KR19990087444A - New disintegrin metalloprotease and use thereof - Google Patents

Abstract

The present invention provides a method for identifying a compound capable of binding to disintegrin protein, and a method for measuring the amount and affinity of a compound capable of binding to disintegrin protein in a sample. The present invention also provides a host cell containing a recombinant expression vector for a disintegrin protein and a recombinant expression vector encoding a disintegrin protein and a human disintegrin metalloprotease protein, fragment or variant thereof useful for this use. The present invention also provides in vivo or ex vivo methods for screening diseases based on osteoarthritis and other metalloproteases, which can be prepared and used in kit form.

Description

New disintegrin metalloprotease and use thereof

Many enzymes degrade structural proteins and are structurally related metalloproteases. Such enzymes include human epidermal fibroblast collagenase, human epidermal fibroblast gelatinase, human saliva collagenase and gelatinase, and human stromelysin. These are zinc-containing metalloprotease enzymes such as angiotensin-converting enzymes and enkephalinases. Collagenase, stromelysin and related enzymes are known as rheumatoid arthritis (Mullins, D. E., et al., Biochim Biophys Acta (1983) 695: 117-214); Osteoarthritis (Henderson, B., et al., Drugs of the Future (1990) 15: 495-508); Metastasis of tumor cells (ibid, Broadhurst, MJ, et al., European Patent Application 276,436 (published 1987), Reich, R., et al., 48 Cancer Res 3307-3312 (1988); and various ulcer diseases such as The ulcer disease is a corneal as a result of alkaline laceration or as a result of infection with Pseudomonas aeruginosa, Acanthamoeba, herpes simplex and baccinia virus. Can happen in

Other diseases characterized by undesired metalloprotease activity include periodontal disease, epidermal vesicles and scleritis. In view of the metalloproteases involved in many diseases, preparation of inhibitors for these enzymes has been attempted. Many such inhibitors are disclosed in the literature. The present invention seeks to provide novel inhibitors, preferably novel inhibitors specific for said protease, which have enhanced activity in treating a disease mediated or regulated by said protease.

Inhibitors of metalloproteases are useful for treating the breakdown of structural proteins, at least in part by causing diseases. Although various inhibitors have been prepared, there is still a need for metalloprotease inhibitor screens for designing a medicament to treat the disease.

Metalloproteases are a large class of proteins and have a wide variety of functions. Disintegrin is a zinc metalloprotease that is abundant in snake venom. Alternative cloning techniques may be used. Mammalian disintegrins are a group of proteins with about 18 known subpopulations. They are known to act as cell adhesion inhibitors and also involved in reproduction (eg fertilization of sperm by sperm, and fusion thereof, and sperm maturation).

These proteases and many others have been found in the field of molecular biology and biochemistry. As a result, Genbank, the gene sequence bank, is provided with a number of metalloprotease sequences, including those believed to encode fragments of disintegrins. For example, GenBank Accession No. Z48444 of February 25, 1994 discloses a murine gene of 2407 bases believed to be murine disintegrin metalloprotease gene; GenBank Accession No. Z48579 of March 2, 1995, discloses a gene partial sequence of 1824 bases believed to be the human disintegrin metalloprotease gene; GenBank Accession No. Z21961 of October 25, 1994 discloses a gene partial sequence of 2397 bases considered to be a bovine zinc metalloprotease gene.

Since metalloproteases are associated with certain disease states, it would be advantageous to use such metalloproteases as tools to detect and ultimately treat the disease and to modulate or devise therapies.

Purpose of the Invention

It is an object of the present invention to provide a method for identifying a compound capable of binding to a disintegrin protein.

It is an object of the present invention to provide a host cell containing a recombinant expression vector for a disintegrin protein and a recombinant expression vector encoding the disintegrin protein.

It is also an object of the present invention to provide a method for screening diseases mediated by metalloproteases such as cancer, arthritis (including ankylosing spondylitis, rheumatoid arthritis, gouty arthritis (gout), inflammatory arthritis, Lyme disease and osteoarthritis) .

The present invention also provides antibodies useful for screening and isolating said protein or as part of targeting a protein.

