Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6489—Metalloendopeptidases (3.4.24)

Definitions

• the invention relates to a novel protein, its fragments and mutants and to its use in detecting and testing drugs for ailments, including osteoarthritis and others characterized by up regulation of metalloproteases.
• a number of enzymes effect the breakdown of structural proteins and are structurally related metalloproteases. These include human skin fibroblast collagenase, human skin fibroblast gelatinase, human sputum collagenase and gelatinase, and human stromelysin. See e.g., S.E. Whitham et al., Comparison of human stromelysin and collagenase by cloning and sequence analysis" Biochem J. 240:913 (1986). See also G.I. Goldberg et al., "Human Fibroblast Collagenase” J Biol. Chem. 261:660 (1986). Metal dependence (e.g., zinc) is a common feature of these structurally related enzymes known as "metalloproteases.”
• Inhibitors of metalloproteases are useful in treating diseases caused, at least in part, by breakdown of structural proteins.
• a variety of inhibitors have been prepared, but there is a continuing need for metalloprotease inhibitor screens to design drugs for treating such diseases.
• Example TapI-2 and 1,10-phenanthroline are known metalloprotease inhibitors. See, e.g., J. Arribas et al., "Diverse Cell Surface Protein Ectodomains Are Shed by a System Sensitive to Metalloprotease Inhibitors", J. Biol. Chem. 271 :11376 (1996).
• Metalloproteases are a broad class of proteins which have widely varied functions. Disintegrins are zinc metalloproteases, abundant in snake venom. Mammalian disintegrins are a family of proteins with about 18 known subgroups. They act as cell adhesion disrupters and are also known to be active in reproduction (for example, in fertilization of the egg by the sperm, including fusion thereof, and in sperm maturation).
• GenBank a repository for gene sequences, provides several sequences of metalloproteases, including some said to encode fragments of disintegrins.
• GenBank accession # Z48444 dated February 25, 1994 discloses 2407 nucleotides of a rat gene said to be a rat disintegrin metalloprotease gene
• GenBank accession # Z48579 dated March 2, 1995 discloses 1824 nucleotides of a partial sequence of a gene said to be a human disintegrin metalloprotease gene
• GenBank accession # Z21961 dated October 25, 1994 discloses 2397 nucleotides of a partial sequence of a gene said to be a bovine zinc metalloprotease gene. Because there is such a wide variety of metalloproteases, there is a continuing need for i) methods that will specifically detect a particular metalloprotease, as well as ii) methods for identifying candidate inhibitors.
• metalloprotease mediated diseases such as cancer, arthropothies (including ankylosing spondolytis, rheumatiod arthritis, gouty arthritis (gout), inflammatory arthritis, Lyme disease and osteoarthrtis).
• This invention provides a method for identifying compounds capable of binding to the disintegrin protein, and determining the amount and affinity of a compound capable of binding to the disintegrin protein in a sample.
• This invention also provides a host cell comprising a recombinant expression vector to the disintegrin protein and a recombinant expression vector encoding to the disintegrin protein and the human disintegrin metalloprotease protein, fragment or mutant thereof, useful for these purposes.
• This invention also provides an in vivo or in vitro method for screening for osteoarthritis and other metalloprotease based diseases, such as cancer, capable of manufacture and use in a kit form.
• the term "gene” refers to a DNA >equence that comprises control and coding sequences necessary for the production of a mature protein or precursor thereof.
• the protein can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired cn/> matic activity is retained.
• oligonucleotide as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ⁇ bonucleotides, usually more than three (3). and typically more than ten (10) and up to one hundred (100) or more (although preferably between twenty and thirty). The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
• the oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, restriction endonuclease digestion reverse transcription, or a combination thereof.
• an end of an oligonucleotide is referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
• a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.
• the former may be called the "upstream” oligonucleotide and the latter the "downstream” oligonucleotide.
• primer refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated.
• An oligonucleotide “primer” may occur naturally, as in a purified restriction digest or may be produced synthetically.
• a primer is selected to be "substantially" complementary to a strand of specific sequence of the template.
• a primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur.
• a primer sequence need not reflect the exact sequence of the template.
• a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being substantially complementary to the strand.
• Non- complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
• Hybridization methods involve the annealing of a complementary sequence to the target nucleic acid (the sequence to be detected).
• the ability of two polymers of nucleic acid containing complementar sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon.
• the initial observations of the "hybridization” process b Marmur and Lane, Proc. Natl. Acad Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology. Nonetheless, a number of problems have prevented the wide scale use of hybridization as a tool in human diagnostics. Among the more daunting problems are: 1) the inefficiency of hybridization; 2) low concentration of specific target sequences in a mixture of genomic DNA; and 3) the hybridization of only partially complementary probes and targets.
• the target sequence must be made accessible to the probe via rearrangements of higher-order structure.
• These higher-order structural rearrangements may concern either the secondary structure or tertiary structure of the molecule.
