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BioAssay: AID 588560

Late stage assay provider results from the probe development effort to identify inhibitors of PAD4: colorimetric biochemical substrate assay to identify inhibitors of PADs 1-4

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PAD4: colorimetric biochemical substrate assay to identify inhibitors of PADs 1-4. ..more
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 Tested Compounds
 Tested Compounds
All(30)
 
 
Active(12)
 
 
Inactive(18)
 
 
 Tested Substances
 Tested Substances
All(30)
 
 
Active(12)
 
 
Inactive(18)
 
 
AID: 588560
Data Source: The Scripps Research Institute Molecular Screening Center (PAD4_INH_SUBSTRATE_SAR_PADS10UM)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-10-19
Hold-until Date: 2012-10-18
Modify Date: 2012-10-18

Data Table ( Complete ):           Active    All
BioActive Compounds: 12
Depositor Specified Assays
Show more
AIDNameTypeComment
463083Summary of the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)summarySummary (PAD4 inhibitors)
485272Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4) (1536 HTS)screeningPrimary screen (PAD4 inhibitors in singlicate)
488796Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Arginine Deiminase 4 (PAD4)screeningConfirmation screen (PAD4 inhibitors in triplicate)
463073Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)screeningPrimary screen (PAD4 inhibitors in singlicate)
492970Fluorescence-based biochemical dose response assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)confirmatoryDose response (PAD4 inhibitors in duplicate)
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 PAD4other
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 selectivityother
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 1-3other
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 inactivationother
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 inactivationother
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 inactivationother
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 inactivationother
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 compoundsconfirmatory
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Paul Thompson, TSRI (Florida)
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 GM079357-01
Grant Proposal PI: Paul Thompson
External Assay ID: PAD4_INH_SUBSTRATE_SAR_PADS10UM

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PAD4: colorimetric biochemical substrate assay to identify inhibitors of PADs 1-4.

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:

late stage, late stage AID, assay provider, protein arginine deiminase type-1, protein arginine deiminase type-2, protein arginine deiminase type-3, protein arginine deiminase type-4, PAD1, PAD2, PAD3, PAD4, rheumatoid arthritis, RA, collagen-induced model of arthritis, 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
Panel Information
Targets
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1PAD11020protein-arginine deiminase type-1 [Homo sapiens] [gi:122056685]
Taxonomy id: 9606
Gene id: 29943
2PAD21020protein-arginine deiminase type-2 [Homo sapiens] [gi:122939159]
Taxonomy id: 9606
Gene id: 11240
3PAD31119protein-arginine deiminase type-3 [Homo sapiens] [gi:122939161]
Taxonomy id: 9606
Gene id: 51702
4PAD493protein-arginine deiminase type-4 [Homo sapiens] [gi:216548487]
Taxonomy id: 9606
Gene id: 23569

§ Panel component ID.
Protocol
Assay Overview:

The purpose of this assay is to determine whether test compounds can inhibit PADs 1-4 using a substrate-based assay. In this assay, the target enzyme (PAD 1, 2, 3 or 4) is pre-incubated with test compound followed by addition of substrate Na-benzoyl-L-arginine ethyl ester HCl (BAEE). The percent activity remaining is determined by measuring the amount of citrulline produced using a standard colorimetric absorbance assay. Test compounds that act as PAD inhibitors will prevent the production of citrulline.

Protocol Summary:

Recombinant PAD1 (Assay 1; 0.2 uM), PAD2 (Assay 2; 0.5 uM), PAD3 (Assay 3; 0.5 uM), or PAD4 (Assay 4; 0.2 uM) in assay buffer (50 mM NaCl, 10 mM CaCl2, 2 mM DTT, 100 mM Tris-HCl, pH 7.6) was treated with test compound (10 uM) or DMSO for 15 minutes at 37 C. Substrate BAEE (10 mM) was added, and the reaction was incubated for 15 minutes at 37 C in 60 uL total reaction volume. The reaction was quenched by flash freezing in liquid nitrogen, and a color developing reagent for detection of the enzymatic byproduct citrulline (COLDER; 200 uL), 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 inhibition for each compound was calculated as follows:

%_Inhibition = 1 - ( ( ABS_Test - Blank ) / Correction_Factor ) / ( ( ABS_DMSO - Blank ) / Correction_Factor )

Where:

ABS_Test is defined as absorbance at 540 nm for PAD4 treated with test compound.
ABS_DMSO is defined as absorbance at 540 nm for PAD4 treated with DMSO.
Blank is defined as the absorbance of a blank sample at 540 nm.
Correction_Factor is defined as the correction factor generated from a standard curve of known concentrations of citrulline.

PubChem Activity Outcome and Score:

The following applies to each panel:

Compounds with greater than or equal to 50% inhibition were considered active. Compounds with less than 50% inhibition were considered inactive.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-67, and 27 -0 for inactive compounds .

PAD1 Score: The PubChem Activity Score range for active compounds is 100-66, and for inactive compounds 52-0.

PAD2 Score: The PubChem Activity Score range for active compounds is 100-61, and for inactive compounds 40-0.

PAD3 Score: The PubChem Activity Score range for active compounds is 100-76, and for inactive compounds 44-0.

PAD4 Score: The PubChem Activity Score range for active compounds is 100-56, and for inactive compounds 43-10.

Overall Outcome and Score:

The overall outcome was active if the compound was active in at least one panel, inactive otherwise.

The overall score is 0 if the compound was inactive, otherwise the score is taken as the fraction of panels where the compound is active, multiplied by 100.

The PubChem Activity Score range for active compounds is 100-25, and for inactive compounds 0-0.

List of Reagents:

Recombinant PADs 1-4 (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 with powder samples of synthetic test compounds.
Categorized Comment
BAO: version: 1.4b1080

BAO: bioassay specification: assay stage: lead optimization

BAO: bioassay specification: assay biosafety level: bsl1

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: enzyme: generic hydrolase

BAO: detection technology: spectrophotometry: absorbance

Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [PAD1]One of Active, Inactive, or Not Tested for inhibition of PAD11protein-arginine deiminase type-1 [Homo sapiens]Outcome
2Score [PAD1]The BioAssay activity ranking score1Integer
3Inhibition at 10 uM [PAD1] (10μM**)Inhibition of recombinant PAD1 upon 10 uM compound treatment as assessed by colorimetric substrate assay using BAEE.1Float%
4Outcome [PAD2]One of Active, Inactive, or Not Tested for inhibition of PAD22protein-arginine deiminase type-2 [Homo sapiens]Outcome
5Score [PAD2]The BioAssay activity ranking score2Integer
6Inhibition at 10 uM [PAD2] (10μM**)Inhibition of recombinant PAD2 upon 10 uM compound treatment as assessed by colorimetric substrate assay using BAEE.2Float%
7Outcome [PAD3]One of Active, Inactive, or Not Tested for inhibition of PAD33protein-arginine deiminase type-3 [Homo sapiens]Outcome
8Score [PAD3]The BioAssay activity ranking score3Integer
9Inhibition at 10 uM [PAD3] (10μM**)Inhibition of recombinant PAD3 upon 10 uM compound treatment as assessed by colorimetric substrate assay using BAEE.3Float%
10Outcome [PAD4]One of Active, Inactive, or Not Tested for inhibition of PAD44protein-arginine deiminase type-4 [Homo sapiens]Outcome
11Score [PAD4]The BioAssay activity ranking score4Integer
12Inhibition at 10 uM [PAD4] (10μM**)Inhibition of recombinant PAD4 upon 10 uM compound treatment as assessed by colorimetric substrate assay using BAEE.4Float%

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: R01 GM079357-01

Classification
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