JP2002518016A - NEK-related and BUB1-related protein kinases - Google Patents

Abstract

  (57) [Summary] The present invention relates to BUB1 and NEK kinase polypeptides, nucleotide sequences encoding the kinase polypeptides, as well as various products and methods useful for the diagnosis and treatment of various kinase-related diseases and conditions. Utilizing the targeted PCR cloning method, the serine / threonine kinase of human BUB1 was identified and its protein structure was predicted. In addition, we used bioinformatic techniques to identify three mammalian members of the NEK-subfamily of STKs and predict their protein structure.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to novel kinase polypeptides, nucleotide sequences encoding the novel kinase polypeptides, and various products and methods useful for the diagnosis and treatment of various kinase-related diseases and conditions.

[0002] The following description of the background of the background the present invention invention, there is provide to aid the understanding of the present invention, also an admission that it is prior art to the present invention, describes a prior art It is not something.

Cell signaling is a fundamental mechanism by which external stimuli, which regulate various cellular processes, are relayed inside the cell. One of the important biochemical mechanisms of signal transduction is the reversible phosphorylation of proteins, which allows the activity of mature proteins to be modulated by altering their structure and function.

[0004] The best characterized protein kinases in eukaryotes phosphorylate proteins at the hydroxyl groups of serine, threonine, and tyrosine residues. These kinases are roughly divided into two groups, one is a kinase specific to phosphorylation of serine and threonine, and the other is a kinase specific to phosphorylation of tyrosine. Some kinases, called "dual-specific kinases", can phosphorylate on tyrosine with serine / threonine residues.

[0005] Protein kinases can also be characterized by their location in the cell. Some kinases are transmembrane receptor-type proteins that can directly change their catalytic activity in response to an external environment, such as ligand binding. Other kinases are non-receptor proteins that lack the transmembrane domain. They can be found in various subcellular fractions from the inner surface of the cell membrane to the nucleus.

[0006] Many kinases are involved in a regulatory cascade in which other kinases whose activities are regulated by their phosphorylation state are included in their substrates. Phosphorylation resulting from activation of such pathways ultimately modulates the activity of some downstream effectors.

[0007] Protein kinases are one of the largest families of eukaryotic proteins with hundreds of known members. These proteins share a domain of 250-300 amino acids that can be subdivided into twelve different subdomains containing a common catalytic core structure. Examination of these conserved protein motifs recently using a PCR-based cloning strategy resulted in a significant increase in the number of known kinases.

[0008] The arrangement of a large number of protein kinase catalytic domain sequences, and subsequent saving analysis, has enabled the separation of related kinases into different derivatives or subfamilies, including: tyrosine kinases, Heavy-specific kinases, and serine / threonine kinases (STK's). The latter subfamily includes cyclic nucleotide dependent kinases, calcium / calmodulin kinases, cyclin dependent kinases (CDK's), MAP kinases, serine-threonine kinase receptors, and other poorly defined subfamilies.

A cell cycle checkpoint is a regulatory pathway that controls the order and timing of cell cycle transitions, ensuring that critical events such as DNA replication and chromosome segregation are completed with a high degree of accuracy. The mitotic checkpoint ensures chromosome meiosis by delaying anaphase until all chromosomes are aligned on the spindle.

[0010] Lack of cell checkpoints renders the genome unstable, thereby affecting the transformation of normal cells into cancer cells. Cancer cells differentiate, increase invasiveness,
And different from normal cells for many important properties, including reduced drug sensitivity.

The CDK plays a central role in regulating mitosis during cell division. The cell division process occurs in four stages. S phase, G2 phase, which is the period during which the chromosome replicates; mitosis; and G1, ie, interphase. The chromosome replicated during mitosis eventually undergoes meiosis, allowing each daughter cell to inherit a complete copy of the genome.
The key mitotic regulator in all eukaryotic cells is cyclin B
Is regulated by STK cdc2, CDK.

[0012] As an overview portion of the Invention The present invention has been identified a novel serine / threonine kinases (STK) is the sequence of the protein is estimated. NEK, a mammal of the NEK family
Was isolated by bioinformatic methods, and human BUB1 kinase was isolated using targeted PCR cloning. The partial or complete sequences of these kinases are provided herein along with an assessment of their classification, putative or deduced structure, and their expression patterns.

