WO2000000161A2

WO2000000161A2 - Methods for identification of tef-3 interacting factors

Info

Publication number: WO2000000161A2
Application number: PCT/US1999/014829
Authority: WO
Inventors: Michael Howard Tindal; Richard Lee Wang
Original assignee: The Procter & Gamble Company
Priority date: 1998-06-30
Filing date: 1999-06-30
Publication date: 2000-01-06
Also published as: NO20006198L; AU5086699A; JP2002519008A; CA2332121A1; IL140058A0; WO2000000161A3; EP1089794A2; NO20006198D0

Abstract

This invention provides in vivo or in vitro methods for screening for inhibitors of TEF-3 activity. The invention further provides methods for identifying TEF-3 interacting factors. The invention still further provides methods for treating matrix metalloprotease-mediated diseases, including those of the musculoskeletal system, and cardiac muscle, as well as growth related skeletal diseases.

Description

METHODS FOR ΓNHJBITING TEF-3 ACTIVITY

CROSS REFERENCE

This application claims priority under Title 35, United States Code 119(e) from Provisional Application Serial No. 60/091,318 filed June 30, 1998.

TECHNICAL FIELD

The invention relates to the protein, TEF-3, its fragments, variants, and splice variants; its use in screening for inhibitors of TEF-3 activity; and treatments for ailments, including osteoarthritis and others characterized by up-regulation of matrix metalloproteases.

BACKGROUND

Gene transcription is the result of the binding and activity of a large and diverse class of proteins known as transcription factors. These factors bind to the promoter/enhancer region of a gene. Gene activity is modulated by the mix of transcription factors bound to a particular promoter, the abundance of the transcription factors, and the extent of post translational modifications to the transcription factors. These modifications include covalent modification, ligand binding, phosphorylation, dephosphorylation, and proteolytic cleavage. The various transcription factors bound to a gene promoter must interact with each other in order to modulate gene activity. Inhibiting the activity of one or more transcription factors is an effective means of inhibiting gene activity and is therefore an attractive therapeutic intervention point for treating diseases involving increased gene expression.

Transcriptional enhancer factors (TEFs) are a family of transcription factors that regulate the expression of tissue specific genes. For example, expression of cardiac myosin heavy chain is dependent on TEF-1 (Gupta, M. P., et al., Mole. Cell. Biochem. (1996) 157: 117-124) and expression of skeletal myosin heavy chain is dependent on TEFR-1 (Yockey, C. E., et al., J. Biol. Chem. (1996) 271(7): 3727-3736). Expression of certain TEFs is highest in heart, skeletal muscle, and lung.

Most pathological conditions are characterized by alterations in gene expression, often as the result of changes in gene transcription. Genes may be either turned on or off, or may be up regulated or down regulated. For example, many forms of cancer are characterized by increased expression of one or more so called oncogenes. Likewise, osteoarthritis (OA) is characterized by increased transcription of the matrix metalloproteases (MMPs).

The expression of a number of enzymes affecting the breakdown of structural proteins is regulated by transcription factors. These include several structurally related metalloproteases, including human skin fibroblast collagenase, human skin fibroblast gelatinase, human sputum collagenase and gelatinase, and MMP-3. These and other 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 such as Krohn's disease and ulcerated cornea.

The matrix metalloprotease (MMP) family consists of matrix-degrading proteases that share sequence homology and common structural domains amongst its members. In general, these enzymes are expressed at low levels and are secreted as zymogens. Upon activation, MMPs are capable of degrading various extracellular matrix (ECM) components, thus providing matrix remodeling as well as destruction when inappropriately activated.

MMPs are responsible for cartilage destruction in degenerative diseases of the hyaline cartilage such as primary osteoarthritis (OA), secondary OA, rheumatoid arthritis (RA), ankylosing spondylitis, gouty arthritis, inflammatory arthritis, Lyme disease, and other arthropathies. For example, osteoarthritis is a slow, progressive degenerative disease of synovial joints. The disease is characterized by destruction of the articular cartilage resulting in the denuding of the articular surface and exposure of the underlying bone. The proximal mediators of the cartilage degeneration are the MMPs. Many studies have demonstrated that the MMP genes are up regulated in OA at both the mRNA and protein levels. Inhibition of the transcription of the MMP genes would inhibit the breakdown of the articular cartilage and would be efficacious in treating OA and other joint diseases characterized by loss of the articular cartilage. Thus an effective therapy would be to inhibit up-regulation of the MMPs at the transcriptional level.

SUMMARY OF THE INVENTION

This invention provides in vivo or in vitro methods for screening for inhibitors of TEF-3 activity. The invention further provides methods for identifying TEF-3 interacting factors. The invention still further provides methods for treating matrix metalloprotease- mediated diseases, including those of the musculoskeletal system, and cardiac muscle, as well as growth related skeletal diseases.

DETAILED DESCRIPTION

It has been discovered, surprisingly, that TEF-3 (formerly referred to as CSTF-1), a member of the TEF family of transcription factors, is expressed in articular chondrocytes. Even more surprisingly, it has been discovered that TEF-3 is part of the transcriptional machinery for expression of the matrix metalloprotease (MMP) genes, particularly collagenase- 1 (MMP-1), stromelysin-1 (MMP-3), and collagenase-3 (MMP- 13).

It is believed that decreasing the level of MMPs produced by chondrocytes will result in a decrease in the rate of cartilage breakdown and thus will be efficacious in treating or preventing OA and other arthropathies. It is further believed that inhibiting TEF-3 activity will decrease the level of MMPs made by chondrocytes. Accordingly, the present invention is directed to methods of screening for pharmaceutically active compounds that inhibit TEF-3 activity. The present invention is further directed to methods of treating or preventing osteoarthritis and other diseases of hyaline cartilage by administering an inhibitor of TEF-3 activity.

