Late 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 PAD4
Name: Late 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 PAD4. ..more
BioActive Compounds: 6
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: PAD4_INH_ABS_BINDING_MODE
Name: Late 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 PAD4.
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specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations).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, powders, protein arginine deiminase type-4, PAD4, 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
The purpose of this assay is to determine whether powder samples of test compounds inhibit PAD4 in a reversible or irreversible manner. In this assay, PAD4 is incubated with test compound or DMSO control followed by dialysis to remove small molecules not tightly bound to the protein. Substrate Na-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. Test compounds that act as PAD4 inhibitors will prevent the production of citrulline. As designed, test compounds that act as irreversible inhibitors will prevent enzyme-probe interactions following dialysis relative to the DMSO (no compound) control. In contrast, compounds that act as reversible inhibitors will show high residual enzyme activity following dialysis.
Recombinant PAD4 (0.6 uM in 100 mM Tris-HCl, pH 7.6, 50 mM NaCl, 2 mM DTT, and 10 mM CaCl2, 400 uL total reaction volume) was pre-incubated with test compound (100 uM) or DMSO control, followed by dialysis in Long-Term Storage Buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 500 mM NaCl, 1 mM DTT, and 10% glycerol) for 20 hours. Residual PAD4 activity was then measured using a colorimetric substrate (Na-Benzoyl-L-arginine ethyl ester hydrochloride; BAEE) assay as follows: After addition of BAEE (10 mM), the reactions were incubated for 15 minutes at 37 C (60 uL total reaction volume). The reactions were quenched by flash freezing in liquid nitrogen, and a color developing reagent for detection of the enzymatic byproduct citrulline (COLDER; 200 uL), which 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, was added. This mixture was incubated for 30 minutes at 95 C and 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.
%_Inhibition = 1 - ( ( ABS_test - Blank ) / X ) / ( ( ABS_DMSO - Blank ) / X )
ABS_test is defined as absorbance at 540 nm for target treated with test compound.
ABS_DMSO is defined as absorbance at 540 nm for target treated with DMSO.
Blank is defined as the absorbance of a blank sample at 540 nm.
X is defined as the correction factor generated from a standard curve of known concentrations of citrulline.
PubChem Activity Outcome and Score:
Compounds resulting in greater than 75% reduction in residual PAD4 activity were considered active; Compounds resulting in less than or equal to 75% reduction in residual PAD4 activity were considered inactive.
Activity score was then ranked by reduction in residual activity, with the greatest reductions in residual activity assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
List of Reagents:
Recombinant PAD4 (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)
This assay was performed by the assay provider with powder samples of synthetic compounds.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)