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BioAssay: AID 588451

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 ..
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 Tested Compounds
 Tested Compounds
All(11)
 
 
Active(10)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(11)
 
 
Active(10)
 
 
Inactive(1)
 
 
AID: 588451
Data Source: Broad Institute (2078-05_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-05

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 10
Related Experiments
AIDNameTypeComment
488894Broad Institute MLPCN Screen of the Prion (PrP 5' Untranslated region) Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
488862Inhibitors of Prion Protein 5' UTR mRNA Measured in Cell-Based System Using Plate Reader - 2078-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
504539Inhibitors of Prion Protein 5' UTR mRNA Measured in Cell-Based System Using Plate Reader - 2078-01_Inhibitor_Dose_CherryPick_Activity_Set2Confirmatorysame project related to Summary assay
504592Inhibitors of Prion Protein 5' UTR mRNA Measured in Cell-Based System Using Plate Reader - 2078-01_Inhibitor_SinglePoint_CherryPick_ActivityConfirmatorysame project related to Summary assay
504932Inhibitors of Prion Protein 5' UTR mRNA Measured in Cell-Based System Using Plate Reader - 2078-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
540255H4 neuroblastoma control for prion reporter construct Measured in Cell-Based System Using Plate Reader - 2078-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
540343Amyloid Precursor 5' UTR neuroblastoma cell reporter line Measured in Cell-Based System Using Plate Reader - 2078-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
588507PrP Counter Screen: GFP reporter Measured in Cell-Based System Using Imaging - 2078-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords: Luciferase

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.
Protocol
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
Comment
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).

NORMALIZATION:
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)

PUBCHEM_ACTIVITY_OUTCOME:
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.

PUBCHEM_ACTIVITY_SCORE:
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.

Note:
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
From ChEMBL:
Assay Type: Binding
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
5ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
6ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_0.000016uM_(%)_TEST1 (1.6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.000030uM_(%)_TEST1 (3e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.000060uM_(%)_TEST1 (6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.00012uM_(%)_TEST1 (0.00012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.00026uM_(%)_TEST1 (0.00026μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.0005uM_(%)_TEST1 (0.0005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.001uM_(%)_TEST1 (0.001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.0021uM_(%)_TEST1 (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.0042uM_(%)_TEST1 (0.0042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.008uM_(%)_TEST1 (0.008μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_0.016uM_(%)_TEST1 (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_0.035uM_(%)_TEST1 (0.035μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_0.068uM_(%)_TEST1 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_0.135uM_(%)_TEST1 (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_0.26uM_(%)_TEST1 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_0.5uM_(%)_TEST1 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_1uM_(%)_TEST1 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_2.1uM_(%)_TEST1 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_4.2uM_(%)_TEST1 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_8uM_(%)_TEST1 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
38ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
39ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
40AC50_Qualifier_TEST2'>','=', or '<'String
41AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
42pAC50_M_TEST2Equal to -1*log10(AC50)String
43Hill_Slope_TEST2The slope at AC50Float
44S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
45Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
46Num_Points_TEST2The number of data points used to generate the plotInteger
47Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
48Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
49Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
50Activity_at_0.000016uM_(%)_TEST2 (1.6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
51Activity_at_0.000030uM_(%)_TEST2 (3e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
52Activity_at_0.000060uM_(%)_TEST2 (6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
53Activity_at_0.00012uM_(%)_TEST2 (0.00012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
54Activity_at_0.00026uM_(%)_TEST2 (0.00026μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
55Activity_at_0.0005uM_(%)_TEST2 (0.0005μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
56Activity_at_0.001uM_(%)_TEST2 (0.001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
57Activity_at_0.0021uM_(%)_TEST2 (0.0021μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
58Activity_at_0.0042uM_(%)_TEST2 (0.0042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
59Activity_at_0.008uM_(%)_TEST2 (0.008μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
60Activity_at_0.016uM_(%)_TEST2 (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
61Activity_at_0.035uM_(%)_TEST2 (0.035μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
62Activity_at_0.068uM_(%)_TEST2 (0.068μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
63Activity_at_0.135uM_(%)_TEST2 (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
64Activity_at_0.26uM_(%)_TEST2 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
65Activity_at_0.5uM_(%)_TEST2 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
66Activity_at_1uM_(%)_TEST2 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
67Activity_at_2.1uM_(%)_TEST2 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
68Activity_at_4.2uM_(%)_TEST2 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
69Activity_at_8uM_(%)_TEST2 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1-R21-NS-064853-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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