US20030166037A1

US20030166037A1 - Aggrecanase-1 and -2 peptide substrates and methods

Info

Publication number: US20030166037A1
Application number: US10/050,200
Authority: US
Inventors: Anne Fourie; Lars Karlsson; Fawn Coles
Original assignee: Ortho McNeil Pharmaceutical Inc
Current assignee: Janssen Pharmaceuticals Inc
Priority date: 2002-01-16
Filing date: 2002-01-16
Publication date: 2003-09-04
Also published as: ES2339758T3; CA2473513A1; EP1474509A4; WO2003062263A2; EP1474509A2; WO2003062263A3; AU2003203028A1; CA2473513C; EP1474509B1; US7312045B2; DE60331414D1; US20050164319A1; ATE458810T1

Abstract

The present invention describes synthetic peptide substrates of the metalloproteases, agggrecanase-1 and/or -2 suitable for assays of enzyme activity. The invention also describes methods using these peptides to discover pharmaceutical agents that modulate these proteases.

Description

FIELD OF THE INVENTION

The present invention describes synthetic peptide substrates of the metalloproteases, aggrecanase-1 and/or -2, suitable for use in assays of enzyme activity. The invention also describes methods using these peptides to discover pharmaceutical agents that modulate these proteases.

BACKGROUND OF THE INVENTION

The disintegrin metalloprotease (or ADAM) family of cell surface proteolytic enzymes is known to play roles in sperm-egg binding and fusion, muscle cell fusion, neurogenesis, modulation of Notch receptor and ligand processing, and processing of the pro-inflammatory cytokine, TNFα (Primakoff and Myles, Trends Genet 16:83-87, 2000). The ADAMs have been shown to consist of pre-, pro-, protease, disintegrin-like-, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains. Members of a novel sub-family of the ADAMs, the ADAMTS proteins, lack the transmembrane domain and contain unique thrombospondin motifs, believed to mediate their binding to the extracellular matrix (Tang and Hong, FEBS Lett. 445:223-225, 1999). Two members of the ADAMTS family, namely ADAMTS-4 and -5 (also referred to as ADAMTS-11), have been shown to be capable of aggrecan cleavage. Aggrecan is the major proteoglycan of cartilage (Abbaszade et al., J. Biol. Chem. 274:23443-23450, 1999; Tortorella et al., Science 284:1664-1666, 1999). As a result, these proteins have been implicated in the cartilage damage associated with osteoarthritis and inflammatory joint disease, and have been named “Aggrecanase-1” (Genbank Accession NM 005099) and “Aggrecanase-2” (Genbank NM 007038), respectively.

Aggrecanases and MMPs have been shown to cleave aggrecan at a number of different sites (Pratta et al., J. Biol. Chem. 275:39096-39102, 2000; Sandy et al., Biochem. J. 351:161-166, 2000; Tortorella et al., J. Biol. Chem. 275:18566-18573, 2000). Products resulting from cleavage of aggrecan at the site Glu373-Ala374, in the interglobular domain of aggrecan, have been shown to accumulate in synovial fluid of patients with osteoarthritis and inflammatory joint disease (Lohmander et al, Arthritis Rheum. 36:1214-22, 1993). Aggrecanase-1 and -2, but not MMPs, are able to cleave aggrecan at this site. A 40 amino acid peptide representing the sequence of aggrecan surrounding the aggrecanase cleavage site (PCT Publication Number WO 00/05256) was able to serve as a substrate for aggrecanase enzymatic activity; however, no peptides less than 40 amino acids in length functioned as suitable substrates for aggrecanase activity, suggesting that shorter substrates, such as substrates of 20 amino acids in length, would not work. Minimum size limits for aggrecanase substrates are consistent with studies suggesting that aggrecanase activity is sensitive to the amino terminal truncation of aggrecan (Horber et al., Matrix Biol. 19:533-543, 2000). Glycosylation of the aggrecan substrate has also been shown to affect aggrecanase activity (Pratta et al., J. Biol. Chem. 275:39096-39012, 2000).

A sensitive and specific assay for the aggregan degrading metalloproteases, suitable for high-throughput screening, would be helpful in identifying inhibitors of these enzymes for potential therapeutic agents against cartilage damage associated with osteoarthritis and inflammatory joint disease. This invention relates to amino acid peptides shorter than 40 amino acids, unrelated to the aggrecan sequence, but containing aggrecanase sensitive sites, and their use in assays suitable for HTS formats.

SUMMARY OF THE INVENTION

The present invention relates to peptides less than 40 amino acids in length having a cleavage site between a glutamic acid on the N-terminal side of the cleavage site and a non-polar or uncharged residue on the C-terminal side of the cleavage site and wherein the peptide is cleavable by an enzyme having an amino acid sequence of SEQ ID NO:8 (Aggrecanase-1) and/or SEQ ID NO:9 (Aggrecanase-2). In one aspect of this embodiment, the peptide comprises the amino acid sequence of SEQ ID NO:3 and SEQ ID NO:4. Preferably the peptide is of natural or synthetic origin. In a preferred aspect of this embodiment, the peptide comprises a detectable label selected from the group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, a fluorescent dye, or a colorimetric indicator. The peptide preferably also comprises a fluorophore and a quencher or acceptor located at opposite ends of the cleavage site of the peptide. In one embodiment, the peptide further comprises an affinity moiety located at opposite ends of the cleavage site of the peptide.

In another embodiment, the invention relates to a method to identify a compound that inhibits Aggrecanase enzymatic activity comprising the steps of: contacting a test compound, an Aggrecanase, and a peptide less than 40 amino acids in length wherein the peptide comprises a cleavage site between a glutamic acid on the N-terminal side of the cleavage site and a non-polar or uncharged amino acid residue on the C-terminal side of the cleavage site and wherein the peptide is cleavable by an enzyme having the amino acid sequence of SEQ ID NO:8.; and detecting cleavage of the peptide, wherein inhibition of peptide cleavage in the presence of a test compound indicates compound inhibition of Aggrecanase enzymatic activity. In a preferred aspect of this embodiment, the method is performed in a single reaction vessel. Preferably the enzyme is selected from the group consisting of Aggrecanase-1 or Aggrecanase-2. Preferably the peptide is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6 and SEQ ID NO:7. Preferably the peptide further comprises a detectable label selected from the group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, a fluorescent dye, or a colorimetric indicator. The peptide preferably further comprises a fluorophore and a quencher or acceptor located at opposite ends of the cleavage site of the peptide. In one aspect of this embodiment, the contacting step further comprises a cell expressing the Aggrecanase.

In another aspect of this invention, the invention relates to a method to detect the ability of a compound to inhibit Aggrecanase-1 or -2 enzymatic activity comprising the steps of: contacting a test compound, an Aggrecanase secreted by a cell, and a peptide having an amino acid sequence selected from the group consisting of SEQ.ID.NO.:3 or SEQ.ID.NO.:4; incubating the compound, enzyme, and peptide to permit enzymatic cleavage of the peptide; and measuring enzymatic cleavage of the peptide wherein the method is conducted in a single reaction vessel without further manipulation. Preferably the peptide comprises a detectable label selected from the group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, a fluorescent dye, or a calorimetric indicator. Also preferably, the peptide comprises a fluorophore and a quencher or acceptor located at opposite ends of the cleavage site of the peptide.

