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 ..
BioActive Compounds: 1854
Keywords: Caspase 6, inhibitors, apoptosis
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.
Caspase 6 enzyme was received from collaborator and stored at -80 C. Stock concentration was at 1.2 mg/ml (43.1 uM).
100 mM Hepes pH 7.5, 10% sucrose, 0.1 % CHAPS, 5 mM DTT (final concentration) was added fresh just prior to assay
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
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)
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.
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.
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.
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
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)