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 ..
BioActive Compounds: 159
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.
Buffer: 100 mM Sodium Acetate, pH 5.5, 5 mM DTT, Triton X-100 concentration: 0.01%.
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.
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.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)