Definitions and Usage of Terms

"Antibody" refers to an antibody or fragment thereof raised and active against TEF-3, a fragment thereof, a variant thereof, or a splice variant thereof. These may be monoclonal or polyclonal. Antibodies can be from any of several animals (preferably rabbit, mouse, horse, or goat) and/or sources (preferably serum for polyclonal antibodies and spleen for monoclonal antibodies). An antibody may be modified in its primary sequence using methods known in the art, yet still retain its activity against TEF-3. All such modifications are contemplated by the term "antibody."

"Antisense" refers to a DNA sequence that is complementary to an mRNA sequence or its corresponding DNA sequence.

"cDNA" or "complementary-DNA" is a DNA product generated by reverse transcription of RNA, particularly mRNA, and its sequence is complementary to the coding strand of DNA.

"Chondrocyte-like cells" refers to an established tissue culture cell line which maintains phenotypic characteristics of primary chondrocytes. Chondrocyte-like cells may be generated from primary isolates of chondrocytes or derived from tumor cell lines. "Compound" refers to a small molecule, peptide, or antibody that is suspected to inhibit TEF-3 activity.

"Coding region" refers to a segment of mRNA or DNA for which protein sequences are encoded by genetic codons as defined in the art.

"Compound screen" refers to methods and screens for: (1) finding compounds that inhibit TEF-3 activity, (2) determining a compound's affinity for TEF-3 or other relevant proteins, and (3) designing or selecting compounds based on the screen. Compound screens include the use of high throughput screening methods. Compound screens further include the use of the three dimensional structure for drug design, preferably "rational drug design", as understood in the art. Use of such screening methods assists the skilled artisan in finding novel structures, whether made by the chemist or by nature, which inhibit TEF-3 activity.

"DNA" or "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in single-stranded or double-stranded helix form and can be either a linear or circular molecule as defined in the art. According to the normal convention, DNA sequence is given in the 5' to 3' direction, the 5' direction is called upstream and 3' downstream.

"Fusion" refers to having two coding sequence operably linked together at the DNA level to encode a hybrid protein contributed by both coding sequences. A "fusion protein" therefore refers to such a hybrid protein.

"Gene" is a segment of DNA involved in producing a polypeptide chain, including regions preceding and following the protein coding region as well as intervening sequences (introns) between individual coding segments (exons).

"Homologue" refers to protein sequences exhibiting 90% identity or greater to SEQ ID NO:2 or to DNA sequences exhibiting 90% identity or greater to SEQ ID NO:l.

"Inhibitor of TEF-3 activity" or "TEF-3 inhibitor" refers to a small molecule, peptide, or antibody that inhibits TEF-3 activity. Inhibitors of TEF-3 activity are believed to be useful in inhibiting the expression of matrix metalloproteases in chondrocytes and other cell types thereby inhibiting extracellular matrix remodeling and/or degradation.

"Messenger RNA (mRNA)" is a single stranded polymeric ribonucleotide sequence commonly generated from DNA by RNA polymerase and its nucleotide sequence corresponds to the sequence of amino acids coding for a protein.

"MMP-3" refers to human stromelysin-1 (GenBank Accession No. P08254) (EC3.4.24.17). "Oligonucleotide" is defined as a short form of DNA molecule comprised of at least two and preferably less than 100 deoxyribonucleotides. Oligonucleotides may be single or double stranded.

"Primer" refers to an oligonucleotide, whether derived from a natural source or produced synthetically, which is capable of acting as a point of initiation of DNA synthesis due to its sequence complementation to the DNA to be synthesized.

"Peptide" refers to protein with less than 100 amino acid residues as defined in the art.

"Promoter fragment" is a DNA regulatory region of a gene capable of binding RNA polymerase and other transcription factors, and initiating the transcription. "DNA recognition element" refers to a DNA sequence or "motif often found in gene promoters that are protein binding sites, and often bind transcription factors. A promoter can contain one or more DNA recognition sequences or cis-acting elements. The promoter is often located numerically by the position of its beginning and ending nucleotide, in reference to the transcription start site being 0. By convention, nucleotides located 5' upstream of 0 are given a negative coordinate in a descending order, and nucleotides down stream from 0 are given a positive numbering in a ascending order.

"Promoter/reporter gene" refers to a recombinant gene comprising a gene promoter DNA fragment ligated to a cDNA which codes for a protein whose activity can be measured. Preferred cDNAs are luciferase and betagalactosidase. "TEF-3 responsive promoter/reporter gene" refers to a promoter/reporter gene wherein the promoter DNA fragment binds TEF-3 and increases transcription of the reporter cDNA.

"Small molecule" refers to chemical entity generated by synthetic or natural methods with a molecular weight equal to or less than 1000 daltons.

"Splice variants" refer to mRNA or protein products that result from alternative usage of mRNA introns and exons. "Splice variants" may occur in various individuals, tissues, or species. These may be detected in a variety of ways, such as varying molecular weight bands detected on Northern blots or Western blots. Hence 2.5 kb, 3.5 kb and 9 kb TEF-3 mRNAs are contemplated where some variants are found in subpopulations of organisms, or in certain tissues in an organism. Such splice variants often retain their activity, and may differ only in molecular weight, by virtue of having amino acids inserted or deleted from the native sequence, such as that found in SEQ ID NO:2. It is likely that such splice variants may exist even between tissues of the same organism. In addition, without being bound by theory, it is likely that such splice variants may impact regulation of biological cascades as well. For example, it is possible that a TEF-3 variant expressed preferentially in a chondrocyte, as opposed to a variant found elsewhere in a body (such as in skeletal muscle), would be particularly effective in regulating a specific matrix metalloprotease, such as MMP-3, or some variants may actually down regulate MMP-3. Where this type of change in activity is found, the variant gene is preferred for antisense gene therapy. Specific variants will be useful in antisense gene therapy.

