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
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.
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specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations).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.
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.
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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).
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
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)
MTT Reagent (Invitrogen)
24-well plates (Costar (Corning),3506 )
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 : Equation: = ( [Response Range] / ( 1 + ( [Concentration] / [CC50] )^[Slope Factor] ) ) + [Background]
Assay: Dictionary: Version: 0.1
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: NIH 3T3
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)