The NEK kinase of the present invention functions in regulating the cell cycle. It is therefore implicated in cancer that these kinases cannot be regulated by overexpression and / or mutation events. Similarly, they also play a role in neurodegenerative and immune disorders through abnormal regulation of cell division, differentiation, and apoptosis. Therefore NEK
Modulators of kinases may play a role in treating or suppressing cancer or neurodegenerative or immune disorders where NEK kinase is abnormally functioning as a result of overexpression or mutational events It is.

[0014] Apart from utilizing them as clinical targets, NE in the regulation of the cell cycle
Further understanding of the role of K-kinase will likely affect issues as diverse as cell differentiation and apoptosis, and this further understanding may be as diverse as aging, nerve regeneration, and abnormal growth. It seems to affect that. Various aspects of the claims of the present invention include inhibitors, probes, antibodies, and hybridomas to the proteins or genes of the present invention, which will facilitate the above studies.

[0015] The human bub1 gene was found to be a checkpoint gene during mitosis. BUB1 biological and clinical therapeutic relevance suggests that expression analysis shows that bub1 transcripts are expressed at very slow levels in normal human tissues but are markedly overexpressed in many tumor cell lines Proven by Overexpression of the bub1 gene in cancer cells suggests that it may be an attractive target for cancer therapy.

[0016] The expression of BUB1 has been shown to regulate the cell cycle and localize to the centromere, indicating that BUB1 is a centromere protein involved in chromosome division during mitosis. Is shown. BU in addition to having the potential to play a role in tumorigenesis
B1 may also be a target for human autoimmune diseases such as CREST syndrome, which are typed by the presence of autoantibodies to the centromere protein.

Accordingly, in a first aspect of the present invention, a BUB1 kinase having an amino acid sequence represented by SEQ ID NO: 2; a NEK4a kinase having an amino acid sequence represented by SEQ ID NO: 8;
Kinase poly selected from the group consisting of NEK4b kinase having the amino acid sequence represented by SEQ ID NO: 10, NEK5 kinase having the amino acid sequence represented by SEQ ID NO: 4, and NEK6 kinase having the amino acid sequence represented by SEQ ID NO: 6 It features an isolated, enriched, or purified nucleic acid molecule encoding a peptide.

“Isolated” with respect to nucleic acids refers to six (preferably 21, more preferably 39) linked together, including DNA and RNA, isolated or synthesized from natural sources. , Most preferably 75) or more nucleotides. In certain embodiments of the invention, longer nucleic acids are preferred, e.g., 300, 6
A nucleic acid consisting of 00, 900 or more nucleotides and / or at least 50 of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 %, 60%, 75%, 90%, 95%, or 99% nucleic acid.

An isolated nucleic acid of the present invention is unique in that it is not found in pure or isolated form in nature. Use of the term "isolated" indicates that the naturally occurring sequence has been separated from its normal cellular (ie, chromosomal) environment. Thus, the sequence may be in a cell-free solution or may be in a different cellular environment. The term does not mean that the sequence is the only nucleotide strand present, but that it is essentially free of the relevant non-nucleotide material (at least about 90-95% pure). And thus are distinguished from isolated chromosomes.

The term “enrichment” as used with respect to nucleic acids, refers to a specific DNA or RNA sequence
The proportion of total DNA or RNA present in the cells or solution concerned is significantly higher (2.
５5 times). This can be done artificially, by selectively reducing the amount of other DNA or RNA present, by selectively increasing the amount of a particular DNA or RNA sequence, or by a combination of both. .
However, it should be noted that the term enriched does not mean that no other DNA or RNA sequences are present, but merely that the relative amounts of the sequences concerned are significantly increased. The term "significant" is used to indicate that the level of increase is useful to those making such an increase, and is generally at least about 2-fold, more preferably It means at least 5 to 10-fold or more increase. The term also does not mean that there is no DNA or RNA from other sources. DNA of other origin may include, for example, DNA from yeast or bacterial genomes, or cloning vectors such as pUC19. The term is distinguished from naturally occurring events, such as viral infections or the growth of tumor types, in which the level of one mRNA may be naturally increased compared to mRNA of another species. . That is, the term is meant to apply only to situations where a person intervenes to increase the proportion of the desired nucleic acid.

