WO2002004613A2 - Isolated polynucleotides encoding human 5'-nucleotide cn-ia and cn-ib, isolated proteins encoded by the same, and methods utilizing the same - Google Patents

Abstract

The isolated polynucleotide sequence identified herein as SEQ ID NO: 1 encodes an human cytosolic 5'-nucleotidase form I (human cN-IA) and encodes the protein human cN-IA of SEQ ID NO:2. The isolated polynucleotide sequence identified herein as SEQ ID NO: 3 encodes a cytosolic 5'-nucleotidase form I (cN-IB) and encodes the protein cN-IB of SEQ ID NO:4. Expression of human cN-IA decreases the efficacy of antineoplastic drugs and increases the toxicity of AZT. Inhibitors of human cN-IA are useful in methods of treating cancer by increasing the efficacy of antineoplastic drugs, and in methods of treating viral infections such as HIV infection and AIDS by decreasing the side effects of AZT.

Description

ISOLATED POLYNUCLEOTIDES ENCODING HUMAN

5'-NUCLEOTIDASES cN-IA and cN-IB, ISOLATED PROTEINS ENCODED

BY THE SAME, AND METHODS UTILIZING THE SAME

Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/216,827, filed July 7, 2000, the disclosure of which is incorporated herein by reference in its entirety.

Federal Support of the Invention

This invention was made with United States government support under grant number 2-RO1-CA34085-13 from the National Institutes of Health. The United States government has certain rights to this invention.

Field of The Invention

This invention relates to isolated polynucleotide sequences that encode human 5'-nucIeotidases. The invention also relates to inhibitors of these enzymes and methods of using the same.

Background of the Invention

Chemotherapy is presently a primary form of conventional cancer treatment. However, one major problem associated with cancer chemotherapy is the ability of tumor cells to develop resistance to the cytotoxic effects of anti-cancer drugs during the course of treatment. Another major complication of both cancer chemotherapy and antiviral chemotherapy is damage to bone marrow cells or suppression of their function. Many cancer patients die of infection or other consequences of hematopoietic failure subsequent to chemotherapy. Chemotherapeutic compounds can also result in subnormal formation of platelets which produces a propensity toward hemorrhage. Inhibition of erythrocyte production can result in anemia. The risk of damage to the hematopoietic system or other important tissues can prevent utilization of doses of chemotherapy compounds high enough to provide good antitumor or antiviral efficacy.

Many antineoplastic or antiviral chemotherapy compounds are nucleoside analogs that substitute for the normal nucleosides in nucleic acids, producing defective RNA or DNA and ultimately causing apoptosis in cancer cells. Among other factors, the therapeutic efficacy of these drugs depends on the accumulation of their triphosphate derivatives in cancer cells. Certain enzymes may potentially remove, retard or otherwise inhibit the efficacy of active drug derivatives by catalytically dephosphorylating them, thus limiting their pharmacological efficacy. These enzymes are thought to contribute to the development of drug resistance by the recipient of the drug during the course of treatment. Accordingly, there is an ongoing need for pharmacological compounds able to inhibit such enzyme-catalyzed drug resistance. Such compounds may be useful as adjuncts to certain anti-cancer treatments.

Acquired Immunodeficiency Syndrome (AIDS) is a progressive and presently fatal disease caused by the infection of the Human Immunodefiency Virus (HIV). One prevalent treatment of AIDS or HIV infection is the administration of 3^'-azido-3^'-deoxythymidine (AZT). AZT was first identified in 1964 (J. P. Horowitz et al., J. Org. Chem., 28, 2076 (1964)), and later found to have antiviral activity (E. DeClerq et al., Biochem. Pharmacol., 29, 1849 (1980)). Methods of treating humans and animals afflicted with AIDS, HIV infections and other viral infections with AZT and its analogs and salts are set forth in U.S. Patent Nos. 4,724,232; 4,874,609; 5,643,891 and 5,885,957, all to Rideout et al.

AZT is commercially available as a powdered form of 3^'-azido-3^'- deoxythymidine. This product is converted by the subject's body to a phosphate form. This phosphorylation process occurs within the cell, where AZT is first converted to a monophosphate then to a diphosphate. The final phosphorylation step results in a triphosphate form of AZT. It is this triphosphate form or "activated" form of AZT which has the most inhibitory ^• effect on vital transcription.

AZT generally prolongs the Iifespan of patients infected with HIV, but unfortunately may also impair hematopoiesis, producing leukopenia and anemia. Other side effects of AZT administration related to the drug's toxicity are documented. The administration of uridine by periodic intravenous injection in order to attenuate AZT toxicity has been proposed. See U.S. Pat. No. 5,077,280 to Sommadossi et al. It has also been reported that deoxycytidine protects normal human bone marrow progenitor cells in vitro against the cytotoxicity of AZT with preservation of antiretroviral activity. See, e.g., Bhalla et al., Blood 74, 1923-1928 (1989).

Recently, studies have demonstrated that a breakdown product of AZT, 3^'-amino-3^'-deoxythymidine (AMT), is five to seven times more toxic to bone marrow cells than AZT. In addition, AMT, which has no antiviral activity, has an antagonistic effect on the anti-HIV action of AZT. The antagonistic effect is apparently a result of competitive binding by AMT of the reverse transcriptase enzyme. AMT is produced at about twenty percent of the level of AZT. Significantly, this catabolite has a half life of 2.7 hours, longer than that of the parent molecule. Because of the longer half life and concomitant longer time present in the body, the product may be responsible for some of the toxic side effects seen with higher doses of AZT. See P.M. Stagg et al., Clin. Pharmacol. Ther. 51 , 668-676 (1993).

An effective and safe male contraceptive remains an elusive goal. Although numerous approaches for achieving male contraception have been tried, no known method is without its significant disadvantages in efficacy, risk and/or compliance. The development of compounds that would achieve a safe means of male-based birth control is desirable.

Several distinct 5'-nucleotidase genes in humans and other species are known. The enzymatic reaction catalyzed by this family of enzymes is as follows:

Nucleoside monophosphates → nucleosides . + phosphate

The term "nucleosides," as used herein, encompasses natural components of the cell as well as various nucleoside analogues that are used in clinic to treat cancer and viral infections. Three 5'-nucleotidases have been cloned: ecto-5'-nucleotidase (e-N), cytosolic 5 -nucleotidase form II (cN-ll), and cytosolic 5'-nucleotidase form I (cN-l). Each of the three δ'-nucleotidases appears to have distinct cellular functions. Ecto-5'-nucleotidase is localized to the cellular membrane, dephosphorylates extracellular nucleoside monophosphates, and is involved in the generation of extracellular adenosine.

In comparison to e-N, the nucleotidases cN-l and cN-ll are cytosolic proteins. cN-ll is responsible for the regulation of purine nucleotide pools such as inosine monophosphate (IMP) and guanosine 5'-monophophate (GMP). The nucleotidase cN-l has been found to be adenosine 5'- monophosphate (AMP)-selective, and has been implicated in adenosine formation during AMP breakdown. Until very recently, the molecular identity of cN-l was unknown, although the IMP/GMP-selective cN-ll and e-N had already been cloned. In 1999, it was reported that a full-length cDNA clone encoding a 40 kDa peptide identified as cN-l was isolated from pigeon heart tissue. See G. B. Sala-Newby et al., J. Biol. Chem. 274, 17789-17793 (1999). This same study found that expression of the cloned pigeon heart cN-l in COS-7 cells caused the production of adenosine during ATP breakdown. A later study with cloned pigeon heart cN-l and human cN-ll found that cN-l and cN-ll played distinctive roles in AMP and IMP breakdown, with cN-l playing a significant role in AMP breakdown to adenosine and cN-ll playing a role in the breakdown of IMP to inosine and GMP to guanosine. See G. B. Sala-Newby et al., J. Biol. Chem. 275, 11666-11671 (2000).

Heretofore, however, a human clone of cN-l had not been isolated or expressed. Moreover, the role of cN-l beyond the production of adenosine, or the clinical use of the enzyme or its inhibitors, has not been described.