"TEF-3" refers to the protein identified in SEQ ID NO:2, a fragment thereof, a homologue thereof, a variant thereof, or a splice variant thereof. Preferably, the TEF-3 is derived from a human source. Without being bound by theory, TEF-3 may regulate, in certain tissues, the expression of matrix metalloprotease genes.

"TEF-3 activity" refers to the ability of TEF-3 to bind to its DNA recognition sequence by itself or in complex with its interacting factors and/or to modulate transcription of TEF-3 responsive genes.

"TEF-3 DNA recognition element" refers to the DNA sequence TTTGGAATG, which is found in a TEF-3 responsive gene promoter.

"TEF-3 interacting factor (TLF)" refers to a protein, a fragment thereof, a homologue thereof, a variant thereof, or a splice variant thereof that interacts with TEF-3 and contributes to TEF-3 activity.

"TEF-3 responsive gene" refers to a gene that contains a TEF-3 binding site in its promoter and whose expression is regulated by TEF-3.

"Transcription factor" is a protein capable of modulating mRNA synthesis during transcription due to its interaction with DNA promoter sequence and the transcription apparatus.

Methods of Screening for Inhibitors of TEF-3 Activity

The invention can be used to find inhibitors of TEF-3 activity. Hence it is useful as a screening tool for rational drug design. Likewise, inhibitors of TEF-3 activity may be found by conventional, random drug screening methods where compounds are assayed for their ability to bind to TEF-3 and/or inhibit its activity. The use of high throughput screening methods and the use of combinatorial compound libraries, both synthetic and natural, are also contemplated by this invention.

TEF-3 inhibitors may be found in cell-based inhibitor assays using a TEF-3 responsive promoter/reporter gene. Such vectors for constructing promoter/reporter genes are well known in the art.

For example a DNA fragment of the MMP-3 promoter (GenBank Accession No. U56422) comprising nucleotides +13 through -486 is ligated to a luciferase cDNA in a vector in proper frame and orientation that the MMP-3 promoter will direct expression of the luciferase cDNA in a cell. The MMP-3 promoter/reporter gene is transfected into SW1353 cells under conditions that promote retention of the MMP-3 promoter/reporter gene in the cells. The cells are plated in 96 well plates and incubated with compound. After 16 hours, the cells are lysed and the inhibitory activity of the compound is determined by observing the decrease in luciferease activity using techniques that are well known in the art.

TEF-3 itself can be used to determine the binding of compounds to the protein. Drug screening using protein targets is used in the art and can be employed using automated, high throughput technologies. In screening, a compound 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 disease. For example one can directly screen for compound binding to TEF-3 by affixing TEF-3 to an affinity matrix using chemical crosslinking methods that are well known in the art. Labeled compounds (e.g., radiolabeled, biotinylated or the like) are added to the bound TEF-3 and the extent of binding is determined using standard techniques. Alternatively one may determine compound binding using competition assay where the compound and a known TEF-3 binding molecule (e.g., TIF, small molecule, antibody, DNA fragment or the like) compete for TEF-3 binding.

In addition, it is apparent to the skilled artisan that fragments of TEF-3 may be used in screening, especially high throughput screening, drug design and the like, and that the entire protein may not be required for the purposes of using the invention for screening. Thus it is clearly contemplated that the skilled artisan will understand that the disclosure of the protein and its uses contemplates useful protein fragments. Such fragments may also be useful in determination of active sites, for DNA binding, phosphorylation, epitope formation, binding sites for proteins and other factors, and the like, each of which are useful in rational drug design and TEF-3 structure determination generally.

The practical considerations of protein expression, purification, yield, stability, solubility, and the like, are considered by the skilled artisan when choosing whether to use a fragment, and which fragment is to be used. For example, soluble fragments are preferred because they provide ready reference and ease of handling, hence a "soluble fragment" is one that is not easily precipitated using, for example, standard ELISA or other screening procedures, yet retains its activity. These are known for other proteins, and finding a useful fragment, by digestion or other means is well within the skill of the artisan. As a result, using routine practices in the art, the artisan can, given this disclosure, practice the invention using fragments of the TEF-3 as well. Specifically contemplated in this context are soluble fragments of TEF-3, which are amenable to larger scale production and isolation.

TEF-3 is speculated to interact with other cofactors for its tissue specific activity. Such cofactors are referred to herein as TEF-3 interacting factors (TIFs). Identification of TIFs presents another way of inhibiting TEF-3 activity. These TIFs can be isolated and identified by a number of available methods using the TEF-3 DNA sequence or its recombinant protein. For example, one can directly screen a cDNA expression library or cellular proteins bound to a supporting material with labeled (e.g., radiolabeled, biotinylated or the like) TEF-3 protein. Another example is to detect protein-protein interaction through a yeast two-hybrid screening system. In the yeast two-hybrid approach, TEF-3 cDNA is fused to a DNA binding (DB) domain of a known transcription factor to form a bait. A random library of cDNA from a desired tissue is linked to a prey construct containing an activation domain (AD) of the transcription factor. When the bait and prey are present and interact in the same cell, the DB and AD are brought into close proximity and a reporter gene is activated. One can also isolate the interacting factors by affinity chromatography, in which TEF-3 protein itself, or antibodies raised against TEF-3 protein, or DNA fragment containing TEF-3 binding site are immobilized on a solid matrix by various standard chemical or physical methods. Protein extracts from desired cells or tissues are allowed to pass through the matrix and TEF-3 interacting factors are retained. A similar approach can be taken in solution, where solution phase TEF-3 is used, and a factor bound to TEF-3 may be immunoprecipitated by TEF-3 specific antibodies.