[0021] It is also advantageous for some purposes that the nucleotide sequence is in purified form. The term "purified" with respect to nucleic acids does not require absolute purity (like a homogeneous preparation). Instead, the sequence is meant to be relatively purer than in its natural environment (as compared to the natural level, this level must be at least 2-5 times greater, for example, in mg / ml) ). Individual clones isolated from the cDNA library may be purified to electrophoretic homogeneity. The DNA molecules claimed herein obtained from these clones contain the total D
It can be obtained directly from NA or total RNA. The cDNA is not naturally occurring, but rather is preferably obtained by manipulation of partially purified naturally occurring material (messenger RNA). Construction of a cDNA library from mRNA involves the production of synthetic material (cDNA), and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of cells having the cDNA library. Thus, cD from mRNA
By a process that involves the construction of an NA library and the isolation of different cDNA clones,
About ^106- fold purification of the original message is obtained. Thus, at least one order of magnitude of purification, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude are clearly contemplated.

The “kinase polypeptide” has 32 amino acids (SEQ ID NO: 2) described in the amino acid sequence of SEQ ID NO: 2.
Preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids, 33 (preferably 40,
More preferably 45, most preferably 55) or more contiguous amino acids,
Or 8 (preferably 15, more preferably 20, most preferably 30) or more consecutive amino acids described in the amino acid sequence of SEQ ID NO: 6, SEQ ID NO:
8 (preferably 40, more preferably 45, and most preferably 55) or more consecutive amino acids described in the amino acid sequence of SEQ ID NO: 8, 33 (preferably 40) , More preferably 45, most preferably 55) or more contiguous amino acids, or functional derivatives thereof, as described herein. In certain aspects, polypeptides consisting of 100, 200, 300 or more amino acids are preferred. The kinase polypeptide may be encoded by a full-length nucleic acid sequence, or may be encoded by any portion of the full-length nucleic acid sequence, as long as the polypeptide retains a functional activity. Good. It is well known in the art that the degeneracy of the genetic code allows so many different nucleic acid sequences to encode the same amino acid sequence. It is also well known in the art that conservative changes in the amino acid sequence can result in proteins or polypeptides that retain their original function. In each case, all substitutions are intended to be included by this disclosure.

The amino acid sequence comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or the corresponding full-length amino acid sequence or a fragment thereof. A substantially similar arrangement.

SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10
A sequence substantially similar to the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 is preferably at least 90% identical ( More preferably it has at least 95%, most preferably 99-100%).

“Identity” refers to the property of a sequence whose similarity or relatedness is to be measured.
Identity is calculated by dividing the number of identical residues by the total number of residues and gaps and dividing the resulting quotient by 100.
Is measured by multiplying by A "gap" is a space in an arrangement that is the result of an amino acid addition or deletion. Thus, two copies of the exact same sequence are
Sequences that have 100% identity and are less conserved, and that have deletions, additions, or substitutions, will have a lower degree of identity. One skilled in the art will recognize several computer programs to determine sequence identity using standard parameters, such as Gapped BLAST or PSI-BLAST (Altschul (
Altschul et al., (1990) Nucleic Acids Res. 25: 3389-3402), BLAST (Altschul et al., (1990) J. Mol. Biol. 215: 403-410), and Smith Waterman (Smith (1981) J. Mol. Biol. 147: 195-1.
97) may be available.

