Assays and peptide substrate for determining aggrecan degrading metallo protease activity

This invention is directed to assays to determine the presence or absence of proteins that exhibit aggrecanase or ADMP activity. This invention also relates to peptides that acts as a substrates for ADMPs, their use in various assays to determine the presence or absence of ADMP activity, and their use as inhibitors of ADMP activity.

FIELD OF THE INVENTION
 This invention is directed to various assays for determining aggrecanase or
 aggrecan degrading metallo protease (ADMP) activity. This invention also
 relates to a peptide that acts as a substrate for ADMPs, its use in
 various assays to determine the presence or absence of (ADMP) activity,
 and its use as an inhibitor of ADMP activity.
 BACKGROUND OF THE INVENTION
 Aggrecan is the major proteoglycan of cartilage and provides this tissue
 with its mechanical properties of compressibility and elasticity. In
 arthritic conditions one of the earliest changes observed in cartilage
 morphology is the depletion of aggrecan [Mankin et al. (1970) J. Bone
 Joint Surg. 52A, 424-434], which appears to be due to an increased rate of
 degradation.
 The aggrecan molecule is composed of two N-terminal globular domains, G1
 and G2, which are separated by an approximately 150 residue interglobular
 domain (IGD), followed by a long central glycosaminoglycan (GAG)
 attachment region and a C-terminal globular domain, G3 [Hardingham et al.
 (1992) in Articular Cartilage and Osteoarthritis: Aggrecan, The
 Chondroitin Sulfate/Keratan Sulfate Proteoglycan from Cartilage (Kuettner
 et al.) pp. 5-20, Raven Press, New York and Paulson et al. (1987) Biochem.
 J. 245, 763-772]. These aggrecan molecules interact through the G1 domain
 with hyaluronic acid and a link protein to form large molecular weight
 aggregates which are trapped within the cartilage matrix [Hardingham et
 al. (1972) Biochim. Biophys. Acta 279, 401-405, Heinegard et al. (1974) J.
 Biol. Chem. 249, 4250-4256, and Hardingham, T. E. (1979) Biochem. J. 177,
 237-247]. Loss of aggrecan from cartilage in arthritic conditions involves
 proteolytic cleavage of the aggrecan core protein within the IGD,
 producing a N-terminal G-1 fragment that remains bound to hyaluronic acid
 and the link protein within the matrix, releasing a large C-terminal
 GAG-containing aggrecan fragment that diffuses out of the cartilage
 matrix. Loss of the C-terminal fragment results in cartilage deficient in
 its mechanical properties. This deficiency arises because the GAGs which
 are present on the C-terminal portion of the aggrecan core protein are the
 components of aggrecan that impart the mechanical properties to the
 molecule through their high negative charge and water binding capacity.
 Two major sites of proteolytic cleavage have been identified within the
 IGD, one between amino acid residues Asn.sup.341 -Phe.sup.342 and the
 other between amino acid residues Glu.sup.373 -Ala.sup.374 (human sequence
 enumeration). Although G1 fragments formed by cleavage at the Asn.sup.341
 -Phe.sup.342 site and at the Glu.sup.373 -Ala.sup.374 site have been
 identified within articular cartilage [Flannery et al. (1992) J. Biol.
 Chem. 267, 1008-1014], the only N-terminus identified on GAG-containing
 aggrecan C-terminal aggrecan fragments in synovial fluids of patients with
 osteoarthritis [Sandy et al. (1992) J. Clin. Invest. 69, 1512-1516],
 inflammatory joint disease [Lohmander et al. (1993) Arthritis Rheum. 36,
 1214-1222] and in the media from cartilage explant and chondrocyte
 cultures stimulated with interleukin-1 or retinoic acid [Sandy et al.
 (1991) J. Biol. Chem. 266, 8198., Sandy et al. (1991) J. Biol. Chem. 266,
 8683-8685., Leulakis et al. (1992) Biochem. J. 264, 589-593., Ilic et al.
 (1992) Arch. Biochem. Biophys. 294, 115-122., Lark et al. (1995) J. Biol.
 Chem. 270, 2550-2556. ] is ARGSVIL, indicating that they were formed by
 cleavage between amino acid residues Glu.sup.373 -Ala.sup.374. These
 observations suggest that cleavage at this site may be responsible for
 cartilage degradation.
 Although many matrix metalloproteases (MMP-1, -2, -3, -7, -8, -9 and 13)
 have been shown to cleave in vitro at the Asn.sup.341 -Phe.sup.342 site,
 digestion of aggrecan with a number of these purified proteases has not
 resulted in cleavage at the Glu.sup.373 -Ala.sup.374 site [Fosang et al.
 (1992) J. Biol. Chem. 267, 19470-19474., Flannery et al. (1992) J. Biol.
 Chem. 267, 1008-1014., Fosang et al. (1993) Biochem. J. 295, 273-276.,
 Fosang et al. (1996) FEBS Lett. 380, 17-20., Flannery et al. (1993)
 Orthop. Trans. 17, 677., and Fosang et al. (1994) Biochem. J. 305,
 347-351]. Therefore, cleavage at this site has been attributed to a novel,
 proteolytic activity, "aggrecanase".