• Secondary structure is determined by intramolecular bonding. In the case of DNA or RNA targets this consists of hybridization within a single, continuous strand of bases (as opposed to hybridization between two different strands). Depending on the extent and position of intramolecular bonding, the probe can be displaced from the target sequence preventing hybridization.
• the DNA fragment containing the target sequence is usually in relatively low abundance in genomic DNA. This presents great technical difficulties: most conventional methods that use oligonucleotide probes lack the sensitiv ity necessary to detect hybridization at such low levels.
• K. B. Mullis et al. U.S. Patent Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, describe a method for increasing the concentration of a segment of a target sequence in a mixture of any DNA without cloning or purification.
• This process for amplifying the target sequence (which can be used in conjunction with the present invention to make target molecules) consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase.
• the two primers are complementary to their respective strands of the double stranded target sequence.
• the mixture is denatured and the primers then allowed to annealed to their complementary sequences within the target molecule.
• the primers are extended with a polymerase so as to form a new pair of complementary strands.
• the steps of denaturation, primer annealing, and primer extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle;” there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
• the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
• PCR Polymerase Chain Reaction
• any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.
• the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
• the PCR amplification process is known to reach a plateau concentration of specific target sequences of approximately 10"° M.
• a typical reaction volume is 100 ⁇ l, which corresponds to a yield of 6 x 10 ⁇ double stranded product molecules.
• complementarity it is important for some diagnostic applications to determine whether the hybridization represents complete or partial complementarity. For example, where it is desired to detect simply the presence or absence of pathogen DNA or RNA (such as from a virus, bacterium, fungi, mycoplasma, protozoan) it is only important that the hybridization method ensures hybridization when the relevant sequence is present; conditions can be selected where both partially complementary probes and completely complementary probes will hybridize. Other diagnostic applications, however, may require that the hybridization method distinguish between partial and complete complementarity. It may be of interest to detect genetic polymorphisms. For example, human hemoglobin is composed, in part, of four polypeptide chains.
• Two of these chains are identical chains of 141 amino acids (alpha chains) and two of these chains are identical chains of 146 amino acids (beta chains).
• the gene encoding the beta chain is known to exhibit polymorphism.
• the normal allele encodes a beta chain having glutamic acid at the sixth position.
• the mutant allele encodes a beta chain having valine at the sixth position.
• This difference in amino acids has a profound (most profound when the individual is homozygous for the mutant allele) physiological impact known clinically as sickle cell anemia. It is well known that the genetic basis of the amino acid change involves a single base difference between the normal allele DNA sequence and the mutant allele DNA sequence.
• the probe will hybridize to both the normal and variant target sequence.
• Hybridization regardless of the method used, requires some degree of complementarity between the sequence being assayed (the target sequence) and the fragment of DNA used to perform the test (the probe). (Of course, one can obtain binding without any complementarity but this binding is nonspecific and to be avoided.)
• nucleic acid sequence refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "antiparallel association.”
• Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
• nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
• T m melting temperature
• Other references include more sophisticated computations which take structural as well as sequence characteristics into account for the calculation of T m .
• probe refers to a labeled oligonucleotide which forms a duplex structure with a sequence in another nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the other nucleic acid.
• label refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Such labels can be added to the oligonucleotides of the present invention.
• substantially single-stranded when used in reference to a nucleic acid substrate means that the substrate molecule exists primarily as a single strand of nucleic acid in contrast to a double-stranded substrate which exists as two strands of nucleic acid which are held together by inter-strand base pairing interactions.
• sequence variation refers to differences in nucleic acid sequence between two nucleic acid templates.
• a wild-type structural gene and a mutant form of this wild-type structural gene may vary in sequence by the presence of single base substitutions and/or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another.
• a second mutant form of the structural gene may exist.
• This second mutant form is said to vary in sequence form both the wild-type gene and the first mutant form of the gene. It should be noted that, while the invention does not require that a comparison be made between one or more forms of a gene to detect sequence variations, such comparisons are possible with the oligo/ solid support matrix of the present invention using particular hybridization conditions as described in U.S. Pat. Appl. Ser. No. 08/231,440, hereby incorporated by reference.
• Oligonucleotide primers matching or complementary to a gene sequence refers to oligonucleotide primers capable of facilitating the template-dependent synthesis of single or double-stranded nucleic acids.
• Oligonucleotide primers matching or complementary to a gene sequence may be used in PCRs, reverse transcriptase-PCR (RT-PCRs) and the like.
• a "consensus gene sequence” refers to a gene sequence which is derived by comparison of two or more gene sequences and which describes the nucleotides most often present in a given segment of the genes; the consensus sequence is the canonical sequence.
• the terms “protein” and “protease” refer to metalloprotease.
• metalloprotease refers to a native metal dependent protease, a fragment thereof, a mutant or homologue which still retains its function.
• the invention contemplates metalloproteases (or “disintegrins”) from differing species, and those prepared by recombinant methods, in vitro methods, or standard peptide synthesis.
• the protein is a human disintegrin or mutant thereof.
