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BioAssay: AID 651628

Late 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 selectivity

Name: Late 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 selectivity. ..more
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 Tested Compounds
 Tested Compounds
All(15)
 
 
Active(7)
 
 
Inactive(8)
 
 
 Tested Substances
 Tested Substances
All(15)
 
 
Active(7)
 
 
Inactive(8)
 
 
 Related BioAssays
 Related BioAssays
AID: 651628
Data Source: The Scripps Research Institute Molecular Screening Center (ANTITARGET_INH_FLUO_GELBASEDABPP_SELECTIVITY)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-10-05
Hold-until Date: 2013-06-14
Modify Date: 2013-06-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 7
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)
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
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 PAD4Othersame 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: ANTITARGET_INH_FLUO_GELBASEDABPP_SELECTIVITY

Name: Late 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 selectivity.

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-4, PAD4, rheumatoid arthritis, RA, collagen-induced model of arthritis, activity-based protein profiling, ABPP, gel-based ABPP, chloroacetamide-rhodamine, CA-Rh, selectivity, anti-target, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Assays
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1Soluble Proteome (10 uM)411
2Soluble Proteome (50 uM)69
3Membrane Proteome (10 uM)69
4Membrane Proteome (50 uM)78

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this assay is to assess compound selectivity in a complex proteome using a gel-based activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction with a cysteine-reactive chloroacetamide-rhodamine (CA-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as anti-target inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.
Protocol Summary:
Soluble or membrane proteome (50 uL of 1 mg/ml in DPBS) of MCF7 human cancer cells was treated with 10 uM or 50 uM test compound (1 uL of a 50x stock in DMSO). Test compounds were incubated for 1 hour at 25 C, followed by the addition of 10 uM CA-Rh (1 uL of 50x stock in DMSO). The reactions was incubated for 1 hour at 25 C, quenched with 4x SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the anti-target bands relative to a DMSO-only (no compound) control. Bands were counted as anti-targets if greater than or equal to 50% inhibition was observed.
%_Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Where:
Test_Compound is defined as target enzyme treated with test compound.
High_Control is defined as target enzyme treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with one or more anti-targets were considered active. Compounds with no observed anti-targets were considered inactive.
Activity score was ranked by observed anti-targets, with compounds with the greatest number of anti-targets given the highest score.
Soluble Proteome (10 uM) Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
Soluble Proteome (50 uM) Score: The PubChem Activity Score range for active compounds is 100-60, and for inactive compounds 0-0.
Membrane Proteome (10 uM) Score: The PubChem Activity Score range for active compounds is 100-40, and for inactive compounds 0-0.
Membrane Proteome (50 uM) Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
Overall Outcome and Score:
Compounds that were active in any assay were considered active. Compounds inactive in all assays were considered inactive.
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:
MCF7 soluble and membrane proteome (supplied by Assay Provider)
CA-Rh (supplied by Assay Provider)
DPBS (Cellgro, 20-031-CV)
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
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [Soluble Proteome (10 uM)]One of Active, Inactive, or Not Tested1Outcome
2Score [Soluble Proteome (10 uM)]BioAssay activity score.1Integer
3Qualifier [Soluble Proteome (10 uM)]Qualifier identifies if the number of anti-targets is greater than reported value.1String
4Anti-targets [Soluble Proteome (10 uM)] (10μM**)Number of anti-targets as assessed by gel-based ABPP with CA-Rh in MCF7 soluble proteome at 10 uM compound concentration.1Integer
5Outcome [Soluble Proteome (50 uM)]One of Active, Inactive, or Not Tested2Outcome
6Score [Soluble Proteome (50 uM)]BioAssay activity score.2Integer
7Qualifier [Soluble Proteome (50 uM)]Qualifier identifies if the number of anti-targets is greater than reported value.2String
8Anti-targets [Soluble Proteome (50 uM)] (50μM**)Number of anti-targets as assessed by gel-based ABPP with CA-Rh in MCF7 soluble proteome at 50 uM compound concentration.2Integer
9Outcome [Membrane Proteome (10 uM)]One of Active, Inactive, or Not Tested3Outcome
10Score [Membrane Proteome (10 uM)]BioAssay activity score.3Integer
11Qualifier [Membrane Proteome (10 uM)]Qualifier identifies if the number of anti-targets is greater than reported value.3String
12Anti-targets [Membrane Proteome (10 uM)] (10μM**)Number of anti-targets as assessed by gel-based ABPP with CA-Rh in MCF7 membrane proteome at 10 uM compound concentration.3Integer
13Outcome [Membrane Proteome (50 uM)]One of Active, Inactive, or Not Tested4Outcome
14Score [Membrane Proteome (50 uM)]BioAssay activity score.4Integer
15Qualifier [Membrane Proteome (50 uM)]Qualifier identifies if the number of anti-targets is greater than reported value.4String
16Anti-targets [Membrane Proteome (50 uM)] (50μM**)Number of anti-targets as assessed by gel-based ABPP with CA-Rh in MCF7 membrane proteome at 50 uM compound concentration.4Integer

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

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