In yet another aspect of this invention, the invention relates to a method to identify a compound capable of inhibiting Aggrecanase activity comprising the steps; providing a peptide comprising an affinity moiety, an amino acid sequence selected from a group consisting of SEQ.ID.NO.:3 SEQ.ID.NO.:4 and a detectable label, said affinity moiety and label located on opposite sides of a cleavage site encoded by the amino acid sequence; contacting the peptide with an affinity capture coated solid phase support for sufficient time to bind a portion of the peptide; washing the support to remove unbound peptide; contacting a solution comprising a test compound and functional enzyme with the peptide bound solid phase support for sufficient time to allow enzymatic cleavage of the peptide, thereby releasing the peptide and detectable label into the solution; and measuring changes in the quantity of the detectable label as a result of compound modulation of expected enzymatic function. Preferably the enzyme is selected from the group consisting of Aggrecanase-1 and -2. Also preferably the peptide comprises a detectable label selected from the group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, a fluorescent dye, or a colorimetric indicator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the domain structures of (A) full-length Aggrecanase-1 protein, (B) full-length Aggrecanase-2 protein and (C) the recombinant truncated forms used in a preferred protease assay of this invention. [0009]
FIG. 2 illustrates the relative activities of Aggrecanase-1(A) and -2 (B) for 56 different FRET peptides, A1 to H7. In FIG. 2, every other peptide is numbered. [0010]
FIG. 3 provides the kinetic analysis of the relative affinities of Aggrecanase-2 for cleavage of 2 different peptides [0011]
FIG. 4 illustrates the use of the Aggrecanase-1 and -2 peptide cleavage assays to identify inhibitory compounds. FIG. 4A is a comparison of inhibition of Aggrecanase-1 proteolytic activity by compounds A and B. FIG. 4B provides the IC50 analysis for inhibition of Aggrecanase-2 by inhibitory compounds, A, B and C.[0012]

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of this invention, the invention relates to peptide substrates useful to measure the enzymatic activity of Aggrecanase-1 and/or -2 metalloproteases. Using the peptide substrates identified in this invention it is possible to find others that are capable of being cleaved by the preferred truncated Aggrecanase-1 and -2 enzymes of this invention. Preferred recombinant truncated forms of human Aggrecanase-1 and -2 (i.e., Aggrecanase lacking some portion of the complete native sequence), in this invention were creating using the pro- and protease domains and optionally included a FLAG epitope tag, as provided in schematic in FIG. 1 (and provided as nucleic acid encoding the truncated Aggrecanse, see SEQ ID Nos: 1 and 2 respectively). These recombinant truncated enzymes were produced from Sf9 cells infected with a recombinant baculovirus construct, and purified by affinity chromatography. A number of substrates were identified by screening a collection of 56 potential peptide substrates. Two different peptide sequences were found that were particularly preferred for their ability to be cleaved by Aggrecanase-2. One peptide sequence was a good substrate for both Aggrecanase-1 and Aggrecanase-2. This latter peptide was used to optimize an assay in a format suitable for high throughput screening, which was then used for the identification of small molecule inhibitors of Aggrecanase-1 and -2 as potential therapeutic compounds. [0013]
The amino acid sequence of the most preferred peptides is provided in single letter code in Table 1. [0014]

TABLE 1

Relative activities of AGGRECANASE-1 AND -2 for 2 different FRET peptides

Relative proteolytic

Activity

SEQ ID NO: Peptide name Peptide sequence Agg-1 Agg-2

3 FasL1 Aedans-E -KELAELRESTS-Dabcyl-K * *****

4 29CD23 Aedans-E -ADLSSFKSQEL-Dabcyl-K n.d. *****
These peptides and the other peptides of this invention demonstrating aggrecanase substrate activity are useful in assays to discover new pharmaceutical drugs that alter the activity of Aggrecanase-1 and/or -2. [0015]
The invention also relates to assays using the peptides of this invention to detect compounds that inhibit Aggrecanase enzymatic activity. In one aspect of this embodiment, the assay is a homogeneous in vitro protein-based assay to detect compound modulation of Aggrecanase-1 and/or -2 enzymatic activity. [0016]
The term “homogeneous” refers to an assay conducted in a single vessel where there is no further reagent manipulation after the reaction reagents are placed in a vessel. A preferred method comprises the steps of; [0017]
1) combining a test compound with an Aggrecanase and a peptide substrate, [0018]
2) incubating the compound, enzyme, and substrate for a time sufficient to detect substrate cleavage; and [0019]
3) detecting substrate cleavage. [0020]
In a preferred embodiment, the detecting step comprises detecting a change in the level of substrate cleavage. Preferably the change in the level of substrate cleavage is compared to the change in the level of substrate cleavage in a reaction vessel containing Aggrecanase and peptide substrate in the presence of a control test compound that has a known capacity or no capacity to inhibit Aggrecanase activity or alternatively in a reaction vessel without test compound. [0021]
In a preferred embodiment, the peptide substrate is selected from SEQ ID NO:3 (E5 in FIG. 2) or SEQ ID NO:4 (G7 in FIG. 2). [0022]
Other preferred peptides that can serve as peptides substrates in the assays of this invention for Aggrecanase-2 include, but are not limited to: [0023]