Method of Treating Disorders by Inhibiting TEF-3 Activity

In one aspect of the invention, a method of treating MMP-mediated disorders by administering to a subject an inhibitor of TEF-3 activity is provided. "Subject" refers to a human or other mammal experiencing or at risk of experiencing an MMP-mediated disorder. Inhibitors of TEF-3 activity are administered to a subject in a safe and effective amount. As used herein, "safe and effective amount" means an amount of an inhibitor sufficient to significantly induce a positive modification in the condition to be treated, but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. A safe and effective amount of an inhibitor will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the particular pharmaceutically-acceptable carrier utilized, and like factors within the knowledge and expertise of the attending physician. As used herein, an "MMP-mediated disorder" is a disorder that involves unwanted or elevated MMP activity in the biological manifestation of the disorder; in the biological cascade leading to the disorder; or as a symptom of the disorder. Thus, "involvement" of the MMP includes:

1. The unwanted or elevated MMP activity as a "cause" of the disorder or biological manifestation, whether the activity is elevated genetically, by infection, by autoimmunity, by trauma, by biomechanical causes, by lifestyle [e.g. obesity], or by some other cause;

2. The MMP as part of the observable manifestation of the disorder. That is, the disorder is measurable in terms of the increased MMP activity. From a clinical standpoint, unwanted or elevated MMP levels indicate the disorder, however, MMPs need not be the "hallmark" of the disorder; or

3. The unwanted or elevated MMP activity is part of the biochemical or cellular cascade that results or relates to the disorder. In this respect, inhibition of the MMP activity interrupts the cascade, and thus controls the disease.

TEF-3 activity may be inhibited by several methods, which are described in detail below: Small Molecules and/or peptides

In a preferred embodiment of the invention, inhibition of TEF-3 activity is achieved by administering an inhibitor that interferes with the binding of TEF-3 to the MMP promoter gene thereby preventing transcription. Such inhibitors include small molecules and/or peptides that bind to the DNA binding site on the MMP promoter gene and prevent TEF-3 from binding to the site. Other inhibitors include small molecules and/or peptides that bind to the TEF-3 protein itself and prevent TEF-3 from binding to an MMP promoter fragment.

In another embodiment of the invention, inhibition of TEF-3 activity is achieved by administering a small molecule or peptide that interferes with the binding of TEF-3 to a TIF thereby preventing transcription. Such inhibitors include small molecules and/or peptides that bind to TEF-3 or to a TLF.

Antibodies to TEF-3

Antibodies to TEF-3, both monoclonal and polyclonal, are useful for detecting TEF-3, and hence have several useful purposes. These include detection of TEF-3 up regulation in disease, therapeutic treatments of diseases in which TEF-3 is involved, purification of TEF-3, delivery of therapeutic agents to TEF-3 and development of drug screens for the binding of molecules to TEF-3.

For purposes of purification of TEF-3, the antibody of the invention can also be used in immunoprecipitation, or preferably conjugated to solid supports. These conjugates can be used as affinity reagents for the purification of the TEF-3. For example, these antibodies, when conjugated to a suitable chromatography material are useful in isolating the TEF-3. Separation methods using affinity chromatography are known in the art, and are within the purview of the skilled artisan.

In development of drug screens for the binding of molecules to TEF-3, the antibody can be used to provide information as to epitopes on TEF-3 that are to be targeted. An antibody can be used to identify the TEF-3 sites essential for activity, and hence useful to design inhibitors of the TEF-3. For example, an antibody that may also be used to identify a DNA binding site on TEF-3 is useful in a similar way. In addition, there are likely domains on TEF-3 that interact with other transcription promoting or inhibiting factors necessary for regulating gene promoter activity. The utility of designing a molecule that effectively perturbs or disturbs this interaction is apparent to the skilled artisan: such a strategy provides a very specific drug for promoting or inhibiting gene expression.

In addition, the antibody of the invention is useful in Western blotting and other identifying techniques, hence it is useful in ELISA, capture assays, sandwich assays, scintillation proximity assay, and the like.

For purposes of detection of TEF-3 up regulation in disease, the antibody of the invention is directly conjugated to a label. A biopsy sample of articular cartilage is retrieved during arthroscopic surgery and subjected to histological analysis commonly used in the art. The histological sections are reacted with labeled TEF-3 antibody using standard techniques. High levels of antibody binding indicate TEF-3 up-regulation.

Without being bound by theory, expression of genes, including the gene of the invention 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. Hence an antibody raised to the TEF-3 would operate as a screening tool for such enhanced expression, indicating disease.

Antibodies may be made by several methods, for example, the TEF-3, TEF-3 variants, or fragments of TEF-3 may be injected into suitable (e.g., mammalian) subjects including mice, rabbits, goats, horses, and the like. The TEF-3, variants, or fragments may be coupled to a hapten to enhance its antigenicity. 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 cells of the immunized animal and immortalizing the antibody-producing cells, followed by identifying the suitable antibody producers using standard immunoassay techniques.

Gene therapy

Without being bound by theory it is thought that the TEF-3 is up-regulated during osteoarthritis in tissues. The skilled artisan will recognize that if up-regulation is a cause of the onset of osteoarthritis, then interfering with the activity of this gene may be useful in treating osteoarthritis. This is done by any of several methods, including gene (i.e., antisense) therapy.