 In addition to the Glu.sup.373 -Ala.sup.374 bond within the interglobular
 domain of aggrecan, four potential aggrecanase-sensitive sites have been
 identified within the C-terminus of the aggrecan core protein [Loulakis et
 al. (1992) Biochem. J. 264, 589-593. and Sandy et al. (1995) Acta Orhtop
 Scand (Suppl 266) 66, 26-32]. Although cleavage at these sites which are
 not within the interglobular domain would not be expected to release the
 major portion of the aggrecan molecule from the matrix, they may be
 involved in earlier processing of aggrecan within the matrix.
 It follows from the foregoing considerations that a sensitive and specific
 assay that can detect such aggrecanase activity of aggrecan degrading
 metallo proteases (ADMPs) would be beneficial in helping to identify
 inhibitors of members of the ADMP family, which could serve as potential
 therapeutic agents for treating aggrecanase-related disorders cited above.
 SUMMARY OF THE INVENTION
 A preferred embodiment of the invention provides assays that determine the
 presence of aggrecan degrading metallo protease (ADMP) activity.
 A preferred embodiment of the invention provides an assay using purified
 native aggrecan or recombinant aggrecan as the substrate and monitoring
 product generation via a direct enzyme-linked immunosorbent assay (ELISA)
 using neoepitope antibodies to detect the new N-terminus or new C-terminus
 on aggrecan fragments formed by specific cleavage at an ADMP-sensitive
 site in the aggrecan core protein.
 A preferred embodiment of the invention provides peptides that have been
 found to act as substrates for the family of aggrecan degrading metallo
 proteases (ADMPs). One peptide, based on the human aggrecan sequence
 around the Ala373-Glu374 ADMP-sensitive site has the sequence:
 QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL (SEQ ID No:1)
 A second peptide, based on the bovine aggrecan sequence around the
 Ala373-Glu374 ADMP-sensitive site has the sequence:
 QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPE (SEQ ID NO:2) Both peptides are
 capable of being cleaved at this specific recognition site by members of
 the family of ADMP proteins. A third peptide, based on the human aggrecan
 sequence around the Alal1714-Glyl1715 ADMP-sensitive site has the
 sequence:
 ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGT (SEQ ID NO:3)
 and is capable of being cleaved at this specific recognition site by the
 family of ADMP proteins.
 A preferred embodiment of the invention provides assay formats and methods
 of utilizing these peptide substrates for the detection and quantification
 of ADMP activity.
 A preferred embodiment of the invention provides a modified version of the
 peptide substrates and a method for their use as an inhibitor of ADMP
 activity.

DETAILED DESCRIPTION OF THE INVENTION
 A family of aggrecan degrading metallo protease (ADMP) proteins cleave the
 aggrecan core protein at the Glu.sup.373 -Ala.sup.374 peptide bond and
 thus exhibit the enzymatic activity referred to as "aggrecanase" activity
 [Flannery et al. (1992) J. Biol. Chem. 267, 1008-1014]. The presence of
 ADMP enzymatic activity can be determined by monitoring the production of
 aggrecan fragments generated exclusively by cleavage at the Glu.sup.373
 -Ala.sup.374 peptide bond within the aggrecan core protein. These aggrecan
 fragments are detected by using neoepitope antibodies to the new
 N-terminus or new C-terminus on fragments produced by specific cleavage at
 this ADMP-sensitive site. The neoepitope antibodies used encompase but are
 not limited to, the BC-3 monoclonal antibody (Hughes, C. E., et al.,
 Biochem. J. 306:799-804, 1995) as first described in U.S. Provisional
 Patent Application Serial No. 60/006,684 and subsequently described in
 U.S. patent application Ser. No. 08/743,439.
 ADMP activity may also be detected by monitoring the production of
 fragments formed by cleavage at alternative ADMP-sensitive sites using
 neoepitope antibodies to the new C-terminus or to the new N-terminus
 generated by ADMP-specific cleavage at these sites. Alternative sites in
 the aggrecan core protein encompass, but are not limited to, the
 E1545-G1546, E1714-G1715, E1819-A1820, or E1919-L1920 bond (numbering
 based on the human aggrecan core protein sequence.
 A preferred assay format involves using purified native aggrecan or
 recombinant aggrecan as the substrate with product detection via a direct
 enzyme-linked immunosorbent assay (ELISA), herein referred to as the
 "Problot assay", using neoepitope antibodies to the new C-terminus or new
 N-terminus on aggrecan fragments generated upon specific cleavage at
 ADMP-sensitive sites within the aggrecan core protein. Alternative sites
 in the aggrecan core protein encompass, but are not limited to, the
 E1545-G1546, E1714-G1715, E1819-A1820, or E1919-L1920 bond (numbering
 based on the human aggrecan core protein sequence). These human aggrecan
 ADMP-senstitive cleavage sites are conserved in aggrecan from various
 animal species although the absolute numbering based on the sequence of
 the aggrecan core protein may vary from species to species. Conserved
 amino acid sequences in various species around conserved ADMP-sensitive
 sites are shown below.