The present invention in a preferred embodiment features an isolated, enriched or purified nucleic acid molecule encoding a kinase polypeptide comprising the nucleotide sequence: That is, (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, (b) nucleotide A nucleotide sequence that is the complement of the sequence (a), (c)
A nucleotide sequence that hybridizes to the nucleotide molecule (a) under high stringency conditions and encodes a naturally occurring kinase polypeptide; (d)
Positions 1 to 785, 786 to 1028, or 1029 to 1085 of SEQ ID NO: 2, 1 of SEQ ID NO: 4
-31, 32-283, or 284-302, or the absence of one or more but not all of the segments of amino acid residues consisting of positions 1-22 or 23-302 of SEQ ID NO: 6. Except for the nucleotide sequence encoding the kinase polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, (e) nucleotide sequence ( d) a nucleotide sequence that is the complementary sequence, (f) positions 1 to 785, 786 to 1028, or 1 of SEQ ID NO: 2.
SEQ ID NO: 029 to 1085, SEQ ID NO: 4 derived from amino acid residue consisting of positions 1-31 to 32, 283 to 283, or 284 to 302, or SEQ ID NO: 6 derived from amino acid residue consisting of positions 1 to 22 or 23 to 302 : 2, a nucleotide sequence encoding a kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 6, (g) a nucleotide sequence that is the complement of the nucleotide sequence (f), (h) an N-terminal SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 6 except for the absence of one or more, but not all, of the domain, catalytic domain, C-terminal catalytic domain, C-terminal domain, and C-terminal tail It is a nucleotide sequence that encodes a polypeptide having the amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10, or (i) a nucleotide sequence that is a complementary sequence to the nucleotide sequence (h). Preferably, for BUB1 kinase, this domain is selected from the group consisting of an N-terminal domain, a catalytic domain, a C-terminal catalytic domain, and a C-terminal tail, and comprises NEK4a kinase, NEK4b kinase, NEK5 kinase or
For NEK6 kinase, this domain is selected from the group consisting of an N-terminal domain, a catalytic domain, a C-terminal catalytic domain, and a C-terminal domain.

The term “complement” refers to two nucleotides that can form a number of convenient interactions with one another. For example, adenine is complementary to thymine because they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary because they can form three hydrogen bonds. A nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all of the nucleotides of the second sequence.

[0028] The term "domain" refers to a region of a polypeptide that contains a particular function. For example, the N-terminal or C-terminal domain of a signaling protein may bind molecules that localize the signaling molecule to different regions of the cell, or bind other signaling molecules that are directly involved in the transmission of certain cellular signals. Functions including but not limited to these can be exhibited. While some domains are expressed away from the rest of the protein and are capable of functioning by themselves, some domains must retain an intact portion of the protein to retain function. The latter are called functional regions of the protein, which also relate to domains.

The term “N-terminal domain” refers to the extra-catalytic region located between methionine, the protein kinase starting material, and the catalytic domain. The N-terminal domain can be identified after Smith Waterman placement of the protein sequence against a non-overlapping protein database to define the N-terminal boundary of the catalytic domain.
Depending on its length, the N-terminal domain may or may not play a regulatory role in kinase function. Examples of protein kinases where the N-terminal domain has been shown to play a regulatory role include CRIB, which is used for Cdc42 and rac binding
PAK65 containing a motif (Burbelo, PD, et al., (1995) J. Biol
Chem. 270, 29071-290740). The N-terminal domain is represented by amino acid residues 1 to 785 of the sequence set forth in SEQ ID NO: 2, positions 1 to 31 of the sequence set forth in SEQ ID NO: 4, SEQ ID NO:
6 and positions 1-42 of the sequence set forth in SEQ ID NO: 8.

The term “catalytic domain” is typically 25-300 amino acids in length and may be derived from a high energy phosphate donor molecule such as ATP or GTP by itself (autophosphorylation) or other proteins. (Exogenous phosphorylation) means the region of protein kinase responsible for performing a phosphate transfer reaction. The catalytic domain of protein kinases is composed of 12 subdomains containing highly conserved amino acid residues and is responsible for the proper folding and catalysis of the polypeptide. Catalytic domains can be identified after Smith Waterman placement of the protein sequence against the non-redundant protein database. The catalytic domain is at amino acid residues 32-283 of the sequence set forth in SEQ ID NO: 4, at positions 23-302 of the sequence set forth in SEQ ID NO: 6, and at positions 43-302 of the sequence set forth in SEQ ID NO: 8. At position 294 and 1-259 of the sequence shown in SEQ ID NO: 10
In the ranks. The C-terminal catalytic domain spans amino acid residues 786 to 1028 of the sequence shown in SEQ ID NO: 2.

As used herein, the term “catalytic activity” defines the rate at which the kinase catalytic domain phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of substrate converted to phosphorylated product as a function of time. Catalytic activity can be measured by the method of the invention by keeping the time constant and determining the concentration of phosphorylated substrate after a certain period of time. Substrate phosphorylation occurs at the active site of the protein kinase. The active site is usually the hollow part where the substrate binds to the protein kinase and is phosphorylated.