For example, use of the MMP-3 gene as a model indicates that transcription of the MMP genes in the chondrocytes of articular cartilage depends on the transcription factor, TEF-3. Inhibition of TEF-3 activity by mutation of its promoter binding site or inhibition of its expression by a TEF-3 antisense oligonucleotide results in decreased expression of MMP-3 in chondrocytes in vitro. TEF-3 activity may be inhibited by over expression of TEF-3 by gene transfer techniques. This induces "squelching" of the transcriptional activity and thereby inhibits or decreases expression of TEF-3 responsive genes. Likewise, introduction of a variant form of TEF-3 may inhibit the activity of TEF-3 responsive genes by inducing a dominant negative phenotype.

Accordingly, the present invention is directed to a method of treating or preventing a matrix metalloprotease-mediated disorder, comprising the steps of: (a) cloning a TEF-3 cDNA into an expression vector in proper frame and orientation for expression in a cell; (b) packaging the expression vector in a suitable host virus; (c) injecting the virus in the diseased area in the body; (d) allowing the cells to take up the expression vector and express the TEF-3; and (e) observing a decrease in MMP expression due to transcriptional squelching.

Inhibition of TEF-3 activity will make an effective therapy for treating degenerative joint diseases. This method utilizes currently available ex vivo and explant techniques. For example, cells from a degrading cartilage specimen (within a joint, such as a knee, hip or the like) may be removed from a patient, manipulated ex vivo using antisense techniques, such as adenovirus, and returned to the patient in the same way as current cartilage graft therapies are employed. The patient would then be able to regenerate the cartilage lost to degradation in the affected joint.

Additionally, over expression of TEF-3, expression of antisense TEF-3, expression of variant TEF-3, in vivo or ex vivo may be accomplished using a tissue-specific promoter for expression of the recombinant gene. In the case of chondrocytes and articular cartilage this may be accomplished using a collagen type II gene promoter.

Accordingly, the present invention is directed to a method of treating or preventing a matrix metalloprotease-mediated disorder, comprising the steps of: (a) cloning a TEF-3 cDNA into an expression vector in proper frame, but in inverse orientation for expression in a cell; (b) packaging the expression vector in a suitable host virus; (c) injecting the virus in the diseased area in the body; (d) allowing the cells to take up the expression vector and express the TEF-3; and (e) observing a decrease in MMP expression due to antisense expression of the TEF-3 gene.

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 are 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 available. For example T4 polynucleotide kinase and λgtl 1 bacteriophage are known materials. In addition Kunkel method mutagenesis and autoradiography are known techniques.

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

Binding of putative transcription factors to gene promoters are conveniently identified by way of electrophoretic mobility retardation assay (EMRA). Double stranded oligonucleotides homologous to the MMP-3 promoter are synthesized and end labeled with [32p] via reaction with T4 polynucleotide kinase and ^2p-ATP. The radioactive oligonucleotides are incubated with nuclear extract proteins prepared from articular chondrocytes and/or other cell types. DNA/protein complexes formed as a result of the incubation are separated from the unbound oligonucleotides by electrophoresis on non denaturing polyacrylamide gels. DNA/protein interactions are identified visually as bands migrating more slowly on the gel than the unbound oligonucleotide following exposure of the dried gel to X-ray film (autoradiography) or by way of a phosphoimager, or other imaging techniques.

The DNA recognition element for a binding protein is identified using EMRAs as above. Double stranded, labeled oligonucleotides containing nucleotide mutations in the putative DNA binding site for the transcription factor are used as probes in EMRAs. A positive result is a reduction in binding of the factor to the DNA probe as recorded by a decrease in intensity of the binding signal on the autoradiograph. Alternatively, competition assays may be used. Unlabeled oligonucleotides containing the putative binding sequence are included in the incubation of the labeled promoter fragment/nuclear extract mix. A positive result is recorded as a reduction in the signal on the autoradiogram.

The cDNA for the transcription factor binding to the MMP-3 promoter fragment may be cloned by screening a bacterial recombinant cDNA expression library (λgtl l bacteriophage in E. coli), prepared using articular chondrocyte cDNA, with the labeled promoter fragment containing the transcription factor binding site. The bacteriophage containing the cDNA that binds to the labeled DNA is purified by several rounds of picking the positive clone followed by replating and rescreening until all clones yield a positive signal. The bacteriophage DNA carrying the transcription factor cDNA is purified and the cDNA insert is subcloned into a plasmid vector (pBlueScript) for propagation in E. coli. The nucleotide sequence of the cDNA is determined using standard techniques.

The amino acid sequence for the transcription factor is deduced from the nucleotide sequence of the cDNA clone. The amino acid sequence is used to prepare synthetic peptides for monoclonal antibody production via standard techniques. The monoclonal antibodies are used to confirm the DNA/protein binding interaction observed on the EMRAs. The antibodies are included in the labeled DNA/nuclear extract incubation mixture. A positive result is recorded on the autoradiogram as an even greater retardation in mobility of the labeled DNA fragment (so called "supershift").

The functional significance of the transcription factor/DNA recognition element binding is determined via transfection of a MMP-3 promoter/luciferase reporter gene construct into chondrocytes. Promoter activity of native MMP-3 promoter fragment is compared to similar constructs containing mutations in the DNA recognition element for the transcription factor. A positive result is determined as a decrease in luciferase expression (recorded as a decrease in light emission) using the variant promoter. Example 2

Inhibition of extracellular matrix remodeling is explored via inhibition of TEF-3 activity in vitro. Using a small molecular weight inhibitor of TEF-3 activity, tissue integrity and proteoglycan is monitored.

