WO2007076302A2

WO2007076302A2 - High throughput assay for modulators of peptidylarginine deiminase activity

Info

Publication number: WO2007076302A2
Application number: PCT/US2006/062141
Authority: WO
Inventors: Brian Glenn Werneburg; Martha Priscilla Brown; Dorothy Mae Freeman; Jeffrey David Yingling
Original assignee: Boehringer Ingelheim International Gmbh; Boehringer Ingelheim Pharma Gmbh & Co. Kg
Priority date: 2005-12-20
Filing date: 2006-12-15
Publication date: 2007-07-05
Also published as: WO2007076302A3

Abstract

The present invention is a high through-put assay for PAD activity. The assay is designed to identify modulators of PAD catalysis for the treatment of multiple sclerosis and rheumatoid arthritis.

Description

HIGH THROUGHPUT ASSAY FOR MODULATORS OF PEPTIDYLARGININE DEIMINASE ACTIVITY

Prior Applications

This application claims benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 60/743,054 that was filed on December 20, 2005.

Background of the Invention

1. Field of the Invention

This invention is an assay for modulators peptidylarginine deiminase (PAD) activity. More specifically, this invention is a high throughput, fluorescence resonance energy transfer (FRET) assay for modulators of the conversion of protein incorporated arginine to citrulline by the process of deimination by PAD.

2. Description of the Prior Art

The peptidylarginine deiminase (PAD, EC 3.5.3.15) family of enzymes is composed of PAD1 , 2, 3, 4 and 5 [Vossenaar et al. (2003) BioEssays 25: 1106-1118]. PAD catalyzes the deimination of arginine amino acid residues incorporated into proteins and peptides. While it is a poor catalyst of L-arginine deimination, it will catalyze the deimination of small arginine analogues, including benzoyl-L-arginine and benzoyl-L- arginine ethyl ester. PAD is associated with the development and progression of the autoimmune disorders multiple sclerosis and rheumatoid arthritis. For example, an increase in PAD2 activity in the central nervous system (CNS) coincides with the development and progression of multiple sclerosis in man [Wood & Moscarello (1989) JBC 264, 5121-5127; Moscarello et al. (1994) J. Clin. Inv. 94, 146-164]. PAD2 catalyzed deimination of myelin proteins, including glial fibrilliary acidic protein (GFAP) and myelin basic protein (MBP), is believed to propagate multiple sclerosis by two mechanisms. First, deimination of myelin proteins leads to the instability and demise of the axon insulating myelin sheath. Second, deimination of myelin proteins results in autoantigen production that mediates a chronic inflammatory response in the diseased state [Berger, T et al. (2003) N. Engl. J. Med. 349, 139-145; Doyle and Mamula (2001) Trends in Immun. 22, 443-449].

PAD catalysis has also been associated with the preclinical diagnosis and onset of rheumatoid arthritis. The detection of circulating auto-reactive anti-citrulline antibodies with the anti-cyclic-citrullinated antibody serological test kit (sold by Eurodiagnostica, Axis-Shield and RDL) is predictive of the disease, and it is 80% effective in diagnosing the disease after onset (99% specific for RA vs. all other rheumatic diseases). PAD2 and PAD4, expressed in macrophages and neutrophils, may both produce citrullinated auto-antigens in the synovia of rheumatoid arthritis patients. In fact, a PAD4 haplotype in the Japanase population has been linked to rheumatoid arthritis susceptibility [Suzuki et al., (2003) Nat. Gen. 34, 395-402].

Thus, potent and specific inhibitors of PAD2 and PAD4 are required to block deimination of myelin sheath proteins associated with multiple sclerosis, and the production of latent auto-antigens associated with multiple sclerosis and rheumatoid arthritis.

However, a robust, specific, high through-put assay does not exist for the identification of potent inhibitors of PAD2 and PAD4. The problem is further complicated by the fact that the specificities of the PAD family members for specific arginylated peptide sequences have never been determined.