As used herein, the term “substrate” refers to a molecule that is phosphorylated by the kinase of the present invention. Kinases phosphorylate substrates on serine / threonine or tyrosine amino acids. The molecule may be another protein or polypeptide.

By “C-terminal domain” is meant the region located between the catalytic domain or the last (closest to the C-terminus) functional domain of the protein kinase and the carboxyl-terminal amino acid residue. A “functional” domain is regulated by amino acid sequence homology to other proteins, or by the presence of amino acid sequences that may give rise to particular structural configurations (eg, N-terminal domains). Or any region of the polypeptide that may play a catalytic role.
The C-terminal domain can be identified using the Smith Waterman arrangement of protein sequences to a non-overlapping protein database to define the C-terminal border of the catalytic domain or of a functional C-terminal extra-catalytic domain. Depending on its length and amino acid composition, the C-terminal domain may or may not play a regulatory role in kinase function. Examples of C-terminal domain regulatory role protein kinases, including heterotrimeric G _b subunit binding site on the C-terminus near P
AK3 (Leeuw, T. et al. (1998) Nature 391, 191-195). In some of the kinases of the invention, the C-terminal domain also contains a catalytic domain (described above). The C-terminal catalytic domain is comprised of amino acid residues 786-1028 of the sequence shown in SEQ ID NO: 2.
Position; positions 284 to 302 of the sequence represented by SEQ ID NO: 4; 29 of the sequence represented by SEQ ID NO: 8
And positions 260-268 of the sequence shown in SEQ ID NO: 10.

As used herein, the term “C-terminal tail” refers to the C-terminal domain of a protein kinase that extends, by homology, beyond the C-terminal amino acid sequence of the closest homolog. . The C-terminal tail can be identified using a Smith-Waterman sequence alignment of the protein sequence against a non-overlapping protein database or by means of multiple sequence alignments of homologous sequences using the DNAStar program Megalin. Depending on the length, the C-terminal tail may or may not play a regulatory role in kinase function. The C-terminal tail is composed of amino acid residues 1029-10 of the sequence shown in SEQ ID NO: 2.
It ranks 85th.

The term “signaling pathway” refers to a molecule that transduces an extracellular signal through a cell membrane into an intracellular signal. This signal can then stimulate a cellular response. The polypeptide molecules involved in the signal transduction process are typically receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, SRC homology 2 and 3 domains, phosphotyrosine binding protein (SRC homology 2 ( SH2) and phosphotyrosine binding (PTB and PH) domain-containing proteins), proline-rich binding protein (SH3 domain-containing protein), nucleotide exchange factors, and transcription factors.

Various low or high stringency hybridization conditions may be used, depending on the specificity and selectivity desired. These conditions are well known to those skilled in the art. Under stringent hybridization conditions, only highly complementary nucleic acid sequences will hybridize. Preferably, such conditions prevent hybridization of nucleic acids having one or more mismatches out of 20 contiguous nucleotides, and more preferably such conditions will prevent one or more mismatches out of 50 contiguous nucleotides. Most preferably, such conditions prevent hybridization of nucleic acids having one or more mismatches out of 100 consecutive nucleotides. In some cases, conditions may prevent hybridization of nucleic acids having five or more mismatches in the full length sequence.

By stringent hybridization assay conditions is meant hybridization assay conditions that are at least stringent as follows: 50% formamide, 5 × SSC, 50 mM NaH ₂ PO ₄ , pH 6.8, 0.5% SDS, 0.1 mg
/ ml sonicated salmon sperm DNA and hybridization overnight at 42 ° C. in 5 × Denhardt's solution; wash at 45 ° C. with 2 × SSC, 0.1% SDS; and 0.2 × SSC, 0.1
Wash at 45 ° C with% SDS. Under the most stringent hybridization assay conditions, the second wash can be performed in 0.1 × SSC at temperatures up to 70 ° C. (herein incorporated by reference in its entirety, including drawings, tables or figures). , Berger et al. (1987) "Guide to Mol.
ecular Cloning Techniques) ", p. 421). However, other applications may require the use of conditions other than a combination of these conditions. Methods for determining the conditions necessary to obtain the desired hybridization are well known to those of skill in the art and can be based on a number of factors, including but not limited to the sequence to be hybridized and the sample to be tested. I have.