Human NITEGE.sup.373 .sup.374 ARGSVILT
 Bovine NITEGE ARGSVILT
 Rat NITEGE ARGNVILT
 Mouse NVTEGE ALGSVILT
 Pig NITEGE ARGTVILT
 Sheep NITEGE ARGNVILT
 Chicken NVTEEE ARGSI
 Horse NITEGE ARGNVILT
 Human ASTASELE.sup.1545 .sup.1546 GRGTIGIS
 Bovine ATTAGELE GRGTIDIS
 Mouse ATTSSELE GRGTIGIS
 Rat ATTASELE GRGTISVS
 Human PTTFKEEE.sup.1714 .sup.1715 GLGSVELS
 Bovine PTTFKEEE GLGSVELS
 Rat PTTFREEE GLGSVELS
 Mouse PTTFREEE GLGSVELS
 Human TQAPTAQE.sup.1819 .sup.1820 AGEGPSGI
 Bovine TQAPTAQE AGEGPSGI
 Rat TLAPTAQE AGEGPSSI
 Mouse TQAPTAQE AGEGPSGI
 Chicken TQTSVAQE VGEGPSGM
 Human TEPTISQE.sup.1919 .sup.1920 LGQRPPVT
 Bovine TEPTVSQE LGQRPPVT
 Rat TEPTVSQE LGHGPSMT
 Mouse TEPTVSQE LGHGPSMT
 Chicken TRPTVSQE LGGETAVT
 Dog TEPTVSQE LAQRPPVT
 Thus, aggrecan from various animal species, including but not limited to,
 bovine, dog, pig, rat, mouse, sheep, horse and chicken may also be used as
 a substrate for detecting ADMP activity.
 The direct ELISA assay employs 96-well filtration plates containing
 polyvinyl-denedifluoride (PVDF) cationically charged membranes. These
 membranes are semi-selective in binding the highly negatively-charged
 aggrecan, which allows for binding of detectable levels of neoepitope
 antibody-reactive aggrecan fragments from solutions containing high levels
 of other proteins.
 Utilizing neoepitope antibodies allows detection of fragments formed
 specifically by ADMP-mediated cleavage even in the presence of other
 proteolytic activities which may be present in crude preparations. Thus,
 the Problot assay can be used to monitor ADMP activity in culture medium
 containing other proteases, as well as to monitor the activity of the
 purified ADMP enzyme. Therefore, this assay has particular use in
 following ADMP activity during purification from tissue or media samples
 as well as for use in enzymatic assays to evaluate inhibitors of the ADMP
 enzyme. The Problot assay can also be used to detect ADMP-generated
 aggrecan fragments in culture media from tissue or cell cultures
 stimulated to induce ADMP-mediated degradation. This assay may also be
 useful for detecting ADMP-generated aggrecan fragments in cartilage,
 synovial fluid, serum, urine or other biological samples from patients
 with ADMP-associated diseases.
 Peptide substrates are commonly employed in a variety of assays to
 determine the presence of enzymes that catalyze the hydrolysis of
 proteins. One skilled in the art would rely on the use of peptide
 substrates that are relatively short in length, generally consisting of
 approximately six to ten amino acids in length. These peptide substrates
 typically encompass amino acid sequences that bracket the known hydrolysis
 site of the natural protein substrates. These peptide substrates,
 including those for matrix metalloproteases, serine proteases, aspartyl
 proteases, and aminopeptidases, are readily available for use in a variety
 of enzymatic assays.
 This invention provides a peptide that has been found to act as a substrate
 for the family of ADMPs. It is commonly known that short peptide sequences
 which contain the proper substrate cleavage site are quite acceptable
 substrates for many proteases (Copeland, R. A., Enzymes: A Practical
 Introduction to Structure, Mechanism and Data Analysis, VCH/Wiley, New
 York, 1996). However, no such peptide, even those containing as many as
 twenty amino acids, has been determined that will act as a suitable
 substrate for ADMPs. The peptides of the instant invention are unique in
 that it was unexpectedly found that these longer, forty amino acid
 sequence acted as very good substrates for ADMPs. One such peptide
 provided by the invention, of the sequence
 QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL (SEQ ID NO:1) comprises a 40
 amino acid segment of the human aggrecan protein that contains the
 ITEGE373-374ARGS cleavage site present in the natural protein substrate,
 aggrecan, and is capable of being cleaved at this specific recognition
 site by the ADMPs. Since the human aggrecan ADMP-senstitive cleavage sites
 are conserved in aggrecan from various animal species, peptides based on
 the amino acid sequence around the ADMP-sensitive cleavage sites from
 other species can also serve as substrates for ADMPs. A peptide substrate,
 similar to SEQ ID NO:l, based on a 41 amino acid segment of the bovine
 aggrecan protein, of the sequence
 QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPE (SEQ ID NO:2)
 containing the E373-A374 cleavage site is also capable of being cleaved at
 this specific recognition site by the ADMPs.
 Cleavage products are easily detected by using neoepitope antibodies to the
 N-terminal or C-terminal fragments produced by specific cleavage at the
 E373-A374 bond, encompasing, but not limited to, the monoclonal antibody
 BC-3 (Hughes, C. E., et al., Biochem. J. 306:799-804, 1995). The BC-3
 antibody recognizes the new N-terminus, ARGS, which is the amino terminal
 portion of one of the product peptides resulting from the ADMP activity of
 the enzyme.
