Disintegrin metalloprotease and its use

Details Disintegrin metalloprotease and its use Disintegrin metalloprotease and its use

1998-02-25

2001-07-03

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S219000, C435S226000

Reexamination Certificate

active

06255064

ABSTRACT:

FIELD OF THE INVENTION
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.
BACKGROUND
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.”
Controlled production and activity of these enzymes plays an important role in the normal development of tissue architecture. In excess, however, these enzymes can cause pathologic destruction of connective tissues. See generally, J. Saus et al., “The Complete Primary Structure of Human Matrix Metalloprotease-3”
J. Biol. Chem.
263:6742 (1988). Many of these are zinc-containing metalloprotease enzymes, as are the angiotensin-converting enzymes and the enkephalinases. Collagenase, stromelysin and related enzymes are important in mediating the symptomatology of a number of diseases, including 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); the metastasis of tumor cells (ibid, Broadhurst, M. J., et al., European Patent Application 276,436 (published 1987), Reich, R., et al., 48 Cancer Res 3307-3312 (1988); and various ulcerated conditions. Ulcerative conditions can result in the cornea as the result of alkali burns or as a result of infection by
Pseudomonas aeruginosa
, Acanthamoeba,
Herpes simplex
and vaccinia viruses.
In fact, measurement of metalloproteases in cancer tissue suggests increased levels of metalloproteases correlate with metastatic potential. See e.g., M. J. Duffy et al., “Assay of matrix metalloproteases types 8 and 9 by ELISA in human breast cancer”
Br. J. Cancer
71:1025 (1995).
Other conditions characterized by undesired metalloprotease activity include periodontal disease, epidermolysis bullosa and scleritis. In view of the involvement of metalloproteases in a number of disease conditions, attempts have been made to prepare inhibitors to these enzymes. A number of such inhibitors are disclosed in the literature. The invention seeks to provide novel inhibitors, preferably specific to this protease, that have enhanced activity in treating diseases mediated or modulated by this protease.
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.
Given the involvement of matrix metalloproteases in a number of disease conditions, attempts have been made to identify inhibitors of these enzymes. For 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).
These proteases and many others are uncovered in molecular biology and biochemistry. As a result, GenBank, a repository for gene sequences, provides several sequences of metalloproteases, including some said to encode fragments of disintegrins. For example, GenBank accession # Z48444 dated Feb. 25, 1994 discloses 2407 nucleotides of a rat gene said to be a rat disintegrin metalloprotease gene; GenBank accession # Z48579 dated Mar. 2, 1995 discloses 1824 nucleotides of a partial sequence of a gene said to be a human disintegrin metalloprotease gene; GenBank accession # Z21961 dated Oct. 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.
It would be advantageous to implicate metalloproteases in specific disease states, and to use these metalloproteases as tools to detect and ultimately cure, control or design cures for such diseases.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide a method for identifying compounds capable of binding to the disintegrin protein.
It is also an object of the present invention to provide a host cell comprising a recombinant expression vector to the disintegrin protein and a recombinant expression vector encoding to the disintegrin protein.
It is also an object of the present invention to provide a method for screening for metalloprotease mediated diseases such as cancer, arthropothies (including ankylosing spondolytis, rheumatiod arthritis, gouty arthritis (gout), inflammatory arthritis, Lyme disease and osteoarthrtis).
It is also an object of the present invention to provide an antibody to the protein useful in the screen, in the isolation of the protein or as a targeting moiety for the protein.
SUMMARY OF THE INVENTION
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.
DETAILED DESCRIPTION
The term “gene” refers to a DNA sequence 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 enzymatic activity is retained.
The term “oligonucleotide” as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, 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.
Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage, 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. As used herein, a nucleic acid sequence, even if

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