• the preferred "native" protein is partially described in Gen Bank accession #Z48579, incorporated herein by reference and referred to in the sequence below.
• Homologue disintegrins include whole proteins with at least 90% homology as understood by the art, or fragments thereof. It is recognized that some interspecies variation may occur including insertions or deletions which may or may not alter function. For example, a rat protein which is 95% homologous to the protein based on the peptide sequence, and a bovine protein (based on DNA sequence) being 97-98% homologous based on the first 300 base pairs are both considered homologues.
• GenBank accession #Z48444 dated February 25, 1994 discloses 2407 bases of a rat gene said to be a rat disintegrin metalloprotease gene;
• GenBank accession #Z21961 dated October 25, 1994 discloses 2397 bases of a partial sequence of a gene said to be a rxmne zinc metalloprotease gene.
• this metalloprotease is a human disintegrin as described below.
• the term "antibody” refers to an antibody to a disintegrin, or fragment thereof. These many be monoclonal or polyclonal, and can be from any of several sources. The invention also contemplates fragments of these antibodies made by any method in the protein or peptide art.
• disease screen refers to a screen for a disease or disease state.
• a disease state is the physiological or cellular or biochemical manifestation of the disease.
• this screen is used on body tissues or fluids of an animal or cell culture, using standard techniques, such as ELISA. It also contemplates "mapping" of disease in a whole body, such as by labeled antibody as described above given systemically: regardless of the detection method, preferable such detection methods include fluorescence, X-ray (including CAT scan), NMR (Including MRI), and the like.
• compound screen is related to the methods and screens related to finding compounds, determining their affinity for the protease, or designing or selecting compounds based on the screen.
• it contemplates the use of the three dimensional structure for drug design, preferable “rational drug design”, as understood by the art.
• the protease is in "essentially pure form", which refers to a protein reasonably free of other impurities, so as to make it useful for experiments or characterization. Use of this screening method assists the skilled artisan in finding novel structures, whether made by the chemist or by nature, which bind to and preferably inhibit the protease.
• inhibitors may be useful in regulating or modulating the activity of the protease, and may be used to thus modulate the biological cascade that they function in. This approach affords new pharmaceutically useful compounds.
• the term “disintegrin” refers to a disintegrin, a fragment thereof, a mutant thereof or a homologue which still retains its function. This term contemplates aggrecanase, and other proteases which are involved in or modulate tissue remodeling. This contemplates disintegrins from differing species, and those prepared by recombinant methods, in vitro methods, or standard peptide synthesis.
• the protein is a human disintegrin or mutant thereof.
• SEQ ID NO:l describes a fragment of that DNA sequence and its transcript and SEQ ID NO:2 describes the protein coded by the gene.
• Homologue disintegrins include whole proteins with at least 90% homology as understood by the art, or fragments thereof.
• a rat protein which is 95% homologous to that of SEQ ID NO:2 based on the amino acid sequence deriv a ;rom the DNA or cDNA sequence containing SEQ ID NO: 1 , and a bovine protein (similarly derived) being 97-98% homologous, are both considered homologues.
• homologous cDNAs cloned from other organisms give rise to homologous proteins.
• proteins may be considered homologues based on the amino acid sequence alone. Practical limitations of amino acid sequencing would allow one to determine that a protein is homologous to another using, for example, comparison of the first 50 amino acids of the protein. Hence 90% homology in would allow for 5 differing amino acids in the chain of the first 50 amino acids of the homologous protein.
• the skilled artisan will appreciate that the degeneracy of the genetic code provides for differing DNA sequences to provide the equivalent transcript, and thus the same protein. In certain cases preparing the DNA sequence, which encodes for the same protein, but differs from the native DNA include;
• fragments of the protein may be used in screening, drug design and the like, and that the entire protein may not be required for the purposes of using the invention.
• the skilled artisan will understand that the disclosure of the protein and its uses contemplates the useful peptide fragments.
• the present invention specifically contemplates the use of less than the entire nucleic acid sequence for the gene and less than the entire amino acid sequence of the protein. Fragments of the protein may be used in screening, drug design and the like, and that the entire protein may not be required for the purposes of using the invention.
• the protein itself can be used to determine the binding activity of small molecules to the protein. Drug screening using enzymatic targets is used in the art and can be employed using automated, high throughput technologies.
• the protein or protease itself can be used to determine the binding activity of small molecules to the protein. Drug screening using enzymatic targets is used in the art and can be employed using automated, high throughput technologies.
• the inhibition of disintegrin activity may be a predictor of efficacy in the treatment of osteoarthritis, and other diseases involving degeneration of articular cartilage and other tissues having matrix degradation, such as tissue remodeling and the like.
• the metalloprotease is up regulated during osteoarthritis in tissues.
• a human disintegrin is up-regulated in human chondrocytes during osteoarthritic conditions.
• Inhibition of signal transduction mechanism is efficacious in disrupting the cascade of events in osteoarthritis and other diseases involving cartilage degeneration.