Summary of the Invention

The present invention identifies a compound capable of binding to disintegrin protein and provides a method for measuring the amount and affinity of a compound capable of binding to disintegrin protein in a sample.

The present invention also provides host cells and disintegrin proteins containing recombinant expression vectors for disintegrin proteins and recombinant expression vectors encoding human disintegrin methylloprotease proteins, fragments or variants thereof useful for this purpose.

The present invention also provides a method for screening diseases, such as cancer, based on osteoarthritis and other metalloproteases, which may be prepared and used in kit form, in vivo or ex vivo.

The present invention relates to its use in the detection and testing of medicaments for novel proteins, fragments and variants thereof and diseases, such as diseases characterized by the upregulation of osteoarthritis and other metalloproteases.

As used herein, the terms "protein", "protease", and "metalloprotease" refer to disintegrin. Preferably it is a human disintegrin as described below.

The term "antibody" refers to an antibody against disintegrin, or a fragment thereof. It may be monoclonal or polyclonal and may be provided from a number of sources. The present invention also deals with fragments of such antibodies made using any method in the field of protein or peptide.

The term "disease screen" means a screen for a disease or disease state. Disease state means that the disease is a physiological or cytological or biochemical condition. Preferably the screen is used for animal body tissues or body fluids or cell cultures using standard techniques such as ELISA. The screen also deals with the "mapping" of the disease throughout the body, with the labeled antibody of the description to the whole body: irrespective of the test method, preferred test methods include fluorescence, X-rays (including CAT scans). , NMR (including MRI), and the like.

The term “compound screen” relates to methods and screens related to finding compounds, measuring their affinity for proteases, or devising or selecting compounds based on the screens. In another embodiment, this relates to the use of three-dimensional structures in drug design, preferably "reasonable drug design," as is known in the art. The protease is preferably a "essentially purified form", ie, a protein that is adequately free from other impurities to be useful for experimentation or characterization. The use of such screening methods helps those skilled in the art to discover novel structures produced or naturally produced by chemists that bind to and preferably inhibit proteases. The "inhibitor" is useful for modulating or altering the activity of the protease and thus can be used to modulate the biological cascade in which the inhibitor acts. This approach provides new compounds that are pharmaceutically useful.

The term "disintegrin" refers to a disintegrin, a fragment thereof, a variant thereof, or a homologue that still retains its function. The term relates to agracanase and other proteases involved in or regulating tissue remodeling. It relates to disintegrins derived from various species, and to disintegrins produced by recombinant methods, ex vivo methods, or standard peptide synthesis. Preferably the protein is human disintegrin or variant thereof. Preferred "native" proteins for the purpose of defining variants of the protein are described in GenBank Accession No. Z48579, incorporated herein by reference and represented by the following sequence. SEQ ID 1 describes a fragment of the DNA sequence and its transcript and SEQ ID 2 describes the coding region of the gene and its transcript. Homologous disintegrins include all, or fragments thereof, proteins having at least 90% identity as known in the art. For example, murine proteins that exhibit 95% homology with those of SEQ ID NO: 1 or SEQ ID NO: 2 based on peptide sequences derived from DNA or cDNA sequences, and bovine proteins (similarly derived proteins) that are 97-98% homologous are all It is considered a homologue. Thus homologous cDNA cloned from other organisms produces homologous proteins.

Likewise, proteins can be considered homologs based solely on amino acid sequences. The real limitation of amino acid sequencing is that it is possible to determine whether proteins are homologous to one another, for example by comparing the first 50 amino acids of a protein. Thus, 90% homology means that there are five different amino acids in the first 50 amino acid chains of the homologous protein.

One skilled in the art will know that due to the degeneracy of the genetic code different DNA sequences provide the same transcript, and thus the same peptide. In some cases, preparing a DNA sequence encoding the same peptide but different from the original DNA involves:

Ease of sequence analysis or synthesis;

Increase in peptide expression; And

-Preference of certain heterogeneous hosts for certain codons.

Such practical considerations are well known and provide embodiments that may be advantageous to the user of the present invention. Thus, it becomes clear that the original DNA is not the only embodiment for which the present invention is intended.

