absence of compound. Mean IC50 values are the average of independent experiments performed in triplicate on three different days.
Comment
Compounds that were inactive were given a PUBCHEM_ACTIVITY_SCORE of 0. Compounds that gave any kind of inhibition were considered active and got a PUBCHEM_ACTIVITY_SCORE of -10*Log_IC50 rounded to the closest whole number.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, IC50.FloatμM
3Log_IC50The logarithm of the Potency (calculated based on Molar Units).Float

* Activity Concentration.
Additional Information
Grant Number: DA024891-01

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