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BioAssay: AID 720566

Turbidometric Biochemical Primary HTS to identify inhibitors of ERp5 Measured in Biochemical System Using Plate Reader - 7002-01_Inhibitor_Dose_CherryPick_Activity

Keywords: PDIA6 protein disulfide isomerase family A, member 6 , ERp5, arterial thrombosis, inhibitors of ERp5, anti-thrombotic, insulin aggregation assay ..more
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 Tested Compounds
 Tested Compounds
All(175)
 
 
Active(4)
 
 
Inactive(171)
 
 
 Tested Substances
 Tested Substances
All(175)
 
 
Active(4)
 
 
Inactive(171)
 
 
AID: 720566
Data Source: Broad Institute (7002-01_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-08-06

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 4
Related Experiments
AIDNameTypeComment
651716Broad Institute Inhibitors of ERp5 Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
651711Turbidometric Biochemical Primary HTS to identify inhibitors of ERp5 Measured in Biochemical System Using Plate Reader - 7002-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
Description:
Keywords: PDIA6 protein disulfide isomerase family A, member 6 , ERp5, arterial thrombosis, inhibitors of ERp5, anti-thrombotic, insulin aggregation assay


Assay Overview:

An assay was set up to monitor and identify inhibitors of the ERp5 mediated aggregation of insulin. The assay measures aggregation of insulin chains by monitoring absorbance at 650 nm. ERp5 is a member of the thiol isomerase family and cleaves disulfide bonds in the beta chain of insulin causing them to aggregate. When ERp5 is inhibited there is a decrease in aggregation and therefore a decrease in absorbance.

Expected Outcome: Active hits are determined as compounds that decrease absorbance by at least 25% of the signal reduction observed in the mean of positive control wells.
Protocol
Recombinant ERp5 produced in E.coli was received from collaborator. Batch 04.06.12 was received in large tubes (16 tubes with 7 ml/tube) with concentration of 4.4 mg/ml.


Assay Buffer:
100 mM Potassium Phosphate, pH 7.0 with 2 mM EDTA
Prepare 1M stock solutions of K2HPO4 (dibasic- 176 g/L)) and KH2PO4 (monobasic 136.09 g/L)
to prepare 1L of assay buffer add:
61.5 ml dibasic 1M solution
38.5 ml monobasic 1M solution
4 ml EDTA (0.5 M stock from Sigma cat # E7889, lot # 021M8719)
896 ml sterile H2O


Prepare Bovine Insulin:
Bovine insulin from SIGMA Cat # I6634 Lot # SLBD2712V: Insulin from Bovine Pancreas. Insulin is stored at - 20 C as lyophilized powder. Reconstitute 100 mg with 10 ml of 0.1 N HCL. Dilute 1:8 with assay buffer for a final concentration of 213 uM. Once reconstituted insulin may be stored at -20 C and thawed once.

ERp5:
ERp5 batch 04.06.12 at 4.4 mg/ml stock solution. Dilute 1:92 in diluted insulin from step above (final concentration 48 ug/ml = 1uM). This is referred to as insulin/ERp5 mix.

DTT:
1M Stock solution in sterile H20. Dilute 1:178 for a working solution of 5.6 mM (this will be diluted 1:10 in the assay well for final concentration of 350 uM).

Positive Control from TCI America Cat # P008 Lot # FHJ01 9,10-Phenanthrenequinone CAS # 84-11-7 MW 208.21
Prepare 10 mM stock of 9,10-Phenanthrenequinone (2.08 mg/ml DMSO)
Dilute 10 mM stock in DMSO to 200 uM by diluting 1:50
This will be diluted 1:10 in well for a final concentration of 20 uM.


Set up Reagents:
One bottle on bioraptr will hold 9,10-Phenanthrenequinone positive control.
Remaining bottles will contain Insulin/ ERp5 reaction mixture (213 uM insulin with 1 uM ERp5).
Combi NL will deliver DTT.

Run:
Dispense 8.0 ul of insulin/ERp5 mix to all wells using the BioraptR.
Dispense 1.0 uL 200 uM 9,10-Phenanthrenequinone to positive control wells using BioraptR.
Move plate to combiNL and deliver 1.0 ul DTT to all wells.
Spin plates in centrifuge 2 min 1000 RPM to break bubbles.
Incubate plate 90 minutes RT.
Read plates on Envision using absorbance (650 nm) protocol on the Envision.

Retest in Dose: 384 well format
Assay components will be pipeted by hand:
Dispense 30.0 ul of insulin/ERp5 mix to all wells.
Dispense 1 uL 1.28 mM 9,10-Phenanthrenequinone to positive control wells.
Pin cherry pick compounds (100 nl) into assay plates
Dispense 1 ul 12.4 mM DTT diluted 1: 32 in well.
Incubate plate 90 minutes RT.
Read plates on Envision using absorbance (650 nm) protocol on the Envision.








Comment
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.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

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).

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.

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)

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.

DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.

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 PubChem:
Assay Format: Biochemical
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_0.0135uM_(%)_TEST1 (0.0135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.015uM_(%)_TEST1 (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.016uM_(%)_TEST1 (0.016μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.042uM_(%)_TEST1 (0.042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.046uM_(%)_TEST1 (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.12uM_(%)_TEST1 (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.135uM_(%)_TEST1 (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.15uM_(%)_TEST1 (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.38uM_(%)_TEST1 (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_0.42uM_(%)_TEST1 (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_1.1uM_(%)_TEST1 (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_1.2uM_(%)_TEST1 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_1.35uM_(%)_TEST1 (1.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_3.5uM_(%)_TEST1 (3.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
31Activity_at_3.8uM_(%)_TEST1 (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32Activity_at_10uM_(%)_TEST1 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33Activity_at_11uM_(%)_TEST1 (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34Activity_at_12uM_(%)_TEST1 (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35Activity_at_30uM_(%)_TEST1 (30μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36Activity_at_35uM_(%)_TEST1 (35μM**)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_0.015uM_(%)_TEST2 (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
51Activity_at_0.046uM_(%)_TEST2 (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
52Activity_at_0.135uM_(%)_TEST2 (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
53Activity_at_0.42uM_(%)_TEST2 (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
54Activity_at_1.2uM_(%)_TEST2 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
55Activity_at_3.8uM_(%)_TEST2 (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
56Activity_at_11uM_(%)_TEST2 (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
57Activity_at_35uM_(%)_TEST2 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R01 HL092125-01A1

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