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_1X%INH_HTSHITS

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 HTS hits 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specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations).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, 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, Na-Benzoyl-L-arginine ethyl ester hydrochloride, BAEE, citrulline, absorbance, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN

Targets

Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		PAD1		34	protein-arginine deiminase type-1 [Homo sapiens] [gi:122056685]		Taxonomy id: 9606 Gene id: 29943
2		PAD2		34	protein-arginine deiminase type-2 [Homo sapiens] [gi:122939159]		Taxonomy id: 9606 Gene id: 11240
3		PAD3		34	protein-arginine deiminase type-3 [Homo sapiens] [gi:122939161]		Taxonomy id: 9606 Gene id: 51702
4		PAD4		34	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 determine whether cherry picked HTS hit compounds can inhibit PADs 1-4 using a substrate-based assay. In this assay, the target enzyme (PAD1, 2, 3 or 4) 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.

Protocol Summary:

Recombinant PAD1 (Assay 1; 0.2 uM), PAD2 (Assay 2; 0.5 uM), PAD3 (Assay 3; 0.5 uM), or PAD4 (Assay4; 0.2 uM) in assay buffer (50 mM NaCl, 10 mM CaCl2, 2 mM DTT, 100 mM Tris-HCl, pH 7.6) was treated with test compound (10 uM) or 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 - ( ( ABS_Test - Blank ) / Correction_Factor ) / ( ( ABS_DMSO - Blank) / Correction_Factor )

Where:

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.
Correction_Factor is defined as the correction factor generated from a standard curve of known concentrations of citrulline.
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 inactive compounds is 100-0. There are no active compounds.

PAD2 Score: The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.

PAD3 Score: The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.

PAD4 Score: The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.

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 inactive compounds is 0-0. There are no active 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 liquid cherry-picked compounds.

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: detection technology: spectrophotometry: absorbance

Grant Number: R01 GM079357-01