• up-regulation is a cause of the onset of arthritis, then interfering with the activity of this gene may be useful in treating osteoarthritis.
• gene therapy i.e., antisense therapy.
• Media, cell extracts or inclusion bodies from mammalian, yeast, insect or eukaryotic cells containing recombinant disintegrin or fragments of the full length protein are used for purification of disintegrin or fragments of disintegrin. Solutions consisting of denatured disintegrin may be refolded prior to purification across successive chromatographic resins or following the final stage of separation. Media, cell extracts, or solubilized disintegrin are prepared in the presence of one or a combination of detergents, denaturants or organic solvents, such as octylglucoside, urea or dimethylsulfoxide, as required.
• Ion exchange and hydrophobic interaction chromatography are used individually or in combination for the separation of recombinant disintegrin from contaminating cell material. Such material is applied to the column and disintegrin is eluted by adjustment of pH, changes in ionic strength, addition of denaturant and/or use of organic solvent. Typically, solutions containing disintegrin are then passed over an antibody affinity column or ligand affinity column for site specific purification of disintegrin.
• the immunoaffinity column contains an antibody specific for disintegrin immobilized on a solid support such as Sepharose 4B
• the column is washed to remove unbound proteins and the disintegrin is eluted via low pH glycine buffer or high ionic strength.
• the ligand affinity column may have specificity for the active site of disintegrin or to a portion of the molecule adjacent or removed from the active site.
• the column is washed and disintegrin is eluted by addition of a competing molecule to the elution buffer.
• a protease inhibitor cocktail containing one or more protease inhibitors, such as benzamidine, leupeptin, phosphoramidon, phenylmethylsulfonyl fluoride, and 1,10-phenanthroline is present throughout the purification procedure.
• Various detergents such as octylthioglucoside and Triton X-
• glycerol may be added to increase disintegrin solubility and stability.
• Final purification of the protein is achieved by gel filtration across a chromatographic support, if required.
• the protease of the invention can be used to find inhibitors of the protease.
• the protease may modulate cellular remodeling and in fact may enhance extracellular matrix remodeling and thus enhance tissue breakdown.
• inhibition of disintegrin provides a therapeutic route for treatment of diseases characterized by these processes.
• a drug compound in screening, can be used to determine both the quality and quantity of inhibition. As a result such screening provides information for selection of actives, preferably small molecule actives, which are useful in treating these diseases.
• synthetic metalloprotease inhibitors such as those used to inhibit the matrix metalloproteases would be used to inhibit extracellular matrix remodeling.
• Metalloproteases can be targeted by conjugating a metalloprotease inhibitor to a to an antibody or fragment thereof. Conjugation methods are known in the art. These antibodies are then useful both in therapy and in monitoring the dosage of the inhibitors.
• the antibody of the invention can also be conjugated to solid supports. These conjugates can be used as affinity reagents for the purification of a desired metalloprotease, preferably a disintegrin.
• the antibody of the invention is directly conjugated to a label.
• the label can be used to detect the presence of relatively high levels of metalloprotease in vivo or in vitro cell culture.
• targeting ligand which specifically reacts with a marker for the intended target tissue
• Methods for coupling the invention compound to the targeting ligand are well known and are similar to those described below for coupling to carrier.
• the conjugates are formulated and administered as described above.
• Antibodies may be made by several methods, for example, the protein may be injected into suitable (e.g., mammalian) subjects including mice, rabbits, and the like. Preferred protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum.
• suitable e.g., mammalian
• Preferred protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum.
• the titers of the immune serum can readily be measured using immunoassay procedures, now standard in the art.
• the antisera obtained can be used directly or monoclonal antibodies may be obtained by harvesting the peripheral blood lymphocytes or the spleen of the immunized animal and immortalizing the antibody-producing cells, followed by identifying the suitable antibody producers using standard immunoassay techniques.
• Polyclonal or monoclonal preparations are useful in monitoring therapy or prophylaxis regimens involving the compounds of the invention.
• Suitable samples such as those derived from blood, serum, urine, or saliva can be tested for the presence of the protein at various times during the treatment protocol using standard immunoassay techniques which employ the antibody preparations of the invention.
• These antibodies can also be coupled to labels such as scintigraphic labels, e.g.,
• Tc-99 or 1-131 using standard coupling methods.
• the labeled compounds are administered to subjects to determine the locations of excess amounts of one or more metalloproteases in vivo.
• a labeled antibody to the protein would operate as a screening tool for such enhanced expression, indicating the disease.
• the ability of the antibodies to bind metalloprotease selectively is thus taken advantage of to map the distribution of these enzymes in situ.
• the techniques can also be employed in histological procedures and the labeled antibodies can be used in competitive immunoassays.
• Antibodies are advantageously coupled to other compounds or materials using known methods.
• materials having a carboxyl functionality the carboxyl residue can be reduced to an aldehyde and coupled to carrier through reaction with side chain amino groups, optionally followed by reduction of imino linkage formed.
• the carboxyl residue can also be reacted with side chain amino groups using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.
• Condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.
• Linker compounds can also be used to effect the coupling; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, 111.
• the present invention contemplates detecting expression of metalloprotease genes in samples, including samples of diseased tissue. It is not intended that the present invention be limited by the nature of the source of nucleic acid (whether DNA or RNA); a variety of sources is contemplated, including but not limited to mammalian (e.g., cancer tissue, lymphocytes, etc.), sources.
• mammalian e.g., cancer tissue, lymphocytes, etc.
• this gene may have a restricted tissue distribution and its expression is up regulated by potential osteoarthritis mediators.
• Enhanced expression of this gene (and hence its protein) for example, in articular chondrocytes provides a marker to monitor the development, including the earliest, asymptomatic stages, and the progression of osteoarthritis.
• an antibody raised to the protein would operate a screening tool for such enhanced expression, indicating the disease.
• antibodies when used in a disease screen, antibodies can be conjugated to chromophore or fluorophore containing materials, or can be conjugated to enzymes which produce chromophores or fluorophores in certain conditions. These conjugating materials and methods are well known in the art. When used in this manner detection of the protein by immunoassay is straightforward to the skilled artisan. Body fluids, (serum, urine, synov lal tluid) for example can be screened in this manner for calibration, and detection of Jistnbution of metalloproteases, or increased levels of these proteases.
• the invention is a useful diagnostic and/or clinical marker for metalloprotease mediate " diseases, such as osteoarthritis or other articular cartilage degenerative diseases or other diseases characterized by degradation or remodeling of extracellular matrix.
• diseases such as osteoarthritis or other articular cartilage degenerative diseases or other diseases characterized by degradation or remodeling of extracellular matrix.
• disease may be treated before the onset of symptom or debilitation.
• the nucleic acid content of cells consists of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
• DNA contains the genetic blueprint of the cell.
• RNA is involved as an intermediary in the production of proteins based on the DNA sequence. RNA exists in three forms within cells, structural RNA (i.e., ribosomal RNA "rRNA”), transfer RNA ("tRNA”), which is involved in translation, and messenger RNA ("mRNA"). Since the mRNA is the intermediate molecule between the genetic information encoded in the DNA, and the corresponding proteins, the cell's mRNA component at any given time is representative of the physiological state of the cell. In order to study and utilize the molecular biology of the cell, it is therefore important to be able to purify mRNA, including purifying mRNA from the total nucleic acid of a sample.
• RNA is complicated by the presence of ribonucleases that degrade RNA (e.g., T. Maniatis et al., Molecular Cloning, pp. 188-190, Cold Spring Harbor Laboratory [1982]). Furthermore, the preparation of amplifiable RNA is made difficult by the presence of ribonucleoproteins in association with RNA. (See, R. J. Slater, In: Techniques in Molecular Biology. J. M. Walter and W. Gaastra, eds., Macmillan, NY, pp. 113-120 [1983]).
• the steps involved in purification of nucleic acid from cells include 1) cell lysis; 2) inactivation of cellular nucleases; and 3) separation of the desired nucleic acid form the cellular debris and other nucleic acid.
• Cell lysis may be achieved through various methods, including enzymatic, detergent or chaotropic agent treatment.
• Inactivation of cellular nucleases may be achieved by the use of proteases and/or the use of strong salts.
• separation of the desired nucleic acid is typically achieved by extraction of the nucleic acid with phenol or phenol-chloroform; this method partitions the sample into an aqueous phase (which contains the nucleic acids) and an organic phase (which contains other cellular components, including proteins).
• the structure of the mRNA molecule may be used to assist in the purification of mRNA from DNA and other RNA molecules.
• poly-A tail or "poly-A track”
• one means of isolating RNA from cells has been based on binding the poly-A tail with its complementary sequence (i.e., oligo-dT), that has been linked to a support such as cellulose.
• oligo-dT complementary sequence
• the hybridized mRNA/oligo-dT is separated from the other components present in the sample through centrifugation or, in the case of magnetic formats, exposure to a magnetic field.
• the mRNA is usually removed from the oligo-dT. However, for some applications, the mRNA may remain bound to the oligo-dT that is linked to a solid support.
• the mRNA is treated with reverse transcriptase to make cDNA.
• the cDNA can be used in primer extension and PCR using the primers described below.
• primer extension and PCR for that matter
• primer extension can be carried out under conditions (so-called "high stringency conditions") such that only complementary nucleic acid will hybridize (as opposed to hybridization with partially complementary nucleic acid). These conditions including annealing at or near the melting temperature of the duplex.
• the invention provides a partial nucleic acid full length protein coding region sequence of a novel disintegrin metalloprotease gene useful for, among other things, the detection of disintegrin metalloprotease gene expression.
• primers directed to a portion of this partial sequence are use to detect the presence or absence of the gene sequence. These primers can be also be used for the identification of a cDNA clone representing the entire gene, allowing for recombinant expression in a host cell of the nucleic acid sequence encoding the disintegrin metalloprotease or fragments (or mutants) thereof.