Summary of the Invention

The present inventors have isolated and characterized two homologous, human cDNA clones of the enzyme cytosolic 5'-nucleotidase form cN-I, identified herein as human cN-IA and cN-IB. cN-IA has previously been shown to confer drug resistance to subjects undergoing nucleoside based anti-cancer therapy. cN-IA has high activity toward pyrimidine monophosphates and is localized in the cytoplasm; accordingly, it satisfies two major conditions that make it a prime candidate for conferring drug resistance. Thus, inhibitors of cN-IA are useful in adjunct treatments with antineoplastic compounds in the treatment of cancer. By increasing endogenous adenosine, cN-IA may also have tumor-promoting functions. Accordingly, in addition to being useful in adjunct therapies to existing anti- cancer treatments, inhibitors of cN-IA are useful in the treatment of cancer by decreasing the generation of endogenous adenosine in tumor cells. Furthermore, the expression of cN-IA has been found to increase the toxicity of AZT in target cells. cN-IA inhibitors are thus useful in decreasing the toxicity of AZT, which is advantageous in the treatment of viral disorders such as HIV infection and AIDS.

The gene identified herein as cN-IB is newly cloned and appears to have an approximately 73% homology with cN-IA at the nucleotide sequence level, and an approximately 80% homology with cN-IA at the protein level. cN-IB is expressed exclusively in testis and has been previously linked with autoimmune-related infertility in humans. This suggests that cN-IB activity is important during spermatogenesis. Inhibitors for cN-IB may thus provide new pharmacological means for male-targeted birth control.

Accordingly, a first aspect of the invention is an isolated polynucleotide encoding human cytosolic 5 -nucleotidase form cN-IA. In a preferred embodiment, the sequence of the isolated polynucleotide is the cDNA sequence set forth herein as SEQ ID NO: 1.

A second aspect of the invention is an isolated polypeptide identified as the human cytosolic 5'-nucleotidase form cN-IA. In a preferred embodiment, the sequence of the isolated polypeptide has the amino acid sequence set forth herein as SEQ ID NO: 2.

A third aspect of the invention is a method of identifying inhibitors of human cytosolic 5'-nucleotidase form cN-IA (such inhibitors being referred to herein as "active compounds.") Such inhibitors may be cN-IA antagonists, antibodies, antisense oligonucleotides and other compounds that decrease the expression or activity of cN-IA. The inhibitors themselves are also an aspect of the invention.

A fourth aspect of the present invention is a method of decreasing the side effects of the drug AZT and its analogs and salts in a subject in need of such treatment by administering to the subject a side-effect inhibiting amount of a cN-IA inhibitor.

A fifth aspect is a method of increasing the efficacy of a cytotoxic or anti-neoplastic drug in a subject in need of such treatment by administering to the subject an efficacy-increasing amount of a cN-IA inhibitor in conjunction with the cytotoxic or antineoplastic.

A sixth aspect of the invention is a pharmaceutical formulation comprising a cN-IA inhibitor in a pharmaceutically acceptable carrier.

A seventh aspect of the present invention is the use of an active compound as described above for the preparation of a medicament for the reduction of the side effects of AZT or for the increase of the efficacy of antineoplastic drugs.

An eighth first aspect of the invention is an isolated polynucleotide encoding cytosolic 5'-nucleotidase form cN-IB. In a preferred embodiment, the sequence of the isolated polynucleotide is the cDNA sequence set forth herein as SEQ ID NO: 3.

A ninth aspect of the invention is an isolated polypeptide identified as the cytosolic 5'-nucleotidase form cN-IB. In a preferred embodiment, the sequence of the isolated polypeptide has the amino acid sequence set forth herein as SEQ ID NO: 4.

A tenth aspect of the invention is a method of identifying inhibitors of cytosolic 5'-nucleotidase form cN-IB (such inhibitors also being referred to herein as "active compounds.") Such inhibitors may be cN-IB antagonists, antibodies, antisense oligonucleotides and other compounds that decrease the expression or activity of cN-IB. The inhibitors themselves are also an aspect of the invention.

An eleventh aspect of the invention is a pharmaceutical formulation comprising a cN-IB inhibitor in a pharmaceutically acceptable carrier.

The human cDNA clone of the enzyme cytosolic 5'-nucleotidase form cN-IA is an important new drug target in a broad range of human malignancies. Development of specific inhibitors of this enzyme may increase the therapeutic efficacy known drugs presently used to treat cancer, as well as decrease the undesirable side effects of AZT. The cDNA clone of the enzyme cytosolic 5'-nucIeotidase form cN-IB is also an important new drug target.

The foregoing and other aspects of the present invention are explained in detail in the specification set forth below.

Brief Description of the Drawings FIG. 1 is a graphical illustration of the genomic sequence and structure of the human cN-IA gene. The exon/intron structure and exon sequences that code for a full-length cN-IA protein are shown. The ATG start codon is indicated in bold and is double underlined. The TAG stop codon is in indicated in bold and single underlined.

FIG. 2 provides the nucleotide sequence of the cDNA of the human cN- IA clone set forth herein as SEQ ID NO:1.

FIG. 3 provides the sequence of the human cN-IA polypeptide set forth herein as SEQ ID NO:2.

FIG. 4A is a graphical illustration of the effect of the enforced expression of human cN-IA in HEK-293 cells treated with the anti-cancer drug Cladrabine (CdA). The concentration of CdA is plotted on the x-axis; the percentage of surviving cells is plotted on the y-axis. Data from cells in which cN-IA was expressed are represented by the triangle data points (red curve), while data from control cells are represented by the square (blue) data points and the circle (green) data points.

FIG. 4B is a graphical illustration of the effect of the enforced expression of human cN-IA in Jurkat cells treated with the anti-cancer drug Cladrabine (CdA). The concentration of CdA is plotted on the x-axis; the percentage of surviving cells is plotted on the y-axis. Data from cells in which human cN-IA was expressed are represented by the triangle data points (red curve), while data from control cells are represented by the square (blue) data points and the circle (green) data points. FIG. 5A is a graphical illustration of the effect of the enforced expression of human cN-IA in HEK-293 cells treated with AZT. The concentration of CdA is plotted on the x-axis; the percentage of surviving cells is plotted on the y-axis. Data from cells in which cN-IA was expressed are represented by the triangle data points (red curve), while control cells are represented by the square (blue) data points and the circle (green) data points.

FIG. 5B is a graphical illustration of the effect of the enforced expression of human cN-IA in Jurkat cells treated with AZT. The concentration of CdA is plotted on the x-axis; the percentage of surviving cells is plotted on the y-axis. Data from cells in which cN-IA was expressed are represented by the triangle data points (red curve), while data from control cells are represented by the square (blue) data points and the circle (green) data points. FIGS. 6A and 6B in combination provide the nucleotide sequence of the cDNA of the cN-IB clone set forth herein as SEQ ID NO:3.

FIG. 7 provides the sequence of the cN-IB polypeptide set forth herein as SEQ ID NO:4.

Detailed Description of the Preferred Embodiments

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by. reference in their entirety.

Except as otherwise indicated, standard methods may be used for the production of cloned genes, expression cassettes, vectors (e.g., plasmids), proteins and protein fragments according to the present invention. Such techniques are known to those skilled in the art (see e.g., Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, NY 1989); F.M. Ausubel et al, eds., Current Protocols In Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, 1991).

Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right. Nucleotides and amino acids are represented herein by three letter code, in accordance with 37 CFR §1.822 and established usage. See, e.g., Patentln User Manual, 99- 102 (Nov. 1990) (U.S. Patent and Trademark Office). Alternatively, amino acids are represented by the one letter code commonly used by those skilled in the art as follows:

A. DEFINITIONS

The terms "cN-l" and "cN-l nucleotidases of the present invention," when used without indication of the subtype (i.e., cN-IA or cN-IB), refer to both cN-IA and cN-IB. For example, a technique, method or activity described in terms of "cN-l" is a technique, method or activity that is equally applicable to cN-IA and cN-IB.

An "allele" or "allelic sequence," as used herein, is an alternative form of the gene encoding cN-IA or cN-IB. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide . bonds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein, means both naturally occurring and synthetic amino acids. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations. Chemical blocking groups or other chemical substituents may also be added.

"Amino acid sequence," as used herein, refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Fragments of human cN-IA or cN- IB preferably retain the biological activity or the immunological activity of cN- IA or cN-IB, respectively. Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence, and like terms, the term is not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

"Amplification", as used herein, refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

As used herein, the term "antibody" refers to intact molecules as well as fragments thereof, such as Fa, F(ab^')2, and Fc, which are capable of ^" binding an epitopic determinant. Antibodies that bind human cN-IA and/or cN- IB polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal can be derived from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).

The term "antigenic determinant," or "epitopic determinant," as used herein, refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three- dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

The term "antisense", as used herein, refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand.

The terms "complementary" or "complementarity," as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A." Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

A "deletion," as used herein, refers to a change in the amino acid or nucleotide sequence and results in the absence of one or more amino acid residues or nucleotides.