 One skilled in the art could readily design peptides of similar size
 encompasing the alternative ADMP-sensitive cleavage sites in the aggrecan
 core protein, encompasing, but not limited to, regions of the molecule
 containing the E1545-G1546, E1714-G1715, E1819-Al820, or E1919-L1920 bond
 (numbering based on the human aggrecan core protein sequence). One such
 peptide provided by the invention, of the sequence
 ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGT (SEQ ID NO:3)
 comprises a 40 amino acid segment of the human aggrecan protein that
 contains the KEEE1714-1715GLGS cleavage site present in the natural
 protein substrate, aggrecan, and is capable of being cleaved at this
 specific recognition site by the ADMPs.
 When a preferred form of a peptide substrate, biotinylated at the carboxy
 terminus or amino terminus, is employed, several streptavidin coated
 supports may be used. These include, but are not limited to microplates,
 metallic and non-metallic beads, and membranes.
 Another preferred assay format involves the direct analysis, by
 high-performance liquid chromatography (HPLC), of the cleavage fragments
 from the substrate that are generated by ADMP activity.
 Another preferred embodiment of the invention provides that a peptide
 substrate of this invention may be reversed in its role. With proper
 modification at the P1 position the substrate may be turned into an
 inhibitor of ADMP activity. Specifically it was found that esterification
 of the P1 glutamic acid residue (GLU.sup.373) of the substrate peptide SEQ
 ID NO:1 or its replacement by glutamine abolish catalytic hydrolysis.
 Unexpectedly, the peptide containing the GLU to GLN substitution at amino
 acid position 373 (the P1-glutamine containing peptide) was shown to be a
 competitive inhibitor of the enzyme. Thus, a carboxylate residue at
 position P1 of the substrate appears to be critical for turnover by ADMPs,
 but exerts less influence over initial substrate binding to the enzyme.
 This feature can be readily exploited by one trained in the art to design
 specific peptide and non-peptide inhibitors of this enzyme.
 DEFINITIONS
 As used herein, the following terms and expressions have the indicated
 meanings.
 The term "aggrecan degrading metallo protease" ("ADMP") activity as
 referred to herein, refers to the enzymatic activity of a family of
 polypeptides which specifically cleave the protein aggrecan within the
 interglobular domain at the Glu.sup.373 -Ala.sup.374 peptide bond, but do
 not readily cleave at the Asn341-Phe342 bond which is preferentially
 cleaved by matrix metalloproteinases.
 The term "amino acid" as used herein means an organic compound containing
 both a basic amino group and an acidic carboxyl group.
 The term "amino acid residue" as used herein means that portion of an amino
 acid (as defined herein) that is present in a peptide.
 The term "peptide" as used herein means a compound that consists of two or
 more amino acids (as defined herein) that are linked by means of a peptide
 bond. The term "peptide" also includes compounds containing both peptide
 and non-peptide components, such as pseudopeptide or peptide mimetic
 residues or other non-amino acid components. Such a compound containing
 both peptide and non-peptide components may also be referred to as a
 "peptide analog".
 The term "peptide bond" means a covalent amide linkage formed by loss of a
 molecule of water between the carboxyl group of one amino acid and the
 amino group of a second amino acid.
 The term "substrate" refers to a molecule that is bound by the active site
 and acted upon by the enzyme.
 The term "solid-phase peptide synthesis" refers to the direct chemical
 synthesis of peptides utilizing an insoluble polymeric support as the
 anchor for the growing peptide, which is built up one amino acid at a time
 using a standard set of reactions in a repeating cycle (Merrifield, R. B.,
 Science 232, 341-347 1986).
 As used herein, the term "TMB" refers to 3,3', 5,5'-tetramethylbenzidine.
 The term "neoepitope antibody" refers to an antibody which specifically
 recognizes a new N-terminal amino acid sequence or new C-terminal amino
 acid sequence generated by proteolytic cleavage but does not recognize
 these same sequences of amino acids when they are present within the
 intact protein.
 As used herein, the cleavage site "E373-374A" refers to the
 ITEGE373-374ARGS bond of human aggrecan as well as to the homologous
 aggrecanase-sensitive cleavage site in aggrecan from various animal
 species, the cleavage site "E1545-1546G" refers to the SELE1545-1546GRGT
 bond of human aggrecan as well as to the homologous aggrecanase-sensitive
 cleavage site in aggrecan from various animal species, the cleavage site
 "E1714-1715G" refers to the KEEE1714-1715GLGS bond of human aggrecan as
 well as to the homologous aggrecanase-sensitive cleavage site in aggrecan
 from various animal species the cleavage site "E1819-1820A" refers to the
 TAQE1819-1820AGEG bond of human aggrecan as well as to the homologous
 aggrecanase-sensitive cleavage site in aggrecan from various animal
 species, the cleavage site "E1919-1920L" refers to the ISQE1919-1920LGQR
 bond of human aggrecan as well as to the homologous aggrecanase-sensitive
 cleavage site in aggrecan from various animal species.
 The term "aggrecan" as used herein refers to the aggregating proteoglycan,
 aggrecan, of cartilage from human or various animal species, as the native
 aggrecan isolated from tissue, as recombinant full-length aggrecan or as a
 recombinant protein representing a portion of the aggrecan molecule.
 As used herein the term "ADMP-susceptible cleavage site" refers to the
 E373-374A bond, the E1545-1546G bond, the E1545-1546G bond, the
 E1819-1820A bond, and the E1919-1920L bond of aggrecan from human and
 various animal species, and to a peptide bond of a protein containing an
 amino acid sequence which has a glutamine in the P1 position and shows at
 least 65% homology with the P1, P2, P3, P1', P2' and P3' amino acids of
 one or more of the ADMP-sensitive sites in the aggrecan molecule.
