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_CMPD21

Name: Late 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 PAD1-4.

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specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations).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, protein arginine deiminase type-4, PAD1, PAD2, PAD3, PAD4, rheumatoid arthritis, RA, collagen-induced model of arthritis, Na-Benzoyl-L-arginine ethyl ester hydrochloride, BAEE, citrulline, absorbance, IC50, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN

Targets

Data Table(Active)    Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		PAD1		1	protein-arginine deiminase type-1 [Homo sapiens] [gi:122056685]		Taxonomy id: 9606 Gene id: 29943
2		PAD2		1	protein-arginine deiminase type-2 [Homo sapiens] [gi:122939159]		Taxonomy id: 9606 Gene id: 11240
3		PAD3	1		protein-arginine deiminase type-3 [Homo sapiens] [gi:122939161]		Taxonomy id: 9606 Gene id: 51702
4		PAD4	1		protein-arginine deiminase type-4 [Homo sapiens] [gi:216548487]		Taxonomy id: 9606 Gene id: 23569

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§ Panel component ID.

Assay Overview:

The purpose of this assay is to assess the potency of test compounds for inhibition of PADs 1-4 using a substrate-based assay. In this assay, the target enzyme is pre-incubated with test compound followed by addition of substrate Na-benzoyl-L-arginine ethyl ester HCl (BAEE). The percent activity remaining is determined by measuring the amount of citrulline produced using a standard colorimetric absorbance assay. Test compounds that act as PAD inhibitors will prevent the production of citrulline. IC50 values for inhibition of recombinant PADs were determined from dose-response curves from 2 trials at each inhibitor concentration in a 7-point dilution series from 0 to 5 uM (PAD1, PAD2, PAD4) or 0 to 2 uM (PAD3).

Protocol Summary:

Recombinant PAD1 (0.2 uM; Assay 1), PAD2 (0.5 uM; Assay 2), PAD3 (0.5 uM; Assay 3), or PAD4 (0.2 uM; Assay 4) in assay buffer (50 mM NaCl, 10 mM CaCl2, 2 mM DTT, 100 mM Tris-HCl, pH 7.6) was treated with test compound (0-5 uM final concentration; stock in DMSO) for 15 minutes at 37 C. Substrate BAEE (10 mM) was added, and the reaction was incubated for 15 minutes at 37 C in 60 uL total reaction volume. The reaction was 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 - ( ( ABStest - Blank ) / Correction_Factor ) / ( ( ABSDMSO - Blank ) / Correction_Factor)

Where:

ABStest is defined as absorbance at 540 nm for target treated with test compound.
ABSDMSO 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.
Correction_Factor is defined as the correction factor generated from a standard curve of known concentrations of citrulline.

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

Fractional_Activity = 1 / ( 1 + ( Conc / IC50 ) )

Where:

Conc is the concentration of inhibitor.
IC50 is the concentration of inhibitor that yields half-maximal activity.

PubChem Activity Outcome and Score:

The following applies to each panel in this assay:

Compounds with IC50 values less than or equal to 1 uM were considered active. Compounds with IC50 values less than 1 uM were considered inactive.

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 50-50. There are no inactive compounds.

List of Reagents:

Recombinant PADs 1-4 (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 with powder compounds.

BAO: version: 1.4b1080

BAO: bioassay specification: assay stage: secondary: selectivity

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: spectrophotometry: absorbance

Grant Number: R01 GM079357-01