The most common method used to assay for PAD activity is the colorimetric detection of citrulline analogue products. In 1983 this assay was used to measure the activity of PAD 2 from rabbit skeletal muscle [Takahara et al. (1983) J. Biochem. 94, 1945-1953]. The assay utilizes the reaction of the citrulline ureido group (of citrulline and small citrulline analogues) with diacetyl monoxime. It is tedious because heat and acid are required [Zarabian et al. (1987) Anal. Biochem. 166, 113- 119; Rothnagel and Rogers (1984) Methods in Enzymology 107, 624-631]. This colorimetric assay was originally designed to assay the conversion of arginine to citrulline in protein and peptide substrates [Rothnagel and Rogers (1984) Method in Enzymology 107, 624-631 ; Boyde and Rahmatullah (1980) Anal. Biochem. 107, 424; Guthohrlein and Knappe (1968) Anal. Biochem. 26, 188; Archibald (1944) J. Biol. Chem. 156, 121]. The assay for citrulline from protein and peptide products requires an additional protein hydrolysis step prior to the standard citrulline colorimetric assay. Both of these PAD assays are labor intensive and not amenable to high through-put. A second method available for PAD 2 (rabbit skeletal muscle) assay measures PAD 2 activity indirectly by the inhibition of trypsin with soybean trypsin inhibitor [Takahara et al. (1985) J. Biol. Chem. 260, 8378-8383]. PAD catalyzed deimination of soybean trypsin inhibitor renders the inhibitor unable to block trypsin catalysis of benzoyl-L- arginine-p-nitroanilide. The residual trypsin inhibiting activity of soybean trypsin inhibitor, after deimination of the inhibitor by PAD from rabbit muscle, is detected and pseudo-first-order rate constants of trypsin inhibition are reported. This assay has limited functionality, because the deimination of only one protein sequence (soybean trypsin inhibitor) can be measured. Furthermore, this assay has never been used in a high through-put screening format and it has only been used to assay one PAD family member (PAD 2). It has not been reported to be a functional assay for other PAD family members.

Currently existing techniques using fluorescence resonance energy transfer (FRET) assays to measure peptidase activity have not been applied to the study and characterization of PAD activity. FRET assays are based on the principle that the excited state energy of a first fluorophore (a "donor") can be transferred to a second fluorophore (an "acceptor") in a non-radiative manner. This transfer of energy is dependent on several factors, including the spectral overlap of the absorbance spectra of the acceptor and the emission spectrum of the donor, and the distance between the two fluorophores. The Forster distance, the distance at which FRET is 50% efficient, is typically 10-100 angstroms for most donor/acceptor pairs, a distance that characterizes the dimensions of many biological molecules. FRET is therefore a method that can probe biological events that involve changes in the proximity of molecules. For example, to monitor the activity of an enzyme of interest, a substrate (i.e., a peptide) is labeled with both a donor and an acceptor and the enzyme of interest is used to cleave the labeled peptide. Enzymatic activity is monitored by detecting the change in FRET due to enzymatic cleavage of the peptide, which cleavage results in a change in the proximity of the FRET donor/acceptor pair.

Summary of the Invention

The present invention is a high through-put assay for PAD activity, the conversion of protein incorporated arginine to citrulline by the process of deimination. The assay is designed to identify modulators (i.e., potential inhibitors) of PAD catalysis for the treatment of multiple sclerosis and rheumatoid arthritis.

This assay provides the flexibility to identify the optimal substrate sequence of each PAD family member, as determined by V_maχ/K_m, so that sensitive assays may be tailored to the specificity of each PAD enzyme for the identification of potent modulators of PAD activity. Since multiple arginine containing protein sequences can be incorporated into the assay, multiple, sensitive, high through-put assays can be created to match the specificity of each PAD enzyme by alteration of the peptide sequence.

Brief Description of the Drawings

Figure 1 is a graph showing the time and concentration dependence of PAD2 catalyzed deimination of IA. Figure 2 is a graph showing a comparison of reducing agents and buffers in the PAD2 catalyzed deimination of IA.

