WO2000005256A1 - Dosages et substrat de type peptides permettant de determiner l'action de metallo-protease degradant l'aggrecan - Google Patents

Dosages et substrat de type peptides permettant de determiner l'action de metallo-protease degradant l'aggrecan Download PDF

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WO2000005256A1
WO2000005256A1 PCT/US1998/015436 US9815436W WO0005256A1 WO 2000005256 A1 WO2000005256 A1 WO 2000005256A1 US 9815436 W US9815436 W US 9815436W WO 0005256 A1 WO0005256 A1 WO 0005256A1
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peptide
aggrecan
admp
activity
bond
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PCT/US1998/015436
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English (en)
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Jeffrey A. Miller
Elizabeth C. Arner
Robert A. Copeland
Gary L. Davis
Michael Pratta
Micky D. Tortorella
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Du Pont Pharmaceuticals Company
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Priority to AU85130/98A priority Critical patent/AU8513098A/en
Priority to PCT/US1998/015436 priority patent/WO2000005256A1/fr
Publication of WO2000005256A1 publication Critical patent/WO2000005256A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • a peptide containing a specific ADMP-susceptible cleavage site 1.
  • a peptide comprising a sequence of amino acids 1-40 of SEQ ID N ⁇ :l.
  • a peptide comprising a sequence of amino acids that is at least 80% identical to the sequence of amino acids 1-40 of SEQ ID NO:l.
  • a peptide of comprising a sequence of amino acids 1-40 of SEQ ID NO: 2.
  • a peptide comprising a sequence that is at least 80% identical to the sequence of amino acids 1-40 of SEQ ID NO: 3.
  • a peptide of claim 2 wherein the linking-moiety is a biotinylated lysine.
  • a product peptide of claim 1 comprising the amino acids from the N-terminus through PI of the ADMP- susceptible cleavage bond.
  • a product peptide of claim 1 comprising the amino acids from PI' of the ADMP-susceptible cleavage bond through C-terminus.
  • a peptide of claims 15 or 16 wherein the peptide is biotinylated is biotinylated.
  • a peptide of claim 18 wherein the linking- moiety is a biotinylated lysine.
  • a peptide comprising a sequence of amino acids 20-40 of claim 3, wherein an additional biotinylated lysine is attached to the C-terminus via a peptide bond, comprising a sequence of amino acids of SEQ ID NO: 5.
  • a peptide comprising a sequence of amino acids 1-20 of claim 3, wherein an additional biotinylated lysine is attached to the N-terminus via a peptide bond, comprising a sequence of amino acids of SEQ ID NO: 6.
  • a method for the determination of the presence of aggrecan-degrading metalloprotease activity comprising:
  • ADMP peptide substrate comprises a covalently-linked linking- moiety.
  • a method for the determination of ADMP activity by quantifying the appearance of a product peptide comprising:
  • a method for assaying compounds for activity against an ADMP comprising:
  • An assay for detecting ADMP activity which comprises:
  • tissue or cell source of ADMPs is cartilage or chondrocytes .
  • 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.
  • 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, Gl 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 Gl 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)
  • 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.
  • 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 34 l-Phe 342 an( - ⁇ t ] e o her between amino acid residues Glu 37 3-Ala 374 (human sequence enumeration) .
  • Gl fragments formed by cleavage at the Asn34l-phe342 site and at the Glu 373 -Ala 374 site have been identified within articular cartilage [Flannery et al. (1992) J.
  • ADMPs aggrecan degrading metallo proteases
  • 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.
  • ADMP aggrecan degrading metallo protease
  • 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) .
  • ADMPs aggrecan degrading metallo proteases
  • One peptide, based on the human aggrecan sequence around the Ala373-Glu374 ADMP-sensitive site has the sequence: QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL (SEQ ID No:l)
  • a second peptide, based on the bovine aggrecan sequence around the Ala373-Glu374 ADMP-sensitive site has the sequence :
  • Both peptides are capable of being cleaved at this specific recognition site by members of the family of
  • a third peptide, based on the human aggrecan sequence around the Alal714-Glyl715 ADMP- sensitive site has the sequence:
  • 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.
