Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-03-27
2003-06-17
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
Reexamination Certificate
active
06579708
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
The drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics”, that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superseding earlier approaches based on “positional cloning”. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
Functional genomics relies heavily on high-throughput DNA sequencing technologies and the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterise further genes and their related polypeptides/proteins, as targets for drug discovery.
SUMMARY OF THE INVENTION
The present invention relates to human uridine kinase, in particular uridine kinase polypeptides and uridine kinase polynucleotides, recombinant materials and methods for their production. Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, cancers, hereinafter referred to as “diseases of the invention”. In a further aspect, the invention relates to methods for identifying agonists and antagonists (e.g., inhibitors) using the materials provided by the invention, and treating conditions associated with uridine kinase imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate uridine kinase activity or levels.
DESCRIPTION OF THE INVENTION
In a first aspect, the present invention relates to human uridine kinase polypeptides. Such polypeptides include:
(a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO:1;
(b) an isolated polypeptide comprising a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
(c) an isolated polypeptide comprising the polypeptide sequence of SEQ ID NO:2;
(d) an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
(e) the polypeptide sequence of SEQ ID NO:2; and
(f) an isolated polypeptide having or comprising a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2;
(g) fragments and variants of such polypeptides in (a) to (f).
Polypeptides of the present invention are believed to be members of the enzymes-kinases family of polypeptides. They are therefore of interest because uridine kinase (UDK) is the rate-limiting enzyme in the pyrimidine nucleoside salvage pathway of all mammalian cells. It catalyzes the phosphorylation of uridine and cytidine to form uridine monophosphate (UMP) and cytidine monophosphate (CMP). A recent study revealed the positive correlation of uridine kinase activity with proliferation and transformation in human ovarian carcinomas and rat hepatomas (1). It showed that in normal rat liver, the Km for uridine was 5.0 mM, while in the rapidly growing hepatoma 3924A the Km was 0.8 mM. UDK activity also increased in tumor tissues. It increased to 1.5- to 2.6-fold in the hepatomas of slow and intermediate growth rates, and increased to 5.1- to 5.8-fold in rapidly growing hepatomas, compared with normal livers. This correlation was also evident in human ovarian cancer patients. The Km of uridine kinase activity for uridine was 0.5 mM in ovarian carcinoma verses 11.5 mM in normal ovary. The kinase activity increased to 5- to 13-fold in ovarian carcinoma over that in normal ovary. Another group reported that the activity of UDK, together with three other enzymes involved in the pyrimidine metabolism, was increased in human colon carcinomas compared to corresponding normal tissues (2). Importantly, uridine kinase is responsible for the activation of a novel antimetabolite, Tas-106 (3′-ethynylcytidine), used in the treatment of cancer. The antitumor effects and specific tissue toxicity of a chemotherapeutic compound, TAS-106, is closely linked to the the level of expression of UDK. Therefore, it is expected that this agent will be most effective in tumors expressing high levels of the enzyme. The availability of the cloned human UDK will allow for the development of tools for the determination of UDK expression levels at the mRNA or protein level by methods such as TaqMan analysis, in situ hybridization, Northern blot and Western blot analysis. These techniques may prove useful in predicting which patients are the best candidates for treatment with TAS-106 based on high expression of the enzyme in their tumors. Taken together, human uridine kinase will have broad application in the diagnosis, anticancer drug discovery and treatment of cancer patients.
The biological properties of the uridine kinase are hereinafter referred to as “biological activity of uridine kinase” or “uridine kinase activity”. Preferably, a polypeptide of the present invention exhibits at least one biological activity of uridine kinase. Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including all allelic forms and splice variants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred variants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acids are inserted, substituted, or deleted, in any combination.
Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO: 2. Preferred fragments are biologically active fragments that mediate the biological activity of uridine kinase, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or immunogenic in an animal, especially in a human.
Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention. The polypeptides of the present invention may be in the form of the “mature” protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence for stability during recombinant production.
Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurring sources, from genetically engineered host cells comprising expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesizers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.
In a further aspect, the present invention relates to uridine kinase polynucleotides. Such polynucleotides include:
(a) an is
Ho Yen Sen
Johnson Randall
Gimmi Edward R.
Kinzig Charles M.
Prouty Rebecca E.
SmithKline Beecham Corporation
Steadman David
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