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BioAssay: AID 686996

VEID(2) R110 Enzymatic Primary HTS to identify Inhibitors of Caspase 6 Measured in Biochemical System Using Plate Reader - 7052-01_Inhibitor_SinglePoint_HTS_Activity_Set2

An assay was set up to monitor cleavage of of a fluorescent labeled peptide caspase susbtrate, Rhodamine 110, bis-(N-CBZ-L-Valyl-L-Glutamyl-L-Isoleucyl-L-Aspartic Acid Amide) (Z-VEID-R110). This caspase substrate is a derivative of the fluorophore rhodamine 110 (R110). Peptides are covalently linked to each of the amino groups on R110, which suppresses the fluorescence of the dye. When the more ..
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 Tested Compounds
 Tested Compounds
All(344314)
 
 
Active(1854)
 
 
Inactive(342467)
 
 
Inconclusive(3)
 
 
 Tested Substances
 Tested Substances
All(348016)
 
 
Active(1854)
 
 
Inactive(346159)
 
 
Inconclusive(3)
 
 
 Related BioAssays
 Related BioAssays
AID: 686996
Data Source: Broad Institute (7052-01_Inhibitor_SinglePoint_HTS_Activity_Set2)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-05-23

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1854
Depositor Specified Assays
AIDNameTypeComment
687025Broad Institute Inhibitors of Caspase 6 Inhibitor Probe Projectsummary
Description:
Keywords: Caspase 6, inhibitors, apoptosis


Assay Overview:
An assay was set up to monitor cleavage of of a fluorescent labeled peptide caspase susbtrate, Rhodamine 110, bis-(N-CBZ-L-Valyl-L-Glutamyl-L-Isoleucyl-L-Aspartic Acid Amide) (Z-VEID-R110). This caspase substrate is a derivative of the fluorophore rhodamine 110 (R110). Peptides are covalently linked to each of the amino groups on R110, which suppresses the fluorescence of the dye. When the peptides are cleaved by the caspase, the substrate is converted first to the fluorescent monoamide and then to R110, with a further increase in fluorescence. The substrate can be used to continuously measure enzyme activity in cell extracts and purified enzyme preparations using a fluorometer or fluorescence microplate reader. When caspase 6 is inhibited there is a decrease in fluorescence as compared with the negative control wells.

Expected Outcome: Active hits are determined as compounds that decrease fluorescence by at least 35 % of the signal observed in the mean of neutral control wells.
Protocol
Caspase 6 enzyme was received from collaborator and stored at -80 C. Stock concentration was at 1.2 mg/ml (43.1 uM).

Assay Buffer:
100 mM Hepes pH 7.5, 10% sucrose, 0.1 % CHAPS, 5 mM DTT (final concentration) was added fresh just prior to assay

Reagents:
VEID-R110-VEID: Caspase 6 substrate, was reconstituted to 10 mM from dry powder with DMSO and stored at -20 C.
VEID-CHO: Caspase inhibitor (positive control) was reconstituted to 10 mM from dry powder with DMSO and stored at -20 C.

Assay Ready Plates:
1536 black, solid bottom plates containing 7.5 ul of compound

Protocol:
Add 4 ul 2.2 nM Caspase-6 to 1536 ARPS using BioRaptR
Add 100 nL VEID-CHO to positive control wells using Combi nL
Incubate Room temp 60 min
Add 2 ul of 24 uM VEID-R110-VEID (Caspase 6 substrate) to all wells
Incubate at room temp for 40 min
Read plate on Envision (FITC settings and filters)
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 35.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid
Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_12.48uM_(%) (12.48μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA035189-01A1

Data Table (Concise)
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