Figure 3 is a graph showing the time dependence of trypsin catalyzed hydrolysis of FRET peptide substrate IA.

Figure 4A and 4B are graphs showing the time dependence of PAD2 catalysis of FRET peptide substrate IA deimination.

Figure 5 is a graph showing PAD2, PAD3 and PAD4 catalysis of FRET peptide substrate IA deimination.

Figure 6 is a graph showing inhibition of PAD2.

Figure 7 is a graph showing inhibition of PAD3.

Figure 8 is a graph showing inhibition of PAD4.

Detailed Description of the Invention

The present invention is a high-throughput assay for the measurement of peptidylarginine deiminase (PAD) activity. The assay makes use of arginylated peptide substrates and the peptidase activity of trypsin. Peptides of variable length and amino acid composition that contain at least one arginine residue flanked by fluorescence resonance energy transfer (FRET) donor (D) and acceptor (A) moieties are novel substrates for the PAD family of enzymes. These substrates described herein are incorporated into a screening assay format that can be applied to identify inhibitors of all PAD family members, including PAD2 and PAD4, which are implicated in the etiology of autoimmunity.

The assay consists of two half reactions as illustrated in Schemes 1 and 2. The first half reaction involves incubation of the peptide substrate I, where (X)j and and (Y)j are amino acid sequences of variable length and composition, more fully defined below, in the presence of PAD activity (Rxn 1 ). The second half reaction involves incubation of the first half reaction product (II) in the presence of trypsin activity (Rxn 2). If arginine is deiminated to citrulline, as in intermediate II, trypsin will not cleave the peptide as shown in Scheme 1.

Scheme 1

PAD Trypsin A-(X)_rArg-(Y)_fD >- A-(X)i-Cit-(Y)j-D *^■ A-(X)i-Cit-(Y)j-D

Rxn 1 Rxn 2

I Il Il

In the presence of active PAD, the arginine is deiminated to citrulline resulting in formation of intermediate II. The overall fluorescence emission intensity of the FRET peptide does not change in this scheme because the distance between the fluorophores does not change. Fluorescence emission by the donor is reduced by FRET from the donor (D) to the acceptor (A). Concurrently, the fluorescence emission from the acceptor is increased due to FRET to the acceptor (A) from the donor (D). Scheme 2

PAD

+ inhibitor Trypsin A~(X)_rArg-(Y)_rD *- A-(X)i-Arg-(Y)j-D *- A-(X)i-Arg + (Y)J-D

Rxn 1 Rxn 2

I I III IV

Inhibition of PAD activity prevents arginine deimination. Thus, substrate I is unchanged or not totally citrullinated by PAD in the presence of an inhibitor so that trypsin may cleave the substrate I to produce III and IV, as illustrated in Scheme 2.

Cleavage of the peptide results in a separation of the FRET donor and acceptor to a distance whereby energy transfer is drastically diminished resulting in both an increase in fluorescence emission from the FRET donor (D) and a decrease in fluorescence emission from the acceptor (A). Thus, inhibitors can be identified and the potency of inhibition can be determined by monitoring the corresponding decrease in FRET acceptor fluorescence emission or the increase in donor fluorescence emission.

The present invention includes peptides which contain an arginine within a consensus sequence for trypsin cleavage and are labeled with an appropriate donor/acceptor pair. The invention includes a variety of donor/acceptor pairs and the identification of the consensus sequence: HQSTRGSGHC. Depending on the probes chosen, the fluorescence of the donor and/or acceptor changes when trypsin cleaves the peptide due to a decrease of FRET. Deimination of the arginine by a PAD enzyme such as PAD2 prevents trypsin from cleaving the peptide, thereby resulting in no change in FRET signal. The FRET signal serves as the observable in this invention. Unless otherwise specified herein, the conditions that can be employed in the FRET assay of the present invention (e.g., pressure, temperature, pH, solvents, and time) may be readily determined by one having ordinary skill in the art.

