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BioAssay: AID 651861

Late 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 1-3

Name: Late 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 1-3. ..more
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AID: 651861
Data Source: The Scripps Research Institute Molecular Screening Center (PAD1-3_INH_ABS_BINDING_MODE)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-12-05
Hold-until Date: 2013-06-14
Modify Date: 2013-06-15

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
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AIDNameTypeComment
463073Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)screeningPrimary screen (PAD4 inhibitors in singlicate, NIH 2K Validation)
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)
492970Fluorescence-based biochemical dose response assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)confirmatoryDose response (PAD5 inhibitors in duplicate)
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 PAD4confirmatoryLate stage dose response (potency of compound 16 against PAD4 in duplicate)
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 PAD4confirmatoryLate stage dose response (potency of compound 15 against PAD4 in duplicate)
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 PAD4confirmatoryLate stage dose response (potency of compound 13 against PAD4 in duplicate)
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 PAD4confirmatoryLate stage dose response (potency of compound 12 against PAD4 in duplicate)
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 PAD4confirmatoryLate stage dose response (potency of compound 11 against PAD4 in duplicate)
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 PAD4confirmatoryLate stage dose response (potency of compound 6 against PAD4 in duplicate)
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 PAD4confirmatoryLate stage dose response (potency of compound 2 against PAD4 in duplicate)
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 PAD4confirmatoryLate stage dose response (potency of compound 18 against PAD4 in duplicate)
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 PAD1-4confirmatoryLate stage dose response (compound 1 potency against PAD1-4 in duplicate)
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 PAD1-3confirmatoryLate stage dose response (streptonigrin potency against PAD1-4 in duplicate)
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 PAD1-4confirmatoryLate stage dose response (compound 3 potency against PAD1-4 in duplicate)
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 PAD1-4confirmatoryLate stage dose response (compound 14 potency against PAD1-4 in duplicate)
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 PAD1-4confirmatoryLate stage dose response (compound 10 potency against PAD1-4 in duplicate)
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 PAD1-4confirmatoryLate stage dose response (compound 21 potency against PAD1-4 in duplicate)
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 1-4otherLate stage assay (ABPP inhibition by HTS hits of PADs 1-4 in singlicate)
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 PAD1-4otherLate stage assay (compound 17 potency against PAD1-4 in duplicate)
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 PAD1-4confirmatoryLate stage dose response (HTS hits potency against PAD1-4 in singlicate)
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 PAD4otherLate stage results (PAD4 inhibitors in singlicate)
588560Late stage assay provider results from the probe development effort to identify inhibitors of PAD4: colorimetric biochemical substrate assay to identify inhibitors of PADs 1-4otherLate stage results (PAD1-4 inhibitors in singlicate)
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 PAD4otherLate stage assay (binding mode of test compounds to PAD4)
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 selectivityotherLate stage assay (compound selectivity)
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: PAD1-3_INH_ABS_BINDING_MODE

Name: Late 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 1-3.

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, powders, protein arginine deiminase type-1, protein arginine deiminase type-2, protein arginine deiminase type-3, PAD1, PAD2, PAD3, rheumatoid arthritis, RA, collagen-induced model of arthritis, Na-Benzoyl-L-arginine ethyl ester hydrochloride, BAEE, citrulline, dialysis, binding mode, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Assays
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1PAD11protein-arginine deiminase type-1 [Homo sapiens] [gi:122056685]
Taxonomy id: 9606
Gene id: 29943
2PAD21protein-arginine deiminase type-2 [Homo sapiens] [gi:122939159]
Taxonomy id: 9606
Gene id: 11240
3PAD31protein-arginine deiminase type-3 [Homo sapiens] [gi:122939161]
Taxonomy id: 9606
Gene id: 51702

§ Panel component ID.
Protocol
Assay Overview:

The purpose of this assay is to determine whether powder samples of test compounds inhibit PADs 1-3 in a reversible or irreversible manner. In this assay, PAD1, 2, and 3 are incubated with test compound or DMSO control followed by dialysis to remove small molecules not tightly bound to the protein. Substrate Na-benzoyl-L-arginine ethyl ester HCl (BAEE) is added, and the percent activity remaining is determined by measuring the amount of citrulline produced using a standard colorimetric absorbance assay. Test compounds that act as PAD1, 2, or 3 inhibitors will prevent the production of citrulline. As designed, test compounds that act as irreversible inhibitors will prevent enzyme-probe interactions following dialysis relative to the DMSO (no compound) control. In contrast, compounds that act as reversible inhibitors will show high residual enzyme activity following dialysis and can be labeled by active site-directed probes.

Protocol Summary:

Recombinant PAD1, recombinant PAD2, and recombinant PAD 3 (0.6 uM in 100 mM Tris-HCl, pH 7.6, 50 mM NaCl, 2 mM DTT, and 10 mM CaCl2, 400 uL total reaction volume), were pre-incubated with test compound (100 uM) or DMSO control, followed by dialysis in 4 L of Long-Term Storage Buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 500 mM NaCl, 500 uM TCEP, and 10% glycerol) for 20 hours. Residual PAD1, 2, and 3 activity was then measured using a colorimetric substrate (Na-Benzoyl-L-arginine ethyl ester hydrochloride; BAEE) assay as follows: After addition of BAEE (10 mM), the reactions were incubated for 15 minutes at 37 C (60 uL total reaction volume). The reactions were 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.

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

Where:
ABS_test is defined as absorbance at 540 nm for target treated with test compound.
ABS_DMSO is defined as absorbance at 540 nm for target 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 in this assay:

Compounds resulting in greater than 75% reduction in residual PAD activity were considered active; compounds resulting in less than or equal to 75% reduction in residual PAD activity were considered inactive.

Overall Outcome and Score:

Compounds active in the PAD1, PAD2 and PAD3 assays were considered active; compounds inactive in any of PAD1, PAD2, or PAD3 assays were considered inactive.

The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.

The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

List of Reagents:

Recombinant PADs 1-3 (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
CaCl2 (Sigma, part C3881)
DTT (RPI, part D110000)
Slide-A-Lyzer dialysis cassettes (Thermo Scientific, part 66333)
EDTA (Fisher, part BP2927)
Glycerol (Fisher, part BP2291)
BAEE (Sigma, part B4500)
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)
Comment
This assay was performed by the assay provider with powder samples of synthetic compounds.
Result Definitions
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [PAD1]The Outcome of Assay 1, one of Active or Inactive1protein-arginine deiminase type-1 [Homo sapiens]Outcome
2Residual Activity [PAD1] (100μM**)Percent of residual PAD1 activity following incubation with 100 uM compound and dialysis as measured by BAEE substrate assay relative to DMSO (no compound) control.1Float%
3Outcome [PAD2]The Outcome of Assay 2, one of Active or Inactive2protein-arginine deiminase type-2 [Homo sapiens]Outcome
4Residual Activity [PAD2] (100μM**)Percent of residual PAD2 activity following incubation with 100 uM compound and dialysis as measured by BAEE substrate assay relative to DMSO (no compound) control.2Float%
5Outcome [PAD3]The Outcome of Assay 3, one of Active or Inactive3protein-arginine deiminase type-3 [Homo sapiens]Outcome
6Residual Activity [PAD3] (100μM**)Percent of residual PAD3 activity following incubation with 100 uM compound and dialysis as measured by BAEE substrate assay relative to DMSO (no compound) control.3Float%

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

Classification
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