qHTS Assay for Promiscuous and Specific Inhibitors of Cruzain (without detergent)
Cruzain is a cysteine protease from the tropical parasite Trypanosoma cruzi. Dual qHTS experiment was performed against the NCGC compound collection in order to 1) identify novel inhibitors of the enzyme and 2) profile the compound collection for promiscuous inhibitors operating via colloidal aggregation (by performing detergent-free and detergent-containing comparison screens). Cruzain was more ..
BioActive Compounds: 4500
Cruzain is a cysteine protease from the tropical parasite Trypanosoma cruzi. Dual qHTS experiment was performed against the NCGC compound collection in order to 1) identify novel inhibitors of the enzyme and 2) profile the compound collection for promiscuous inhibitors operating via colloidal aggregation (by performing detergent-free and detergent-containing comparison screens). Cruzain was assayed by the use of fluorogenic coumarin-based substrate Z-FR-AMC: proteolytic cleavage releases AMC, whose fluorescence is measured at 360 nm excitation and 450 nm emission. Purified cruzain was supplied by the labs of Prof. James McKerrow and Brian Shoichet, UC San Francisco.
This assay had 0.00005% Triton X-100 concentration. See also releated assay "qHTS Assay for Promiscuous and Specific Inhibitors of Cruzain (with detergent)" with 0.01% Triton X-100.
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.
Assay Provider: Shoichet, Brian; University of California, San Fancisco
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
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: 1.5 nM Cruzain final concentration in columns 1, 2, 5-48. Column 1 is neutral (100% activity). Column 2 contained titration of covalent inhibitor K11777, starting concentration 1 mM, then 1:2 dilution in duplicate).
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.
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 activators are likely fluorescent artifacts and are ranked lower than apparent inhibitors.
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.
3. For all active or inconclusive compounds that gave high background fluorescence (RFU > 40 for background read), PUBCHEM_ACTIVITY_SCORE is 1 and 'Phenotype' is set to "Fluorescent".
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, Cruzain, Cruzipain, Aggregation, Chagas disease
Categorized Comment - additional comments and annotations
Assay Type: Functional
From MLP Probe Report:
Probe count: 3
MLP Probe ML# for probe 1: ML091
PubChem Substance ID (SID) for probe 1: 85267411,99206570
PubChem Compound ID (CID) for probe 1: 44143092
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 1260
Target for probe 1: Cruzain
Anti-target for probe 1: Papain
Fold selectivity for probe 1: 40
Grant number for probe 1: DA024891-01
MLP Probe ML# for probe 2: ML092
PubChem Substance ID (SID) for probe 2: 85267412,99206571
PubChem Compound ID (CID) for probe 2: 44143088
Probe type for probe 2: Inhibitor
IC50/EC50 (nM) for probe 2: 100
Target for probe 2: Cruzain
Anti-target for probe 2: Papain
Fold selectivity for probe 2: 3
Grant number for probe 2: DA024891-01
MLP Probe ML# for probe 3: ML093
Grant number for probe 3: DA024891-01
PubMed Publication ID (PMID) for probe 1: 19908842
* Activity Concentration.
Data Table (Concise)