Suitable peptide substrates have the formula (I):

A-(X)_rArg-(Y)_rD

wherein (X)i represents a sequence of i amino acids and (Y)j represents a sequence of j amino acids, and wherein i and j are integers defining the number of amino acids in X and Y respectively. Preferably, the sum of i and j is less than or equal to about 100.

A and D may be chemically bound to any amino acid in (X)j and (Y)j, respectively. The sequences (X)ι and (Y)j may contain any amino acids in any order with the following exceptions: lysine at all positions (unless followed immediately to the right by proline), which would add additional trypsin cleavage sites that would not be sensitive to peptidylarginine deiminase catalysis of citrullination; praline in the j = 1 position, which would prevent trypsin cleavage beyond arginine; and arginine, in addition to the one arginine shown between (X)j and (Y)j, unless it is followed immediately by a proline. The presence of additional arginines not followed by praline in X and Y would require an extended time of incubation for peptidylarginine catalysis of citrullination, prior to trypsin catalysis of cleavage. While not ideal, additional arginines are feasible, if the incubation time is extended as the number of arginine residues increases.

A wide variety of suitable donor (D) and acceptor (A) fluorophores are commercially available. The choice of probe pair is influenced by system constraints as well as by the length and sequence of the peptide used in the desired application. The length and sequence of the peptide will influence the labeling sites for attachment of the probes. The distance between the attachment sites influences the choice of the donor/acceptor pair due to the distance-dependence of FRET. Many donor/acceptor pairs are commercially available. These include, but are not limited to:

5-TAMRA/QSY-7

Dansyl / Eosin

Tryptophan / Dansyl

Fluorescein / Texas Red (rhodamine)

Naphthalene/Dansyl

Dansyl / ODR

BODIPY / BODIPY

Terbium / Thodamine

Dansyl / FITC

Pyrere / Coumarin

IAEDANS / IAF

BPE / Cy5

Europium / Cy5

Europium / APC FITC / TMR IAEDANS / FITC IAF / TMR IAF / EIA Tryptophan / IAEDANS

The optimum distance between A and D depends on the donor acceptor pair used and is typically between 10 and 100 angstroms.

An example of a suitable substrate is shown below as formula (IA).

QSY-7-HQSTRGSGHC-5-TAM RA-KK-NH₂

IA

QSY-7 is commercially available as the succinimidyl ester (formula II), which may be used to label an amino acid with a free amine by formation of an amide bond.

5-TAMRA (5-carboxytetramethylrhodamine) is commercially available as the rnaleimide (formula III), which may be used to label an amino acid with a free sulfhydryl group via conjugation.

A preferred method for using the assay of the present invention generally comprises the following steps:

(a) selection of a suitable donor/acceptor pair by determining their spectral overlap and Forster distance;

(b) demonstration of trypsin cleavage of the labeled peptide in the presence of a test compound {i.e., a potential inhibitor) that is a PAD inhibitor via a decrease in FRET; and/or

(c) demonstration of arginine deimination by PAD2 in the presence of a test compound {i.e., a potential inhibitor) that is not a PAD inhibitor via no change in FRET.

As a preliminary step, the absorbance, fluorescence excitation and fluorescence emission spectra are determined for each fluorescent probe. The absorbance spectrum is measured in order to avoid inner filter effect. The absorbance and fluorescence emission spectra are measured to ensure appropriate spectral overlap of the donor and acceptor. These spectra can be determined using any conventional spectrophotometer and fluorimeter with probe in a suitable assay buffer, as known to one skilled in the art.

The labeled peptide concentration must yield acceptable fluorescence intensities upon trypsin cleavage, which must be determined for each pair since the quantum yields, overlap integrals and Forster distances of different pairs vary. The type of 96 or 384-well plate must be carefully determined in order to minimize nonspecific adsorption of the probe to the walls of the wells. Alternatively, a cuvette may be used. The samples are mixed and allowed to incubate for a pre-determined period of time, preferably between 10 minutes and three hours, prior to FRET measurement. Instrument settings are defined by the fluorescence characteristics of the FRET pair (excitation spectrum, emission spectrum, quantum yield). The required change in intensity is defined by the instrumentation to be used in the screen.