• Preferred primers are primer SEQ ID NO:9 (5 * -AGCCTGTGTC-3') and SEQ ID NO: 10 (5'-AGCCTGTGTCTGAACCACT-3').
• other primers can be readily designed from the sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 1.
• the present invention contemplates, in one embodiment, using electrophoresis to confirm product formation and compare the results between samples.
• the present invention contemplates detection of sequences of the novel disintegrin metalloprotease gene in mixtures of nucleic acid (e.g., cDNA or RT- mRNA).
• SEQ ID NO:l is a fragmentary DNA sequence, and is part of SEQ ID NO:3.
• the first base (Cytosine or C) of SEQ ID NO:l is base 940 of SEQ ID NO:3.
• the DNA sequences are identical where they overlap.
• SEQ ID NO:2 and SEQ ID NO:4 are the expressed amino acid sequences of SEQ ID NO:l and SEQ ID NO:3 respectively.
• the first amino acid of SEQ ID NO:2, Gin is the 309th amino acid in SEQ ID NO:4.
• the two sequences are homologous to the carboxy terminus of the protein.
• SEQ ID NO:7 is a sense strand of DNA provided by differential display experiments.
• the first base of SEQ ID NO:7 corresponds to base 1371 of SEQ ID NO:l, and to base 2310 of SEQ ID NO: . These sequences are homologous for 452 bases, to base 1822 of SEQ ID NO: I and to base 2761 of SEQ ID NO:3.
• the difference in the last two bases of SEQ ID NO:l and SEQ ID NO:3 may be due to errors in sequencing or a common replicatory error found in PCR, or may be part of a cloning vector.
• SEQ ID NO:7 continues some 284 bases beyond the homology, and thus well beyond the terminus of SEQ ID NO:l and SEQ ID NO:3.
• bases 477 to 716 of SEQ ID NO:7 are the SEQ ID NO 6.
• SEQ ID NO 6 is the sense strand of SEQ ID NO:5, which is an antisense strand found via differential display cl9oning. Hence SEQ ID NO: 6 shows the DNA orientation as it would appear in the mRNA. These two sequences are found near the 3' end of this gene. Although bases 452 to the 3' end of SEQ ID NO:7 differ from SEQ ID NO:l and SEQ ID NO:3, SEQ ID NO:7 is nonetheless valid. It is essential to note that the expressed peptide sequence is not affected by this difference. It is likely these bases do not appear in SEQ ID NO:l and SEQ ID NO:3 because of the use of an alternative polyadenylation signal. SEQ ID NO 8 is a novel full length DNA sequence.
• SEQ ID NO:9 is the novel expressed protein of SEQ ID NO:8.
• SEQ ID NO:9 differs from SEQ ID NO:4 in that amino acids 162 (Ser)-213 (Tyr) of SEQ ID NO: 4 is replaced by a single residue, Asn, at position 162 of SEQ ID NO:9. That change is reflected in the DNA by a deletion bases 501-654 for a total of 153 bases, leaving the reading frame intact but changing one residue and deleting the 51 amino acids present in SEQ ID NO:4.
• SEQ ID NO: 10 and SEQ ID NO: l 1 are antisense primers useful in PCR, and are the inverse of the 3' terminus of SEQ ID NO:7, other sequences for primers are discernible by the skilled artisan using sequences refe ⁇ ed to herein.
• EXAMPLES The following non-limiting examples illustrate a preferred embodiment of the present invention, and briefly describe the uses of the present invention. These examples * Sre provided for the guidance of the skilled artisan, and do not limit the invention in any way. Armed with this disclosure and these examples the skilled artisan is capable of making and using the claimed invention. Standard starting materials are used for these examples. Many of these materials are known and commercially av ailable. For example, E. coli CJ236 and JM101 are known strains, pUB H O is a known plasmid and Kunkel method mutagenesis is also well known in the art. In addition certain cell lines and cDNA may be commercially available, for example U-937, available from Clontech Inc., Palo Alto, California.
• Variants may be made by expression systems and by various methods in various hosts, these methods are within the scope of the practice of the skilled artisan in molecular biology, biochemistry or other arts related to biotechnology.
• Example 1 RNA is isolated from unstimulated and interleukin-1 stimulated cultures of normal human articular chondrocytes. The RNA is reverse transcribed into cDNA. The cDNA is subjected to a modified differential display procedure using a series of random primers.
• PCR samples generated from both stimulated and unstimulated chondrocytes are electrophoresed in adjacent lanes on polyacrylamide gels.
• the differentially expressed band is excised from the gel, cloned, and sequenced.
• the differential expression of the gene is confirmed by RNAase protection and nuclear run on experiments.
• Example 2 A novel partial human cDNA coding the protein is cloned from primary cultures of interleukin-1 stimulated human articular (femoral head) chondrocytes, using known methods. The same sequence is found, and the gene completed by screening of human cDNA libraries to obtain full length clones.