  • Figure 1 Shows the activity of a biotinylated form of the 41-mer peptide substrate (41-PS) against ADMP enzymatic activity using the microplate assay format and the inhibition of that activity by a hydroxymate inhibitor compound.
  • Figure 2 Shows the activity of a biotinylated form of the 41-mer peptide substrate (41-PS) against ADMP enzymatic activity using the HPLC assay format.
  • Figure 3 Shows the activity of the biotinylated form of the 41-mer peptide substrate (41-PS) against ADMP enzymatic activity using the microplate assay format and the inhibition of that activity by the 30-mer inhibitor peptide (30-IP) , QTVTWPDMELPLPRNITEGQARGSVILTVK-Biotin, the sequence of which is based upon the sequence of the 41-PS.
  • ADMP aggrecan degrading metallo protease
  • 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 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 Number 60/006,684 and subsequently described in U.S. Patent Application Serial Number 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
  • ELISA enzyme-linked immunosorbent assay
  • the aggrecan core protein encompasses, 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. conserveed amino acid sequences in various species around conserved ADMP-sensitive sites are shown below.
  • 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.
  • PVDF polyvinyl-denedifluoride
  • 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
  • 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 .
  • 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-A1820, or E1919-L1920 bond (numbering based on the human aggrecan core protein sequence) .
  • ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGT (SEQ ID NO: 3)
  • the human aggrecan protein 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 .
  • streptavidin coated supports 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.
  • a peptide substrate of this invention may be reversed in its role. With proper modification at the PI position the substrate may be turned into an inhibitor of ADMP activity. Specifically it was found that esterification of the Pi glutamic acid residue (GLU 373 ) of the substrate peptide SEQ ID NO:l or its replacement by glutamine abolish catalytic hydrolysis. Unexpectedly, the peptide containing the GLU to GLN substitution at amino acid position 373 (the Pl-glutamine containing peptide) was shown to be a competitive inhibitor of the enzyme. Thus, a carboxylate residue at position PI 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.
  • ADMP activity refers to the enzymatic activity of a family of polypeptides which specifically cleave the protein aggrecan within the interglobular domain at the Glu 37 3_Aia 3 74 peptide bond,
  • amino acid as used herein means an organic compound containing both a basic amino group and an acidic carboxyl group.
  • amino acid residue means that portion of an amino acid (as defined herein) that is present in a peptide.
  • 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.
  • 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".
  • 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.
  • substrate refers to a molecule that is bound by the active site and acted upon by the enzyme.
  • 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).
  • TMB 3', 5,5'- tetramethylbenzidine
  • noneoepitope 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
  • 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
  • aggrecan 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.
  • 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 PI position and shows at least 65% homology with the PI, P2, P3, PI', P2' and P3' amino acids of one or more of the ADMP-sensitive sites in the aggrecan molecule.
  • issel bond refers to the peptide bond of a polypeptide that is to be cleaved by a protease.
  • PI refers to the amino acid residue on the N-terminal side of the sissel bond.
  • P2 refers to the amino acid residue adjacent to PI on the N-terminal side of the sissel bond.
  • P3 refers to the amino acid residue adjacent to P2 on the N-terminal side of the sissel bond.
  • Pi 1 refers to the amino acid residue on the C-terminal side of the sissel bond.
  • P2 1 refers to the amino acid residue adjacent to PI' on the C-terminal side of the sissel bond.
  • P3 ' refers to the amino acid residue adjacent to P2 ' on the C-terminal side of the sissel bond.
  • 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 Glu373-Ala374 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).
  • SEQ ID NO:l refers to the peptide sequence QTVTWPDMELPLPRNITEGE-ARGSVILTVKPIFEVSPSPL.
  • SEQ ID NO: 2 refers to the peptide sequence