ID from SEQ

Sequence ID NO

G1 Aedans-EKARVLAEAADabcyl-Kamide 5

B3 Aedans-EKARVLAEAMDabcyl-Kamide 6

C7 Aedans-ERABQQRLKSQDLDabcyl-Kamide 7
Still other peptides tested are provided in Table II. In addition, a variety of peptides can also serve as substrates for Aggrecanase-1 and/or -2 activity. For example, the present set of peptide substrates was selected by identifying other protease substrates known in the art. The peptides included a collection of substrates for other proteases, as well as a number of sequences corresponding to membrane proximal cleavage sites of various proteins postulated to be released by metalloproteases (including those published by Roghani et al., [0024] J. Biol. Chem. 274:3531-340, 1999) for ADAM9/MDC9). Thus, those of ordinary skill in the art could similarly identify other substrates and test them in the assays of this invention using a truncated Aggrecanase as contemplated here.
The term “Aggrecanase” as used herein refers to a truncated enzyme (as shown in FIG. 1) that displays enzymatic cleavage of a peptide substrate, and for which the corresponding full-length enzyme is known to have the capacity to cleave aggrecan. Efficient cleavage of aggrecan depends on multiple interactions between the enzyme and aggrecan. For example, cleavage depends on an intact N-terminal portion of the substrate, aggrecan (Horber et al., [0025] Matrix Biology 19:533-543, 2000). Tortorella et al. (J. Biol. Chem. 275:25791-25797, 2000) showed that cleavage of aggrecan was dependent on the thrombospondin motif in the enzyme, Aggrecanase-1, although both full-length and truncated Aggrecanase-1 could cleave a peptide substrate (quoted as unpublished data). Currently known Aggrecanases are Aggrecanase-1 and -2 (Genbank Accession Nos. NM 005099 and NM 007038 respectively). Nucleic acid encoding the truncated versions of these enzymes used in the assays of this invention are provided here as SEQ ID NOS:1 and 2, corresponding to truncated Aggrecanase-1 and truncated Aggrecanase-2, respectively.
While the Aggrecanases used in this invention are truncated forms of a full length native Aggrecanase provided by the GenBank citations above, other Aggrecanases can be used in this invention as long as they retain their ability to cleave exemplarly peptides SEQ ID NO:3 and SEQ ID NO:4. The Aggrecanases used in this invention can be full length, partial, truncated, chimeric or modified enzymes that still retain their ability to cleave the peptides as described in this invention. It has been demonstrated that Aggrecanase cleavage sites in aggrecan contain glutamic acid on the N-terminal side of the cleavage site (P1 position) and a non-polar or uncharged residue on the C-terminal side of the cleavage site (P1′ position), namely alanine, leucine or glycine (Caterson et al., [0026] Matrix Biology 19:333-344, 2000; Tortorella et al., J. Biol. Chem. 275 18566). As shown later under Kinetic Analysis in Example 2, the truncated Aggrecanase-2 used in the assays described here cleaves the peptides of SEQ ID NOS: 3 and 4 between glutamic acid and leucine residues, consistent with the cleavage specificity of aggrecan cleavage sites.
The term “compound” is used herein in connection with a small molecule, preferably an organic molecule that has the potential to disrupt the specific enzymatic activity of the enzyme. For example, but not to limit the scope of the current invention, compounds may include small organics, synthetic or natural amino acid peptides, proteins, synthetic or natural nucleic acid sequences, or any chemical derivatives of the aforementioned. The term “chemical derivative” describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as [0027] Remington: The Science and Practice of Pharmacy. 1995. Mack Publishing Co. ISBN 0912734051.
The methods described herein are especially useful for high throughput screening (HTS) of compounds to discover compounds that modulate Aggrecanase function. The term “high throughput” refers to an assay design that allows easy analysis of multiple samples simultaneously, and capacity for robotic manipulation. Preferred assays are homogeneous assays. Preferred assays also include assay designs that are optimized to reduce reagent usage in order to achieve the analysis desired. The methods described herein demonstrate highly robust performance and good linearity as a function of enzyme concentration and substrate concentration. For example in the assays of the present invention, at appropriately adjusted enzyme and substrate concentrations, the assay was linear for up to four hours. From FIG. 4A, it can be seen that for kinetic analysis, the signal-to-noise ratio was effectively infinite, as no change in the background (blank, no enzyme) was observed over the time of the assay. For endpoint measurements, the enzyme and substrate concentrations can be adjusted to achieve the desired signal-to-noise ratio. In the example in FIG. 4A, it can be seen that this ratio (control versus blank endpoints) was approximately three. Therefore the amount of reagent used can be varied to utilize a minimum of expensive reagent, such as a recombinant enzyme. [0028]
Examples of assay formats include 96-well or 384-well plates, levitating droplets, and “lab on a chip” microchannel chips used for liquid handling experiments. For example, capillary electrophoresis (CE)-based assays for the activity of proteases have been developed. In this type of system, the assays can be carried out in small volumes (<51 μl). Here both the fluorescent-labeled substrate and product can be monitored by laser-induced fluorescence, based on the ability of CE to rapidly separate the two species. [0029]
It is well known to those in the art that as miniaturization of plastic molds and liquid handling devices are advanced, or as improved assay devices are designed, that greater numbers of samples may be performed using the design of the present invention. Such new assay designs will not limit the scope of the intended assay. [0030]
In another embodiment of the invention, the present invention provides a homogeneous in vitro cell-based method to detect compound modulation of Aggrecanase enzymatic activity. In this embodiment, the cells express Aggrecanase and the peptide substrate and test compound are in contact with Aggrecanase. Aggrecanase is preferably released extracellularly. In a preferred embodiment, the Aggrecanase is an [0031] Aggrecanase 1 or an Aggrecanase 2. The method comprises the steps of:
1) combining a test compound, a cell expressing Aggrecanase, and a peptide substrate; and [0032]
2) detecting enzymatic cleavage of the peptide substrate. [0033]
Alternatively the assays of this invention could be made non-homogeneous. That is, the assay could be modified to require more than one vessel or a wash step requiring that all events to do not take place in a single reaction sample. Such assays can involve, for example, the immobilization of the substrate peptide. One example is the use of an affinity moiety-affinity capture pair such as streptavidin capture of a biotinylated substrate peptide. Affinity capture pairs are well known in the art and include, for example, avidin/biotin, antibody capture of a region of the substrate peptide, and polyhistidine/immobilized nickel. A preferred non-homogeneous method comprises the steps of: [0034]
1) providing a substrate peptide comprising an affinity moiety, an Aggrecanase cleavage site, and a detectable label, said affinity moiety and label located on opposite sides of the cleavage site; [0035]
2) contacting the substrate peptide with an affinity capture coated solid phase support for sufficient time to bind a portion of the peptide; [0036]
3) washing the support to remove unbound peptide; [0037]
4) contacting a solution comprising a test compound and Aggrecanase enzyme with the peptide bound solid phase support for sufficient time to allow enzymatic cleavage of the substrate, thereby releasing the substrate and detectable label into the solution; and [0038]
5) measuring changes in the quantity of the detectable label as a result of compound modulation of expected Aggrecanase enzymatic function. [0039]
In one embodiment, the Aggrecanase is [0040] Aggrecanase 1 and/or 2. In another embodiment, the solution is transferred to a reaction vessel prior to the measuring step. The terms solid phase support, affinity capture, unbound versus bound peptide, and the like are all well-known terms to those of ordinary skill in the art to who this invention pertains and therefore these definitions will not be repeated here.
A change in the quantity of product can be expressed as the total amount of product changing over time (a stop-time assay) or can be kinetic where a change in the enzymatic rate is measured as a function of time. Kinetic assays are preferably measured from the time of initial contact of the enzyme and substrate to a point in time where approximately 50% of the maximum observed product are generated. [0041]
The amount of expected Aggrecanase enzymatic activity can be determined by running, concurrently or separately, an assay using a compound that does not inhibit enzymatic function (i.e., a blank or a control compound), or with a solvent vehicle that has similar properties as that used for the test compound but lacks any test compound, such as DMSO, DMF, or isopropyl alcohol. [0042]
For cell-based assays, the amount of time necessary for cellular contact with the compound is empirically determined, for example, by running a time course with a known Aggrecanase modulator and measuring change as a function of time. [0043]
Cells useful in the cell-based Aggrecanase assays of this invention are those cells that naturally express Aggrecanase, or cells transfected with recombinant Aggrecanase. These cells may be immortalized cell lines or primary culture cells from any mammal, preferably murine, rat, rabbit, monkey, chimpanzee, or human. [0044]
Methods for detecting compounds that modulate Aggrecanase proteolytic activity comprise combining a test compound with an Aggrecanase protein and a suitable labeled substrate and detecting the ability of the enzyme to cleave the substrate in the presence of the compound. Enzymatic cleavage can result in release of the label or release of a labeled peptide fragment that can be distinguished from intact labeled peptide. In one example, the substrate is labeled. A variety of methods for exploiting labeled substrates are known in the art. Examples of different types of labeled substrates include, for example, substrate that is radiolabeled (Coolican et al., [0045] J. Biol. Chem. 261:4170-76, 1986), fluorometric (Twining, Anal. Biochem. 143:30-4, 1984) or colorimetric (Buroker-Kilgore and Wang, Anal. Biochem. 208:387-392, 1993) substrates.
Radioisotopes useful in the present invention include those well known in the art, specifically [0046] ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, and ³³P Radioisotopes are introduced into the peptide by conventional means, such as iodination of a tyrosine residue, phosphorylation of a serine or threonine residue, or incorporation of tritium, carbon or sulfur utilizing radioactive amino acid precursors. Fluorescent resonance energy transfer (FRET)-based methods (Ng and Auld, Anal. Biochem. 183:50-6, 1989) can also be used to detect compounds that modulate Aggrecanase proteolytic activity. Compounds that are activators will increase the rate of substrate degradation resulting in a reduction in substrate as a function of time. Compounds that are inhibitors will decrease the rate of substrate degradation and will result in greater remaining substrate as a function of time.
A preferred assay format useful for the method of the present invention is a FRET-based method using peptide substrates that contain a fluorescent donor with either a quencher or acceptor that are separated by a peptide sequence encoding the Aggrecanase cleavage site. A fluorescent donor is a fluorogenic compound that can absorb energy and transfers a portion of the energy to another compound. Examples of fluorescent donors suitable for use in the present invention include, but are not limited to, coumarins, xanthene dyes such as fluoresceines, rhodols, and rhodamines, resorufins, cyanine dyes bimanes, acridines, isoindols, dansyl dyes, aminophthalic hydrazides such as luminol and isoluminol derivatives, aminonapthalimides, aminobenzofurans, aminoquinolines, dicanohydroquinones, and europium and terbium complexes and related compounds. A quencher is a compound that reduces the emission from the fluorescent donor when it is appropriately proximally located to the donor. Preferred quenchers do not generally re-emit the energy in the form of fluorescence. Examples of quenching moieties include indigos, benzoquinones, anthraquinones, azo compounds, nitro compounds, indoanilines, and di- and triphenylmethanes. [0047]
A FRET method using a donor/quencher pair measures increased emission from the fluorescent donor as a function of Aggrecanase enzymatic activity upon the peptide substrate. Therefore a test compound that antagonizes Aggrecanase will generate an emission signal between two control samples—a low (basal) fluorescence from the FRET peptide alone and a higher fluorescence from the FRET peptide digested by the activity of enzymatically active Aggrecanase. An acceptor is a fluorescent molecule that absorbs energy from the fluorescent donor and re-emits a portion of the energy as fluorescence. An acceptor is a specific type of quencher that enables a separate mechanism to measure Aggrecanase proteolytic efficacy. Methods that use a donor/acceptor pair measure a decrease in acceptor emission as a function of Aggrecanase enzymatic activity upon the peptide substrate. Therefore a test compound that antagonizes Aggrecanase will generate an emission signal between two control samples—a higher basal fluorescence from the FRET peptide alone and a lower fluorescence from the FRET peptide digested by the activity of enzymatically active Aggrecanase. Examples of acceptors useful in the methods of the present invention include, but are not limited to, coumarins, fluoresceins, rhodols, rhodamines, resorufins, cyanines, difluoroboradiazindacenes, and phthalcyanines. FRET peptides can also be used for zymography (see PCT publication number WO 01/94377 to Fourie et al.) following SDS polyacrylamide gel electrophoresis. [0048]
The following examples illustrate the present invention without, however, limiting the same thereto. All references are incorporated herein by reference. [0049]