 The term "sissel bond" refers to the peptide bond of a polypeptide that is
 to be cleaved by a protease. The term "P1" as used herein refers to the
 amino acid residue on the N-terminal side of the sissel bond. The term
 "P2" as used herein refers to the amino acid residue adjacent to P1 on the
 N-terminal side of the sissel bond. The term "P3" as used herein refers to
 the amino acid residue adjacent to P2 on the N-terminal side of the sissel
 bond. The term "P1'" as used herein refers to the amino acid residue on
 the C-terminal side of the sissel bond. The term "P2'" as used herein
 refers to the amino acid residue adjacent to P1' on the C-terminal side of
 the sissel bond. The term "P3'" as used herein refers to the amino acid
 residue adjacent to P2' on the C-terminal side of the sissel bond.
 The term "BC-3 antibody" refers to a monoclonal antibody that reacts
 specifically with the newly-formed amino-terminal sequence ARGS on
 fragments produced by proteolytic cleavage at the Glu.sup.373 -Ala.sup.374
 aggrecan cleavage site, but does not recognize this same sequence of amino
 acids when they are present within the intact interglobular domain of the
 protein (Hughes, C. E., et al., Biochem. J. 306:799-804, 1995).
 The term "SEQ ID NO:1" refers to the peptide sequence
 QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL. The term "SEQ ID NO:2" refers
 to the peptide sequence QTVTWPDVELPLPRNITEGE-ARGSVILTAKPDFEVSPTAPE. The
 term "SEQ ID NO:3" refers to the peptide sequence
 ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGT. The term "41-PS" and "SEQ ID
 NO:4" refer to the peptide sequence:
 QTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPL-(BIOTINYL)K. The term "SEQ ID
 NO:5" refers to the peptide sequence: ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K.
 The term "SEQ ID NO:6" refers to the peptide sequence:
 K(BIOTINYL)-QTVTWPDMELPLPRNITEGE. The term "30-IP" and "SEQ ID NO:7" refer
 to the peptide sequence QTVTWPDMELPLPRNITEGQARGSVILTV-(BIOTINYL)K.
 The invention can be further understood by the following examples. These
 examples provide an illustration of embodiments of the invention and
 should not be construed to limit the scope of the invention which is set
 forth in the appended claims. In the following examples all methods
 described are conventional unless otherwise specified.
 Example 1
 Microplate Assay Format for Detection of ADMP Activity
 The substrate and product peptides were prepared in the following manner. A
 41 amino acid form (41-PS) SEQ ID NO:4 of the peptide substrate SEQ ID
 NO:1 was prepared by solid phase peptide synthesis. The peptide was
 prepared commercially (Quality Controlled Biochemicals, Inc. Hopkinton,
 Mass.) as a biotin conjugate by adding an additional lysine residue at the
 carboxy terminus of the peptide SEQ ID NO:1. Biotin was then covalently
 attached through the lysine .epsilon.-amino side chain. 41-PS sequence:
 QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K (SEQ ID NO:4) A 21
 amino acid peptide representing the product of ADMP-mediated cleavage of
 the 41-PS containing the ARGS N-terminus was prepared in a similar manner
 and had the following sequence:
 ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K (SEQ ID NO:5)
 The substrate and product peptide microplates were prepared in the
 following manner. A 0.1 mM stock of 41-PS was made by dissolving it in
 distilled water. From this a working solution of 7.times.10.sup.-8 M 41-PS
 was prepared in 1.times.PBS, 0.05% Tween 20. Aliquots of 100 .mu.L of this
 solution were added to the microplate wells of a streptavidin coated
 microtiter strip plate (DUPONT, NEN Products, Catalog # NEF-711). The
 solutions were allowed to sit at 25.degree. C. overnight in order for the
 biotin moiety of the peptides to bind to the streptavidin which was coated
 on the microplate. The plate was washed three times with 200 .mu.L of
 1.times.PBS, 0.05% Tween 20, after which the plate was inverted, blotted
 dry, sealed and stored at 4.degree. C.
 The assay was performed in the following manner. Microplate strips (eight
 wells each) were rinsed once with 100 .mu.L of 1.times.Assay Buffer (Assay
 Buffer consists of: 50 mM Tris, pH 7.5, 10 mM CaCl.sub.2, and 100 mM NaCl)
 and blotted dry. Reactions were prepared in duplicate in a final volume of
 100 .mu.L, containing: 50 .mu.L of 2.times.Assay Buffer (100 mM Tris, pH
 7.5, 20 mM CaCl.sub.2, and 200 mM NaCl), 25 .mu.L of a hydroxymate
 inhibitor compound (final concentrations consisting of 5.0, 1.0, 0.75,
 0.5, 0.25, 0.125, 0.05, 0.001, and 0.0 .mu.M), and 25 .mu.L of soluble
 ADMP (0.05 .eta.M). The microplate strips were incubated for 3 hours at
 37.degree. C. The microplate wells were then washed six times with 200
 .mu.L of 1.times.PBS, 0.05% Tween 20 using a Denley Well Wash 4 micro
 plate washer.