The example below illustrates the method used to measure PAD2 activity using the substrate of formula (IA).

The FRET peptide IA was diluted into assay buffer (20 mM TRIS pH 7, 10 mM CaCI₂, 5 mM TCEP (neutral), 5% DMSO ) to a suitable stock concentration, 50OnM. IA (500 nM) was aliquoted into a microtiter plate, followed by 1 , 10, and 50 nM of PADII. A ¹/4-area flat bottom non-binding surface black polystyrene (Corning 3686) microtiter plate provided the most robust signal. The fluorescence intensity of each component was measured in a plate reader with the appropriate settings for the fluorophores chosen. In this example, a Biosystems LJL Analyst AD Platereader was utilized with filters appropriate for the TAMRA/QSY-7 pair found on IA. The fluorescence signal from the IA-PAD2 mixture was stable for at least 1 hr. After the addition of trypsin (500 nM) to the IA-PAD2 mixture, the fluorescence intensity was proportional to the amount of IA cleaved by trypsin (Figure 1 ).

The assay described above was modified in order to allow for its use in an ultrahigh- throughput screening mode. The modifications included altering the reducing agent from TCEP to DTT (Figure 2). The buffer chosen for use in high throughput screening mode contained 25 mM MOPS (pH 7.2), 200 μM CaCI₂, 0.01 % CHAPS, 5 mM DTT. The final reaction contained 250 pM PAD2 and 250 nM IA in a plate format using an LJL Analyst platereader with the standard rhodamine filters (Excitation 530nm/Emission 580nm).

In the final assay format, 500 nM of the FRET peptide IA was incubated with 100 nM trypsin. The fluorescence intensity increased as a function of time (Figure 3), indicative of the peptide cleavage activity of trypsin. The time-course of the assay was determined by incubating 1 nM PAD2 with 50OnM FRET peptide for defined time periods. This was followed by the addition of trypsin and a subsequent incubation of 10 min (Figure 4). The time-course of the assay for other members of the PAD family was also measured (Figure 5).

Use of the assay to detect the inhibition of PAD2 catalyzed deimination of IA substrate is illustrated in the following example (Figure 6). PAD2, IA and potential inhibitor or test compound A (10 nM -10 microM) were allowed to incubate for 30 min in 25 mM MOPS (pH 7.2), 200 microM CaCI₂, 0.01% CHAPS and 5 mM DTT prior to the addition of 400 nM trypsin (10 min). The fluorescence intensity was measured as described above. The IC₅O (110 nM) was determined from a plot of % residual activity vs. [compound A]. The inhibition of PAD3 and PAD4 activity by compound A are shown, respectively, in Figures 7 and 8.

Time and concentration dependence of PAD2 catalysis of IA deimination.

Referring to Fig. 1 , PAD2 (1 , 10 and 50 nM) was incubated with 500 nM peptide substrate IA (QSY^-HQSTRGSGHC-S-TAMRA-KK-NH₂) for one hr in the following buffer: 20 mM Tris (pH 7.0), 10 mM CaCI₂, 5 % DMSO and 5 mM TCEP (neutral). Trypsin (500 nM) was added to the IA-PAD2 mixture after the 1 hr incubation. Fluorescence was quantified with an LJL Analyst using the standard rhodamine filter set (Excitation 550 nm/Emission 580 nm).

PAD2 catalyzed deimination of IA. Comparison of reducing agents and buffers.

Referring to Fig. 2, PAD2 (20 nM) was incubated with 500 nM IA (QSY-7- HQSTRGSGHC-5-TAMRA-KK-NH2) for 2 hr and 45 min at 25 ⁰C in the presence of 0.2 mM TCEP or 5 mM DTT in the following buffers: 25 mM TES (pH 7.2) and 10 mM CaCI₂; 25 mM Tris (pH 7.0) and 10 mM CaCI₂; 25 mM MOPS (pH 7.2) and 10 mM CaCI₂; and 25 mM HEPES (7.5) and 10 mM CaCI₂. Trypsin (33 nM) was added subsequently and the cleavage reaction was allowed to proceed for 2 hr at 25 ⁰C. Fluorescence was quantified with an LJL Analyst using the standard rhodamine filter set (Excitation 530 nm/Emmision 580 nm) and the relative fluorescence units were plotted vs. buffer conditions.

