AID	Name	Type	Comment
2389	qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-1: Summary	summary
2494	Confirmation Assay for Allosteric/Competitive Inhibitors of Caspase-1	confirmatory

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 caspases (Cysteine Aspartyl Protease) comprise a related family of 14 dimeric proteases that are critical mediators of apoptosis (programmed cell death) and inflammation. The caspases represent an important class of drug targets for stroke, ischemia, cancer and inflammatory diseases. Comparison of crystal structures of procaspases and caspases have revealed that the subunits of the homodimer are composed of subdomains which have highly conserved architecture and mechanism, and show large structural transitions between procaspase and active conformers. One goal of this project was to identify compounds that act as allosteric inhibitors via stabilizing the procaspase form of the enzyme. Previous work in Well's laboratory has shown that such allosteric inhibitors can be identified and these act by binding at the dimer interface of enzyme (Hardy, Lam et al., 2004).

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 slope of the linear regression of fluorescent intensity versus time (the rate). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity. Active compounds showed concentration-dependent decreases in the measured rate. Inconclusive compounds had appreciable concentration-dependent effects on both the measured rate and the interpolated basal fluorescence intensity at the start of the reaction but where the basal fluorescent intensity was marginal (e.g. < 10-fold). Inconclusive compounds also encompass highly fluorescent compounds (>100-fold increases in fluorescent intensities values relative to controls) where artificial concentration-dependent decreases in the rate occurred at high compound concentrations due to fluorescent interferences.


Keywords: Caspase 1, proteases, profluorescent, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC

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 "Rate-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. Rate-Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.