EXAMPLE 1

Generation of Truncated Recombinant Enzyme

Aggrecanase proteins usually comprise: an N-terminal pro-domain and a metalloprotease domain, followed by the disintegrin domain, cysteine-rich domain, epidermal growth factor repeat, thrombospondin repeats and a spacer region, as illustrated in FIG. 1. For production of biologically active and soluble ADAMTS proteins (Aggrecanase-1 and -2), PCR products containing the pro- and protease domains and a C-terminal FLAG epitope (used for immuno-detection and purification) were cloned into pFastBac1 (GibcoBRL) vectors using standard techniques. The DNA sequences of truncated [0050] Aggrecanase 1 and 2 used in the methods of this invention are provided as SEQ ID NOS:1 and 2 respectively. The protein sequences corresponding to these DNA sequences are provided as SEQ ID NOS: 8 and 9.
In order to generate large quantities of protein for biological testing and assay development, Sf9 cells were infected with pFastBac (GibcoBRL) containing the coding sequences for truncated Aggrecanase-1 or -2. [0051]
Recombinant baculovirus for truncated Aggrecanase-1 or -2 expression was generated from the pFastBac1 construct described above using the Bac-to-Bac system (Gibco BRL). Sf9 cells were infected with baculovirus and the medium was collected after 72 hours. The medium was concentrated 10-fold by ultrafiltration, and exchanged to TBS (Tris Buffered Saline) by repeated addition and re-concentration. The supernatant was centrifuged for one hour at 15000×g, filtered through a 0.45 μM filter to remove debris, and incubated, with mixing, overnight at 4° C. with M2-αFlag-agarose (Sigma). The resin was loaded into a column and washed with TBS, followed by elution of the bound material with 0.1M Glycine (pH 3.5) and immediate neutralization by addition of 12.5 μl/ml of 2M Tris-HCl, [0052] pH 8. The supernatant from the infection (before and after incubation with M2-αFlag-agarose) and fractions from the purification were analyzed by SDS-PAGE followed by staining and Western blotting. By SDS-PAGE, fractions containing the immunopurified Aggrecanase-1 or -2 protein contained a protein band with an apparent molecular weight of about 30 kDa. Western analysis indicated that the M2αFlag (Sigma) antibody identified a 30 kDa band in the infection supernatant before, but not after, anti-FLAG agarose adsorption. The immunoreactive protein was also present in eluted fractions. This protein was then used to test potential substrate peptides.

EXAMPLE 2

Fret Assay: Peptide Substrate Screening

Fifty-six different peptides were synthesized to test for protease activity (see Table 3 below). The peptides included a collection of substrates for other proteases, as well as a number of sequences corresponding to membrane proximal cleavage sites of various proteins postulated to be released by metalloproteases (including those published by (Roghani et al., [0053] J. Biol. Chem. 274:3531-340, 1999) for ADAM9/MDC9). In order to use the principle of fluorescence resonance energy transfer, or FRET, the peptides were labeled at the C-terminus with Dabcyl and at the N-terminus with Aedans (or vice versa). Thus cleavage of the peptides were monitored by the increase in Aedans fluorescence at 460 nm (excitation 360 nm) as a result of the decrease in proximity of the Dabcyl quencher. The assay was performed by diluting the Aggrecanase-1 (approximately 2.5 to 5 μg of protein, 85 to 167 picomoles, SEQ ID NO:8) or Aggrecanase-2 (approximately 0.5 to 1 μg of protein, 17 to 33 picomoles, SEQ ID NO:9), in assay buffer (50 mM HEPES pH 7.5, 10 mM CaCl₂, 0.1M NaCl and 0.05%(w/v) Brij-35 detergent (Sigma).
The reaction was initiated by the addition of peptide substrate to a final concentration of 100 uM for Aggrecanase-1 and 50 uM for Aggrecanase-2. The assays were typically run for 60 minutes at room temperature and the slope of the kinetic increase in fluorescence was determined to calculate the rate of the reaction. [0054]
FIG. 2 illustrates the relative activities for the 56 different peptides, A1 to H7 (only every alternate peptide is numbered in FIG. 2) expressed in arbitrary, but relative units. Aggrecanase-1 and -2 both showed the highest activity for peptide E5 (FasL1). Aggrecanase-2, but not Aggrecanase-1, also showed high activity for cleavage of peptide G7 (29CD23). Peptide D7 (16 amino acids) corresponds to the sequence within aggrecan containing the Glu373-Ala374 aggrecanase cleavage site. Neither Aggrecanase-1 nor Aggrecanase-2 showed any activity on this peptide, consistent with findings that peptides corresponding to this region of aggrecan, and shorter than 40 amino acids do not function as substrates for aggrecanases (PCT Publication Number WO 00/05256; Horber et al., [0055] Matrix Biology 19:533-543, 2000).
Peptide E5 (SEQ ID NO:3) was also shown in similar screening assays to be a suitable substrate for the metalloproteases MMP7 and MMP 13 (Chemicon, Cat. #CC1059 and CC068 respectively). [0056]
Kinetic Analysis of the Affinity of Aggrecanase-1 and -2 for Cleavage of 4 Different Peptides [0057]
To confirm the screening assay, Aggrecanase-2 was further analyzed for its rate of catalysis using 2 different peptides. The assay was performed by diluting the Aggrecanase-2 in assay buffer (50 mM HEPES pH 7.5, 10 mM CaCl[0058] ₂, 0.1M NaCl and 0.05% Brij-35). As illustrated in FIG. 3, the reaction was initiated by the addition of substrate (FasL1 or 29CD23) to different final concentrations for analysis of affinities. The assay was run for 60 minutes at room temperature. FIG. 3 illustrates the proteolytic activity (in relative fluorescence units per minute) as a function of peptide concentration for peptides FasL1 and 29CD23. The curves were fitted to the data with the program Grafit (Erithacus Software Lmited). The results of these analyses are provided in Table 2. The Vmax and Km for each substrate were calculated by non-linear fitting of the data. The cleavage site for Aggrecanase-2 within each peptide was determined by LC-MS analysis to be between a glutamic acid and leucine residues in each case, as indicated in Table 2 by a carot within each peptide sequence. These results indicate that the cleavage by the truncated Aggrecanase-2 has the same specificity as the full-length enzyme, namely glutamic acid in the P1 position and a non-polar residue in the P1′ position. However, these are clearly not the only requirements for efficient cleavage, as a number of the 56 peptides tested have similar residues and were not cleaved by the aggrecanases.

TABLE 2

K_mand V_mof Aggrecanase-2 for peptides

(X = Aedans-E; Z = Dabcyl-K; rfu = relative fluorescence units)

PEPTIDE CLEAVAGE SITE K_m V_m

FasL1 X-KELAE{circumflex over ( )}LRESTS-Z 80 μM 2.8 rfu/min

29CD23 X-ADLSSFKSQE{circumflex over ( )}L-Z 40 μM 0.6 rfu/min

TABLE 3


WELL	SEQUENCE	SEQ. ID NO.