The term "nucleic acid derivative," as used herein, refers to the chemical modification of a nucleic acid encoding or complementary to cN-IA or the encoded cN-IA, or alternatively to a chemical modification of a nucleic acid encoding or complementary to cN-IB or the encoded cN-IB. Such modifications include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative encodes a polypeptide which retains the biological or immunological function of the natural molecule. A derivative polypeptide is one which is modified by glycosylation, pegylation, or any similar process which retains the biological or immunological function of the polypeptide from which it was derived.

The term "homology", as used herein, refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity). In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence. The term "hybridization", as used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing. The term "hybridization complex", as used herein, refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).

An "insertion" or "addition", as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occurring molecule.

By "nucleic acid' or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of . the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al., Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger, et al., J. Am. Chem. Soc, 110:4470 (1988); and Pauwels, et al., Chemica Scripta, 26:141 (1986)); phosphorothioate (Mag, et al., Nucleic Acids Res., 19:1437 (1991); and U.S.

Patent No. 5,644,048); phosphorodithioate (Briu, et al., J. Am. Chem. Soc,

111 :2321 (1989)); O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc, 114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207 (1996), all of which are incorporated by reference)). Other analog nucleic acids include those with positive backbones (Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic backbones (see U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 , and 4,469,863); Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991); Letsinger, et al., J. Am. Chem. Soc, 110:4470 (1988); Letsinger, et al., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research," (eds. Y.S. Sanghui and P. Dan Cook); Mesmaeker, et al., Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J. Biomolecular NMR, 34:17 (1994); Tetrahedron Lett, 37:743 (1996)) and non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins, et al., Chem. Soc. Rev., (1995) pp. 169-176). These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. The nucleic acids may be single . stranded or double stranded; as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.

"Nucleic acid sequence" and "polynucleotide" are used interchangeably herein to refer to an oligonucleotide, nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.

The term "oligonucleotide" refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides, which can be used in PCR amplification or a hybridization assay, or a microarray. As used herein, oligonucleotide is substantially equivalent to the terms "oligomers", and "probes," as commonly defined in the art.

The terms "stringent conditions" or "stringency", as used herein, refer to the conditions for hybridization as defined by the nucleic acid, salt, and temperature. These conditions are well known in the art and may be altered in order to identify or detect identical or related polynucleotide sequences. Numerous equivalent conditions comprising either low or high stringency depend on factors such as the length and nature of the sequence (DNA, RNA, base composition), nature of the target (DNA, RNA, base composition), milieu (in solution ^"or immobilized on a solid substrate), concentration of salts and other components (e.g., formamide, dextran sulfate and/or polyethylene glycol), and temperature of the reactions (within a range from about 5° C. below the melting temperature of the probe to about 20° C. to 25° C. below the melting temperature). One or more factors may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.

A "substitution", as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

"Transformation," or "transfection" as defined and used interchangeably herein, describes a process by which exogenous DNA enters and changes a . recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, microinjection, CaCI₂-mediated uptake of nucleic acid into a cell, heat shock, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted . DNA or RNA for limited periods of time.

B. ISOLATED HUMAN cN-IA AND cN-IB AND POLYNUCLEOTIDES ENCODING THE SAME; VECTORS AND PROTEIN EXPRESSION

Human cN-IA, as used herein, refers to the amino acid sequences of substantially purified human cN-IA, and may be natural, synthetic, semi- synthetic, or recombinant. In a preferred embodiment, the human cN-IA of the present invention has the amino acid sequence set forth herein as SEQ ID NO:2 (see FIG. 3). Human cN-IA of the present invention may be encoded by an isolated polynucleotide, a preferred embodiment of which is cDNA with the nucleotide sequence set forth herein as SEQ ID NO:1.

Polynucleotides of the present invention also include those coding for proteins homologous to, and having essentially the same biological properties as, the proteins disclosed herein, and particularly the DNA disclosed herein as SEQ ID NO:3 and encoding the polypeptide cN-IB provided herein as SEQ ID NO:4. This definition is intended to encompass natural allelic sequences thereof. Thus, polynucleotides that hybridize to DNA disclosed herein as SEQ ID NO:3 (or fragments or derivatives thereof which serve as hybridization probes as discussed below) and which code on expression for a protein of the present invention (e.g., a protein according to SEQ ID NO:4), are also an aspect of the invention. cN-IB, as used herein, refers to the amino acid sequences of substantially purified cN-IB, and may be natural, synthetic, semi-synthetic, or recombinant. In a preferred embodiment, the cN-IB of the present invention has the amino acid sequence set forth herein as SEQ ID NO:4 (see FIG. 7). cN-IB of the present invention may be encoded by an isolated polynucleotide, a preferred embodiment of which is cDNA with the nucleotide sequence set forth herein as SEQ ID NO:3.

Polynucleotides of the present invention include those coding for proteins homologous to, and having essentially the same biological properties as, the proteins disclosed herein, and particularly the DNA disclosed herein as SEQ ID NO:3 and encoding the polypeptide cN-IB provided herein as SEQ ID NO:4. This definition is intended to encompass natural allelic sequences thereof. Thus, polynucleotides that hybridize to DNA disclosed herein as SEQ ID NO:3 (or fragments or derivatives thereof which serve as hybridization probes as discussed below) and which code on expression for a protein of the present invention (e.g., a protein according to SEQ ID NO:4), are also an aspect of the invention.

Conditions which will permit other polynucleotides that code on expression for a protein of the present invention to hybridize to the DNA of SEQ ID NO:1 or SEQ ID NO: 3 disclosed herein can be determined in accordance with known techniques. For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% formamide with 5x Denhardt's solution, 0.5% SDS and 1x SSPE at 37°C; conditions represented by a wash stringency of 40-45% formamide with 5x Denhardt's solution, 0.5% SDS, and 1x SSPE at 42°C; and conditions represented by a wash stringency of 50% formamide with 5x Denhardt's solution, 0.5% SDS and 1x SSPE at 42°C, respectively) to DNA of SEQ ID NO:1 or SEQ ID NO:3 disclosed herein in a standard hybridization assay. In general, sequences which code for proteins of the present invention and which hybridize to the DNA of SEQ ID NO:1 or SEQ ID NO:3 disclosed herein will be at least 75% homologous, 85% homologous, and even 95% homologous or more with SEQ ID NO:1 or SEQ ID NO:3, respectively. Further, polynucleotides that code for proteins of the present invention, or polynucleotides that hybridize to that as SEQ ID NO:1 or SEQ ID NO:3, but which differ in codon sequence from SEQ ID NO:1 or SEQ ID NO:3 due to the degeneracy of the genetic code, are also an aspect of this invention. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same protein or peptide, is well known in the literature. See, e.g., U.S. Patent No. 4,757,006 to Toole et al. at Col. 2, Table

1.

Although nucleotide sequences which encode human cN-IA and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring human cN-IA under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding cN-IA or its derivatives possessing a substantially different codon usage. The same is true for nucleotide sequences that encode cN-IB. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding human cN-IA and its derivatives or cN-IB and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences, or fragments thereof, which encode human cN-IA and its derivatives, and/or which encode cN-IB and its derivatives, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding human cN-IA or cN-IB any fragment thereof.

The nucleotide sequences as disclosed herein in SEQ ID NO:1 and SEQ ID NO:3 can be used to generate hybridization probes which specifically bind to the polynucleotide (i.e., cDNA) of the present invention or to mRNA to determine the presence of amplification or overexpression of the proteins of the present invention.

The production of cloned genes, recombinant DNA, vectors, transformed host cells, proteins and protein fragments by genetic engineering is well known. See, e.g., U.S. Patent No. 4,761 ,371 to Bell et al. at Col. 6 line 3 to Col. 9 line 65; U.S. Patent No. 4,877,729 to Clark et al. at Col. 4 line 38 to Col. 7 line 6; U.S. Patent No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line 12; and U.S. Patent No. 4,879,224 to Wallner at Col. 6 line 8 to Col. 8 line 59. (Applicant specifically intends that the disclosure of all patent references cited herein be incorporated herein in their entirety by reference). A vector is a replicable nucleic acid (preferably, DNA) construct. Vectors may be used herein either to amplify DNA encoding the proteins of the present invention or to express the proteins of the present invention. An expression vector is a replicable DNA construct in which a DNA sequence encoding the proteins of the present invention is operably linked to suitable control sequences capable of effecting the expression of proteins of the present invention in a suitable host. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants.