  • SEQ ID NO: 3 refers to the peptide sequence
  • ITFVDTSLVEVTPTTFKEEE-GLGSVELSGLPSGELGVSGT The term “41- PS” and “SEQ ID NO:” refer to the peptide sequence: QTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPL- (BIOTINYL) K.
  • SEQ ID NO: 5" refers to the peptide sequence: ARGSVILTVKPIFEVSPSPL- (BIOTINYL) K.
  • SEQ ID NO: 6 refers to the peptide sequence: K (BIOTINYL) - QTVTWPDMELPLPRNITEGE.
  • 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:l was prepared by solid phase peptide synthesis.
  • the peptide was prepared commercially (Quality Controlled Biochemicals, Inc. Hopkinton, MA) as a biotin conjugate by adding an additional lysine residue at the carboxy terminus of the peptide SEQ ID NO:l. Biotin was then covalently attached through the lysine ⁇ -amino side chain.
  • 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
  • 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 7xl0 -8 M 41-PS was prepared in
  • Microplate strips (eight wells each) were rinsed once with 100 ⁇ L of IX Assay Buffer (Assay Buffer consists of: 50 mM Tris, pH 7.5, 10 mM CaCl 2 , and 100 mM NaCl) and blotted dry. Reactions were prepared in duplicate in a final volume of 100 ⁇ L, containing: 50 ⁇ L of 2X Assay Buffer (100 mM Tris, pH 7.5, 20 mM CaCl 2 , and 200 mM
  • 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 ⁇ M
  • 25 ⁇ L of soluble ADMP 25 ⁇ L
  • the microplate strips were incubated for 3 hours at 37 °C.
  • the microplate wells were then washed six times with 200 ⁇ L of IX PBS, 0.05% Tween 20 using a Denley Well Wash 4 micro plate washer.
  • a BC-3 antibody solution was prepared by adding 4 ⁇ 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
  • the secondary (detection) antibody solution was prepared by adding 4 ⁇ 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 ⁇ L of this
  • ADMP activity can easily be followed by this method and inhibition of ADMP activity can be monitored.
  • the IC 50 for the inhibition of ADMP by the hydroxamate inhibitor tested was 0.413 ⁇ M.
  • HPLC High Performance Liquid Chromatography
  • the HPLC assay is performed in the following manner.
  • the reaction buffer contains 50 mM HEPES, 10 mM CaCl 2 , 100 mM NaCl and 0.05% Brij-35, pH 7.5.
  • a 10 ⁇ L portion of the reaction mixture was injected onto a reverse-phase HPLC C ⁇ 8 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 (Figure 2) .
  • a peptide inhibitor was prepared based upon the sequence of the 40 amino acid peptide SEQ ID NO:l, but designed such that it contained a Glu to Gin substitution at the PI of the Glu373-Ala374 bond.
  • Example 3 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 ⁇ M.
  • ADMP activity was inhibited as shown in Figure 3.
  • the IC 50 for the inhibition of ADMP activity by 30-IP was 11 ⁇ M.
  • 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 ⁇ L in 0.05 M Tris, pH 7.6, containing 0.1 M NaCl and 10 mM CaCl 2 - Reactions were incubated for 4 hr at 37°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, MA) was prewet with 50 ⁇ L per well 70% ethanol, incubated for 30 seconds at room temperature then flushed two times each with 200 ⁇ L of purified H2O. The plate was then coated with aggrecan equivalent to 36 ⁇ g of glycosaminoglycan (GAG) as detected by the dimethyl methylene blue dye assay
  • Membranes were then blocked with 150 ⁇ 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 ⁇ L of IX TBS buffer, allowing 20 seconds of contact with membrane per wash.
  • 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 ⁇ g GAG in 150 uL 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 °C for 1 hour.
  • Buffer B comprises 50 mM sodium acetate, 100 mM NaCl, pH 6.5
  • Buffer B were added and allowed to incubate an additional 2 hours at 37 °C. Enzyme solution was filtered out and membranes rinsed one time with 200 ⁇ L of Buffer A. 150 ⁇ 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 ⁇ L Buffer A allowing membrane contact for 20 seconds per wash.
  • a unit of ADMP activity is defined as that resulting in product produced equivalent to 1 ug BSA peptide per hour at 37°C.
  • DMSO dimethyl sulfoxide
  • ADMP anti-oxidant-proliferative protein
  • compounds are prepared as 10 mM stocks in dimethyl sulfoxide (DMSO), water or other solvents and diluted to appropriate concentrations in water.
  • Drug 50 ⁇ L was added to 50 ⁇ L of 2 mg/mL aggrecan substrate and 50 ⁇ l of ADMP (40 units/ml) and brought to a final volume of 200 ⁇ L iby addition of 50 ⁇ l of 0.2 M Tris, pH 7.6, containing 0.4 M NaCl and 40 mM CaCl 2 .
  • the reaction mixture was incubated for 4 hr at 37 °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.

