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BioAssay: AID 889

qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-7

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 more ..
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 Tested Compounds
 Tested Compounds
All(73822)
 
 
Active(15)
 
 
Inactive(71551)
 
 
Inconclusive(2265)
 
 
 Tested Substances
 Tested Substances
All(74940)
 
 
Active(15)
 
 
Inactive(72645)
 
 
Inconclusive(2280)
 
 
 Related BioAssays
 Related BioAssays
AID: 889
Data Source: NCGC (CASP513)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-16
Modify Date: 2010-07-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 15
Description:
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 7 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 7 was assayed using the profluorescent substrate Ac-DEVD-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 7.
Protocol
NCGC Assay Protocol Summary:
Caspase 7 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 7 inhibitor Ac-DEVD-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-DEVD-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 7, proteases, profluorescent, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Baseline-Curve_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 signficant, but below 80% of control.String
6Baseline-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7Baseline-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Baseline-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Baseline-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Baseline-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
11Baseline-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Baseline-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Baseline-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Baseline-Activity at 0.0007359453 uM (0.000735945μM**)% Activity at given concentration.Float%
15Baseline-Activity at 0.00165 uM (0.00164557μM**)% Activity at given concentration.Float%
16Baseline-Activity at 0.00368 uM (0.0036795μM**)% Activity at given concentration.Float%
17Baseline-Activity at 0.00823 uM (0.00822737μM**)% Activity at given concentration.Float%
18Baseline-Activity at 0.018 uM (0.0183964μM**)% Activity at given concentration.Float%
19Baseline-Activity at 0.041 uM (0.0411343μM**)% Activity at given concentration.Float%
20Baseline-Activity at 0.092 uM (0.0919764μM**)% Activity at given concentration.Float%
21Baseline-Activity at 0.206 uM (0.205659μM**)% Activity at given concentration.Float%
22Baseline-Activity at 0.460 uM (0.459854μM**)% Activity at given concentration.Float%
23Baseline-Activity at 1.028 uM (1.02823μM**)% Activity at given concentration.Float%
24Baseline-Activity at 2.299 uM (2.29913μM**)% Activity at given concentration.Float%
25Baseline-Activity at 5.141 uM (5.14085μM**)% Activity at given concentration.Float%
26Baseline-Activity at 11.49 uM (11.495μM**)% Activity at given concentration.Float%
27Baseline-Activity at 25.70 uM (25.7027μM**)% Activity at given concentration.Float%
28Baseline-Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
29Rate-Curve_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 signficant, but below 80% of control.String
30Rate-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
31Rate-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
32Rate-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
33Rate-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
34Rate-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
35Rate-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
36Rate-Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
37Rate-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
38Rate-Activity at 0.0007359453 uM (0.000735945μM**)% Activity at given concentration.Float%
39Rate-Activity at 0.00165 uM (0.00164557μM**)% Activity at given concentration.Float%
40Rate-Activity at 0.00368 uM (0.0036795μM**)% Activity at given concentration.Float%
41Rate-Activity at 0.00823 uM (0.00822737μM**)% Activity at given concentration.Float%
42Rate-Activity at 0.018 uM (0.0183964μM**)% Activity at given concentration.Float%
43Rate-Activity at 0.041 uM (0.0411343μM**)% Activity at given concentration.Float%
44Rate-Activity at 0.092 uM (0.0919764μM**)% Activity at given concentration.Float%
45Rate-Activity at 0.206 uM (0.205659μM**)% Activity at given concentration.Float%
46Rate-Activity at 0.460 uM (0.459854μM**)% Activity at given concentration.Float%
47Rate-Activity at 1.028 uM (1.02823μM**)% Activity at given concentration.Float%
48Rate-Activity at 2.299 uM (2.29913μM**)% Activity at given concentration.Float%
49Rate-Activity at 5.141 uM (5.14085μM**)% Activity at given concentration.Float%
50Rate-Activity at 11.49 uM (11.495μM**)% Activity at given concentration.Float%
51Rate-Activity at 25.70 uM (25.7027μM**)% Activity at given concentration.Float%
52Rate-Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
53Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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