Time dependence of trypsin catalyzed hydrolysis of FRET peptide substrate IA.

Referring to Fig. 3, Trypsin (100 nM) was incubated with 500 nM IA (QSY-7- HQSTRGSGHC-5-TAMRA-KK-NH2) at various times in the following buffer: 25 mM MOPS (pH 7.2), 200 microM CaCI₂, 0.01% CHAPS and 5 mM DTT. Fluorescence was quantified with an LJL Analyst using the standard rhodamine filter set (Excitation 530 nm/Emission 580 nm).

Time dependence of PAD2 catalysis of FRET peptide substrate IA deimination.

Referring to Figs. 4A and 4B, PAD2 (1 nM) was incubated with 500 nM IA (QSY-7- HQSTRGSGHC-5-TAMRA-KK-NH2) at various times in the following buffer: 25 mM MOPS (pH 7.2), 200 microM CaCI₂, 0.01% CHAPS and 5 mM DTT. Subsequent to PAD2 catalysis, 100 nM trypsin was added to the reaction; the solution was incubated for an additional 10 min. Fluorescence was quantified with an LJL Analyst using the standard rhodamine filter set (Excitation 530 nm/Emission 580 nm).

PAD2, PAD3 and PAD4 catalysis of FRET peptide substrate IA deimination.

Referring to Fig. 5, PAD2 (250 pM), PAD3 (5 nM) and PAD4 (500 pM) were incubated with 250 nM IA (QSY-7-HQSTRGSGHC-5-TAMRA-KK-NH₂) in separate reactions at various times in the following buffer: 25 mM MOPS (pH 7.2), 200 microM CaCI2, 0.01 % CHAPS and 5 mM DTT. Subsequent to PAD catalysis, 400 nM trypsin was added to the reaction; the solution was incubated for an additional 10 min. Fluorescence was quantified with an LJL Analyst using the standard rhodamine filter set (Excitation 530 nm/Emission 580 nm).

Inhibition of PAD2.

Referring to Fig. 6, inhibition of PAD2 (250 pM) catalyzed deimination of IA (250 nM; described in Figures 1-3) was measured in the presence of compound A (10 microM - 10 nM). PAD2, FRET peptide substrate IA and compound A were allowed to incubate for 30 min in 25 mM MOPS (pH 7.2), 200 microM CaCI₂, 0.01 % CHAPS and 5 mM DTT prior to the addition of 400 nM trypsin (10 min). Fluorescence was quantified with an LJL Analyst using the standard rhodamine filter set (Excitation 530 nm/Emission 580 nm). The IC50 (110 nM) was determined from a plot of % residual activity vs. [compound A].

Inhibition of PAD3.

Referring to Fig. 7, Inhibition of PAD3 (5 nM) catalyzed deimination of the FRET peptide substrate (250 nM; described in Figures 1-3) was measured in the presence of compound A (10 microM - 10 nM). PAD3, FRET peptide substrate IA and compound A were allowed to incubate for 30 min in 25 mM MOPS (pH 7.2), 200 microM CaCI₂, 0.01 % CHAPS and 5 mM DTT prior to the addition of 400 nM trypsin (10 min). Fluorescence was quantified with an LJL Analyst using the standard rhodamine filter set (Excitation 530 nm/Emission 580 nm). The IC₅₀ (1 ,200 nM) was determined from a plot of % residual activity vs. [compound A].

Inhibition of PAD4.