A sample of interlukin-1 stimulated bovine nasal cartilage is grown in a 1 μM solution of a small molecular weight inhibitor of TEF-3 activity. 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 culture media. The result is consistent with the inhibited MMP activity.

It is contemplated that similar assays using cells stimulated with retinoic acid, fibronectin, TGF-b or E. coli-LPS (lipopolysaccharide) produce similar results.

Example 3

Cells known to transcribe MMP genes are treated with an inhibitor of TEF-3 activity. Control cells that have not been treated with inhibitors of TEF-3 activity are compared. Proteins released from the cell are measured by standard methods. Specifically, the metalloprotease activity is monitored via literature methods. The amount of metalloprotease released is correlated (inversely proportional) to or altered by the amount of inhibitor of TEF-3 activity used to treat the cells.

Example 4

TEF-3 binding is measured with a small molecule TEF-3 inhibitor in mammalian chondrocyte cell culture. Inhibition of TEF-3 results in the inhibition of phenotypic changes, including changes in cell shape, associated with such interactions. Binding is measured via competitive assay, using cellular changes in shape visible via microscopy. The inhibitor inhibits such cellular changes. The osteoarthritis phenotype, characterized by increased matrix synthesis and accelerated matrix metalloprotease activity does not occur. Assayed gene expression, and changes in mitotic activity, are consistent with this result.

Example 5

Candidate inhibitors of TEF-3 gene expression are screened in vitro. Interleukin-1 stimulated cultures of normal human articular chondrocytes are exposed in vitro to candidate inhibitors. MMP-3 RNA is isolated and reverse transcribed into cDNA. The cDNA is subjected to polymerase chain reaction (PCR) amplification using specific oligonucleotide primers. PCR samples generated from both chondrocytes exposed to inhibitors and uninhibited chondrocytes are electrophoresed in adjacent lanes on polyacrylamide gels. Reduced levels of PCR product identifies an inhibitor.

Example 6

Papain induced cartilage degradation induced using the method of (D.G. Murry (1964) Arthritis Rheu. 7: 211-219.) is monitored in mice. The experimental group has a daily 2 mg/kg dose of a TEF-3 inhibitor administered systemically via oil bolus. The experimental group is more ambulatory and appears to recover from the papain treatment. Upon biopsy of the affected joint, MMP-3 expression is minuscule compared to the control group.

Similar results are expected with iodoacetate, or other chemically induced in vivo arthritis models, as well as surgically induced models, such as meniscectomy, ligament transection which may include the anterior (cranial) cruciate ligament, the posterior cruciate ligament, the medial collateral ligament, the lateral collateral ligament. Any of the above, alone or in combination may be used.

Example 7

Six spontaneously arthritic guinea pigs (Hartley) are treated with a TEF-3 inhibitor, and monitored along with a control group as in Example 6. The experimental group moves about while the control group is more restive. Similar results are expected with other similar animal models such as C57 black mice or aged Fisher rats.

Cartilage from these animals is removed, and mRNA prepared from the cells and reverse transcribed into cDNA. A sense oligomer of 20 bases from base 311 to base 330 and an antisense oligomer of 20 bases from base 680 to base 699 are used in a low stringency PCR experiment that provided a fragment of a homologous TEF-3 cDNA from the guinea pig.

Example 8

A 500 base pair (bp) fragment of the human MMP-3 promoter is used as template for PCR to generate a 150 bp fragment of the MMP-3 promoter comprising base pairs +13 through -137 that has a Bgl II restriction endonuclease site on both ends. The 5' sense primer has the sequence

GGGAGATCTCTGCCTCCTTGTAGGTCCAACCTCGGG and the 3' antisense primer has the sequence AGATCTTGTATCATCCTACTTTG. The PCR conditions are, a 3 minute pre elt at 94°C followed by 25 cycles of 30 seconds at 94°C, 30 seconds at 45°C, 1 minute at 72°C. This is followed by 8 minutes at 72°C and then 4°C until the samples are retrieved. PCR is accomplished using the GeneAmp XL PCR kit (Perkin-Elmer). Using the Bgl II restriction sites, this MMP-3 promoter fragment is subcloned into the pGL2-Basic (Promega) luciferase expression vector using standard techniques. The MMP-3 promoter fragment is in proper orientation and frame to direct the expression of luciferease in eucaryotic cells. This plasmid is then cotransfected into the human chondrosarcoma cell line SW1353 (ATCC) along with the plasmid pcDNA3.1/Zeo (InVitrogen) which contains a zeocin antibiotic resistance gene. Cells that stably integrate the plasmid DNAs are selected for by culturing the cells in the presence of 600 μg/mL zeocine. Zeocin resistant SW1353 colonies are transferred to tissue culture flasks. The cells are tested for expression of the luciferase protein using the LucLite reporter gene assay kit (Packard)

Example 9

For high throughput screening of chemical libraries, the stably transfected SW1353 cells are plated in 96 well tissue culture plates at 70% confluence and incubated for 24 hours at 37°C. The cells are then incubated for an additional 16 hours in the presence of the test compound at 10 μM concentration. The cells are then tested for decreases in luciferase activity relative to no compound control cells using the LucLite reporter gene assay kit (Packard).