Vectors but are not limited to plasmids, viruses (e.g., adenovirus, cytomegalovirus), phage, retroviruses and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination). The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. Expression vectors preferably contain a promoter and RNA binding sites which are operably linked to the gene to be expressed and are operable in the host organism.

DNA regions are operably linked or operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase. Transformed host cells are cells which have been transformed or transfected with vectors containing polynucleotides coding for cN-l of the present invention need not, but preferably do, express cN-l. Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic organism cells. Cultures of cells derived from multi-cellular organisms are a desirable host for recombinant protein synthesis. In principal, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, including insect cells. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture (Academic Press, Kruse and Patterson, eds.) (1973). Examples of useful host cell lines are VERO and HeLa cells, Jurkat cells, HEK-293 cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with a ribbsome binding site, RNA splice site (if intron-containing genomic DNA is used), a polyadenylation site, and a transcriptional termination sequence.

The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Patent No. 4,599,308. The early and late promoters are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. See Fiers et al., Nature 273, 113 (1978). Further, the protein promoter, control and/or signal sequences, may also be used, provided such control sequences are compatible with the host cell chosen. An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculorivus expression vector (e.g., vectors derived from Autographa calif ornica MNPV, Trichoplusia n/ MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed to make proteins useful in carrying out the present invention, as described in U.S.

Patents Nos. 4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus expression vector comprises a baculovirus genome containing the gene to be expressed inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding cN-IA and/or cN-IB may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing human cN-IA in infected host cells (Logan, J. and Shenk, T. (1984) Proc Natl. Acad. Sci. 81,3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and the chimeric protein DNA. An example of a suitable selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase. See U.S. Pat. No. 4,399,216. Such markers are proteins, generally enzymes, that enable the identification of transformant cells, i.e., cells which are competent to take up exogenous DNA. Generally, identification is by survival or transformants in culture medium that is toxic, or from which the cells cannot obtain critical nutrition without having taken up the marker protein.

Prokaryote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. Coli) or Bacilli. Higher eukaryotic • cells include established cell lines of mammalian origin as described below. Exemplary host cells are £. Coli W3110 (ATCC 27,325), E. Coli B, £. Coli X1776 (ATCC 31 ,537), E Coli 294 (ATCC 31 ,446). A broad variety of suitable prokaryotic and microbial vectors are available. E. Coli is typically transformed using pBR322. See Bolivar et al., Gene 2, 95 (1977). Promoters most commonly used in recombinant microbial expression vectors include the beta-laciamase (penicillinase) and lactose promoter systems (Chang et al.,

Nature 275, 615 (1978); and Goeddel et al., Nature 281 , 544 (1979), a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057

(1980) and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983). The promoter and Shine- Dalgamo sequence (for prokaryotic host expression) are operably linked to the DNA of the present invention, i.e., they are positioned so as to promote transcription of the messenger RNA from the DNA.

Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host. Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (1978); and Goeddel et al., Nature 281, 544 (1979), a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983). While these are commonly used, other microbial promoters are suitable. Details concerning nucleotide sequences of many have been published, enabling a skilled worker to operably ligate them to DNA encoding the protein in plasmid or viral vectors (Siebenlist et al., Cell 20, 269 (1980). The promoter- and Shine-Dalgamo sequence (for prokaryotic host expression) are operably linked to the DNA encoding the desired protein, i.e., they are positioned so as to promote transcription of the protein messenger RNA from the DNA.

Eukaryotic microbes such as yeast cultures may be also transformed with suitable human cN-IA encoding vectors. See e.g., U.S. Patent No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or anautonomously replicating sequence (ARS), a promoter, DNA encoding the desired protein, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 (1980). This plasmid contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phospho-glycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17 , 4900 (1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.

In general, those skilled in the art will appreciate that minor deletions or substitutions may be made to the amino acid sequences of peptides of the present invention without unduly adversely affecting the activity thereof. Thus, peptides containing such deletions or substitutions are a further aspect of the present invention. In peptides containing substitutions or replacements of amino acids, one or more amino acids of a peptide sequence may be replaced by one or more other amino acids wherein such replacement does not affect the function of that sequence. Such changes can be guided by known similarities between amino acids in physical features such as charge density, hydrophobicity/hydrophilicity, size and configuration, so that amino acids are substituted with other amino acids having essentially the same functional properties. For example: Ala may be replaced with Val or Ser; Val may be replaced with Ala, Leu, Met, or lie, preferably Ala or Leu; Leu may be replaced with Ala, Val or lie, preferably Val or lie; Gly may be replaced with Pro or Cys, preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replaced with Pro or Cys, preferably Cys; His may be replaced with Phe or Gin, preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferably His or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe or Trp; Trp may be replaced with Phe or Tyr, preferably

Tyr; Asn may be replaced with Gin or Ser, preferably Gin; Gin may be replaced with His, Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced with Gin, Thr, Pro, Cys or Ala; Thr may be replaced with Gin or Ser, preferably Ser; Lys may be replaced with Gin or Arg; Arg may be replaced with Lys, Asp or Glu, preferably Lys or Asp; Asp may be replaced with Lys, Arg, or Glu, preferably Arg or Glu; and Glu may be replaced with Arg or Asp, preferably Asp. Once made, changes can be routinely screened to determine their effects on function with enzymes. As noted above, the present invention provides isolated and purified cN-IA proteins and cN-IB proteins, such as mammalian (or more preferably human) cN-IA and cN-IB. Such proteins can be purified from host cells which express the same, in accordance with known techniques, or even manufactured synthetically. Nucleic acids of the present invention, constructs containing the same and host cells that express the encoded proteins are useful for making proteins of the present invention.

Specific initiation signals may also be used to achieve more efficient translation of sequences encoding human cN-IA and/or cN-IB. Such signals . include the ATG initiation codon and adjacent sequences. In cases where sequences encoding cN-IA, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. The same is true for cN-IB. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature. See e.g., D. Scharf et al., Results Probl. Cell Differ. 20,125-162 (1994).

In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a

"prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure the correct modification and processing of the foreign protein. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express cN-IA and/or cN-IB may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for one to two days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55,121-131).

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a cN-l nucleotidase is inserted within a marker gene sequence, transformed cells containing sequences encoding the cN-l can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a cN-l nucleotidase under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

Alternatively, host cells that contain the nucleic acid sequences encoding the cN-l nucleotidases of the present invention and express the same may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA- RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.

The presence of polynucleotide sequences encoding cN-l nucleotidases of the present invention can be detected by DNA-DNA or DNA- RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding the cN-l nucleotidases. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding cN-l nucleotidases to detect transformants containing DNA or RNA encoding the cN-l nucleotidases.

A variety of protocols for detecting and measuring the expression of cN-l nucleotidases of the present invention, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal- based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on the cN-l is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158, 1211-1216).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding human cN-IA include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding human cN-IA, or any fragments thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as 77, 73, or SP6 and labeled nucleotides. 7hese procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio)). Suitable reporter molecules or labels, which may be used for ease of detection, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic compounds as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Host cells transformed with nucleotide sequences encoding cN-l nucleotidases of the present invention may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. 7he . protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode cN-l nucleotidases of the present invention may be designed to contain signal sequences which direct secretion of the cN-l through a prokaryotic or eukaryotic cell membrane. Other constructions may be used to join sequences encoding a cN-l to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and cN-l may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a cN-l nucleotidase and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3, 263-281) while the enterokinase cleavage site provides a means for purifying cN-l nucleotidases from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of cN-l nucleotidases of the present invention may be produced by direct peptide synthesis using solid-phase techniques (Merrifield J., (1963) J. Am. Chem. Soc. 85, 2149- 2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments of human cN-IA may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

C. INHIBITORS OF HUMAN cN-IA, cN-IB AND METHODS OF IDENTIFYING AND MAKING THE SAME Although not wishing to be bound to any particular theory of the invention, the inventors have determined that an undesirable function of cN-IA is based in its ability to remove active monophosphate derivatives of cytotoxic or antineoplastic drugs, thus decreasing their clinical efficacy. Correspondingly, drug resistance developed in the course of neoplastic disease (i.e., cancer) may involve increased activity of cN-IA, with the likely natural function of this enzyme being the control the levels of certain deoxynucleotide pools used in DNA synthesis.

Furthermore, while again, while not wishing to be bound to any particular theory of the invention, a different mechanism appears to play a role in the interaction of cN-IA and antiviral drugs such as AZT. In this interaction, jthe expression of cN-IA unexpectedly causes increased toxicity of AZT. It appears that AZT in its non-phosphorylated form, the level of which is which is increased by cN-IA, may have unrecognized toxic side effects that are unrelated to its primary anti-HIV function. Additionally, increased expression of human cN-IA may decrease the efficacy of anti-HIV potential of AZT.