 A BC-3 antibody solution was prepared by adding 4 .mu.L of BC-3 antibody
 (0.405 mg/mL in PBS) to 2 mL of antibody dilution buffer (DB), which
 consisted of: 0.1 g BSA (Boehringer Mannheim Catalog 238-031), 10 mL
 1.times.PBS, and 10 .mu.L Tween 20. 100 .mu.L of this solution was added
 to each well. The microplate strips were incubated for 1 hour at
 25.degree. C. The microplate wells were washed six times with 200 .mu.L of
 1.times.PBS, 0.05% Tween 20.
 The secondary (detection) antibody solution was prepared by adding 4 .mu.L
 of Goat anti-Mouse-HRP antibody conjugate (Pierce Cat#31430) (0.8 mg/mL in
 PBS) to 2 mL of antibody dilution buffer (DB). 100 .mu.L of this solution
 was added to each well. The microplate strips were incubated for 1 hour at
 25.degree. C. The microplate wells were washed six times with 200 .mu.L of
 1.times.PBS, 0.05% Tween 20. TMB Substrate(100 .mu.L per well, DAKO Cat
 S1600) was added and the microplate strips incubated at 25.degree. C. for
 15 minutes. The reaction was quenched with 100 .mu.L of 1N HCl. The
 optical density was read at 450nm using a Molecular Devices Spectromax 250
 microplate reader. By coating the jplate with the product peptide, one can
 create a standard curve which can be used to convert optical density
 values to units of activity.
 ADMP activity can easily be followed by this method and inhibition of ADMP
 activity can be monitored. The IC.sub.50 for the inhibition of ADMP by the
 hydroxamate inhibitor tested was 0.413 .mu.M.
 Example 2
 HPLC Assay for Detection of ADMP Activity
 The High Performance Liquid Chromatography (HPLC) instrument used for the
 assay was from Hewlett-Packard, model number HP1090, equipped with a HP
 ChemStation. A 250.times.4.6 mm Vydac C.sub.18 column with 10 .mu.
 particle size was obtained from The Separations Group, Tesperia, Calif.;
 HEPES from Research Organics, Cleveland, Ohio; Brij-35 from Technicon
 Corp., Tarrington, N.Y.; Other chemicals from Sigma, St. Louis, Mo.; the
 lyophilized peptide substrate 41-PS,
 QTVTWPDMELPLRNITEGEARGSVILTVKPIFEVSPSPL-(BIOTINYL)K (SEQ ID NO:4), which
 includes the ADMP E373-A374 cleavage site, was obtained from Quality
 Controlled Biochemicals, Inc. Hopkinton, Mass. A 21 amino acid product
 peptide (SEQ ID NO:5) with the sequence, ARGSVILTVKPIFEVSPSPL-(BIOTINYL)K,
 used as a standard for quantitation of product formation, was obtained
 from the same vendor.
 The HPLC assay is performed in the following manner. The reaction buffer
 contains 50 mM HEPES, 10 mM CaCl.sub.2, 100 mM NaCl and 0.05% Brij-35, pH
 7.5. 30 .mu.M 41-PS was incubated with 1 unit ADMP activity (1 unit=the
 amount of ADMP activity resulting in 1 pMole of 21-mer peptide product
 produced per hr at 37.degree.) at 37.degree. C. for 2.5 hours and then the
 reaction was quenched with 50 mM EDTA. A 10 .mu.L portion of the reaction
 mixture was injected onto a reverse-phase HPLC C.sub.18 column. The
 peptides were eluted with a mobile phase of 0.1% trifluoroacetic acid and
 a 25-45% acetonitrile gradient in 20 minutes. UV absorbance was measured
 at 220nm and peak integration was performed on a Hewlett-Packard HP
 ChemStation. The 21-mer product peptide was used as a standard for
 quantitation of product formation.The 41-PS and 21-mer product are well
 separated with retention times of 14.2 and 10.5 minutes, respectively. A
 standard curve was prepared using the 21-mer peptide to allow quantitation
 of product formation. Effect of incubation time was evaluated and found to
 be linear over the timecourse of the assay (FIG. 2).
 Example 3
 Inhibition of ADMP Activity by a Peptide Inhibitor
 A peptide inhibitor was prepared based upon the sequence of the 40 amino
 acid peptide SEQ ID NO:1, but designed such that it contained a Glu to Gln
 substitution at the P1 of the Glu373-Ala374 bond. This peptide (30-IP) of
 the sequence:
 QTVTWPDMELPLPRNITEGQARGSVILTVK-Biotin (SEQ ID NO:7) was prepared with the
 n-terminus acetylated and the c-terminal residue present as the amide
 (Quality Controlled Biochemicals, Inc., Hopkinton, Mass.). This peptide
 was employed in a microplate assay as described in Example 1 wherein the
 30-IP inhibitor was substituted for the hydroxamate inhibitor used in that
 example. The 30-IP inhibitor was employed at final concentrations of 0.01,
 0.1, 1.0, 3.0, 5.0, 10.0, 30.0 and 100.0 .mu.M.
 ADMP activity was inhibited as shown in FIG. 3. The IC.sub.50 for the
 inhibition of ADMP activity by 30-IP was 11 .mu.M.
 Example 4
 Problot Assay
 This example describes a method for analyzing ADMP enzymatic activity and
 inhibitors of this activity by monitoring cleavage at the E373-A374 bond
 using the BC-3 antibody to detect fragments with the new N-terminus, ARGS.