Example 10

Recombinant TEF-3 is produced in and purified from bacteria. A full length cDNA for TEF-3 with a Bam HI restriction endonuclease site at the 5' end and an Eco RI restriction endonuclease site at the 3 ' end is digested with those restriction endonucleases. The plasmid expression vector pET32c (Novagen) is digested with same restriction enzymes. The TEF-3 cDNA is ligated into the expression vector using the FastLink ligation system (Epicentre) in proper frame and orientation for expression using the gene promoter present in the plasmid. The plasmid is introduced into the AD494(DE3) strain of E. coli (Novagen). Bacteria that have taken up the plasmid DNA are selected for by culturing the bacteria on agar plates that contain the antibiotic ampicillin. The ampicillin resistant bacteria are grown in LB broth under standard laboratory conditions. Expression of the TEF-3 is induced by adding 1 mM isopropylthiogalactoside (LPTG) to the culture broth. Sixteen hours after the addition of the LPTG, the bacteria are harvested and the TEF-3 purified to homogenity. Example 11

Compounds are screened for TEF-3 inhibition by their binding to TEF-3. Recombinant TEF-3 or chemically synthesized fragments of TEF-3 are bound to 96 well polystyrene plates in a solution containing 1 mg/ml TEF-3 or TEF-3 fragment for 30 minutes at room temperature. Unbound protein is removed by 3 washes with phosphate buffered saline (PBS). Non specific binding sites are blocked by a 30 minute incubation at room temperature using PBS containing 1 mg/ml bovine serum albumin (BSA) followed by 3 washes with PBS. Radiolabeled compounds at 100 μM concentration in PBS are added to the plates and incubated for 30 minutes at room temperature. The plates are washed 3 times with PBS and the amount of bound compound is determined by scintillation counting of the radioactivity.

Example 12

Compounds are also screened by inhibition of TLF binding. Recombinant TEF-3 or chemically synthesized fragments of TEF-3 are bound to 96 well polystyrene plates in a solution containing 1 mg/ml TEF-3 or TEF-3 fragment for 30 minutes at room temperature. Unbound protein is removed by 3 washes with PBS. Non specific binding sites are blocked by a 30 minute incubation at room temperature using PBS containing 1 mg/ml BSA followed by 3 washes with PBS. Test compounds are added to the plates at a concentration of 100 μg/ml along with ³⁵S-labeled recombinant TEF-3 TLF at a concentration of 100 μg/ml in PBS and incubated at room temperature for 30 minutes. The plates are washed 3 times with PBS. The amount of bound TLF is determined in a scintillation counter. Inhibition of TIF binding is determined by comparison to controls that did not contain test compound. Identified compounds that bind TEF-3 are optimized for high affinity binding by using medicinal chemistry to synthesize closely related analogs and retesting in the above assay.

Example 13

Compounds are also screened by inhibition of TEF-3 antibody binding. Recombinant TEF-3 or chemically synthesized fragments of TEF-3 are bound to 96 well polystyrene plates in a solution containing 1 mg/ml TEF-3 or TEF-3 fragment for 30 minutes at room temperature. Unbound protein is removed by 3 washes with PBS. Non specific binding sites are blocked by a 30 minute incubation at room temperature using PBS containing 1 mg/ml BSA followed by 3 washes with PBS. Test compounds are added to the plates at a concentration of 100 μg/ml along with ³⁵S-labeled TEF-3 antibody at a concentration of 100 μg/ml in PBS and incubated at room temperature for 30 minutes. The plates are washed 3 times with PBS. The amount of bound antibody is determined in a scintillation counter. Inhibition of TLF binding is determined by comparison to controls that did not contain test compound. Identified compounds that bind TEF-3 are optimized for high affinity binding by using medicinal chemistry to synthesize closely related analogs and retesting in the above assay.

Example 14

Compounds are also screened by inhibition of DNA binding to TEF-3. Recombinant TEF-3 or chemically synthesized fragments of TEF-3 are bound to 96 well polystyrene plates in a solution containing 1 mg/ml TEF-3 or TEF-3 fragment for 30 minutes at room temperature. Unbound protein is removed by 3 washes with phosphate buffered saline (PBS). Non specific binding sites are blocked by a 30 minute incubation at room temperature using PBS containing 1 mg/ml bovine serum albumin (BSA) followed by 3 washes with PBS. Test compounds are added to the plates at a concentration of 100 μg/ml along with ³⁵S-labeled DNA fragment that contains the TEF-3 DNA recognition element TTTGGAATG at a concentration of 100 μg/ml in PBS and incubated at room temperature for 30 minutes. The plates are washed 3 times with PBS. The amount of bound DNA fragment is determined in a scintillation counter. Inhibition of DNA fragment binding is determined by comparison to controls that did not contain test compound. Identified compounds that bind TEF-3 are optimized for high affinity binding by using medicinal chemistry to synthesize closely related analogs and retesting in the above assay.

Example 15

Compounds are tested in vitro for their ability to slow the rate of interleukin-1 stimulated cartilage breakdown, using bovine nasal cartilage as described by Price et al. (Arthritis and Rheumatism (1999) 42(1): 137-147). Compounds are incubated throughout the assay time period with the cartilage plugs at compound concentrations of 0, 1 ηg/ml, 10 ηg/ml, 100 ηg/ml, 1 μg/ml, 10 μg/ml, and 100 μg/ml. Proteoglycan breakdown and collagen breakdown are determined as described.

Example 16

Compounds are tested for their ability to slow the rate of cartilage breakdown in vivo using the spontaneous osteoarthritis model in Hartley guinea pigs as described by Bendele and Hulman (Arthritis and Rheumatism (1988) 31 : 561-565). Compounds are dosed once daily at 1, 10, and 100 mg/kg body weight for 6 months by subcutaneous injection of the test compound in PBS. Animals are dosed starting at 3 months of age. Cartilage degradation of the stifle joints is determined as described by comparison to control animals that received only PBS injections.

All references described herein are hereby incorporated by reference.

