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) inhibition by HTS hits of PADs 1-4
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) inhibition by HTS hits of PADs 1-4. ..more
BioActive Compounds: 35
Depositor Specified Assays
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: PAD1-4_INH_ABS_96_2XIC50_CMPD14
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) inhibition by HTS hits of PADs 1-4.
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specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations).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.
late stage, late stage AID, assay provider, cherry picks, protein arginine deiminase type-1, protein arginine deiminase type-2, protein arginine deiminase type-3, protein arginine deiminase type-4, PAD1, PAD2, PAD3, PAD4, rheumatoid arthritis, RA, collagen-induced model of arthritis, activity-based protein profiling, ABPP, gel-based ABPP, rhodamine-conjugated F-amidine, RFA, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
§ Panel component ID.
The purpose of this assay is to determine whether cherry picked HTS hit compounds can inhibit PADs 1-4 in a gel-based activity-based proteomic profiling (ABPP) assay. In this assay, the target enzyme (PAD1, 2, 3 or 4) is incubated with test compound followed by reaction with a rhodamine-conjugated F-amidine (RFA) 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 PAD 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.
Recombinant PAD1 (Assay 1), PAD2 (Assay 2), PAD3 (Assay 3), or PAD4 (Assay4) (10 uM) in assay buffer (50 mM NaCl, 10 mM CaCl2, 2 mM DTT, 100 mM HEPES, pH 8.0) was treated with test compound (10 uM) or DMSO for 15 minutes at 37 C. RFA was added to a final concentration of 1 uM. The reaction was incubated for 30 minutes in the dark at 37 C, quenched with 2x 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 target enzyme (PAD1, 2, 3, or 4) band relative to a DMSO-only (no compound) control.
%_Inhibition = ( 1 -( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control -IOD_Low_Control ) ) * 100
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 greater than or equal to 50% inhibition were considered active. Compounds with less than 50% inhibition were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.
PAD1 Score: The PubChem Activity Score range for active compounds is 100-71 and for inactive compounds 63-0.
PAD2 Score: The PubChem Activity Score range for active compounds is 100-54 and for inactive compounds 51-0.
PAD3 Score: The PubChem Activity Score range for active compounds is 100-86 and for inactive compounds 74-0.
PAD4 Score: The PubChem Activity Score range for active compounds is 100-54 and for inactive compounds 50-0.
Overall Outcome and Score:
The overall outcome was active if the compound was active in at least one panel, inactive otherwise.
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 50-25 and for inactive compounds 0-0.
List of Reagents:
Recombinant PADs 1-4 (supplied by Assay Provider)
RFA (supplied by Assay Provider)
NaCl (Sigma, part S6546)
CaCl2 (Sigma, part C3881)
DTT (RPI, part D110000)
HEPES (Sigma, part 54457)
BAO: version: 1.4b1080
BAO: bioassay specification: assay stage: compound profiling
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: detection technology: fluorescence: fluorescence intensity
** Test Concentration. § Panel component ID.