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BioAssay: AID 651887

Late 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 compounds

Name: Late 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 compounds. ..more
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 Tested Compounds
 Tested Compounds
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Inactive(1)
 
 
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Inactive(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 651887
Data Source: The Scripps Research Institute Molecular Screening Center (NIH3T3_INH_ABS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-12-07
Hold-until Date: 2013-06-13
Modify Date: 2013-06-15

Data Table ( Complete ):           View All Data
Tested Compound:
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
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 inactivationOthersame 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
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: NIH3T3_INH_ABS

Name: Late 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 compounds.

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
Protocol
Assay Overview:
The purpose of this assay is to assess compound cytotoxicity in mouse embryonic fibroblast NIH-3T3 cells using a colormetric MTT assay. In this assay, NIH-3T3 cells are divided into culture plates and are initially treated with test compound, DMSO (vehicle control), or 100% Triton-X (positive control). After incubation of the compounds for 3 days, cell viability is assessed by addition of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, the MTT reagent. The relative number of living cells is determined by the reduction of the yellow-colored MTT reagent in living cells, quantified by measuring absorbance at 570 nm. By design, test compounds that are cytotoxic will have a decreased number of living cells to reduce the MTT reagent resulting in concomitant increase in yellow-colored MTT and decrease in purple-colored formazan. Percent inhibition is calculated relative to a DMSO (vehicle control).
Protocol Summary:
NIH 3T3 cells were treated with test compound for 72 hours in a 24 well plate. The MTT reagent 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was then added to a final concentration of 0.5 mg/mL and the cells were further incubated for 4 hours. The culture medium was removed and DMSO was added to dissolve the formazan dye. The absorbance of the formazan-containing solution was then measured at 570 nm using a spectrophotometer.
The % Viable Cells for each well was calculated as follows:
%_Viable_Cells = ( Absorbance_Test_Compound - Absorbance_Low_Control ) / ( Absorbance_High_Control - Absorbance_Low_Control ) * 100
Where:
Test Compound is defined as wells containing cell in the presence of test compound.
High Control is defined as wells containing cells treated with DMSO.
Low Control is defined as wells containing cells treated with Triton-X.
CC50 values for cell growth inhibition were determined from dose-response curves, using 6-point dilution series from 5 uM to 100 uM. A four parameter equation describing a dose-response curve was fitted with GraFit software.
PubChem Activity Outcome and Score:
Compounds with an CC50 value of less than or equal to 5 uM were considered active (cytotoxic) and compounds with an CC50 value of greater than 5 uM were considered inactive (non-cytotoxic).
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
NIH/3T3 cells (provided by Assay Provider)
DMEM Media (CellGro 10-013-CV)
FBS (CellGro)
MTT Reagent (Invitrogen)
24-well plates (Costar (Corning),3506 )
Comment
This assay was performed by the assay provider with powder samples of synthetic compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: = ( [Response Range] / ( 1 + ( [Concentration] / [CC50] )^[Slope Factor] ) ) + [Background]
Assay: Dictionary: Version: 0.1
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: NIH 3T3
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1CC50*The value for concentration at which 50% of surviving cells are observed; CC50 shown in micromolar.FloatμM
2Response RangeResponse range of the curve fitFloat
3Slope FactorSlope Factor of the curve fitFloat
4BackgroundBackground of the curve fitFloat
5Viable Cells at 5.00 uM (5μM**)Percent of viable cells following incubation with 5.00 uM test compound relative to DMSO (no compound) control.Float%
6Viable Cells at 2.5 uM (12.5μM**)Percent of viable cells following incubation with 12.5 uM test compound relative to DMSO (no compound) control.Float%
7Viable Cells at 25.0 uM (25μM**)Percent of viable cells following incubation with 25.0 uM test compound relative to DMSO (no compound) control.Float%
8Viable Cells at 50.0 uM (50μM**)Percent of viable cells following incubation with 50.0 uM test compound relative to DMSO (no compound) control.Float%
9Viable Cells at 75.0 uM (75μM**)Percent of viable cells following incubation with 75.0 uM test compound relative to DMSO (no compound) control.Float%
10Viable Cells at 100 uM (100μM**)Percent of viable cells following incubation with 100 uM test compound relative to DMSO (no compound) control.Float%

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

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