While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of this invention.

Claims

WHAT IS CLAIMED IS:

1. A method for determining whether a compound is an inhibitor of TEF-3 activity, comprising the steps of:

(a) culturing cells containing a TEF-3 responsive gene which comprises a TEF-3 responsive gene promoter and a reporter cDNA;

(b) incubating the cells with a sample of the compound: and

(c) monitoring the cells for a response from said reporter.

2. The method of Claim 1 wherein the promoter fragment is derived from a matrix metalloprotease gene.

3. The method of Claim 2 wherein the promoter fragment is derived from the MMP- 3 gene.

4. The method of Claim 3 wherein the cells are human chondrocyte cells or human chondrocyte-like cells.

5. The method of Claim 4 wherein the method is a high throughput screening method.

6. The method of Claim 5 wherein the reporter cDNA codes for luciferase.

7. The method of Claim 6 wherein the human chondrocyte-like cells are SW1353.

8. The method of Claim 7 wherein the promoter fragment comprises nucleotides -1 through -150 of the MMP-3 gene.

9. The method of Claim 7 wherein the promoter fragment comprises nucleotides -1 through -499 of the MMP-3 gene.

10. A method for producing recombinant TEF-3 in a bacterial or eucaiyotic cell, comprising:

(a) generating a cDNA coding TEF-3;

(b) placing the cDNA into an expression vector;

(c) introducing the vector containing the cDNA into the cell;

(d) observing synthesis of TEF-3 in the cell; and

(e) purifying the TEF-3 to homogeneity.

11. A method for determining whether a compound is an inhibitor of TEF-3 activity, comprising the steps of:

(a) labeling the compound;

(b) incubating the labeled compound with a TEF-3 prepared by the method of Claim 10 that is bound to a solid support: and

(c) determining the amount of labeled compound that binds the TEF-3.

12. A method for determining whether a compound is an inhibitor of TEF-3 activity, comprising the steps of:

(a) binding a TEF-3 prepared by the method of Claim 10 to a solid support;

(b) incubating the bound TEF-3 with the compound and a TEF-3 binding protein;

(c) determining the inhibition of binding of the TEF-3 binding protein.

13. The method of Claim 12 wherein the method is a high throughput screening method.

14. The method of Claim 13 wherein the TEF-3 binding protein is a TEF-3 interacting factor.

15. The method of Claim 13 wherein the TEF-3 binding protein is an antibody.

16. A method for determining whether a compound is an inhibitor of TEF-3 activity, comprising the steps of:

(a) binding a TEF-3 prepared by the method of Claim 10 to a solid support;

(b) incubating the bound TEF-3 with the compound and a DNA fragment containing the TEF-3 binding site; and

(c) determining the inhibition of binding of the DNA fragment.

17. The method of Claim 16 wherein the DNA fragment is derived from an MMP promoter.

18. The method of Claim 17 wherein the DNA fragment is derived from the MMP-3 promoter.

19. The method of Claim 17 wherein the DNA fragment contains the sequence TTTGGAATG.

20. A method of identifying a TEF-3 interacting factor, comprising the steps of:

(a) collecting different tissues, cells or body fluid samples from an animal or population thereof;

(b) preparing a cellular or nuclear extract thereof;

(c) exposing said extracts to a TEF-3 prepared by the method of Claim 10;

(d) immunoprecipitating with an antibody specific to TEF-3 : and

(e) identifying the TEF-3 -bound factor.

21. A TEF-3 interacting factor identified by the method of Claim 20.

22. A method of identifying a TEF-3 interacting factor, comprising the steps of:

(b) preparing a cellular or nuclear extract thereof;

(c) exposing said extracts to a TEF-3 prepared by the method of Claim 10 that is bound to a solid support; and

(d) identifying the factor.

23. A TEF-3 interacting factor identified by the method of Claim 22.

24. A method of identifying a TEF-3 interacting factor, comprising the steps of:

(b) preparing a cellular or nuclear extract thereof; (c)separating the components of said extract;

(d) identifying said factor by exposing said components to a labeled TEF-3; and (e) detecting the factor by observing binding to labeled TEF-3.

25. A TEF-3 interacting factor identified by the method of Claim 24.

26. A method of identifying a TEF-3 interacting factor, comprising the steps of:

(a) preparing a cDNA library in an expression vector;

(b) introducing said cDNA into cells;

(c) growing cells containing said cDNA;

(d) probing a sample of said cells using labeled TEF-3; and

(e) detecting the factor by observing binding to labeled TEF-3 within said sample.

27. A TEF-3 interacting factor identified by the method of Claim 26.

28. A method of identifying a TEF-3 interacting factor, comprising the steps of:

(a) fusing a first cDNA coding TEF-3 with a DNA binding domain of a known transcription factor;

(b) fusing a second cDNA of a random library with a DNA activation domain of said transcription factor;

(c) introducing said fused first cDNA and said fused second cDNA into a yeast cell containing a promoter/reporter gene that binds said transcription factor;

(d) observing a response of said promoter/reporter gene; and

(e) isolating the cDNA from said random library whose gene product gave the response.

29. The method of Claim 24 wherein the reporter cDNA codes for beta-galactosidase.

30. A TEF-3 interacting factor identified by the method of Claim 29.

31. An antibody, or fragment thereof, to TEF-3.

32. A method of treating or preventing a matrix metalloprotease-mediated disorder, comprising administering to a subject an inhibitor of TEF-3 activity.

33. The method of Claim 32, wherein the matrix metalloprotease-mediated disorder is a degenerative joint disorder.

34. The method of Claim 33, wherein the matrix metalloprotease-mediated disorder is osteoarthritis.