Accordingly, inhibitors of human cN-IA are useful in decreasing the toxic side effects and increasing the efficacy of AZT administration.

The newly cloned cN-IB is expressed exclusively in testis and has been previously linked with autoimmune-related infertility in humans. This suggests that cN-IB activity is important during spermatogenesis. Development of inhibitors for cN-IB may provide new pharmacological means for male- targeted birth control.

By the term "inhibitor," is meant any compound or molecule that interferes with, or decreases, or eliminates, or reduces or retards the activity of a cN-l nucleotidase of the present invention. Accordingly, an inhibitor may be a compound or molecule that inhibits the expression of the gene encoding human cN-IA and/or a gene encoding cN-IB (i.e., a compound or molecule that interacts with the gene or polynucleotide itself, thus inhibiting translation or translation of the gene). Alternatively, the inhibitor may be a compound or molecule that interacts with the protein itself, in such a way as to inhibit the protein's activity. Accordingly, an inhibitor of a cN-l nucleotidase may be a nucleoside or analog or derivative thereof, a nucleotide or analog or derivative thereof, an antisense oligonucleotide, an antibody, or any other compound of molecule that inhibits the activity of the cN-l nucleotidase. Certain inhibitors of cN-IA are known. For example, substrate and product specificity studies were used to develop inhibitors of cN-IA from rabbit myocardium. In one particular study, pyrimidine nucleotide and pyrimidine nucleoside analogs were developed as inhibitors. In particular, phosphonate analogs of thymidine and thymidine monophosphate (TMP) and its analogs, such as the 5'-phosphonate of 3'-deoxythmidine (ddT), were found to be particularly efficient inhibitors of cN-IA. Additionally, pyrimidine nucleoside analogs were good inhibitors of cN-IA, in particular 5-ethynyl-2',3'- dideoxyuridine (5-ethynyl-ddU) and substituted forms thereof. Yet another known inhibitor of cN-IA is thymidine 5'-phosphonate (TCP). In general, 2', 3'- dideoxyribonucleosides (e.g., ddT, ddU and ddC are potent inhibitors of cN- IA, and more potent than their analogous 2',3'-deoxyribonucleoside compounds. See E.P. Garvey et al., Biochemistry 25, 9043-9051 (1998); E. P. Garvey et al., Arch. Biochem. Biophys. 364, 235-240 (1999), the disclosures of which are incorporated herein by reference in their entirety. The cN-l nucleotidases of the present invention are useful as immunogens for making antibodies as described herein, and these antibodies and proteins provide "specific binding pairs." Such specific binding pairs are useful as components of a variety of immunoassays and purification techniques, as is known in the art. The protein of the present invention is of known amino acid sequence as disclosed herein, and hence is useful as a molecular weight markers in determining the molecular weights of proteins of unknown structure.

Antibodies that specifically bind to the proteins of the present invention (i.e., antibodies which bind to a single antigenic site or epitope on the proteins) may be useful for a variety of purposes. Antibodies to cN-l may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with a cN-l nucleotidase of the present invention or any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette- Guerin) and Corynebacterium parvum are especially preferable. It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to cN-l nucleotidases have an amino acid sequence consisting of at least five amino acids and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of cN-l . amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.

Monoclonal antibodies to cN-IA may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.

See, e.g., Kohler, G. et al. (1975) Nature, 256, 495-497; Kozbor, D. et al.

(1985) J. Immunol. Methods 81, 31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 62,109-

120.

In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81, 6851-

6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; Takeda, S. et al.

(1985) Nature 314, 452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce cN-IA-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton D.

R. (1991) Proc. Natl. Acad. Sci. 88,11120-3).

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. See, e.g.,

Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci, 86, 3833-3837; Winter, G. et al.

(1991) Nature 349,:293-299.

Antibody fragments which contain specific binding sites for a cN-l nucleotidase of the present invention may also be generated. For example, such fragments include, but are not limited to, the F(ab^')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the

F(ab^')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. See Huse, W. D. et al. (1989) Science 254,1275-1281.

Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between cN-IA and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering cN-IA epitopes is preferred, but a competitive binding assay may also be employed.

Antibodies may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies may likewise be conjugated to detectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵l, ³¹l), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.

Antisense oligonucleotides to polynucleotides encoding human cN-IA and nucleic acids that express the same may be made in accordance with conventional techniques. See, e.g., U.S. Patent No. 5,023,243 to Tullis; U.S. Patent No. 5,149,797 to Pederson et al. The length of the antisense oligonucleotide (i.e., the number of nucleotides therein) is not critical so long as it binds selectively to the intended location, and can be determined in accordance with routine procedures. In general, the antisense oligonucleotide will be from 8, 10 or 12 nucleotides in length up to 20, 30, or 50 nucleotides in length. Such antisense oligonucleotides may be oligonucleotides wherein at least one, or all, or the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues may be modified as described. In another non- limiting example, such antisense oligonucleotides are oligonucleotides wherein at least one, or all, of the nucleotides contain a 2' loweralkyl moiety ^e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For example, every other one of the nucleotides may be modified as described. See also P. Furdon et al., Nucl. Acids Res. 17, 9193-9204 (1989); S. Agrawal et al., Proc. Natl. Acad. Sci. USA 87, 1401-1405 (1990); C. Baker et al.,

Nucleic Acids Res. 18, 3537-3543 (1990); B. Sproat et al., Nucleic Acids Res. 17, 3373-3386 (1989); R. Walder and J. Walder, Proc. Natl. Acad. Sci. USA 85, 5011-5015 (1988).

In one embodiment of the present invention, the cN-l proteins, nucleic acids, variants, modified proteins, cells and/or transgenics containing the cN-l nucleic acids or proteins are used in screening assays to identify inhibitors of the cN-l. Identification of the cN-l nucleotidases provided herein permits the design of drug screening assays for compounds that bind or interfere with the binding to the cN-l protein and for compounds which modulate cN-l activity. In one embodiment of the invention, these methods comprise combining the cN-l protein and a candidate bioactive compound, and determining the binding of the candidate compound to the cN-l protein. In other embodiments, further discussed below, binding interference or bioactivity is determined. The terms "candidate bioactive compound" or "candidate compound" as used herein describe any molecule, e.g., protein, small organic molecule, carbohydrates (including polysaccharides), polynucleotide, lipids, etc. Generally a plurality of assay mixtures are run in parallel with different compound concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection. In addition, positive controls, i.e. the use of compounds known to alter human cN-IA activity, may be used.

Candidate compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds.

Candidate compounds comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate compounds often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate compounds are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate compounds may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological compounds may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.

In a preferred embodiment, a library of different candidate bioactive compounds are used. Preferably, the library should provide a sufficiently structurally diverse population of randomized compounds to effect a probabilistically sufficient range of diversity to allow binding to a particular target. Accordingly, an interaction library should be large enough so that at least one of its members will have a structure that gives it affinity for the target. In general, a diversity of 10⁷-10⁸ different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organ-ism. Published in vitro selection techniques have also shown that a library size of 10⁷to 10⁸ is sufficient to find structures with affinity for the target. For example, a library of all combinations of a peptide 7 to 20 amino acids in length, has the potential to code for 20⁷ (10⁹) to 20²⁰. Thus, with libraries of 10⁷to 10⁸ different molecules the present methods allow a "working" subset of a theoretically complete interaction library for 7 amino acids, and a subset of shapes for the 20²⁰ library. Thus, in a preferred embodiment, at least 10⁶, preferably at least 10⁷, more preferably at least 10⁸ and most preferably at least 109 different sequences are simultaneously analyzed in the subject methods. Preferred methods maximize library size and diversity.

In one embodiment, the candidate bioactive compounds are proteins. In another preferred embodiment, the candidate bioactive compounds are naturally occurring proteins or fragments of naturally occurring proteins.

Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of prokaryotic and eukaryotic proteins may be made for screening in the systems described herein. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.

In another embodiment, the candidate bioactive compounds are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous compounds.

