Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Paul Thompson, University of South Carolina
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 GM079357-01
Grant Proposal PI: Paul Thompson
External Assay ID: PAD4_INH_FLUO_2X%IC50

Name: Fluorescence-based biochemical dose response assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4).

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specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations).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:

assay provider, powders, dose response, protein arginine deiminase, rheumatoid arthritis, RA, collagen-induced model of arthritis, CIA, Na-Benzoyl-L-arginine ethyl ester hydrochloride, BAEE, citrulline, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN

Assay Overview:

The purpose of this assay is to determine potency of compounds identified as having inhibitory activity against recombinant PAD4. In this assay, recombinant PAD4 is pre-incubated with test compounds, followed by the addition of the substrate, Na-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE). The percent activity remaining is determined by measuring the amount of citrulline produced using a standard assay that measures changes in citrulline produced. Test compounds that act as PAD4 inhibitors will prevent the production of citrulline. IC50 values for inhibition of recombinant PAD4 were determined from dose-response curves from 2 trials at each inhibitor concentration in an 8-point dilution series from 0 to 100 uM.

Protocol Summary:

Recombinant PAD4 (0.2 uM in Assay Buffer (50 mM Tris-HCl pH 7.6, 50 mM NaCl, 10 mM CaCl2, and 2 mM DTT) was incubated with DMSO or test compound for 15 minutes at 37 C before the addition of BAEE at a final concentration of 10 mM in 60 uL total reaction volume. The reaction was incubated for 15 minutes at 37 C, quenched by flash freezing in liquid nitrogen, and 200 uL of a color developing reagent (COLDER), 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.

The percent activity remaining was fit to the following equation using GraFit (version 5.0.11):

Fractional Activity = 1 / ( 1 + ( [I] / IC50 ) )

Where:

[I] is the concentration of inhibitor.
IC50 is the concentration of inhibitor that yields half-maximal activity.

In cases where the highest concentration tested (100 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 100 uM. Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

PubChem Activity Outcome and Score:

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 88-0.

List of Reagents:

Recombinant PAD4 (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
CaCl2 (Sigma, part C3881)
DTT (RPI, part D110000)
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)
NaCl (Sigma, part S6546)
BAEE (Sigma, part B4500)
96-well plates (BD Falcon, part 353228)

This assay was performed by the assay provider.

Grant Number: R01 GM079357-01