Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4) (1536 HTS)
Name: Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4) (1536 HTS). ..more
BioActive Compounds: 1334
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_FP_1536_1X%INH PRUN
Name: Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Arginine Deiminase 4 (PAD4) (1536 HTS).
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specifically inactivates PAD4, reduces disease severity and incidence in the collagen-induced model of arthritis (CIA) (unpublished observations).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.
protein arginine deiminase 4 , PAD4, rheumatoid arthritis, RA, collagen-induced model of arthritis, CIA, fluorescence polarization, FP, fluorescence, rhodamine-conjugated Cl-amidine, RCA, rhodamine-conjugated F-amidine, ABPP, primary screen, 1536, HTS, high throughput screen, uHTS, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that act as inhibitors of protein arginine deiminase 4 (PAD4). In this biochemical assay, PAD4 is labeled in an active site-directed manner by the fluorescent ABPP probe rhodamine-conjugated Cl-amidine (RCA) in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. The assay is performed by incubating test compounds with PAD4 for a defined period, followed by addition of the rhodamine-conjugated Cl-amidine probe and measurement of fluorescence polarization at a specific time point. As designed, test compounds that act as PAD4 inhibitors will prevent PAD4-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization in the well. Compounds were tested in singlicate at a final nominal concentration of 6.36 uM.
Prior to the start of the assay, 4 uL of Assay Buffer (50 mM HEPES pH 7.6, 150 mM NaCl, 10 mM CaCl2, 0.01% Pluronic F-127, and 1 mM TCEP) containing 1.25 uM of PAD4 protein were dispensed into 1536 microtiter plates. Next, 32 nL of test compound in DMSO or DMSO alone (0.64% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 C.
The assay was started by dispensing 1.0 uL of 375 nM RCA probe in Assay Buffer to all wells. Plates were centrifuged and incubated for 20 hours at 37 C. Prior to reading, plates were equilibrated at room temperature for 5 minutes. Fluorescence polarization was read on Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm). Fluorescence polarization was read for 30 seconds for each polarization plane (parallel and perpendicular).
Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):
FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )
Raw1 is defined as the S channel.
Raw2 is defined as the P channel.
The percent inhibition for each compound was calculated as follows:
100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Low_Control is defined as wells containing PAD4, RCA and DMSO.
Test_Compound is defined as wells containing PAD4 in the presence of test compound and RCA.
High_Control is defined as wells containing DMSO, RCA but, no PAD4 protein.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-5, and for inactive compounds 5-0.
List of Reagents:
Recombinant PAD4 (supplied by Assay Provider)
RCA probe (supplied by Assay Provider)
HEPES (Cellgro, part 25-060-Cl)
NaCl (Sigma, part S6546)
CaCl2 (Fisher, C79)
TCEP (Sigma, part C4706)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Corning, part 7261)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. Compound SR-01000800927-1 (PubChem SID: 49725089) was not screened.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)