In yet another embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

In a preferred embodiment, the candidate bioactive compounds are nucleotides or nucleosides or the derivatives thereof, with pyrimidine nucleotides and nucleosides and analogs thereof being particularly preferred. In another preferred embodiment, the candidate bioactive compounds are organic chemical moieties, a wide variety of which are known in the art. Generally, in a preferred embodiment of the methods herein, for example for binding assays, the cN-l nucleotidase or the candidate compound is non-diffusibly bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.). The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, TEFLON^®, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included. The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block important sites on the protein when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or compound on the surface, etc. Following binding of the protein or compound, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety. Also included in this invention are screening assays wherein solid supports are not used; examples of such are described below. In a preferred embodiment, the cN-IA protein is bound to the support, and a candidate bioactive compound is added to the assay. Alternatively, the candidate compound is bound to the support and the cN-IA protein is added. Novel binding compounds include specific antibodies, non-natural binding compounds identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for compounds that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays, and the like.

The determination of the binding of the candidate bioactive compound to the cN-l protein may be done in a number of ways. In a preferred embodiment, the candidate bioactive compound is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of the cN-l proteins to a solid support, adding a labeled candidate compound (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as is known in the art.

By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures. The label can directly or indirectly provide a detectable signal.

In some embodiments, only one of the components is labeled. For example, the proteins (or proteinaceous candidate compounds) may be labeled at tyrosine positions using ¹²⁵l, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using ¹²⁵l for the proteins, for example, and a fluorophor for the candidate compounds.

In a preferred embodiment, the binding of the candidate bioactive compound is determined through the use of competitive binding assays. In jhis embodiment, the competitor is a binding moiety known to bind to the target molecule (i.e., a cN-l protein), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the bioactive compound and the binding moiety, with the binding moiety displacing the bioactive compound. This assay can be used to determine candidate compounds which interfere with binding between cN-l proteins and its biological binding partners. "Interference of binding" as used herein means that native binding of the cN-l proteins differs in the presence of the candidate compound. The binding can be eliminated or can be with a reduced affinity.

Therefore, in one embodiment, interference is caused by, for example, a conformation change, rather than direct competition for the native binding site.

In one embodiment, the candidate bioactive compound is labeled. Either the candidate bioactive compound, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4° and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

In a preferred embodiment, the. competitor is added first, followed by the candidate bioactive compound. Displacement of the competitor is an indication that the candidate bioactive compound is binding to the cN-l protein and thus is capable of binding to, and potentially modulating, the activity of the cN-l protein. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the compound. Alternatively, if the candidate bioactive compound is labeled, the presence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the bioactive compound is bound to the cN-l protein with a higher affinity. Thus, if the candidate bioactive compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate compound is capable of binding to the cN-l protein.

In a preferred embodiment, the methods comprise differential screening to identity bioactive compounds that are capable of modulating the activity of the cN-l protein. Such assays can be done with the cN-l protein or cells comprising said cN-l protein. In one embodiment, the methods comprise combining a cN-l protein and a competitor in a first sample. A second sample comprises a candidate bioactive compound, a cN-l protein and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an compound capable of binding to the cN-l protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the compound is capable of binding to the cN-l protein.

Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native cN-l protein, but cannot bind to modified cN-l protein. The structure of the cN-l protein may be modeled, and used in rational drug design to synthesize compounds that interact with that site. Drug candidates that affect cell cycle bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein. Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the compound to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled compound determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.

A variety of other reagents may be included in the screening assays.

These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial compounds, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding. Screening for compounds that modulate the activity of the cN-l protein may also be done. In a preferred embodiment, methods for screening for a bioactive compound capable of modulating the activity of cN-l protein comprise the steps of adding a candidate bioactive compound to a sample of a cN-IA protein (or cells comprising a cN-l protein) and determining an alteration in the biological activity of the cN-l protein. "Modulating the activity of a cN-l protein" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment, the candidate compound should both bind to the cN-IA protein (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of cN-l protein.

Thus, in this embodiment, the methods comprise combining a cN-l protein and a candidate bioactive compound, and evaluating the effect on the bioactivity of the cN-l protein. By "cN-l protein activity" or grammatical equivalents herein is meant at least one of the cN-l protein's biological activities, including, but not limited to, the protein's ability to catalyze the generation of intracellular adenosine from adenosine monophosphate. In a preferred embodiment, the activity of the cN-l protein is decreased.

Thus, bioactive compounds that are antagonists are preferred in some embodiments. In a preferred embodiment, the invention provides methods for screening for bioactive compounds capable of modulating the activity of a cN- IA protein. The methods comprise adding a candidate bioactive compound, as defined above, to a cell comprising cN-l protein. Preferred cell types include almost any cell as described above. The cells contain a recombinant nucleic acid that encodes a cN-IA protein. In a preferred embodiment, a library of candidate compounds are tested on a plurality of cells. Detection of cN-IA activity may be done as will be appreciated by those in the art. There are a number of parameters that may be evaluated or assayed to allow the detection of alterations in cN-IA bioactivity.

The measurements can be determined wherein all of the conditions are the same for each measurement, or under various conditions, with or without bioactive compounds, etc. For example, measurements of cN-IA activity can be determined in a cell or cell population wherein a candidate bioactive compound is present and wherein the candidate bioactive compound is absent. In another example, the measurements of cN-IA activity are determined wherein the condition or environment of the cell or populations of cells differ from one another. For example, the cells may be evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological compounds including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).

By a "population of cells" or "library of cells" herein is meant at least two cells, with at least about 10³ being preferred, at least about 10⁶ being particularly preferred, and at least about 10⁸ to 10⁹ being especially preferred. The population or sample can contain a mixture of different cell types from either primary or secondary cultures although samples containing only a single cell type are preferred, for example, the sample can be from a cell line, particularly tumor cell lines. In a preferred embodiment, cells that are replicating or proliferating are used; this may allow the use of retroviral vectors for the introduction of candidate bioactive compounds. Alternatively, non- replicating cells may be used, and other vectors (such as adenovirus and lentivirus vectors) can be used. In addition, although not required, the cells are compatible with dyes and antibodies. Preferred cell types for use in the invention include, but are not limited to, mammalian cells, including animal (rodents, including mice, rats, hamsters and gerbils), primates, and human cells, particularly including tumor cells of all types, including breast, skin, lung, cervix, colon, rectal, leukemia, brain, etc.

The proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the cN-l protein can be identified. Genetic systems have been described to detect protein- protein interactions. The first work was done in yeast systems, namely the "yeast two-hybrid" system. The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene. Subsequent work was done in mammalian cells. See Fields et al., Nature 340, 245

(1989); Vasavada et al., Proc. Natl. Acad. Sci. USA 88, 10686 (1991); Fearon et al., Proc. Natl. Acad. Sci. USA 89, 7958 (1992); Dang et al., Mol. Cell. Biol. 11 , 954 (1991); Chien et al., Proc. Natl. Acad. Sci. USA 88, 9578 (1991); and U.S. Patent Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463. In general, two nucleic acids are transformed into a cell, where one is a

"bait," such as the gene encoding the cN-l protein or a portion thereof, and the other encodes a test candidate. Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed. Expression of the indicator indicates when a test candidate binds to the cN-l protein. Using the same system and the newly-identified protein, the reverse procedure can be performed; namely, a cN-l nucleotidase of the present invention provided herein can be used to identify new baits, or compounds which interact with a cN-l. Additionally, the two-hybrid system can be used wherein a test candidate is added in addition to the bait and the cN-l protein encoding nucleic acids to determine compounds which interfere with the bait.

In this way, bioactive compounds are identified. Bioactive compounds

(i.e., compounds) with pharmacological activity are those compounds that are able to enhance, inhibit or interfere with the activity of human cN-IA. The compounds having the desired pharmacological activity may be administered in a pharmaceutically acceptable carrier (i.e., a pharmaceutical formulation) to a host or subject, as described in more detail below.

D. METHODS OF USING cN-IA AND cN-IB INHIBITORS

The present invention is suitable for both medical and veterinary uses. Suitable subjects include, but are not limited to, mammalian and avian subjects. More preferred subjects are mammalian subjects such as humans, monkeys, pigs, cattle^ dogs, horses, cats, sheep, and goats. The most preferred subjects are human subjects. The present methods are preferably carried out with inhibitors of human cN-l nucleotidases, but alternatively may be carried out with inhibitors of cN-l nucleotidases originally isolated from other sources, including rabbit, rat, monkey, avian and purely synthetic sources. As set forth above and illustrated in more detail below, the expression of cN-IA decreases the efficacy of antineoplastic drugs (also referred to herein as chemotherapeutic, anticancer or cytotoxic drugs).. The expression of human cN-IA has also been found to decrease the toxic side effects of the antiviral drug AZT. Accordingly, inhibitors of cN-IA are useful in increasing the efficacy of antineoplastic drugs in methods of treating cancer, and decreasing the toxic side effects of AZT in methods of treating viral infections such as HIV infection and AIDS. Inhibitors of cN-IA are referred to herein generally as "active compounds."