• Example 3 The cloned DNA of example 2 is placed in pUBl 10 using known methods. This plasmid is used to transform E. coli and provides a template for site- directed mutagenesis to create new mutants. Kunkel method mutagenesis was performed altering the Gin 1 to Ala.
• Example 4 [125j] disintegrin antibody is prepared using IODOBEADS (Pierce, Rockford, IL; immobilized chloramine-T on nonporous polystyrene beads). Lyophilized antibody (2 ⁇ g) is taken up in 50 ⁇ l of 10 mM acetic acid and added to 450 ⁇ l of phosphate- buffered saline (PBS) (Sigma, St. Louis. MO) on ice. To the tube is added 500 ⁇ Curie of 125j (Amersham, Arlington Heights, IL) (2200Ci/mmol) in 5 ⁇ l, and one IODOBEAD. The reaction is incubated on ice for 10 min with occasional shaking. The reaction is then terminated by removal of the reaction from the IODOBEAD. To remove unreacted 125j 5 e mixture is applied to a PD-10 gel filtration column.
• IODOBEADS Pierce, Rockford, IL; immobilized chloramine-T on nonporous polystyrene beads
• Example 5 A fluorogenic disintegrin metalloprotease substrate peptide (Bachem, Guelph Mills, King of Prussia, Pa) is mixed with the disintegrin and change in the fluorescence is evaluated at 2 min, as a control. Then the fluorogenic peptide is mixed with the disintegrin in the presence of the compound (metalloprotease inhibitor) in evaluation in a separate run, with evaluation at various time points over 2 to 12 hours. Data are evaluated using standard methodology to provide relative binding of the evaluated compound.
• 0.5 ml of synovial fluid from the left knee of a patient is withdrawn and tested for elevated levels disintegrin by ELISA. The results indicate higher than normal disintegrin level.
• the patient is prescribed a prophylactic dose of a disintegrin inhibitor administered orally over time or is administered an injection of same in the left knee before leaving the clinician's office.
• Example 7 Inhibition of extracellular matrix remodeling is explored via inhibition of disintegrin metalloprotease activity.
• synthetic metalloprotease inhibitor such as those used to inhibit the matrix metalloproteases, tissue integrity and proteoglycan is monitored.
• a sample of IL-I stimulated bovine nasal cartilage derived articular cartilage is grown in a 1 micromolar solution of a small molecular weight disintegrin inhibitor. The experiment is controlled and compared to an identical culture grown with no inhibitor.
• the assay of the culture after 7 days shows that the inhibited culture has less tissue breakdown and less proteoglycan present in the serum of the culture. The result is consistent with the inhibited aggrecanase activity. Inhibition of aggrecanase would inhibit tissue breakdown and reduce the release of proteoglycan.
• Cells known to contain disintegrin are treated with a serine protease. Proteins released from the cell are measured by standard methods. Specifically the metalloprotease activity is monitored v ia literature methods. The amount of metalloprotease released is correlated to the amount of serine protease used to treat the cells. Increases, versus control, in src tyrosine kinase activity are measured by Western blot analysis of intracellular proteins using monoclonal antibodies specific for phosphotyrosine following cleavage and release of the disintegrin metalloprotease. Controls are cells that have not been treated with serine protease. src tyrosine kinase activity in the cell (or is it cell culture) is measured by literature methods.
• Example 9 Integrin binding is measured with a peptide containing the sequence RGD. Inhibition of intercellular adhesion molecules, or extracellular matrix components results in the inhibition of phenotypic changes, including changes in cell shape, associated with such interactions. Integrin binding is measured via competitive assay, using cellular changes in shape visible via microscopy. The peptide inhibits such cellular changes.
• the RGD peptide inhibits cellular changes in chondrocytes.
• the osteoarthritis phenotype characterized by increased matrix synthesis and accelerated matrix metalloprotease activity does not occur.
• Other readily assayable cellular changes can be used to monitor this result, including gene expression, changes in mitotic activity, and the like.
• Example 10 A small molecular weight metalloprotease inhibitor is used to treat a tissue culture according to the method of Example 7. The release of TNF- ⁇ from the cell membrane is measured by literature methods. The inhibitor of Example 7 also decreases the amount of TNF- ⁇ secreted from the cell membrane.
• disintegrin metalloprotease activity will result in the inhibition of a disintegrin associated inflammation cascade and secretase activity. It is contemplated that monitoring the release of cytokines or IL-1 from the cell membrane, and the like, will produce the same result.
• RNA is isolated from unstimulated and interleukin-1 stimulated cultures of normal human articular chondrocytes.
• the RNA is reverse transcribed into cDNA.
• the cDNA is subjected to amplification (PCR) using the above-named primers.
• PCR samples generated from both stimulated and unstimulated chondrocytes are electrophoresed in adjacent lanes on polyacrylamide gels.
• a differentially expressed band i.e., a band found only in the stimulated cells and not expressed at significant or detectable levels in the unstimulated cells
• the partial sequence is shown in SEQ ID NO:5.
• the sequence is found to exhibit approximately 60% homology to a rat metalloprotease (see above).