A1	(Aedans)EHSDAVFTDNYTR(Dabcyl)K-amide	10

B1	(Aedans)EAEN(Dabcyl)K-amide	11

C1	(Aedans)EGRHIDNEEDI(Dabcyl)K-amide	12

D1	(Aedans)EGNAFNNLD(Dabcyl)K-amide	13

E1	(Aedans)EYTPNNEIDSF(Dabcyl)K-amide	14

F1	(Aedans)EQLRMKLP(Dabcyl)K-amide	15

G1	(Aedans)EKARVLAEAA(Dabcyl)K-amide	5

H1	(Aedans)ERGFFYTP(Dabcyl)K-amide	16

A2	(Aedans)EVTEGPIP(Dabcyl)K-amide	17

B2	(Aedans)EPLFYEAP(Dabcyl)K-amide	18

C2	(Aedans)ELPMGALP(Dabcyl)K-amide	19

D2	(Aedans)EKPAALFFRL(Dabcyl)K-amide	20

E2	(Aedans)ELYENKPRRPYIL(Dabcyl)K-amide 21

F2	(Aedans)ESEVNLDAEF(Dabcyl)K-amide	22

G2	(Aedans)ESQNYPIVQ(Dabcyl)K-amide	23

H2	(Aedans)EKPIEFFRL(Dabcyl)K-amide	24

A3	(Aedans)EKPAEFFAL(Dabcyl)K-amide	25

B3	(Aedans)EKARVLAEAM(Dabcyl)K-amide	6

C3	(Aedans)EKPAKFFRL(Dabcyl)K-amide	26

D3	R(Aedans)EIPFHLVIHT(Dabcyl)KR	27

E3	(Aedans)EMAPGAVHLPQ(Dabcyl)K-amide	28

F3	(Aedans)EPLAQAVRSSS(Dabcyl)K-amide	29

G3	(Aedans)EPPVAASSLRN(Dabcyl)K-amide	30

H3	(Aedans)EPQIENVKGTE(Dabcyl)K-amide	31

A4	(Aedans)ESLPVQDSSSV(Dabcyl)K-amide	32

B4	(Aedans)EVHHQKLVFFA(Dabcyl)K-amide	33

C4	(Dabcyl)KRGVVNASSRLAK(Aedans)E-amide	34

D4	(Dabcyl)KLVLASSSF(Aedans)E-amide	35

E4	(Dabcyl)KSNRLEASSRSSP(Aedans)E-amide	36

F4	(Aedans)EDEMEE(Abu)ASHLPY(Dabcyl)K-amide	37

G4	(Aedans)EAGPRGMAGQFSH(Dabcyl)K-amide	38

H4	(Dabcyl)KRPLGLAR(Aedans)E-amide	39

A5	(Aedans)EGYYSRDMLV(Dabcyl)K-amide	40

B5	(Aedans)EQKLDKSFSMI(Dabcyl)K-amide	41

C5	(Aedans)EPSAAQTARQTTP(Dabcyl)K-amide	42

D5	(Aedans)EPGAQGLPGVG(Dabcyl)K-amide	43

E5	(Aedans)EKELAELRESTS(Dabcyl)K-amide	3

F5	(Dabcyl)GLRTNSFS(Aedans)	44

G5	(Dabcyl)RGVVNASSRLA(Aedans)	45

H5	Ac-ED(Aedans)KPILFFRLGK(Dabcyl)E-amide	46

A6	(Aedans)EMHTASSLEKQIG(Dabcyl)K-amide	47

B6	(Aedans)ERFAQAQQQLP(Dabcyl)K-amide	48

C6	(Aedans)EKKENSFEMQGDQ(Dabcyl)K-amide	49

D6	(Dabcyl)LAQAVRSSSR(Aedans)	50

E6	(Aedans)ERTAAVFRP(Dabcyl)K-amide	51

F6	(Aedans)ERVRRALP(Dabcyl)K-amide	52

G6	(Aedans)ESFPRMFSD(Dabcyl)K-amide	53

H6	(Aedans)EEYLESFLERP(Dabcyl)K-amide	54

A7	(Aedans)ERPKPQQFFGLM(Dabcyl)K-amide	55

B7	(Aedans)EHGDQMAQKSQST(Dabcyl)K-amide	56

C7	(Aedans)ERAIEQQRLKSQDL(Dabcyl)K-amide	7

D7	(Aedans)ERNITEGEARGSVIL(Dabcyl)K-amide	57

E7	(Aedans)EAGQRLATAM(Dabcyl)K-amide	58

F7	(Aedans)EVGLMGKLRALNS(Dabcyl)K-amide	59

G7	(Aedans)EADLSSFKSQEL(Dabcyl)K-amide	4

H7	(Aedans)EKEDGEARASTS(Dabcyl)K-amide	60

EXAMPLE 3

Drug Screening Assay

Aggrecanase-1 (2.5 to 5 μg of protein, 85 to 167 picomoles) was diluted in assay buffer (50 mM HEPES pH 7.5, 10 mM CaCl[0060] ₂, 0.1M NaCl, 0.05% Brij-35). Samples were prepared containing putative inhibitors A (Chen et al. Biorg. Med. Chem. Lett. 6(13):1601-1606, 1996) or B (Bailey, et al. Biorg. Med. Chem. Lett. 9(21):3165-3170, 1999), shown below, at a final concentration of 7.5 micromolar. The final %DMSO in the assay was 3% and it was determined experimentally that this concentration was not detrimental to the activity of the enzyme. The reaction was initiated by the addition of FasL1 peptide substrate to a final concentration of 225 μM and readings were taken at one-minute intervals, for a total of 200 minutes at room temperature.
The assay was always performed at enzyme and substrate concentrations where the activity was linearly related to enzyme concentration and where the increase in fluorescence (reaction rate) was linear for at least the time of the assay. From FIG. 4A, it can be seen that for kinetic analysis, the signal-to-noise ratio is effectively infinite, as no change in the background (blank, no enzyme) is observed over the time of the assay. For endpoint measurements, the enzyme and substrate concentrations could be adjusted to achieve the desired signal-to-noise ratio. In the example in FIG. 4A, it can be seen that this ratio (control versus blank endpoints) was approximately three. [0061]
FIG. 4A shows that inhibitors A and B completely inhibited Aggrecanase-1 enzyme activity (results are comparable to blank [no enzyme]). [0062]
IC50 Analysis for Inhibition of Aggrecanase-2 by Inhibitors A, B, and C [0063]
Aggrecanase-2 (0.5 to 1 μg of protein, 17 to 33 picomoles) was diluted in assay buffer (50 mM HEPES pH 7.5, 10 mM CaCl[0064] ₂, 0.1M NaCl, 0.05% Brij-35). Samples were prepared containing Inhibitor A, B or C (shown above) at final concentrations ranging from 0.1 to 12.5 μM (final DMSO concentration of 1.5%). Duplicate assays were run for each concentration of Inhibitor A, B and C (purchased from Peptides International, TAPI-0, Cat. No. INH 3850-P1) for 60 minutes at room temperature. The reaction was initiated by the addition of FasL1 peptide substrate to a final concentration of 225 μM. The reaction rates over 60 minutes at room temperature, in the absence (control) and presence of various concentrations of the inhibitor, were determined by linear regression of the data points. The reaction rate data in FIG. 4B were fitted by non-linear regression using the program Grafit (Erithacus Software). The IC50s for inhibition of Aggrecanase-2 by Inhibitors A, B and C, were 118±5, 38±8, and 102±23 nM, respectively.
1 60 1 1359 DNA Homo sapiens misc_feature (1)..(1359) truncated Aggrecanase 1 1 gaattcgcca tgtcccagac aggctcgcat cccgggaggg gcttggcagg gcgctggctg 60 tggggagccc aaccctgcct cctgctcccc attgtgccgc tctcctggct ggtgtggctg 120 cttctgctac tgctggcctc tctcctgccc tcagcccggc tggccagccc cctcccccgg 180 gaggaggaga tcgtgtttcc agagaagctc aacggcagcg tcctgcctgg ctcgggcacc 240 cctgccaggc tgttgtgccg cttgcaggcc tttggggaga cgctgctact agagctggag 300 caggactccg gtgtgcaggt cgaggggctg acagtgcagt acctgggcca ggcgcctgag 360 ctgctgggtg gagcagagcc tggcacctac ctgactggca ccatcaatgg agatccggag 420 tcggtggcat ctctgcactg ggatggggga gccctgttag gcgtgttaca atatcggggg 480 gctgaactcc acctccagcc cctggaggga ggcaccccta actctgctgg gggacctggg 540 gctcacatcc tacgccggaa gagtcctgcc agcggtcaag gtcccatgtg caacgtcaag 600 gctcctcttg gaagccccag ccccagaccc cgaagagcca agcgctttgc ttcactgagt 660 agatttgtgg agacactggt ggtggcagat gacaagatgg ccgcattcca cggtgcgggg 720 ctaaagcgct acctgctaac agtgatggca gcagcagcca aggccttcaa gcacccaagc 780 atccgcaatc ctgtcagctt ggtggtgact cggctagtga tcctggggtc aggcgaggag 840 gggccccaag tggggcccag tgctgcccag accctgcgca gcttctgtgc ctggcagcgg 900 ggcctcaaca cccctgagga ctcggaccct gaccactttg acacagccat tctgtttacc 960 cgtcaggacc tgtgtggagt ctccacttgc gacacgctgg gtatggctga tgtgggcacc 1020 gtctgtgacc cggctcggag ctgtgccatt gtggaggatg atgggctcca gtcagccttc 1080 actgctgctc atgaactggg tcatgtcttc aacatgctcc atgacaactc caagccatgc 1140 atcagtttga atgggccttt gagcacctct cgccatgtca tggcccctgt gatggctcat 1200 gtggatcctg aggagccctg gtccccctgc agtgcccgct tcatcactga cttcctggac 1260 aatggctatg ggcactgtct cttagacaaa ccagaggctc cattgcatct gcctgtgact 1320 ggggactaca aggacgacga tgacaagggg taggtcgac 1359 2 1516 DNA Homo sapiens misc_feature (1)..