Chemotherapeutic compounds, are classified into a number of diverse groups. The vast majority of these compounds act as cytotoxic drugs, and each member of a specific group is postulated to typically exert its cytotoxic effects through a similar biological mechanism. cN-IA is the first adenosine and pyrimidine-specific δ'-nucleotidase that may be directly involved in the metabolism of drugs that are structurally based on adenosine (e.g., 2-chloro- 2'-deoxyadenosine (Cladribine), ara-A , 2-fluoro-ara-AMP (Fludarabine)) or deoxycytidine (ara-C, Cytarabine). These drugs are administered to patients as nucleosides, and only after transformation into triphosphate derivatives are they cytotoxic to cancer cells.

Accordingly, one aspect of the invention is a method of increasing the efficacy of antineoplastic drugs (i.e., in the course of cancer treatment) by administering a cN-IA inhibitor in conjunction with an antineoplastic drug. Suitable antineoplastic drugs include, but are not limited to, Fludarabine, Ara- A, Cladribine, Cytarabine, Vidarabine, Ara-C, Adriamycin, 5-FU, Fluorodeoxyuridine, 5'-deoxyfluorouridine, UFT, S-1 Capecitabine, Deoxycytidine, Cytosine Arabinoside, 5-Azacytosine, Gemcitabine, 5-

Azacytosine-Arabinoside, 6-Mercaptopurine, 6-Thioguanine, Azathioprine,

Allopurinol, Pentostatin, 2-chloro-adenosine, daunorubicin, vinblastine, etoposide, methotrexate, 5-fluorouracyl, chlorambucil, cisplatin, and hydroxyurea, with Fludarabine, Cladribine, and Cytarabine being preferred. A jnore complete listing of suitable antineoplastic drugs useful in the practice of the present invention is set forth in U.S. Patent No. 5,919,816 to Hausheer et al., the disclosure of which is incorporated by reference in its entirety.

The term "cancer" as used herein is intended to encompass cancers of any origin, including both tumor-forming and non-tumor forming cancers. The term "cancer" has its understood meaning in the art, for example, an uncontrolled growth of tissue that has the potential to spread to distant sites of the body (i.e., metastasize). As used herein, the term "cancer cell" is also intended to encompass those cells referred to as "pre-cancerous," i.e., cells that contain mutated or damaged DNA or other components, which mutations or damage are likely to cause the cell to develop into a cancer cell. Exemplary cancers include osteosarcomas, angiosarcomas, fibrosarcomas and other sarcomas; leukemias (e.g., acute lymphoblastic leukemia (ALL) acute myelogenous leukemia (AML)); sinus tumors; ovarian, uretal, bladder, prostate and other genitourinary cancers; colon, esophageal and stomach cancers and other gastrointestinal cancers; lung cancers; lymphomas; myelomas; pancreatic cancers; liver cancers; breast cancers; kidney cancers; endocrine cancers; skin cancers; melanomas; angiomas; and brain or central nervous system (CNS) cancers. Tumors or cancers, as defined herein, may be any tumor or cancer, primary or secondary, which is recognized by cytotoxic cells (for example, macrophages) and which induces the tumoricidal effect of the cells upon contact. See, e.g., Alexander and Evans, Nature New Biology 232:76 (1971). The term "tumor" is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multi-cellular organism. Tumors can be malignant or benign. Preferably, the inventive methods disclosed herein are used to treat malignant tumors. The inventive methods can be used to treat both the primary cancer and to prevent metastasis.

By the terms "treating cancer" or "treatment of cancer," it is intended that the severity of the cancer is reduced or the cancer is partially or entirely eliminated, or that tumor size is reduced or that the tumor is partially or entirely eliminated, as compared to that which would occur in the absence of treatment. Alternatively, these terms are intended to mean that metastasis of the cancer is reduced or eliminated, as compared to that which would occur in Jhe absence of treatment. The term "treating cancer" may also mean that the rate of cell proliferation is decreased, as compared to that which would occur in the absence of treatment.

Active compounds of the present method are also useful for reducing (decreasing, ameliorating, etc.) the toxic side effects associated with the administration of AZT (also referred to as azidothymidine or zidovudine). Such side effects include anemia, granulocytopenia, mypathy, lactic acidosis, hepatomegaly, headache, nausea, diarrhea and other undesirable conditions. Accordingly, one aspect of the present invention is a method of reducing the toxic side effects of AZT in a subject in need to such treatment by administering AZT in conjunction with an inhibitor of cN-IA.

AZT is commercially available as a powdered form of 3^'-azido-3'- deoxythymidine. Methods of treating humans and animals afflicted with AIDS, HIV infections and other viral infections with AZT and its analogs and salts are set forth in U.S. Patent Nos. 4,724,232; 4,874,609; 5,643,891 and 5,885,957, all to Rideout et al., the disclosures of which are incorporated herein by reference in their entirety. As used herein, the term "AZT" includes not only the pure form of the drug, but also the phosphorylated forms, analogs, derivatives, and salts thereof. The present method is useful when AZT is used in the treatment of any disorder or viral infection in which AZT is indicated as a therapeutic, and is particularly useful in the treatment of HIV infection or AIDS.

As used herein, the term "in conjunction with" means sufficiently close in time in administration such that the active compound (the inhibitor of cN-IA) has the desired effect on either the antineoplastic agent or the AZT. That is, the active compound is administered sufficiently close in time such that the active compound is increases the efficacy of the antineoplastic drug, or decreases the toxicity of the AZT. The active compound and drug may this be administered concurrently, (simultaneously), or may be administered as two or more events occurring within a short time period before or after each other (sequentially).

Simultaneous administration may be carried out by mixing or otherwise combining the active compound and the drug prior to administration, or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration. The nucleotidase cN-IB has been linked to autoimmune-related infertility in humans, and likely plays a role in spermatogenesis. Accordingly, identification of inhibitors of the protein is useful for the development of, for example, pharmacological methods of male contraception, and/or treatment (either inhibition or enhancement) of fertility in humans and other mammals.

E. PHARMACEUTICAL FORMULATIONS

Pharmaceutical formulations of the present invention comprise active compounds (i.e., cN-IA or cN-IB inhibitors) with pharmacological activity (as identified using methods of the present invention) in a pharmaceutically acceptable carrier. Suitable pharmaceutical formulations include those suitable for inhalation, oral, rectal, topical, (including buccal, sublingual, dermal, vaginal and intraocular), parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular) and transdermal administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art. The most suitable route of administration in any given case may depend upon the anatomic location of the condition being treated in the subject, the nature and severity of the condition being treated, and the particular pharmacologically active compound which is being used. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art.

In the manufacture of a medicament according to the invention (the "formulation"), pharmacologically active compounds or the physiologically acceptable salts thereof (the "active compounds") are typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.5% to 99% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which formulations may be prepared by any of the well known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory therapeutic ingredients.

Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing compound(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

Formulations for oral administration may optionally include enteric coatings known in the art to prevent degradation of the formulation in the stomach and provide release of the drug in the small intestine. Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending compounds and thickening compounds. The formulations may be presented in unit\dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising a compound of Formula (I), or a salt thereof, in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt. When the compound or salt is substantially water- insoluble, a sufficient amount of emulsifying compound which is physiologically acceptable may be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. One such useful emulsifying compound is phosphatidyl choline. .

Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture. Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof. Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g.,

Pharmaceutical Research 3, 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Further, the present invention provides liposomal formulations of the compounds disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free. When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques. . Of course, the liposomal formulations containing the pharmaceutically active compounds identified with the methods described herein may be lyophilized to produce a lyophilizate which may be reconstituted with a - pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension. Other pharmaceutical formulations may be prepared from the water- insoluble compounds disclosed herein, or salts thereof, such as aqueous base emulsions. In such an instance, the formulation will contain a sufficient amount of pharmaceutically acceptable emulsifying compound to emulsify the desired amount of the compound or salt thereof. Particularly useful emulsifying compounds include phosphatidyl cholines, and lecithin. In addition to the pharmacologically active compounds, the pharmaceutical formulations may contain other additives, such as pH- adjusting additives. In particular, useful pH-adjusting compounds include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the compositions may contain microbial preservatives.

Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use. Of course, as Indicated, the pharmaceutical formulations of the present invention may be lyophilized using techniques well known in the art.