In addition, fragments of proteins can be used for screening, drug design, and the like, and it will be apparent to those skilled in the art that the entire peptide will not be required to utilize the present invention. Thus, it will be apparent to one skilled in the art that the disclosure of peptides and their use will allow for prediction of useful peptide fragments.

Practical considerations such as protein expression, purification yield, stability, and solubility are considered by those skilled in the art when selecting fragments to be used and when selecting the fragments to be used. As a result, given the above disclosure, one of ordinary skill in the art can practice the invention using fragments of proteins as well.

The protein or protease itself can be used to measure the binding activity of small molecules that bind to the protein. Pharmaceutical screening using enzyme targets is used in the art and can be applied using automated high throughput techniques.

Inhibition of disintegrin activity makes it possible to predict the therapeutic efficacy of osteoarthritis and other diseases involving degeneration of articular cartilage.

Gene therapy

Without being bound by theory, metalloproteases are believed to upregulate tissue osteoarthritis. It has been surprisingly found that human disintegrin is up-regulated in human chondrocytes during osteoarthritis. Inhibition of the signal transduction mechanism is effective in disrupting cascade in osteoarthritis and other diseases involving cartilage degeneration. If up-regulation is the cause of the onset of arthritis, those skilled in the art will recognize that inhibiting the gene activity would be useful for treating osteoarthritis.

This is done via any of a number of methods including gene (ie antisense) therapy.

Inhibitors of disintegrin

Proteases of the invention can be used to find inhibitors of proteases. Thus, it is useful as a screening tool or for rational drug design. Without being bound by theory, proteases can regulate cell remodeling and indeed enhance tissue degradation by enhancing extracellular matrix remodeling. Thus, inhibition of disintegrin provides a means of treating the disease characterized by the above method.

In screening, pharmaceutical compounds can be used to determine the quality and amount of inhibition. As a result, the screening provides information for the selection of active agents, preferably small molecule actives, useful for treating the disease.

In therapy, inhibition of disintegrin metalloprotease activity through binding of low molecular weight, synthetic metalloprotease inhibitors, such as when used to inhibit matrix metalloprotease, may be used to inhibit extracellular matrix remodeling. .

Antibody to Protein

Metalloproteases having activity at particularly undesirable positions (eg organs or certain types of cells) can be targeted by conjugating metalloprotease inhibitors with antibodies or fragments thereof. Conjugation methods are known in the art.

Antibodies of the invention may be conjugated to a solid support. The conjugate can be used as a purification affinity reagent of the desired metalloprotease, preferably disintegrin.

In another aspect, the antibodies of the invention can be conjugated directly to a label. As the antibody binds to the metalloprotease, the label can be used to confirm the presence of relatively high concentrations of the metalloprotease in vivo or in cell culture.

In addition, metalloprotease inhibiting compounds may be conjugated to antibodies. Conventional bonding methods are known in the art. The antibodies are useful for both treatment and for monitoring the dosage of the inhibitor.

For example, a target ligand that specifically reacts with a marker for the intended target tissue can be used. Methods of coupling a compound of the invention to a target ligand are already known and are analogous to the methods of coupling to a carrier described below. The conjugate is prepared and administered as described above.

Preparation and Use of Antibodies:

Antibodies can be prepared by a variety of methods, for example, proteins can be injected into suitable (ie, mammalian) subjects, including mice, rabbits, and the like. Preferred protocols involve repeated injection of the immunogen in the presence of an adjuvant according to a schedule that promotes the production of antibodies in the serum. Titers of immune serum can be readily determined using immunoassay assays, which are now standard techniques in the art.

The antisera obtained can be used directly, or monoclonal antibodies can be obtained by taking peripheral blood lymphocytes or spleen from immunized animals, immunizing antibody-forming cells, and then using standard immunological assay techniques to form appropriate antibody constructs. It can be obtained by confirming.

Polyclonal or monoclonal preparations are useful for monitoring therapies or prophylaxis involving the compounds of the present invention. Suitable samples, such as those derived from blood, serum, urine, or saliva, can be tested for the presence of proteins at various time points during a treatment protocol using standard immunological assay techniques using the antibody preparations of the invention.