 Samples containing ADMP activity (10 units/ml) were incubated with 500 nM
 bovine aggrecan monomer in a final volume of 200 .mu.L in 0.05 M Tris, pH
 7.6, containing 0.1 M NaCl and 10 mM CaCl.sub.2. Reactions were incubated
 for 4 hr at 37.degree. C., quenched with 20 mM EDTA, and analyzed for
 aggrecan fragments with the new N-terminus, ARGS, generated by specific
 ADMP-mediated cleavage using the Problot assay.
 The Immobolin PVDF membrane plate (#MAIPN4550; Millipore Corp., Bedford,
 Mass.) was prewet with 50 .mu.L per well 70% ethanol, incubated for 30
 seconds at room temperature then flushed two times each with 200 .mu.L of
 purified H.sub.2 O. The plate was then coated with aggrecan equivalent to
 36 .mu.g of glycosaminoglycan (GAG) as detected by the dimethyl methylene
 blue dye assay [Farndale R. W.,et al., (1982) Conn. Tiss. Res. 9, 247-248.
 ] in 150 .mu.L of 50 mM carbonate-bicarbonate buffer, pH 9.6, overnight at
 4.degree. C. with gentle agitation. The coating solution was then filtered
 off of the plate using a vacuum manifold and membranes were washed once
 with 200 .mu.L of Buffer A (Buffer A comprises 20 mM Tris, 500 mM NaCl, pH
 7.5) allowing 20 seconds of contact with the membrane. Membranes were then
 blocked with 150 .mu.L of 5% BSA/TBS solution for 1 hour at room
 temperature with gentle agitation. The blocking solution was filtered off
 of the plate and the membranes washed one time with 200 .mu.L of
 1.times.TBS buffer, allowing 20 seconds of contact with membrane per wash.
 Removal of the glycosaminoglycan (GAG) side chains from aggrecan is
 necessary for the BC-3 antibody to recognize the epitope on the core
 protein. Therefore, to remove GAGs from the bound aggrecan, samples were
 treated with deglycosylation enzymes as follows: 0.01 units chondroitinase
 ABC (#EC4.2.2.4; Seikaguku Co., Kogyo, Japan) per 10 .mu.g GAG in 150
 .mu.L of Buffer B (Buffer B comprises 50 mM sodium acetate, 100 mM NaCl,
 pH 6.5) was added to each well and incubated at 37.degree. C. for 1 hour.
 Following incubation the enzyme solution was filtered out of the plate and
 0.01 units chondroitinase ABC per 10 .mu.g GAG, 0.01 units keratanase I
 (#EC3.2.1.103; Co., Kogyo, Japan) per 10 .mu.g GAG, and 0.0005 units
 keratanase II (Seikaguku Co., Kogyo, Japan) per 10 .mu.g GAG in 150 .mu.L
 Buffer B were added and allowed to incubate an additional 2 hours at
 37.degree. C. Enzyme solution was filtered out and membranes rinsed one
 time with 200 .mu.L of Buffer A.
 150 .mu.L of BC-3 antibody was added at a 1:500 dilution in 1% BSA in
 Buffer A and incubated for 1 hour at room temperature with gentle
 agitation. BC-3 antibody was removed and membranes washed three times each
 with 200 .mu.L Buffer A allowing membrane contact for 20 seconds per wash.
 Next 150 .mu.L of goat anti-mouse IgG AP conjugate (#S3721; Promega,
 Madison, WI) was added at a 1:2500 dilution in 1% BSA/TBS buffer and
 allowed to incubate for 1 hour at room temperature with gentle agitation.
 The secondary antibody was prefiltered prior to use, using 0.22 .mu.M
 syringe filters to remove aggregates which cause high background.
 Following the incubation, secondary antibody was removed and wells were
 washed three times each with 200 .mu.L Buffer A allowing contact for 20
 seconds per wash. Then 100 .mu.L of p-NPP AP substrate solution (#
 50-80-00; Kirkegaard & Perry Lab., Gaithersburg, MD) was added to each
 well and incubated in the dark at room temperature for 30 minutes. The
 solution was then filtered into a corresponding ELISA plate. The filter
 plate was washed with 100 .mu.L of 500 mM EDTA and the wash was combined
 with the corresponding samples in the ELISA plate. Absorbance of the
 samples was read at 405 nm (Thermomax plate reader).
 By coating the plate with the target peptide, ARGS, linked to BSA
 representing the ADMP-generated product, one can create a standard curve
 which can be used to convert optical density values to units of activity.
 A unit of ADMP activity is defined as that resulting in product produced
 equivalent to 1 .mu.g BSA peptide per hour at 37.degree. C.
 To evaluate inhibition of ADMP activity, compounds are prepared as 10 mM
 stocks in dimethyl sulfoxide (DMSO), water or other solvents and diluted
 to appropriate concentrations in water. Drug (50 .mu.L) was added to 50
 .mu.L of 2 mg/mL aggrecan substrate and 50 .mu.l of ADMP (40 units/ml) and
 brought to a final volume of 200 .mu.L iby addition of 50 .mu.l of 0.2 M
 Tris, pH 7.6, containing 0.4 M NaCl and 40 mM CaCl.sub.2. The reaction
 mixture was incubated for 4 hr at 37.degree. C., quenched with 20 mM EDTA
 and analyzed for ADMP-generated products. A sample containing enzyme and
 substrate without drug was included as a positive control and enzyme
 prequenched with EDTA served as a measure of background.