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  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne des dosages qui permettent de déterminer la présence ou l'absence de protéines ayant une action d'aggrécanase ou ADMP. Cette invention concerne également des peptides qui jouent le rôle de substrats pour les ADMP, l'utilisation de ces peptides dans divers dosages permettant de déterminer la présence ou l'absence d'action ADMP, ainsi que l'utilisation de ces peptides en qualité d'inhibiteurs d'action ADMP.
PCT/US1998/015436 1998-07-24 1998-07-24 Dosages et substrat de type peptides permettant de determiner l'action de metallo-protease degradant l'aggrecan WO2000005256A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU85130/98A AU8513098A (en) 1998-07-24 1998-07-24 Assays and peptide substrate for determining aggrecan degrading metallo proteaseactivity
PCT/US1998/015436 WO2000005256A1 (fr) 1998-07-24 1998-07-24 Dosages et substrat de type peptides permettant de determiner l'action de metallo-protease degradant l'aggrecan

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1998/015436 WO2000005256A1 (fr) 1998-07-24 1998-07-24 Dosages et substrat de type peptides permettant de determiner l'action de metallo-protease degradant l'aggrecan

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WO2000005256A1 true WO2000005256A1 (fr) 2000-02-03

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003062263A2 (fr) * 2002-01-16 2003-07-31 Ortho Mc Neil Pharmaceutical, Inc. Substrats peptidiques aggrecanase-1 et -2 et procedes associes
EP1476545A2 (fr) * 2002-01-31 2004-11-17 Wyeth Molecules d'aggrecanase
WO2005124352A2 (fr) * 2004-06-18 2005-12-29 InViTek Gesellschaft für Biotechnik & Biodesign mbH Procede de determination des activites de l'aggrecanase

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993022429A1 (fr) * 1992-04-29 1993-11-11 Shriners Hospitals For Crippled Children Nouvelles compositions et nouveaux procedes pour detecter et traiter l'arthrose chez l'homme
WO1996001847A1 (fr) * 1994-07-07 1996-01-25 The University Of Melbourne Methodes et compositions relatives aux proteines de proteoglycane utilisees pour le diagnostic de la decomposition des cartilages
WO1997025437A1 (fr) * 1996-01-04 1997-07-17 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Procede de dosage d'enzymes proteolytiques recourant a des substrats a fluorescence attenuee
EP0785274A1 (fr) * 1996-01-18 1997-07-23 Hoechst Aktiengesellschaft Un substrat recombinant artificiel rAGG-1 et l'aggrecan naturel d'examen de l'activité protéilytique de 'aggrecanase' dans des systèmes de cultures cellulaires

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993022429A1 (fr) * 1992-04-29 1993-11-11 Shriners Hospitals For Crippled Children Nouvelles compositions et nouveaux procedes pour detecter et traiter l'arthrose chez l'homme
WO1996001847A1 (fr) * 1994-07-07 1996-01-25 The University Of Melbourne Methodes et compositions relatives aux proteines de proteoglycane utilisees pour le diagnostic de la decomposition des cartilages
WO1997025437A1 (fr) * 1996-01-04 1997-07-17 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Procede de dosage d'enzymes proteolytiques recourant a des substrats a fluorescence attenuee
EP0785274A1 (fr) * 1996-01-18 1997-07-23 Hoechst Aktiengesellschaft Un substrat recombinant artificiel rAGG-1 et l'aggrecan naturel d'examen de l'activité protéilytique de 'aggrecanase' dans des systèmes de cultures cellulaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE MPSRCH GENBANK 1 January 1900 (1900-01-01), XP002100469, Database accession no. P13608 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003062263A2 (fr) * 2002-01-16 2003-07-31 Ortho Mc Neil Pharmaceutical, Inc. Substrats peptidiques aggrecanase-1 et -2 et procedes associes
WO2003062263A3 (fr) * 2002-01-16 2004-01-15 Ortho Mc Neil Pharmaceutical I Substrats peptidiques aggrecanase-1 et -2 et procedes associes
EP1476545A2 (fr) * 2002-01-31 2004-11-17 Wyeth Molecules d'aggrecanase
EP1476545A4 (fr) * 2002-01-31 2006-11-29 Wyeth Corp Molecules d'aggrecanase
WO2005124352A2 (fr) * 2004-06-18 2005-12-29 InViTek Gesellschaft für Biotechnik & Biodesign mbH Procede de determination des activites de l'aggrecanase
WO2005124352A3 (fr) * 2004-06-18 2006-06-29 Invitek Biotechnik & Biodesign Procede de determination des activites de l'aggrecanase

Also Published As

Publication number Publication date
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