The antibody can also be coupled to a label such as a scintography label such as technetium 99 or I-131 using standard coupling methods. Administration of the labeled compound to a subject identifies the location of excess one or more metalloproteases in vivo. Thus, labeled antibodies to proteins serve as a tool to screen for such enhanced expression indicative of disease.

The ability of antibodies to selectively bind metalloproteases is thus used to in situ map the distribution of these enzymes. The technique can also be used in histological methods and labeled antibodies can be used in antagonistic immunoassays.

Antibodies are advantageously coupled to other compounds or substances using known methods. For example, a substance with carboxyl functionality, the carboxyl moiety, may be aldehyde reduced and coupled to the carrier via reaction with side chain amino groups, followed by optionally reducing the imino bonds formed. Carboxylic moieties can also react with side chain amino groups using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents. Linker compounds may also be used to perform the coupling; Homodifunctional and heterodifunctional linkers are both Pierce Chemical Company, Rockford, Ill. It is marketed by.

The antibody is useful for protein separation when conjugated to a suitable chromatography material. Separation methods using affinity chromatography are well known in the art and are within the understanding of those skilled in the art.

Disease markers

Without being bound by theory, the expression of a gene, preferably said gene, exhibits limited tissue distribution and its expression is upregulated by potential osteoarthritis mediators. Increasing the expression of such genes (and proteins thereof) in articular chondrocytes, for example, provides markers to monitor the development and progression of osteoarthritis, including initially, asymptomatic stages. Thus, antibodies generated against proteins will act as a screening tool for such increased expression indicative of disease.

Moreover, when used in disease screens, antibodies can be conjugated to chromophores or fluorophore containing materials or to enzymes that produce chromophores or fluorophores under certain conditions. . Such bonding materials and methods are well known in the art. If used in this manner, the detection of proteins by immunological assays will be straightforward for those skilled in the art. For example, body fluids can be screened in this manner for the distribution of metalloproteases, or for the measurement and detection of increased amounts of the proteases.

When used in this manner, the present invention is a useful diagnostic and / or clinical marker for diseases mediated by metalloproteases such as osteoarthritis or other articular cartilage degenerative diseases. If a disease is found, it can be treated before symptoms or debilitation begins.

Moreover, the antibodies can be used to target diseased tissue for detection or treatment as described above.

The following non-limiting examples illustrate preferred embodiments of the present invention and briefly describe the use of the present invention. These examples are provided to guide those skilled in the art and do not limit the invention in any way. Having the above disclosure and examples will enable those skilled in the art to make and use the claimed invention.

Standard starting materials are used in this example. Many such materials are known and commercially available. For example, E. coli CJ236 and JM101 are known strains, pUB110 is a known plasmid and Kunkel method mutagenesis is also well known in the art.

Variants can be made by various methods and expression systems in various hosts, which methods are within the experience of one skilled in the art of molecular biology, biochemistry or other fields related to biotechnology.

Example 1

RNA is isolated from unstimulated and normal human joint chondrocyte culture stimulated with interleukin-1. RNA is reverse transcribed into cDNA. A modified fractional display procedure is performed using a series of random primers with cDNA.

PCR samples generated from stimulated and unstimulated chondrocytes are electrophores side by side on a polyacrylamide gel. Fractionally expressed bands are cut out of the gel, cloned and sequenced. RNase protection and a nuclear run are performed to confirm the differential expression of genes.

Example 2

The novel partial human cDNA encoding the protein is cloned from primary cultures of human femoral head chondrocytes stimulated with interleukin-1 using known methods.

The same sequence was found and the human cDNA library was screened to complete the gene to obtain a clone of intact length.

Example 3

The cloned DNA of Example 2 is placed in pUB110 using known methods.

The plasmid is used to transform E. coli and serve as a template for site-specific mutagenesis to generate new variants. Kunkel's method mutagenesis is performed by altering GLN 1 ALA.

Example 4

[ ¹²⁵ I] disintegrin antibodies are prepared using IODOBEADS (Pierce, Rockford, IL; immobilized chloriamne-T on nonporous polystyrene beads). Lyophilized antibody (2 μg) is taken up in 50 μl of 10 mM acetic acid and added to 450 μl of phosphate-buffered saline (PBS) (Sigma, St. Louis, MO) on ice. To the tube is added 500 μCurie of ¹²⁵ I (Amersham, Arlington Heights, IL) (2200 Ci / mmol) in 5 μl, and one IODOBEAD. Incubate on ice for 10 minutes with occasional shaking. The reaction is then terminated by removing the reactants from IODOBEAD. To remove unreacted ¹²⁵ I, a PD-10 gel filtration column is applied to the mixture.