(1516) truncated Aggrecanse-2 2 gtcgacgcag cgcactatgc tgctcgggtg ggcgtccctg ctgctgtgcg cgttccgcct 60 gcccctggcc gcggtcggcc ccgccgcgac acctgcccag gataaagccg ggcagcctcc 120 gactgctgca gcagccgccc agccccgccg gcggcagggg gaggaggtgc aggagcgagc 180 cgagcctccc ggccacccgc accccctggc gcagcggcgc aggagcaagg ggctggtgca 240 gaacatcgac caactctact ccggcggcgg caaggtgggc tacctcgtct acgcgggcgg 300 ccgcaggttc ctcttggacc tggagcgaga tggttcggtg ggcattgctg gcttcgtgcc 360 cgcaggaggc gggacgagtg cgccctggcg ccaccggagc cactgcttct atcggggcac 420 agtggacggt agtccccgct ctctggctgt ctttgacctc tgtgggggtc tcgacggctt 480 cttcgcggtc aagcacgcgc gctacaccct aaagccactg ctgcgcggac cctgggcgga 540 ggaagaaaag gggcgcgtgt acggggatgg gtccgcacgg atcctgcacg tctacacccg 600 cgagggcttc agcttcgagg ccctgccgcc gcgcgccagc tgcgaaaccc ccgcgtccac 660 accggaggcc cacgagcatg ctccggcgca cagcaacccg agcggacgcg cagcactggc 720 ctcgcagctc ttggaccagt ccgctctctc gcccgctggg ggctcaggac cgcagacgtg 780 gtggcggcgg cggcgccgct ccatctcccg ggcccgccag gtggagctgc ttctggtggc 840 tgacgcgtcc atggcgcggt tgtatggccg gggcctgcag cattacctgc tgaccctggc 900 ctccatcgcc aataggctgt acagccatgc tagcatcgag aaccacatcc gcctggccgt 960 ggtgaaggtg gtggtgctag gcgacaagga caagagcctg gaagtgagca agaacgctgc 1020 caccacactc aagaactttt gcaagtggca gcaccaacac aaccagctgg gagatgacca 1080 tgaggagcac tacgatgcag ctatcctgtt tactcgggag gatttatgtg ggcatcattc 1140 atgtgacacc ctgggaatgg cagacgttgg gaccatatgt tctccagagc gcagctgtgc 1200 tgtgattgaa gacgatggcc tccacgcagc cttcactgtg gctcacgaaa tcggacattt 1260 acttggcctc tcccatgacg attccaaatt ctgtgaagag acctttggtt ccacagaaga 1320 taagcgctta atgtcttcca tccttaccag cattgatgca tctaagccct ggtccaaatg 1380 cacttcagcc accatcacag aattcctgga tgatggccat ggtaactgtt tgctggacct 1440 accacgaaag cagatcctgg gcggggacta caaggacgac gatgacaagg ggtagaagct 1500 tgtcgagaag tactag 1516 3 11 PRT Artificial Sequence peptide substrate 3 Lys Glu Leu Ala Glu Leu Arg Glu Ser Thr Ser 1 5 10 4 11 PRT Artificial Sequence Peptide substrate 4 Ala Asp Leu Ser Ser Phe Lys Ser Gln Glu Leu 1 5 10 5 10 PRT Artificial sequence Peptide substrate 5 Glu Lys Ala Arg Val Leu Ala Glu Ala Ala 1 5 10 6 10 PRT Artificial Sequence Peptide Substrate 6 Glu Lys Ala Arg Val Leu Ala Glu Ala Met 1 5 10 7 13 PRT Artificial Sequence Peptide substrate 7 Glu Arg Ala Glu Gln Gln Arg Leu Lys Ser Gln Asp Leu 1 5 10 8 447 PRT Homo sapiens MISC_FEATURE (1)..(447) truncated Aggrecanase 1 8 Met Ser Gln Thr Gly Ser His Pro Gly Arg Gly Leu Ala Gly Arg Trp 1 5 10 15 Leu Trp Gly Ala Gln Pro Cys Leu Leu Leu Pro Ile Val Pro Leu Ser 20 25 30 Trp Leu Val Trp Leu Leu Leu Leu Leu Leu Ala Ser Leu Leu Pro Ser 35 40 45 Ala Arg Leu Ala Ser Pro Leu Pro Arg Glu Glu Glu Ile Val Phe Pro 50 55 60 Glu Lys Leu Asn Gly Ser Val Leu Pro Gly Ser Gly Thr Pro Ala Arg 65 70 75 80 Leu Leu Cys Arg Leu Gln Ala Phe Gly Glu Thr Leu Leu Leu Glu Leu 85 90 95 Glu Gln Asp Ser Gly Val Gln Val Glu Gly Leu Thr Val Gln Tyr Leu 100 105 110 Gly Gln Ala Pro Glu Leu Leu Gly Gly Ala Glu Pro Gly Thr Tyr Leu 115 120 125 Thr Gly Thr Ile Asn Gly Asp Pro Glu Ser Val Ala Ser Leu His Trp 130 135 140 Asp Gly Gly Ala Leu Leu Gly Val Leu Gln Tyr Arg Gly Ala Glu Leu 145 150 155 160 His Leu Gln Pro Leu Glu Gly Gly Thr Pro Asn Ser Ala Gly Gly Pro 165 170 175 Gly Ala His Ile Leu Arg Arg Lys Ser Pro Ala Ser Gly Gln Gly Pro 180 185 190 Met Cys Asn Val Lys Ala Pro Leu Gly Ser Pro Ser Pro Arg Pro Arg 195 200 205 Arg Ala Lys Arg Phe Ala Ser Leu Ser Arg Phe Val Glu Thr Leu Val 210 215 220 Val Ala Asp Asp Lys Met Ala Ala Phe His Gly Ala Gly Leu Lys Arg 225 230 235 240 Tyr Leu Leu Thr Val Met Ala Ala Ala Ala Lys Ala Phe Lys His Pro 245 250 255 Ser Ile Arg Asn Pro Val Ser Leu Val Val Thr Arg Leu Val Ile Leu 260 265 270 Gly Ser Gly Glu Glu Gly Pro Gln Val Gly Pro Ser Ala Ala Gln Thr 275 280 285 Leu Arg Ser Phe Cys Ala Trp Gln Arg Gly Leu Asn Thr Pro Glu Asp 290 295 300 Ser Asp Pro Asp His Phe Asp Thr Ala Ile Leu Phe Thr Arg Gln Asp 305 310 315 320 Leu Cys Gly Val Ser Thr Cys Asp Thr Leu Gly Met Ala Asp Val Gly 325 330 335 Thr Val Cys Asp Pro Ala Arg Ser Cys Ala Ile Val Glu Asp Asp Gly 340 345 350 Leu Gln Ser Ala Phe Thr Ala Ala His Glu Leu Gly His Val Phe Asn 355 360 365 Met Leu His Asp Asn Ser Lys Pro Cys Ile Ser Leu Asn Gly Pro Leu 370 375 380 Ser Thr Ser Arg His Val Met Ala Pro Val Met Ala His Val Asp Pro 385 390 395 400 Glu Glu Pro Trp Ser Pro Cys Ser Ala Arg Phe Ile Thr Asp Phe Leu 405 410 415 Asp Asn Gly Tyr Gly His Cys Leu Leu Asp Lys Pro Glu Ala Pro Leu 420 425 430 His Leu Pro Val Thr Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly 435 440 445 9 492 PRT Homo sapiens MISC_FEATURE (1)..(492) truncated Aggrecanse-2 9 Met Leu Leu Gly Trp Ala Ser Leu Leu Leu Cys Ala Phe Arg Leu Pro 1 5 10 15 Leu Ala Ala Val Gly Pro Ala Ala Thr Pro Ala Gln Asp Lys Ala Gly 20 25 30 Gln Pro Pro Thr Ala Ala Ala Ala Ala Gln Pro Arg Arg Arg Gln Gly 35 40 45 Glu Glu Val Gln Glu Arg Ala Glu Pro Pro Gly His Pro His Pro Leu 50 55 60 Ala Gln Arg Arg Arg Ser Lys Gly Leu Val Gln Asn Ile Asp Gln Leu 65 70 75 80 Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly Arg 85 90 95 Arg Phe Leu Leu Asp Leu Glu Arg Asp Gly Ser Val Gly Ile Ala Gly 100 105 110 Phe Val Pro Ala Gly Gly Gly Thr Ser Ala Pro Trp Arg His Arg Ser 115 120 125 His Cys Phe Tyr Arg Gly Thr Val Asp Gly Ser Pro Arg Ser Leu Ala 130 135 140 Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val Lys His 145 150 155 160 Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg Gly Pro Trp Ala Glu Glu 165 170 175 Glu Lys Gly Arg Val Tyr Gly Asp Gly Ser Ala Arg Ile Leu His Val 180 185 190 Tyr Thr Arg Glu Gly Phe Ser Phe Glu Ala Leu Pro Pro Arg Ala Ser 195 200 205 Cys Glu Thr Pro Ala Ser Thr Pro Glu Ala His Glu His Ala Pro Ala 210 215 220 His Ser Asn Pro Ser Gly Arg Ala Ala Leu Ala Ser Gln Leu Leu Asp 225 230 235 240 Gln Ser Ala Leu Ser Pro Ala Gly Gly Ser Gly