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BioAssay: AID 2161

qHTS Assay for Inhibitors of Papain: Counterscreen for Cruzain Assay

The following papain assay is a counterscreen for a related protein cruzain (AID: 1478). Cruzain is a cysteine protease from the tropical parasite Trypanosoma cruzi. Activity against papain was used to help prioritize chemical series active against cruzain. Papain is a closely related cysteine protease known to utilize the same Z-FR-AMC fluorogenic substrate as cruzain. The fluorogenic assay more ..
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 Tested Compounds
 Tested Compounds
All(393)
 
 
Active(159)
 
 
Inactive(235)
 
 
 Tested Substances
 Tested Substances
All(413)
 
 
Active(171)
 
 
Inactive(242)
 
 
AID: 2161
Data Source: NCGC (PAPN374)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-11-24

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 159
Depositor Specified Assays
AIDNameTypeComment
1478qHTS Assay for Promiscuous and Specific Inhibitors of Cruzain (with detergent)confirmatoryCruzain with detergent
1476qHTS Assay for Promiscuous and Specific Inhibitors of Cruzain (without detergent)confirmatoryCruzain without detergent
504656Inhibitors of Cruzain: Trypanosoma Cruzi Growth Inhibition Assayconfirmatory
2413Probe Summary: Docking False Negative Inhibitors of Cruzainsummary
2737Confirmation Competitive Binding Assay for Inhibitors of Cruzainconfirmatory
2383Probe Development Summary of Inhibitors of Cruzainsummary
2158Confirmation qHTS Assay for Inhibitors of Cruzainconfirmatory
2249Probe Development Summary of Promiscuous Inhibitors (Artifacts) of Cruzainsummary
Description:
Assay Provider: Shoichet, Brian; University of California, San Fancisco
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]

The following papain assay is a counterscreen for a related protein cruzain (AID: 1478). Cruzain is a cysteine protease from the tropical parasite Trypanosoma cruzi. Activity against papain was used to help prioritize chemical series active against cruzain. Papain is a closely related cysteine protease known to utilize the same Z-FR-AMC fluorogenic substrate as cruzain. The fluorogenic assay used for cruzain was utilized here as well, with the exception of the reducing agent, which was 5 mM cystein here instead of DTT. Fluorogenic coumarin-based Z-FR-AMC substrate was used; proteolytic cleavage releases AMC, whose fluorescence is measured at 360 nm excitation and 450 nm emission. Purified papain was supplied by the labs of Profs. James McKerrow and Brian Shoichet, UC San Francisco. This assay had 0.01% Triton X-100 concentration.
Protocol
Buffer: 100 mM Sodium Acetate, pH 5.5, 5 mM DTT, Triton X-100 concentration: 0.01%.

Reagents/Controls:
Buffer in columns 3 and 4 as negative control (no enzyme).
Substrate solution: 2 uM final concentration of Z-FR-AMC (catalog number I-1160, Bachem) dispensed throughout the plate.
Enzyme: 12 nM Papain final concentration.

Assay Steps:
Three uL of enzyme or buffer were dispensed to 1536-well Greiner black solid bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then 1 uL of substrate solution was added to start the reaction. The plates were immediately transferred to and read 4 times every 30 seconds for 2 min on ViewLux High-throughput CCD imager (Perkin-Elmer) using 360 nm excitation and 450 nm emission fluorescence protocol. The fluorescence intensity difference between the third and the first time points (time lapse of 60 seconds) was used to compute reaction progress. Data was normalized to neutral (no compound) and negative (no enzyme) controls.
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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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, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
5Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
6Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
7Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
8Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
9Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
10Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
11Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
12Compound QCSource of compound quality control including compound purification.String

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

Data Table (Concise)
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