qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-1: Spectroscopic Profiling in AFC Spectral Region
The rate of false hits is dramatically reduced by the qHTS approach (Inglese et al, PNAS, 103, 1147 (2006)) as spurious high- or low-response wells are quickly revealed when plotted in the context of concentration response. Occasionally, the artifactual effect may originate from the compounds own spectral or other biophysical properties which generally tend to track its assay concentration. more ..
BioActive Compounds: 779
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: 1 X01 MH078950-01
PI Name: Dr. James Wells,
Dr. Enrique Perez-Paya, Dr.Janice Williams, Dr. Dennis Wolan, Mr. Brandon Butler
NCGC Assay Overview:
The rate of false hits is dramatically reduced by the qHTS approach (Inglese et al, PNAS, 103, 1147 (2006)) as spurious high- or low-response wells are quickly revealed when plotted in the context of concentration response. Occasionally, the artifactual effect may originate from the compounds own spectral or other biophysical properties which generally tend to track its assay concentration. Pre-screening/profiling the library with respect to the compounds spectral properties should help one exclude false positives early on. As part of a kinetic enzyme experiment with caspase 1, baseline fluorescence was measured for each compound in the library in order to flag potential false positives in the assay. A standard curve of 7-amino-4-trifluoromethyl-coumarin (AFC) present on each plate was used to compute a normalized fluorescence response for each compound at each concentration.
The biochemical assay was configured using purified caspase 1 at a high enzyme concentration to promote formation of the homodimers and thus facilitate the identification of allosteric inhibitors. The overall goal was to identify reversible inhibitors with better drug-like properties than the currently available set of aspartyl-containing peptidomimetics that covalently bind the active site. Caspase 1 was assayed using the profluorescent substrate Ac-WEHD-AFC. After initiation of the assay with substrate the plates were rapidly read using an automated robotic system (Kalypsys, Inc.) to maintain consistent timing. A kinetic mode of detection was used where the initial rate was collected (estimated final product formation was ~10%). Compounds were screened as a concentration-titration series that ranged from 57 uM to 0.7 nM. Below is the protocol used for caspase 1.
NCGC Assay Protocol Summary:
Caspase 1 was prepared in buffer (50 mM HEPES pH 7.5, 50 mM KCl, 200 mM NaCl, 10 mM DTT, 0.1% CHAPS) at a concentration of 66.6 nM and 3 uL was dispensed to all wells using black solid Kalypsys 1536-well plates. 20 nL of DMSO containing compounds was added using a pin-tool (Kalypsys Inc.) to columns 5-48. Then 20 nL of DMSO solution from a control plate was added to columns 1-4. Controls were: Column 1, 16 point titration with each concentration in duplicate (1:1 dilutions in DMSO; final starting concentration was 57 uM) of the caspase 1 inhibitor Ac-WEHD-CHO (Alexis Biochemicals); Column 2, a 16 point titration with each concentration in duplicate of the free AFC fluorophore prepared in DMSO (Alexis Biochemicals), final starting was 40 uM; Column 3 neutral (DMSO only) control; Column 4: DMSO alone, to serve as a negative control (no substrate was added). Then 1 uL of 20 uM the substrate Ac-WEHD-AFC (Alexis Biochemicals) prepared in the same buffer was dispensed to all wells except columns 2 and 4 and the plates were immediately transferred (< 1 min) to the Viewlux. The plates were then exposed using 405 nm excitation/520 nm emission filters for 4 sec and read at 20 sec intervals for 3 min. Final enzyme concentration was 50 nM and the final substrate concentration was 5 uM.
Concentration-response curves were fitted to the data calculated from intercept of the linear regression of fluorescent intensity versus time (the baseline). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity. Active (fluorescent) compounds showed concentration-dependent increase in the measured baseline.
Keywords: Caspase 1, proteases, profluorescent, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
For further details on project on assay, see PMID: 15314233 and 16682620
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 ranked higher than compounds that showed apparent inhibition.
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. ** Test Concentration.
Data Table (Concise)