Bookmark and Share
BioAssay: AID 492970

Fluorescence-based biochemical dose response assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)

Name: Fluorescence-based biochemical dose response assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4). ..more
_
   
 Tested Compounds
 Tested Compounds
All(10)
 
 
Active(1)
 
 
Inactive(9)
 
 
 Tested Substances
 Tested Substances
All(10)
 
 
Active(1)
 
 
Inactive(9)
 
 
AID: 492970
Data Source: The Scripps Research Institute Molecular Screening Center (PAD4_INH_FLUO_2X%IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2010-12-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
Show more
AIDNameTypeComment
463073Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)Screeningdepositor-specified cross reference: Primary screen (PAD4 inhibitors in singlicate, NIH 2K Validation)
463083Summary of the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)Summarydepositor-specified cross reference: Summary (PAD4 inhibitors)
485272Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4) (1536 HTS)Screeningdepositor-specified cross reference: Primary screen (PAD4 inhibitors in singlicate)
488796Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Arginine Deiminase 4 (PAD4)Screeningdepositor-specified cross reference: Confirmation screen (PAD4 inhibitors in triplicate)
588416Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 16 against PAD4Confirmatorydepositor-specified cross reference
588417Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 15 against PAD4Confirmatorydepositor-specified cross reference
588418Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 13 against PAD4Confirmatorydepositor-specified cross reference
588419Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 12 against PAD4Confirmatorydepositor-specified cross reference
588420Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 11 against PAD4Confirmatorydepositor-specified cross reference
588421Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical assay to assess potency of compound 6 against PAD4Confirmatorydepositor-specified cross reference
588422Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical assay to assess potency of compound 2 against PAD4Confirmatorydepositor-specified cross reference
588423Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 18 against PAD4Confirmatorydepositor-specified cross reference
588438Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 1 against PAD1-4Confirmatorydepositor-specified cross reference
588462Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of streptonigrin against PAD1-3Confirmatorydepositor-specified cross reference
588471Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 3 against PAD1-4Confirmatorydepositor-specified cross reference
588472Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 14 against PAD1-4Confirmatorydepositor-specified cross reference
588484Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 10 against PAD1-4Confirmatorydepositor-specified cross reference
588486Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 21 against PAD1-4Confirmatorydepositor-specified cross reference
588487Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition by HTS hits of PADs 1-4Otherdepositor-specified cross reference
588488Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of HTS hits against PAD1-4Otherdepositor-specified cross reference
588490Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to assess potency of compound 17 against PAD1-4Confirmatorydepositor-specified cross reference
588559Late stage assay provider results from the probe development effort to identify inhibitors of Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to identify inhibitors of PAD4Otherdepositor-specified cross reference
588560Late stage assay provider results from the probe development effort to identify inhibitors of PAD4: colorimetric biochemical substrate assay to identify inhibitors of PADs 1-4Otherdepositor-specified cross reference
651627Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate dialysis assay to assess binding mode of test compounds to PAD4Otherdepositor-specified cross reference
651628Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) assessment of compound selectivityOtherdepositor-specified cross reference
651861Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate dialysis assay to assess binding mode of test compounds to PADs 1-3Otherdepositor-specified cross reference
651865Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to determine rate constants of test compounds for PAD 2 inactivationOtherdepositor-specified cross reference
651866Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to determine rate constants of test compounds for PAD 3 inactivationOtherdepositor-specified cross reference
651867Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to determine rate constants of test compounds for PAD 4 inactivationOtherdepositor-specified cross reference
651868Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to determine rate constants of test compounds for PAD 1 inactivationOtherdepositor-specified cross reference
651887Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): cell-based absorbance-based assay to assess cytotoxicity of test compoundsConfirmatorydepositor-specified cross reference
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Paul Thompson, University of South Carolina
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 GM079357-01
Grant Proposal PI: Paul Thompson
External Assay ID: PAD4_INH_FLUO_2X%IC50

Name: Fluorescence-based biochemical dose response assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4).

Description:

Rheumatoid Arthritis (RA) is a chronic and progressive autoimmune disorder that affects about one percent of the US population (1). Existing therapies treat the symptoms of the disease but not the underlying cause, and are associated with numerous side effects (2). The activity of Protein Arginine Deiminase 4 (PAD4), one of four known active PAD isozymes, is increased in RA; where it is thought to generate a subset of antigens that the immune system recognizes as foreign (3). Genetic, serological, and biochemical evidence suggests that dysregulated PAD4, and potentially PAD2, activities play a role in both the onset and progression of RA (1). Cl-amidine, a compound that specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations). However, because Cl-amidine inhibits all of the PAD isozymes with equipotency, it is unclear whether the observed reduction in disease severity is due to the inhibition of single or multiple PADs. This is particularly relevant because both PAD 2 and 4 are overexpressed in the joints of patients with RA (4). Thus, the identification of PAD selective inhibitors would facilitate the characterization of their individual contributions to the onset and progression of RA and represent a promising novel therapeutic approach for RA.

References:

1. Vossenaar, E.R., et al., PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays, 2003. 25(11): p. 1106-18.
2. Smolen, J.S. and G. Steiner, Therapeutic strategies for rheumatoid arthritis. Nat Rev Drug Discov, 2003. 2(6): p. 473-88.
3. Vossenaar, E.R., et al., Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages. Ann Rheum Dis, 2004. 63(4): p. 373-81.
4. Lundberg, K., et al., Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity. Arthritis Res Ther, 2005. 7(3): p. R458-67.

