Late stage assay provider results from the probe development effort to identify inhibitors of Protein Arginine Deiminase 4 (PAD4): colorimetric biochemical substrate assay to determine rate constants of test compounds for PAD 3 inactivation
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 determine rate constants of test compounds for PAD 3 inactivation. ..more
BioActive Compound: 1
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: PAD3_INH_ABS_RATE_CONSTANT
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 determine rate constants of test compounds for PAD 3 inactivation.
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specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations).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-3, PAD3, rheumatoid arthritis, RA, collagen-induced model of arthritis, N-a-benzoyl-L-arginine amide HCl, BAA, citrulline, potency, first-order rate constant, observed rate constant, rate constant, reaction velocity, inactivation rate, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to more rigorously determine the potency of test compounds as inhibitors of human recombinant PAD3 using a biochemical substrate assay. The k_inact(app)/KI value for PAD3 is determined by measuring the rate of human recombinant PAD3 enzyme inactivation as a function of time at a single inhibitor concentration. In this assay, PAD3 is pre-incubated with test compound or DMSO control, followed by a biochemical determination of residual enzymatic activity. Substrate N-a-benzoyl-L-arginine amide HCl (BAA) is added, and the percent activity remaining is determined by measuring the amount of citrulline produced using a standard colorimetric absorbance assay. As designed, test compounds that act as PAD3 inhibitors will prevent the production of citrulline.
The Inactivation Reaction proceeded by incubating test compounds (10 uM) with purified PAD3 (5.0 uM) for various time points between 0 and 60 minutes at 37 C (60 uL total volume in 100 mM Tris-HCl, pH: 7.6, 10 mM CaCl2, and 2 mM DTT). After the designated time point, an aliquot (6 uL) of the Inactivation Reaction was added to pre-incubated Reaction Buffer containing N-a-benzoyl-L-arginine amide HCl (BAA; 10 mM final) to measure the residual activity. The Reaction Mixtures (60 uL total volume) were pre-incubated at 37 C for 10 minutes before adding aliquots from the Inactivation Reaction. The final reaction proceeded for 15 minutes at which point the reaction was stopped by flash freezing in liquid N2. 200 uL of a color-developing reagent that detects the product citrulline was added; the COLDER solution 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. This mixture was incubated for 30 minutes at 95 C, at which point 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 data obtained were fit to the following equation using Grafit version 5.0.11.
v = v0 * e^( -k(obs) * t )
v is the velocity
vo is the initial velocity
k(obs) is the observed pseudo-first-order rate constant for inactivation,
t is time
The k_inact(app)/KI was determined by dividing the inactivation rate (kobs) by the concentration of inhibitor.
PubChem Activity Outcome and Score:
Compounds with a k_inact(app)/KI value greater than or equal to 1500/(min*M) were considered active.
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
Recombinant PAD3 (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
From BioAssay Depositor:
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: bioassay specification: assay stage: secondary: selectivity
BAO: detection technology: spectrophotometry: absorbance
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: version: 1.4b1080
Assay Format: Biochemical
** Test Concentration.
Data Table (Concise)