The therapeutically effective dosage of any specific pharmacologically active compound identified by methods of the invention, the use of which compounds is in the scope of present invention, will vary somewhat from compound to compound, and subject to subject, and will depend upon the condition of the patient and the route of delivery.

The following Examples are provided to illustrate the present invention, and should not be construed as limiting thereof.

EXAMPLE 1 MTT Assay: Determination of Cell Death

The MTT assay of cell cytotoxicity was used to test the effect of the anti-cancer compound 2-chloro-deoxyadenosine (CdA) and AZT on HEK-293 (a highly transfectable cell line derived from human embryo kidney [Graham et al., J. Gen. Virol. 36,59-74, 1977)), and Jurkat cells (a T-cell leukemia cell line). This assay measures cell survival by monitoring the ability of surviving cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).

In the examples set forth below, HEK-293 and Jurkat cells are transfected with expression vectors comprising SEQ ID NO:1 (i.e., vectors - expressing human cN-IA in the cells) and control vectors (i.e., vectors not expressing human cN-IA). Cells are exposed to variable concentrations of tested drug for 24 hours, after which MTT is added at concentration of 50 ug/100 μL of culture medium. Cells are further incubated for one to four hours, pelleted and then resuspended in 100 μL DMSO to dissolve the reduced tetrazόlium salt.

The cytotoxic effect of the test drug is quantitated by measuring light absorption at 595 nm using a standard plate reader. Decreased absorption is a measure of cell death, and is plotted on the y axis of a graph as a percentage (%) of surviving cells (i.e., percentage of control) versus the increasing concentration of drug used, plotted on the x-axis.

EXAMPLE 2 Expression Of cN-IA Decreases The Efficacy Of Antineoplastic Drugs

The MTT assay described in Example 1 was performed using two nucleoside-derived drugs as test drugs: the anti-cancer drug Cladribine (CdA) and AZT, used to treat HIV infection. As shown in FIGS. 4A and 4B, enforced expression of cN-IA caused a significant decrease of cell-killing potential of CdA (Cladribine), especially in the Jurkat T-cell leukemic cell line (see FIG. 4B). This result confirms the function of this enzyme in deactivation of drugs used in clinic and suggest a potential for cN-IA inhibitors as adjunct therapies in leukemia and other cancers.

EXAMPLE 3

Expression Of cN-IA Increases The Toxicity Of AZT

Using the MTT assay of Example 1 , it was found that enforced expression of cN-IA unexpectedly caused increased toxicity in target cells. As shown in FIGS. 5A and 5B, AZT has significantly higher cytotoxicity in the presence of elevated expression of cN-IA that is especially evident for HEK- 293 cells, which are of epithelial origin (see FIG. 5A). Based on these results, it appears that the use of cN-IA inhibitors may serve as a viable option to decrease AZT toxic side effects.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. An isolated polynucleotide encoding human cN-IA, wherein the polynucleotide is selected from the group consisting of:

(a) DNA having the nucleotide sequence of SEQ ID NO:1;

(b) polynucleotides that encode human cN-IA and hybridize to DNA of (a) above under stringent conditions; and

(c) polynucleotides that encode human cN-IA and differ from the DNA of (a) or (b) above due to the degeneracy of the genetic code.

2. The isolated polynucleotide according to Claim 1 that encodes human cN-IA having the amino acid sequence of SEQ ID NO:2.

3. An expression vector comprising the isolated polynucleotide according to Claim 1.

4 A cell comprising the expression vector according to Claim 3.

5. An isolated protein encoded by a polynucleotide according to Claim 1.

6. An isolated protein encoded by a polynucleotide of Claim 1 having the amino acid sequence of SEQ ID NO:2.

7. An antibody that specifically binds to a protein encoded by the polynucleotide according to Claim 1.

8. An antibody according to Claim 7, wherein the antibody is a polyclonal antibody.

9. An antibody according to Claim 7, wherein said antibody is a monoclonal antibody.

10. An antisense oligonucleotide complementary to the polynucleotide of Claim 1 and having a length sufficient to hybridize thereto under physiological conditions and to decrease the expression of the protein encoded by the polynucleotide of Claim 1.

11. A DNA encoding a oligonucleotide of Claim 10.

12. An expression vector comprising an antisense oligonucleotide according to Claim 11.

13. A method for producing human cN-IA protein comprising:

(a) culturing a host cell containing an expression vector containing a polynucleotide sequence according to Claim 1 under conditions suitable for the expression of the protein; and

(b) recovering the protein from the host cell culture.

14. The method according to Claim 13, wherein the human cN-IA protein has the sequence SEQ ID NO: 2.

15. A method for screening for a compound capable of inhibiting the activity of human cN-IA, comprising: a) combining human cN-IA and a candidate bioactive compound; and b) determining the effect of said candidate bioactive compound on the activity of said cN-IA.

16. A method for screening for a compound capable of inhibiting the activity of human cN-IA comprising: a) adding a candidate compound to a cell comprising an expression vector encoding human cN-IA; and b) determining the effect of said candidate compound on said cell.

17. A method according to Claim 16, wherein a library of candidate compounds is added to a plurality of cells comprising an expression vector encoding human cN-IA.

18. A method of increasing the efficacy of an antineoplastic drug in a subject in need of such treatment, comprising administering to the subject the antineoplastic drug in conjunction with a cN-IA inhibitor in an efficacy- increasing amount.

19. The method of Claim 18, wherein said cN-IA inhibitor is selected from the group consisting of phosphonate analogs of thymidine monophosphate and pyrimidine nucleoside analogs.

20. The method of Claim 18, wherein the antineoplastic drug is selected from the group consisting of Fludarabine, Cladribine, and Cytarabine.

21. A method of treating cancer in a subject in need of such treatment, comprising administering to the subject an antineoplastic drug in conjunction with an inhibitor of cN-IA in an amount sufficient to increase the efficacy of the antineoplastic drug.

22. The method of Claim 21 , wherein the antineoplastic drug is selected from the group consisting of Fludarabine, Cladribine, and Cytarabine.

23. The method of Claim 21 , wherein said cN-IA inhibitor is selected from the group consisting of phosphonate analogs of thymidine monophosphate and pyrimidine nucleoside analogs..

24. A method of decreasing the toxic side effects of AZT in a subject in need of such treatment, comprising administering AZT to the subject in conjunction with an inhibitor of cN-IA in an amount sufficient to decrease the side effects of AZT.

25. The method of Claim 24, wherein said cN-IA inhibitor is selected from the group consisting of phosphonate analogs of thymidine monophosphate and pyrimidine nucleoside analogs.

26. An isolated polynucleotide encoding cN-IB, wherein the polynucleotide is selected from the -group consisting of:

(a) DNA having the nucleotide sequence of SEQ ID NO:3;

(b) polynucleotides that encode cN-IB and hybridize to DNA of (a), above under stringent conditions; and

(c) polynucleotides that encode cN-IB and differ from the DNA of (a) or (b) above due to the degeneracy of the genetic code.

27. The isolated polynucleotide according to Claim .26 that encodes cN-IB having the amino acid sequence of SEQ ID NO:4.

28. An expression vector comprising the isolated polynucleotide according to Claim 26.

29. A cell comprising the expression vector according to Claim 28.

30. An isolated protein encoded by a polynucleotide according to Claim 26.

31. An isolated protein encoded by a polynucleotide of Claim 26 having the amino acid sequence of SEQ ID NO:4.

32. An antibody that specifically binds to a protein encoded by the polynucleotide according to Claim 26.

33. An antibody according to Claim 32, wherein the antibody is a polyclonal antibody.

34. An antibody according to Claim 32, wherein said antibody is a monoclonal antibody.

35. A method for producing cN-IB protein comprising:

(a) culturing a host cell containing an expression vector containing a polynucleotide sequence according to Claim 26 under conditions suitable for the expression of the protein; and

(b) recovering the protein from the host cell culture.

36. The method according to Claim 35, wherein the cN-IB protein has the sequence SEQ ID. NO: 4.

37. A method for screening for a compound capable of inhibiting the activity of cN-IB, comprising-: a) combining cN-IB and a candidate bioactive compound; and b) determining the effect of said candidate bioactive compound on the activity of said cN-IB.

38. A method for screening for a compound capable of inhibiting the activity of cN-IB comprising: a) adding a candidate compound to a cell comprising an expression vector encoding cN-IB; and b) determining the effect of said candidate compound on said cell.

39. A method according to Claim 38, wherein a library of candidate compounds is added to a plurality of cells comprising an expression vector encoding cN-IB.