Pro Gln Thr Trp Trp 245 250 255 Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu 260 265 270 Leu Val Ala Asp Ala Ser Met Ala Arg Leu Tyr Gly Arg Gly Leu Gln 275 280 285 His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His 290 295 300 Ala Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val 305 310 315 320 Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys Asn Ala Ala Thr 325 330 335 Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly 340 345 350 Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr Arg Glu 355 360 365 Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly Met Ala Asp Val 370 375 380 Gly Thr Ile Cys Ser Pro Glu Arg Ser Cys Ala Val Ile Glu Asp Asp 385 390 395 400 Gly Leu His Ala Ala Phe Thr Val Ala His Glu Ile Gly His Leu Leu 405 410 415 Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu Glu Thr Phe Gly Ser 420 425 430 Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu Thr Ser Ile Asp Ala 435 440 445 Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr Ile Thr Glu Phe Leu 450 455 460 Asp Asp Gly His Gly Asn Cys Leu Leu Asp Leu Pro Arg Lys Gln Ile 465 470 475 480 Leu Gly Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly 485 490 10 13 PRT Artificial sequence Peptide substrate 10 Glu His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg 1 5 10 11 4 PRT Artificial sequence Peptide substrate 11 Glu Ala Glu Asn 1 12 11 PRT Artificial Sequence Peptide Substrate 12 Glu Gly Arg His Ile Asp Asn Glu Glu Asp Ile 1 5 10 13 9 PRT Artificial Sequence Peptide substrate 13 Glu Gly Asn Ala Phe Asn Asn Leu Asp 1 5 14 11 PRT Artificial Sequence Peptide substrate 14 Glu Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe 1 5 10 15 8 PRT Artificial sequence Peptide substrate 15 Glu Gln Leu Arg Met Lys Leu Pro 1 5 16 8 PRT Artificial sequence Peptide substrate 16 Glu Arg Gly Phe Phe Tyr Thr Pro 1 5 17 8 PRT Artificial Sequence Peptide substrate 17 Glu Val Thr Glu Gly Pro Ile Pro 1 5 18 8 PRT Artificial Sequence Peptide substrate 18 Glu Pro Leu Phe Tyr Glu Ala Pro 1 5 19 8 PRT artificial sequence peptide substrate 19 Glu Leu Pro Met Gly Ala Leu Pro 1 5 20 9 PRT Artificial Sequence Peptide substrate 20 Glu Lys Pro Ala Ala Phe Phe Arg Leu 1 5 21 13 PRT Artificial sequence peptide substrate 21 Glu Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu 1 5 10 22 10 PRT Artificial sequence peptide substrate 22 Glu Ser Glu Val Asn Leu Asp Ala Glu Phe 1 5 10 23 9 PRT Artificial Sequence Peptide Substrate 23 Glu Ser Gln Asn Tyr Pro Ile Val Gln 1 5 24 9 PRT Artificial Sequence Peptide Substrate 24 Glu Lys Pro Ile Glu Phe Phe Arg Leu 1 5 25 9 PRT Artificial sequence Peptide substrate 25 Glu Lys Pro Ala Glu Phe Phe Ala Leu 1 5 26 9 PRT Artificial sequence peptide substrate 26 Glu Lys Pro Ala Lys Phe Phe Arg Leu 1 5 27 10 PRT artificial sequence peptide substrate 27 Glu Ile Pro Phe His Leu Val Ile His Thr 1 5 10 28 11 PRT artificial sequence peptide substrate 28 Glu Met Ala Pro Gly Ala Val His Leu Pro Gln 1 5 10 29 11 PRT Artificial sequence peptide substrate 29 Glu Pro Leu Ala Gln Ala Val Arg Ser Ser Ser 1 5 10 30 11 PRT artificial sequence peptide substrate 30 Glu Pro Pro Val Ala Ala Ser Ser Leu Arg Asn 1 5 10 31 11 PRT artificial sequence peptide substrate 31 Glu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu 1 5 10 32 11 PRT artificial sequence peptide substrate 32 Glu Ser Leu Pro Val Gln Asp Ser Ser Ser Val 1 5 10 33 11 PRT artificial sequence peptide substrate 33 Glu Val His His Gln Lys Leu Val Phe Phe Ala 1 5 10 34 13 PRT artificial sequence peptide substrate 34 Lys Arg Gly Val Val Asn Ala Ser Ser Arg Leu Ala Lys 1 5 10 35 9 PRT artificial sequence peptide substrate 35 Lys Leu Val Leu Ala Ser Ser Ser Phe 1 5 36 13 PRT artificial sequence peptide substrate 36 Lys Ser Asn Arg Leu Glu Ala Ser Ser Arg Ser Ser Pro 1 5 10 37 13 PRT Artificial Sequence peptide substrate 37 Glu Asp Glu Met Glu Glu Xaa Ala Ser His Leu Pro Tyr 1 5 10 38 13 PRT Artificial sequence peptide substrate 38 Glu Ala Gly Pro Arg Gly Met Ala Gly Gln Phe Ser His 1 5 10 39 8 PRT Artificial Sequence peptide substrate 39 Lys Arg Pro Leu Gly Leu Ala Arg 1 5 40 10 PRT Artificial Sequence peptide substrate 40 Glu Gly Tyr Tyr Ser Arg Asp Met Leu Val 1 5 10 41 11 PRT artificial sequence peptide substrate 41 Glu Gln Lys Leu Asp Lys Ser Phe Ser Met Ile 1 5 10 42 12 PRT artificial sequence peptide substrate 42 Glu Pro Ser Ala Ala Gln Thr Ala Arg Gln His Pro 1 5 10 43 11 PRT artificial sequence peptide substrate 43 Glu Pro Gly Ala Gln Gly Leu Pro Gly Val Gly 1 5 10 44 8 PRT artificial sequence peptide substrate 44 Gly Leu Arg Thr Asn Ser Phe Ser 1 5 45 11 PRT artificial sequence peptide substrate 45 Arg Gly Val Val Asn Ala Ser Ser Arg Leu Ala 1 5 10 46 10 PRT artificial sequence peptide substrate 46 Lys Pro Ile Leu Phe Phe Arg Leu Gly Lys 1 5 10 47 13 PRT artificial sequence peptide substrate 47 Glu Met His Thr Ala Ser Ser Leu Glu Lys Gln Ile Gly 1 5 10 48 11 PRT artificial sequence peptide substrate 48 Glu Arg Phe Ala Gln Ala Gln Gln Gln Leu Pro 1 5 10 49 13 PRT artificial sequence peptide substrate 49 Glu Lys Lys Glu Asn Ser Phe Glu Met Gln Gly Asp Gln 1 5 10 50 10 PRT artificial sequence peptide substrate 50 Leu Ala Gln Ala Val Arg Ser Ser Ser Arg 1 5 10 51 9 PRT artificial sequence peptide substrate 51 Glu Arg Thr Ala Ala Val Phe Arg Pro 1 5 52 8 PRT artificial sequence peptide substrate 52 Glu Arg Val Arg Arg Ala Leu Pro 1 5 53 9 PRT artificial sequence peptide substrate 53 Glu Ser Phe Pro Arg Met Phe Ser Asp 1 5 54 11 PRT artificial sequence peptide substrate 54 Glu Glu Tyr Leu Glu Ser Phe Leu Glu Arg Pro 1 5 10 55 12 PRT artificial sequence peptide substrate 55 Glu Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 56 13 PRT artificial sequence peptide substrate 56 Glu His Gly Asp Gln Met Ala Gln Lys Ser Gln Ser Thr 1 5 10 57 15 PRT artificial sequence peptide substrate 57 Glu Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu 1 5 10 15 58 10 PRT artificial sequence peptide substrate 58 Glu Ala Gly Gln Arg Leu Ala Thr Ala Met 1 5 10 59 12 PRT artificial sequence peptide substrate 59 Glu Val Gly Leu Met Gly Lys Arg Ala Leu Asn Ser 1 5 10 60 12 PRT artificial sequence peptide substrate 60 Glu Lys Glu Asp Gly Glu Ala Arg Ala Ser Thr Ser 1 5 10