Keywords:

assay provider, powders, dose response, protein arginine deiminase, rheumatoid arthritis, RA, collagen-induced model of arthritis, CIA, Na-Benzoyl-L-arginine ethyl ester hydrochloride, BAEE, citrulline, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:

The purpose of this assay is to determine potency of compounds identified as having inhibitory activity against recombinant PAD4. In this assay, recombinant PAD4 is pre-incubated with test compounds, followed by the addition of the substrate, Na-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE). The percent activity remaining is determined by measuring the amount of citrulline produced using a standard assay that measures changes in citrulline produced. Test compounds that act as PAD4 inhibitors will prevent the production of citrulline. IC50 values for inhibition of recombinant PAD4 were determined from dose-response curves from 2 trials at each inhibitor concentration in an 8-point dilution series from 0 to 100 uM.

Protocol Summary:

Recombinant PAD4 (0.2 uM in Assay Buffer (50 mM Tris-HCl pH 7.6, 50 mM NaCl, 10 mM CaCl2, and 2 mM DTT) was incubated with DMSO or test compound for 15 minutes at 37 C before the addition of BAEE at a final concentration of 10 mM in 60 uL total reaction volume. The reaction was incubated for 15 minutes at 37 C, quenched by flash freezing in liquid nitrogen, and 200 uL of a color developing reagent (COLDER), which consists of solution A (80 mM diacetyl monoxime and 2 mM thiosemicarbazide) and solution B (3 M H3PO4, 6 M H2SO4, and 2 mM NH4Fe(SO4)2) in a 1:3 ratio, was added. This mixture was incubated for 30 minutes at 95 C and the absorbance was measured at 540 nm. The amount of product produced was determined by comparison to a standard curve with known concentrations of citrulline.

The percent activity remaining was fit to the following equation using GraFit (version 5.0.11):

Fractional Activity = 1 / ( 1 + ( [I] / IC50 ) )

Where:

[I] is the concentration of inhibitor.
IC50 is the concentration of inhibitor that yields half-maximal activity.

In cases where the highest concentration tested (100 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 100 uM. Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

PubChem Activity Outcome and Score:

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 88-0.

List of Reagents:

Recombinant PAD4 (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
CaCl2 (Sigma, part C3881)
DTT (RPI, part D110000)
2,3-butanedione monooxime (Sigma, part B0753)
Thiosemicarbazide (Sigma, part T33405)
NH4Fe(SO4)2 (Sigma, part F1668)
H2SO4 (Sigma, part 258105)
H3PO4 (Fisher, part A260)
NaCl (Sigma, part S6546)
BAEE (Sigma, part B4500)
96-well plates (BD Falcon, part 353228)
Comment
This assay was performed by the assay provider.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be greater than its listed IC50 concentrationString
2Average IC50*The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.FloatμM
3Standard DeviationThe standard deviation of the IC50 value.Float
4Inhibition at 0 uM [1] (0μM**)The value for percent inhibition of PAD4 at 0 uM compound; replicate one.Float%
5Inhibition at 1 uM [1] (1μM**)The value for percent inhibition of PAD4 at 1 uM compound; replicate one.Float%
6Inhibition at 2.5 uM [1] (2.5μM**)The value for percent inhibition of PAD4 at 2.5 uM compound; replicate one.Float%
7Inhibition at 5 uM [1] (5μM**)The value for percent inhibition of PAD4 at 5 uM compound; replicate one.Float%
8Inhibition at 10 uM [1] (10μM**)The value for percent inhibition of PAD4 at 10 uM compound; replicate one.Float%
9Inhibition at 25 uM [1] (25μM**)The value for percent inhibition of PAD4 at 25 uM compound; replicate one.Float%
10Inhibition at 50 uM [1] (50μM**)The value for percent inhibition of PAD4 at 50 uM compound; replicate one.Float%
11Inhibition at 100 uM [1] (100μM**)The value for percent inhibition of PAD4 at 100 uM compound; replicate one.Float%
12Inhibition at 0 uM [2] (0μM**)The value for percent inhibition of PAD4 at 0 uM compound; replicate two.Float%
13Inhibition at 1 uM [2] (1μM**)The value for percent inhibition of PAD4 at 1 uM compound; replicate two.Float%
14Inhibition at 2.5 uM [2] (2.5μM**)The value for percent inhibition of PAD4 at 2.5 uM compound; replicate two.Float%
15Inhibition at 5 uM [2] (5μM**)The value for percent inhibition of PAD4 at 5 uM compound; replicate two.Float%
16Inhibition at 10 uM [2] (10μM**)The value for percent inhibition of PAD4 at 10 uM compound; replicate two.Float%
17Inhibition at 25 uM [2] (25μM**)The value for percent inhibition of PAD4 at 25 uM compound; replicate two.Float%
18Inhibition at 50 uM [2] (50μM**)The value for percent inhibition of PAD4 at 50 uM compound; replicate two.Float%
19Inhibition at 100 uM [2] (100μM**)The value for percent inhibition of PAD4 at 100 uM compound; replicate two.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R01 GM079357-01

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