 IC.sub.50 values for inhibitors of ADMP enzymatic activity determined using
 the Problot assay with the BC-3 antibody for analysis of product
 generation by cleavage at the E373-A374 bond correlated (r.sup.2 =0.99)
 with those determined using a BC-3 Western blot analysis to detect product
 formation.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 7
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 40
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: PEPTIDE
 &lt;222&gt; LOCATION: (1)..(40)
 &lt;223&gt; OTHER INFORMATION: This 40 amino acid segment of the human
 aggrecan protein contains the ITEGE373-374ARGS cleavage site
 present in the natural protein substrate, aggrecan, and is capable
 of being cleaved at this specific recognition site by the ADMPs.
 &lt;400&gt; SEQUENCE: 1
 Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu Pro Arg Asn Ile
 1 5 10 15
 Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr Val Lys Pro Ile
 20 25 30
 Phe Glu Val Ser Pro Ser Pro Leu
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 41
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: PEPTIDE
 &lt;222&gt; LOCATION: (1)..(41)
 &lt;223&gt; OTHER INFORMATION: This peptide substrate is based on a 41
 amino
 acid segment of the bovine aggrecan protein and contains the
 E373-A374 cleavage site and is also capable of being cleaved at
 this specific recognition site by the ADMPs.
 &lt;400&gt; SEQUENCE: 2
 Gln Thr Val Thr Trp Pro Asp Val Glu Leu Pro Leu Pro Arg Asn Ile
 1 5 10 15
 Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr Ala Lys Pro Asp
 20 25 30
 Phe Glu Val Ser Pro Thr Ala Pro Glu
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 3
 &lt;211&gt; LENGTH: 40
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: PEPTIDE
 &lt;222&gt; LOCATION: (1)..(40)
 &lt;223&gt; OTHER INFORMATION: This 40 amino acid segment of the human
 aggrecan protein contains the KEEE1714-1715GLGS cleavage site
 present in the natural protein substrate, aggrecan, and is capable
 of being cleaved at this specific recognition site by the ADMPs.
 &lt;400&gt; SEQUENCE: 3
 Ile Thr Phe Val Asp Thr Ser Leu Val Glu Val Thr Pro Thr Thr Phe
 1 5 10 15
 Lys Glu Glu Glu Gly Leu Gly Ser Val Glu Leu Ser Gly Leu Pro Ser
 20 25 30
 Gly Glu Leu Gly Val Ser Gly Thr
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 4
 &lt;211&gt; LENGTH: 41
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: PEPTIDE
 &lt;222&gt; LOCATION: (1)..(41)
 &lt;223&gt; OTHER INFORMATION: This sequence was prepared as a biotin
 conjugate by adding a Lys residue at the carboxy terminus of the
 peptide SEQ ID NO1. Biotin was then covalently attached through
 the lysine amino side chain, i.e., there is a biotinylated lysine
 residue at position 41.
 &lt;400&gt; SEQUENCE: 4
 Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu Pro Arg Asn Ile
 1 5 10 15
 Thr Glu Gly Glu Ala Arg Gly Ser Val Ile Leu Thr Val Lys Pro Ile
 20 25 30
 Phe Glu Val Ser Pro Ser Pro Leu Xaa
 35 40
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: PEPTIDE
 &lt;222&gt; LOCATION: (1)..(21)
 &lt;223&gt; OTHER INFORMATION: This 21 amino acid peptide representing the
 product of ADMP mediated cleavage of SEQ ID NO4 containing the
 ARGS N-terminus was prepared in a similar manner to SEQ ID NO4
 and has a biotinylated lysine residue at position 21.
 &lt;400&gt; SEQUENCE: 5
 Ala Arg Gly Ser Val Ile Leu Thr Val Lys Pro Ile Phe Glu Val Ser
 1 5 10 15
 Pro Ser Pro Leu Xaa
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 21
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: PEPTIDE
 &lt;222&gt; LOCATION: (1)..(21)
 &lt;223&gt; OTHER INFORMATION: This 21 amino acid peptide representing the
 product of ADMP mediated cleavage of SEQ ID NO4 was prepared in a
 similar manner to SEQ ID NO4 and has a biotinylated lysine
 residue at position 1.
 &lt;400&gt; SEQUENCE: 6
 Xaa Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu Pro Arg Asn
 1 5 10 15
 Ile Thr Glu Gly Glu
 20
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 7
 &lt;211&gt; LENGTH: 30
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: PEPTIDE
 &lt;222&gt; LOCATION: (1)..(30)
 &lt;223&gt; OTHER INFORMATION: This peptide inhibitor was prepared based
 upon
 the sequence of the 40 amino acid peptide SEQ ID NO1, but
 designed such that it contained a Glu to Gln substitution at the
 P1 of the Glu373-Ala374 bond, and has a biotinylated lysine
 residue at position 30.
 &lt;400&gt; SEQUENCE: 7
 Gln Thr Val Thr Trp Pro Asp Met Glu Leu Pro Leu Pro Arg Asn Ile
 1 5 10 15
 Thr Glu Gly Gln Ala Arg Gly Ser Val Ile Leu Thr Val Xaa
 20 25 30