Claims

What is claimed is:

1. A peptide less than 40 amino acids in length having a cleavage site between a glutamic acid on the N-terminal side of the cleavage site and a non-polar or uncharged residue on the C-terminal side of the cleavage site and wherein the peptide is cleavable by an enzyme having the amino acid sequence of SEQ ID NO:8 and/or SEQ ID NO:9.

2. The peptide of claim 1 wherein the peptide comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

3. The peptide of claim 1 wherein the peptide is of natural or synthetic origin.

4. The peptide of claim 1 wherein the peptide comprises a detectable label selected from the group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, a fluorescent dye, or a colorimetric indicator.

5. The peptide of claim 1 wherein the peptide comprises a fluorophore and a quencher or acceptor located at opposite ends of the cleavage site of the peptide.

6. The peptide of claim 4 wherein the peptide further comprises an affinity moiety located at opposite ends of the cleavage site of the peptide.

7. A method to identify a compound that inhibits Aggrecanase enzymatic activity comprising the steps of:

contacting a test compound, an Aggrecanase, and a peptide less than 40 amino acids in length wherein the peptide comprises a cleavage site between a glutamic acid on the N-terminal side of the cleavage site and a non-polar or uncharged amino acid residue on the C-terminal side of the cleavage site and wherein the peptide is cleavable by an enzyme having an amino acid sequence corresponding to SEQ ID NO:8 and/or SEQ ID NO:9; and

detecting cleavage of the peptide wherein inhibition of peptide cleavage in the presence of a test compound indicates compound inhibition of Aggrecanase enzymatic activity.

8. The method of claim 7 wherein the method is conducted in a single reaction vessel.

9. The method of claim 7 wherein the enzyme is selected from the group consisting of Aggrecanase-1 and -2.

10. The method of claim 7 wherein the peptide is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6 and SEQ ID NO:7.

11. The method of claim 7 wherein the peptide further comprises a detectable label selected from the group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, a fluorescent dye, or a colorimetric indicator.

12. The method of claim 11 wherein the peptide further comprises a fluorophore and a quencher or acceptor located at opposite ends of the cleavage site of the peptide.

13. The method of claim 7 wherein the contacting step further comprises a cell expressing the Aggrecanase.

14. A method to detect the ability of a compound to inhibit Aggrecanase-1 or -2 enzymatic activity comprising the steps of:

contacting a test compound, an Aggrecanase secreted by a cell, and a peptide having an amino acid sequence selected from the group consisting of SEQ.ID.NO.:3 or SEQ.ID.NO.:4;

incubating the compound, enzyme, and peptide to permit enzymatic cleavage of the peptide; and

measuring enzymatic cleavage of the peptide wherein the method is conducted in a single reaction vessel without further manipulation.

15. The method of claim 14 wherein the peptide comprises a detectable label selected from the group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, a fluorescent dye, or a colorimetric indicator.

16. The method of claim 14 wherein the peptide comprises a fluorophore and a quencher or acceptor located at opposite ends of the cleavage site of the peptide.

17. A method to identify a compound capable of inhibiting Aggrecanase activity comprising the steps;

providing a peptide comprising an affinity moiety, an amino acid sequence selected from a group consisting of SEQ.ID.NO.:3 or SEQ.ID.NO.:4 and a detectable label, said affinity moiety and label located on opposite sides of a cleavage site encoded by the amino acid sequence;

contacting the peptide with an affinity capture coated solid phase support for sufficient time to bind a portion of the peptide;

washing the support to remove unbound peptide;

contacting a solution comprising a test compound and functional enzyme with the peptide bound solid phase support for sufficient time to allow enzymatic cleavage of the peptide, thereby releasing the peptide and detectable label into the solution; and

measuring changes in the quantity of the detectable label as a result of compound modulation of expected enzymatic function.

18. The method of claim 17 wherein the enzyme is selected from the group consisting of Aggrecanase-1 and -2.

19. The method of claim 17 wherein the peptide comprises a detectable label selected from the group consisting of ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, a fluorescent dye, or a calorimetric indicator.