Caspase 9 Counterscreen for Selectivity Fluorescent Measured in Biochemical System Using Plate Reader - 7052-06_Inhibitor_Dose_CherryPick_Activity
Keywords: Caspase 9, Caspase 6, inhibitors, allosteric inhibitors, neurodegenerative disease, selectivity ..more
BioActive Compounds: 26
Keywords: Caspase 9, Caspase 6, inhibitors, allosteric inhibitors, neurodegenerative disease, selectivity
An assay was set up to monitor cleavage of of a fluorescent labeled peptide Caspase 9 substrate, Z-LEHD-R110(rhodamine 110, bis-(N-CBZ-L-leucyl-L-glutamyl-L-histidinyl-L-aspartic acid amide) . 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 9 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 in a dose dependent manner. Compounds that show at least ten fold selectivity for caspase 6 over all other caspases tested (Caspases 1, 3, 7, 8 and 9) are considered hits and continue into next round of testing.
Caspase 9 was received from collaborator.
100 mM MES, pH 6.5
10% PEG 8000
5 mM DTT (Note: DTT should be added fresh just prior to running assay)
R110, bis LEHD rhodamine 110, bis-(rhodamine 110, bis-(N-CBZ-L-leucyl-L-glutamyl-L-histidinyl-L-aspartic acid amide).
Compound plates for pinning: starting concentration 10 mM with ten, 3 fold dilutions (B32 layout).
Add 20 ul 157 nM Caspase-9 to wells of 384 well solid black plates.
Pin 100 nl compound into wells using Cybio pin tool.
Incubate at room temperature for 60 min.
Add 10 ul of 150 uM LEHD-R110 (Caspase 9 substrate) to all wells for a final concentration of 50 uM in the well.
Incubate at room temp for 40 min
Read plate on Envision (FITC settings and filters)
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
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.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)