• the sequence is found to exhibit approximately 85% homology to a human metalloprotease (see Gen Bank Accession #Z48597, see Figure 2).
• Cancer tissue is tested for metalloprotease gene expression.
• the above-named primers are used in PCR on extracted nucleic acid from the sample. High levels of transcripts suggest metastatic potential.
• Candidate inhibitors of metalloprotease gene expression are screened in vitro. Interleukin-1 stimulated cultures of normal human articular chondrocytes are exposed in vitro to candidate inhibitors. The RNA is isolated and reverse transcribed into cDNA. the cDNA is subjected to amplification (PCR) using the above-named primers. PCR samples generated from both chondrocytes exposed to inhibitors and uninhibited chondrocytes are electrophoreses in adjacent lanes on polyacrylamide gels. Reduced levels of PCR product identifies an inhibitor.
• Example 14 Drug Screen For Metalloprotease Inhibitors Candidate inhibitors of the metalloprotease itself are screened in vitro. The culture supernatant of Interleukin-1 stimulated cultures of normal human articular chondrocytes are assayed on suitable metalloprotease substrates (e.g., matrix proteins) in the presence and absence of candidate inhibitors. Known inhibitors are used as controls (e.g., 1,10-phenanthroline available commercially from Sigma Co., St. Louis). Reduced levels of substrate (e.g., fluorogenic disintegrin metalloprotease substrate) degradation identifies an inhibitor.
• substrate e.g., fluorogenic disintegrin metalloprotease substrate
• a 1400 BP clone is isolated via standard screening techniques from U-937, a monocyte-like cell cDNA line library .
• the initial sequence is a truncated clone, missing a portion of the 5' end.
• the 5 * end is generated using 5' R.A.C.E. (Rapid
• Primers SEQ ID NO:9 (5'-AGCCTGTGTC-3') and SEQ ID NO: 10 (5'- AGCCTGTGTCTGAACCACT-3') are used in differential display of mRNA (ddrd- PCR). 2-5 ng of sscDNA is used in the PCR. The reaction is precooled 0.2 ⁇ l thin- walled tubes on ice. Each tube containing, 50mM TrisHCl (pH 8.5), 50mM KC1, 1.5 mM MgCl2 ImM of each dNTP, 2-5 ng of sscDNA, lOpmoles of each primer above, 05.
• ⁇ -p33 dCTP 10 ⁇ Ci/ ⁇ l, Amersham
• water 20 ⁇ l.
• the mixture is subjected to 35 cycles of denaturation (94°C for 30 sec), annealing (36°C for 30 sec.) and extension (72°C for 1 min.) using a Perkin-Elmer System 2400 Thermal Cycler (Perking-Elmer, Norwalk, CT).
• IL-1 treated chondrocytes expressed the mRNA associated with this gene, while the untreated (no IL-1) control chondrocytes expressed no detectable mRNA.
• Example 17 Assay system amenable to high throughput screening
• the protease activity of disintegrin is measured in a kinetic enzyme inhibition assay using a fluorescent substrate. Using cloned disintegrin enzyme, and a small MW fluorescently labeled protein as the substrate. Enzyme activity is quantified by measurement of fluorescence after cleavage of the substrate molecule at room temperature. This assay simple and very easy to automate. Using standard techniques, this assay is adapted to 96 or 384 well plates.
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• CAA AAA CAC CAG CGT GCC AAA AGA GCA _7_ 7 ⁇ A CAT GAA GAC CAA TTT 193 Gin Lys His Gin Arg Ala Lys Arg Ala Vi. - His Glu Asp Gin Phe 540 545 550
• GCT GAA AAA AAT ACT TGT CAG CTT TAT ATT CAG ACT GAT CAT TTG TTC 865 Ala Glu Lys Asn Thr Cys Gin Leu Tyr He Gin Thr Asp His Leu Phe 760 765 770 775
• GAG CTC TAT GAA AAC ATT GCT GAA TGG ATT GTG GCT CAT TGG TGG GCA 2209 Glu Leu Tyr Glu Asn He Ala Glu Trp He Val Ala His Trp Trp Ala 1210 1215 1220
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• GCCAATGTAC ATACCTTGTT ATATGCAGAC A7J7AT77C7 7ACGTACACT GTACTTCTGT 120 GTGCAATTGT AAACAGAAAT TGCAATATGG ATGTTTCTTT GTATTATAAA ATTTTTCCGC 180
• MOLECULE TYPE DNA (genomic)
• ATCCTCAGAC CAGGTGGAAT TACTTAAATT TTAAAGCCTG AAAATTCCAA TTTGGGGGTG 300
• MOLECULE TYPE DNA (genomic)
• GGT CCT CTA GCT AGG CTT AAA AAA GCA ATT TTT AGT CCA GAG CTC TAT 2017 Gly Pro Leu Ala Arg Leu Lys Lys Ala He Phe Ser Pro Glu Leu Tyr 1455 1460 1465
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomi: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

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