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BioAssay: AID 651865

Late 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 inactivation

Name: Late 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 inactivation. ..more
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AID: 651865
Data Source: The Scripps Research Institute Molecular Screening Center (PAD2_INH_ABS_RATE_CONSTANT)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-12-06
Hold-until Date: 2013-06-14
Modify Date: 2013-06-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
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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)
492970Fluorescence-based biochemical dose response assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4)Confirmatorydepositor-specified cross reference: Dose 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 PAD4Confirmatorydepositor-specified cross reference: Late 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 PAD4Confirmatorydepositor-specified cross reference: Late 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 PAD4Confirmatorydepositor-specified cross reference: Late 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 PAD4Confirmatorydepositor-specified cross reference: Late 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 PAD4Confirmatorydepositor-specified cross reference: Late 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 PAD4Confirmatorydepositor-specified cross reference: Late 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 PAD4Confirmatorydepositor-specified cross reference: Late 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 PAD4Confirmatorydepositor-specified cross reference: Late 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 PAD1-4Confirmatorydepositor-specified cross reference: Late 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 PAD1-3Confirmatorydepositor-specified cross reference: Late 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 PAD1-4Confirmatorydepositor-specified cross reference: Late 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 PAD1-4Confirmatorydepositor-specified cross reference: Late 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 PAD1-4Confirmatorydepositor-specified cross reference: Late 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 PAD1-4Confirmatorydepositor-specified cross reference: Late 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 1-4Otherdepositor-specified cross reference: Late 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 PAD1-4Otherdepositor-specified cross reference: Late 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 PAD1-4Confirmatorydepositor-specified cross reference: Late 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 PAD4Otherdepositor-specified cross reference: Late 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 1-4Otherdepositor-specified cross reference: Late 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 PAD4Otherdepositor-specified cross reference: Late 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 selectivityOtherdepositor-specified cross reference: Late stage assay (compound selectivity)
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 1-3Othersame project related to Summary assay
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 inactivationOthersame project related to Summary assay
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 inactivationOthersame project related to Summary assay
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 inactivationOthersame project related to Summary assay
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 compoundsConfirmatorysame project related to Summary assay
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: PAD2_INH_ABS_RATE_CONSTANT

Name: Late 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 inactivation.

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-2, PAD2, rheumatoid arthritis, RA, collagen-induced model of arthritis, N-a-benzoyl-L-arginine ethyl ester HCl, BAEE, citrulline, potency, first-order rate constant, observed rate constant, rate constant, reaction velocity, inactivation rate, 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 more rigorously determine the potency of test compounds as inhibitors of human recombinant PAD2 using a biochemical substrate assay. The k_inact(app)/KI value for PAD2 is determined by measuring the rate of human recombinant PAD2 enzyme inactivation as a function of time at a single inhibitor concentration. In this assay, PAD2 is pre-incubated with test compound or DMSO control, followed by a biochemical determination of residual enzymatic activity. Substrate N-a-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. As designed, test compounds that act as PAD2 inhibitors will prevent the production of citrulline.
Protocol Summary:
The Inactivation Reaction proceeded by incubating test compounds (10 uM) with purified PAD2 (5 uM) for various time points between 0 and 60 minutes at 37 C (60 uL total volume in 100 mM Tris-HCl, pH: 7.6, 10 mM CaCl2, and 2 mM DTT). After the designated time point, an aliquot (6 uL) of the Inactivation Reaction was added to pre-incubated Reaction Buffer containing N-a-benzoyl-L-arginine ethyl ester HCl (BAEE; 10 mM final) to measure the residual activity. The Reaction Mixtures (60 uL total volume) were pre-incubated at 37 C for 10 minutes before adding aliquots from the Inactivation Reaction. The final reaction proceeded for 15 minutes at which point the reaction was stopped by flash freezing in liquid N2. 200 uL of a color-developing reagent that detects the product citrulline was added; the COLDER solution 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. This mixture was incubated for 30 minutes at 95 C, at which point 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 data obtained were fit to the following equation using Grafit version 5.0.11.
v = v0 * e^( -k(obs) * t )
Where:
v is the velocity
v0 is the initial velocity
k(obs) is the observed pseudo-first-order rate constant for inactivation
t is time
The k_inact(app)/KI was determined by dividing the inactivation rate (kobs) by the concentration of inhibitor.
PubChem Activity Outcome and Score:
Compounds with a k_inact(app)/KI value greater than or equal to 1500/(min*M) were considered active.
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 PAD2 (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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1k_inact(app)/KIThe value of kinact(app)/KI calculated by dividing the observed first-order rate constant by the concentration of inhibitorInteger
2Residual Activity at 2 min (10μM**)Percent of residual PAD2 activity after incubation with 10 uM test compound for 2 minutes.Float%
3Residual Activity at 6 min (10μM**)Percent of residual PAD2 activity after incubation with 10 uM test compound for 6 minutes.Float%
4Residual Activity at 10 min (10μM**)Percent of residual PAD2 activity after incubation with 10 uM test compound for 10 minutes.Float%
5Residual Activity at 15 min (10μM**)Percent of residual PAD2 activity after incubation with 10 uM test compound for 15 minutes.Float%
6Residual Activity at 20 min (10μM**)Percent of residual PAD2 activity after incubation with 10 uM test compound for 20 minutes.Float%
7Residual Activity at 30 min (10μM**)Percent of residual PAD2 activity after incubation with 10 uM test compound for 30 minutes.Float%

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

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