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BioAssay: AID 923

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 ..
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 Tested Compounds
 Tested Compounds
All(73910)
 
 
Active(779)
 
 
Inactive(71928)
 
 
Inconclusive(1204)
 
 
 Tested Substances
 Tested Substances
All(75028)
 
 
Active(789)
 
 
Inactive(73033)
 
 
Inconclusive(1206)
 
 
 Related BioAssays
 Related BioAssays
AID: 923
Data Source: NCGC (CASP120.2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-20
Modify Date: 2010-07-12

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 779
Related Experiments
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AIDNameTypeProbeComment
587qHTS Assay for Spectroscopic Profiling in Texas Red Spectral RegionOther depositor-specified cross reference
588qHTS Assay for Spectroscopic Profiling in Resorufin Spectral RegionOther depositor-specified cross reference
589qHTS Assay for Spectroscopic Profiling in 4-MU Spectral RegionOther depositor-specified cross reference
590qHTS Assay for Spectroscopic Profiling in A350 Spectral RegionOther depositor-specified cross reference
591qHTS Assay for Spectroscopic Profiling in A488 Spectral RegionOther depositor-specified cross reference
592qHTS Assay for Spectroscopic Profiling in A647 Spectral RegionOther depositor-specified cross reference
593qHTS Assay for Spectroscopic Profiling in Fluorescein Spectral RegionOther depositor-specified cross reference
594qHTS Assay for Spectroscopic Profiling in Rhodamine Spectral RegionConfirmatory depositor-specified cross reference
888Concentration-Response Counterscreen for Redox Active Inhibitors of Caspase-1: CatalaseConfirmatory depositor-specified cross reference
896Confirmation Concentration-Response Assay for Allosteric/Competitive Inhibitors of Caspase-1Confirmatory depositor-specified cross reference
2389qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-1: SummarySummary1 depositor-specified cross reference
900qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-1Confirmatory same project related to Summary assay
929Concentration-Response Counterscreen for Redox Active Inhibitors of Caspase-1: CysteineConfirmatory same project related to Summary assay
996Concentration-Response Counterscreen for Redox Active Inhibitors of Caspase-1: SummaryConfirmatory same project related to Summary assay
2494Confirmation Assay for Allosteric/Competitive Inhibitors of Caspase-1Confirmatory same project related to Summary assay
504731Allosteric/Competitive Inhibitors of Caspase 1: Metabolic Stability ProfilingOther same project related to Summary assay
504732Allosteric/Competitive Inhibitors of Caspase 1: Caco-2 Permeability ProfilingOther same project related to Summary assay
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 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.
Protocol
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
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 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
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
5Curve_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
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_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
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0007359453 uM (0.000735945μM**)% Activity at given concentration.Float%
15Activity at 0.00165 uM (0.00164557μM**)% Activity at given concentration.Float%
16Activity at 0.00368 uM (0.0036795μM**)% Activity at given concentration.Float%
17Activity at 0.00823 uM (0.00822737μM**)% Activity at given concentration.Float%
18Activity at 0.018 uM (0.0183964μM**)% Activity at given concentration.Float%
19Activity at 0.041 uM (0.0411343μM**)% Activity at given concentration.Float%
20Activity at 0.092 uM (0.0919764μM**)% Activity at given concentration.Float%
21Activity at 0.206 uM (0.205659μM**)% Activity at given concentration.Float%
22Activity at 0.460 uM (0.459854μM**)% Activity at given concentration.Float%
23Activity at 1.028 uM (1.02823μM**)% Activity at given concentration.Float%
24Activity at 2.299 uM (2.29913μM**)% Activity at given concentration.Float%
25Activity at 5.141 uM (5.14085μM**)% Activity at given concentration.Float%
26Activity at 11.49 uM (11.495μM**)% Activity at given concentration.Float%
27Activity at 25.70 uM (25.7027μM**)% Activity at given concentration.Float%
28Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
29Compound 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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