Assay of luciferase inhibitors in dose Measured in Biochemical System Using Plate Reader - 2078-05_Inhibitor_Dose_DryPowder_Activity
Assay Overview: The build-up of prion proteins with the disease causing conformation (PrPsc) in neuronal cells is associated with cell death and encephalopathy. One possible way to mitigate the progression of prion based diseases, such as Creuzfeld-Jakob disease, is to reduce the levels of PrP in nerve cells. Here the approach is to accomplish this by inhibiting the expression of PrP at the more ..
BioActive Compounds: 10
Assay Overview: The build-up of prion proteins with the disease causing conformation (PrPsc) in neuronal cells is associated with cell death and encephalopathy. One possible way to mitigate the progression of prion based diseases, such as Creuzfeld-Jakob disease, is to reduce the levels of PrP in nerve cells. Here the approach is to accomplish this by inhibiting the expression of PrP at the level of message translation. This assay is a counter screen which uses a biochemical luciferase assay to test if compounds identified in the cell based reporter screen are inhibiting the reporter enzyme (luciferase) activity, an off-target activity.
Expected Outcome: Compounds which specifically interfere only with the translation of the PRP 5'UTR from the primary screen reporter construct should not inhibit this assay. Compounds reported as active
are considered luciferase inhibitors.
2 nM Luciferase is prepared in PBS pH 7.2, 1 mg/mL BSA, 0.1 mM MgCl2, 0.01% NaN3
In a 384 well white assay plate,
+ 30 uL / well 2 nM Luciferase using a combi dispenser
+ 25 nL compounds in DMSO using a pintool transfer
Stand 10 minutes
+ 30 uL / well SteadyGlo reagent
Read luminescence with Envision plate reader
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) 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 (10)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
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.7.0.3) 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)