Referring to Fig. 8, inhibition of PAD4 (500 pM) catalyzed deimination of the FRET peptide substrate (250 nM; described in Figures 1-3) was measured in the presence of compound A (10 microM - 10 nM). PAD4, FRET peptide substrate IA and compound A were allowed to incubate for 30 min in 25 mM MOPS (pH 7.2), 200 microM CaCI₂, 0.01% CHAPS and 5 mM DTT prior to the addition of 400 nM trypsin (10 min). Fluorescence was quantified with an LJL Analyst using the standard rhodamine filter set (Excitation 530 nm/Emission 580 nm). The IC50 (75 nM) was determined from a plot of % residual activity vs. [compound A].

Claims

WE CLAIM:

1. An assay for the measurement of peptidylarginine deiminase (PAD) activity comprising an arginylated peptide substrate.

2. The assay of claim 1 , wherein the arginylated peptide substrate comprises peptides comprising at least one arginine residue flanked by fluorescence resonance energy transfer (FRET) donor (D) and acceptor (A) moieties.

3. The assay of claim 1 , wherein the anginylated peptide substrate comprises an arginine within a consensus sequence for trypsin cleavage and is labeled by fluorescence resonance energy transfer (FRET) donor (D) and acceptor (A) moieties.

4. The assay of claim 3, wherein the consensus sequence is HQSTRGSGHC.

5. A method for identifying inhibitors of PAD with a fluorescence resonance energy transfer (FRET) assay comprising the steps of: selecting of a suitable fluorescence donor D and fluorescence acceptor A; incubating a peptide substrate I, where (X)i and and (Y)j are amino acid sequences of variable length and composition and Arg is arginine, in the presence of PAD activity and a test compound that is a potential PAD inhibitor;

A-(X)_rArg-(Y)_rD _(|)

incubating the product of the first incubation in the presence of trypsin activity, wherein, if the test compound is not a PAD inhibitor, arginine is deiminated to citrulline by the PAD activity in the first incubation and trypsin activity will not cleave the peptide substrate I in the second incubation, and wherein, if the test compound is a PAD inhibitor, arginine is not deiminated to citrulline by the PAD activity in the first incubation and trypsin activity will not cleave the peptide substrate I in the second incubation; and monitoring for a corresponding decrease in FRET acceptor fluorescence emission or the increase in donor fluorescence emission.

6. The method of claim 5, wherein the step of selecting of a suitable fluorescence donor D and fluorescence acceptor A comprises the step of determining spectral overlap and Forster distance of D and A.

7. A peptide substrate for an assay for the measurement of PAD activity comprising the formula (I):

A-(X)_rArg-(Y)_rD

I

wherein

(X)i represents a sequence of i amino acids and (Y)j represents a sequence of j amino acids, i and j are integers defining the number of amino acids in X and Y respectively.

A is an acceptor fluorophore and D is a donor fluorophore; and

(X)i and (Y)_j may contain any amino acids in any order with the following exceptions: lysine at all positions unless followed immediately to the right by proline, proline in the j = 1 position, and arginine, in addition to the one arginine shown between (X)j and (Y)j, unless it is followed immediately by a proline.

8 The peptide substrate of claim 7, wherein the sum of i and j is less than or equal to about 100.

9. The peptide substrate of claim 7, wherein a suitable donor and acceptor fluorophore pair is selected from the group consisting of:

5-TAMRA/QSY-7

Dansyl / Eosin

Tryptophan / Dansyl

Fluorescein / Texas Red (rhodamine)

Naphthalene/Dansyl

Dansyl / ODR

BODIPY / BODIPY

Terbium / Thodamine

Dansyl / FITC

Pyrere / Coumarin

IAEDANS / IAF

BPE / Cy5

Europium / Cy5

Europium / APC

FITC / TMR

IAEDANS / FITC

IAF / TMR IAF / EIA

Tryptophan / IAEDANS

10. The peptide substrate of claim 7, wherein A and D are separated by a distance of between 10 and 100 angstroms.

11. The peptide substrate of claim 7, wherein the peptide substrate is formula (IA).

QSY-7-HQSTRGSGHC-5-TAMRA-KK-NH2

IA