Example 5

Fluorescent peptides (Bachem, Guelph Mills, King of Prussia, PA) are mixed with disintegrin and fluorescence changes are assessed in 2 minutes as a control. The fluorogenic peptide is then mixed with disintegrin in the presence of the compound and evaluated in 2 minutes in a separate experiment. Data is evaluated using standard techniques to indicate the relative binding of compounds evaluated.

Example 6

Increased disintegrin is tested by ELISA on 0.5 ml of synovial fluid extracted from the patient's left knee. Disintegrins higher than normal resulted. The patient is prescribed a prophylactic dose of a disintegrin inhibitor and the same dose is administered by injection into the patient's left knee.

Example 7

Inhibition of extracellular matrix remodeling is investigated through inhibition of disintegrin metalloprotease activity. Tissue integrity and proteoglycans are monitored using low molecular weight synthetic metalloprotease inhibitors, such as inhibitors used to inhibit matrix metalloproteases.

Articular cartilage samples from mice are grown in 1 micromolar solution of low molecular weight disintegrin inhibitors. Experiments are controlled and compared based on the same culture grown without inhibitor.

Assay of the cultures after 7 days showed less tissue degradation in the suppressed cultures and less presence of proteoglycans in the serum of the cultures. The results are consistent with inhibited aggrecanase activity. Inhibition of aggrecanase inhibits tissue degradation and reduces the release of proteoglycans.

Example 8

Inhibition of proteolysis resulting in separation of the disintegrin metalloprotease region in a form bound to the membrane inhibits the "secondary messenger" signaling of the disintegrin molecule bound to the membrane. The secondary messenger signaling results in cell phenotypic changes, gene expression changes, mitotic activity changes, and the like.

Cells known to contain disintegrins are treated with serine proteases. Proteins released from cells are measured by standard techniques. Metalloprotease activity is specifically monitored through the methods described in the literature. The amount of metalloprotease released correlates with the amount of serine protease used to treat the cells.

Compared to the control, increased src tyrosine kinase activity is measured through Western blot analysis of intracellular proteins using phosphotyrosine specific monoclonal antibodies followed by cleavage and release of disintegrin metalloproteases. The control is cells not treated with serine protease.

Src tyrosine kinase activity in cells (or cell culture) is measured via the methods described in the literature. Isolation of the metalloprotease portion of the disintegrin is also monitored through the methods described in the literature. There is a direct correlation between separation of metalloprotease moieties and increased intracellular src tyrosine kinase activity. The results are consistent with the stimulation of disintegrin-mediated cell signaling by stimulation of the src tyrosine kinase cascade.

Example 9

Inhibition of intracellular junctional molecules, or extracellular matrix components, as described in Example 8, results in inhibition of phenotypic changes, including changes in cell morphology, associated with such interactions.

Integrin binding is measured with a peptide containing the sequence RGD using the protocol of Example 8. Integrin binding is measured via a comparative assay, using cell shape changes through the microscope. As in Example 8, the peptide inhibits cellular changes.

The results are consistent with the competition or blocking of the interaction of disintegrins. RGD peptides inhibit cellular changes in chondrocytes. There is no osteoarthritis phenotype characterized by increased matrix synthesis and accelerated matrix metalloprotease activity. Other assay-friendly cellular changes, including changes in gene expression, mitotic activity, and the like, can be used to monitor the results.

Example 10

Low molecular weight metalloprotease inhibitors are used to treat tissue culture according to the method of Example 7. Release of TNF-α from the cell membrane is measured via the method described in the literature. Inhibitors of Example 7 also reduce the amount of TNF-α secreted from the cell membrane.

This is consistent with the theory that inhibition of disintegrin metalloprotease activity will result in inhibition of inflammatory cascade and secretase activity associated with disintegrin. It is believed that monitoring such as release of cytokines or IL-1 from cell membranes will produce the same results.

All references in this text are incorporated by reference.

While specific embodiments of the invention have been described, those skilled in the art will recognize that various modifications and changes can be made without departing from the spirit and scope of the invention. It is intended that the appended claims cover all such modifications as the scope of the invention.

(1) General Information:

(i) Applicants: Tyndall, Michael H

Hacky, Tariq M

(ii) Name of the Invention: Use of novel disintegrin metalloproteases, variants thereof, fragments and the like

(iii) number of sequences: 4

(iv) Contact Information:

(A) To: The Procter & Gamble Company

(B) Street: Mason-Montgomery Road 8700

(C) Poetry: Mason

(D) State: Ohio

(E) country: United States

(F) ZIP: 45040-9462

(v) computer readable form:

(A) Medium type: floppy disk

(B) Computer: IBM PC Compatible

(C) working system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.30

(vi) Current application data:

(A) Application number: US 60 / 012,679

(B) Application date: March 1, 1996

(C) Classification:

(viii) Patent Attorney / Agent Information:

(A) Name: Hake, Richard A.

(B) Registration Number: 37,343

(C) Reference / List No .: 5980

(ix) Communications Information:

(A) Phone: 513 / 622-0087

(B) Fax: 513 / 622-0270

(2) information about SEQ ID NO: 1

(i) the nature of the sequence:

(A) Sequence length: 1961 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: DNA (genomic)

(ix) sequence characteristics:

(A) Characteristic symbols: CDS

(B) Presence Location: 2..1474

(xi) SEQ ID NO: SEQ ID NO: 1:

(2) Information about SEQ ID NO:

(i) the nature of the sequence:

(A) Length of sequence: 491 amino acids

(B) Type of sequence: amino acid

(D) topology: linear

(ii) Type of molecule: protein

(xi) SEQ ID NO: SEQ ID NO: 2:

(2) Information about SEQ ID NO:

(i) the nature of the sequence:

(A) Length of sequence: 2763 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) topology: linear

(ii) Type of molecule: DNA (genomic)

(ix) sequence characteristics

(A) Characteristic symbols: CDS

(B) Presence Location: 17..2414

(xi) SEQ ID NO: SEQ ID NO: 3:

(2) Information about SEQ ID NO:

(i) the nature of the sequence:

(A) Sequence length: 799 amino acids

(B) Type of sequence: amino acid

(D) topology: linear

(ii) Type of molecule: protein

(xi) SEQ ID NO: SEQ ID NO: 4:

Claims (22)

A DNA fragment encoding human disintegrin that is differentially expressed during arthritis progression, and can be used for screen disintegrin antagonism, drug design and screening. The human disintegrin according to claim 1 having solubility and molecular weight useful as a pharmaceutical screening agent. Human disintegrin according to claim 1 in purified form. A method for screening a compound capable of binding to human disintegrin, comprising the disintegrin of claim 1. The screening kit for compounds which can bind to the human disintegrin containing the disintegrin of Claim 1. An antibody against human disintegrin, or a fragment thereof according to claim 1. A method for screening a disease mediated by a metalloprotease, comprising administering an antibody according to claim 6 and observing the effect thereof. 8. The method of screening osteoarthritis according to claim 7, comprising administering an antibody and observing the effect thereof. 8. The method of claim 7, wherein the blood, synovial fluid or other body fluid is screened. A screening kit for osteoarthritis, comprising the antibody to human disintegrin of claim 6, or a fragment thereof. The screening method of claim 4, wherein the screening method is useful for determining relative efficacy in treating osteoporitis. DNA encoding the disintegrin according to claim 1 (SEQ ID NO: 2). An expression vector or plasmid containing the DNA of claim 12. A cell containing the DNA of claim 12. A cell containing the expression vector or plasmid of claim 13. 15. The cell of claim 14 wherein the DNA is heterologous to said cell. The inhibitor of human disintegrin of claim 2. A method of treating a disease associated with disintegrin activity. The disintegrin of claim 2, wherein the disintegrin is agrocanase. The method of claim 18, wherein the disease is arthrosis. The method of claim 20, wherein the disease is osteoarthritis. The disintegrin of claim 2, which modulates tissue remodeling or tissue degradation.