In another preferred embodiment, the invention features an isolated, enriched or purified nucleic acid molecule encoding a kinase polypeptide, further comprising a vector or promoter effective to initiate transcription in a host cell. I do. The invention also features a recombinant nucleic acid, preferably in a cell or organism. The recombinant nucleic acid is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or a functional derivative thereof, and is effective for initiating transcription in a host cell. It may include a vector or promoter. Or
A recombinant nucleic acid can include a transcription initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a kinase polypeptide, and a transcription termination region functional in a cell. Combinations of specific vectors and host cells are described below.

The term “vector” refers to a single-stranded or double-stranded circular nucleic acid molecule that can be transfected into a cell and that can replicate within the cell genome or independently of the cell genome. . Circular double-stranded nucleic acid molecules can be cleaved and thereby linearized by treatment with restriction enzymes. A combination of knowledge about nucleic acid vectors, restriction enzymes, and nucleic acid sequences that are cleaved by restriction enzymes
It is readily available to those skilled in the art. The nucleic acid molecule encoding the kinase can be inserted into the vector by cutting the vector with a restriction enzyme and ligating the two pieces together.

The term “transfect” defines a number of methods for inserting a nucleic acid vector or other nucleic acid molecule into a cell organism. These methods involve a variety of techniques that treat cells with high concentrations of salts, electric fields, detergents, or DMSO to render the outer membrane or cell wall permeable to relevant nucleic acid molecules, or various viral traits. Including the use of introduction methods.

[0041] As used herein, "promoter" refers to a nucleic acid sequence necessary for the expression of a gene sequence. The promoter region will vary from organism to organism, but is well known to those skilled in the art for different organisms. For example, in prokaryotes, the promoter region includes both the promoter, which directs the initiation of RNA transcription, as well as a DNA sequence that, when transcribed into RNA, signals a initiation of synthesis. Such regions usually include 5 'non-coding sequences involved in the initiation of transcription and translation, such as TATA boxes, capping structures, CAAT sequences, and the like.

[0042] In a preferred embodiment, the isolated nucleic acid is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, the amino acid sequence of a functional derivative thereof, or the sequence thereof. Number: 2 at least 35, 40, 45, 50, 60, 75, 100
, 200 or 300 contiguous amino acids, or at least 35 of SEQ ID NO: 4
, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids, or at least 10, 15, 20, 30, 40 of SEQ ID NO: 6 Pieces, 45 pieces, 5
0, 60, 75, 100, 200, or 300 contiguous amino acids, or at least 35, 40, 45, 50, 60, 75, 100 of SEQ ID NO: 8, Encodes 200 or 300 contiguous amino acids, or at least 35, 40, 45, 50, 60, 75, 100, 200 or 300 contiguous amino acids of SEQ ID NO: 10 , SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO:
Including, consisting essentially of, or consisting of, the nucleic acid sequences set forth in 9. The BUB1 kinase polypeptide comprises or comprises at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO: 2. Consists essentially of or consists of those amino acids. NEK kinase polypeptide comprises at least 35 of SEQ ID NO: 4,
40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids, at least 10, 15, 20, 30, 40, 45 of SEQ ID NO: 6 , 50, 60, 75, 100, 200, or 300 contiguous amino acids, at least 35, 40, 45, 50, 60, 75, 100 of SEQ ID NO: 8 , 200 or 300 contiguous amino acids, or at least 35, 40, 45, 50, 60, 75 of SEQ ID NO: 10
Comprises, consists essentially of, or consists of 1, 100, 200, or 300 contiguous amino acids. Nucleic acids may be isolated from natural sources by cDNA cloning or subtractive hybridization. Natural sources may be mammals, preferably humans, blood, sperm, or tissue, and nucleic acids may be synthesized using the triester method or an automated DNA synthesizer.

The term “mammal” preferably refers to mice, rats, rabbits, guinea pigs,
It refers to organisms such as sheep and goats, more preferably cats, dogs, monkeys and apes, most preferably humans.

In still other preferred embodiments, the nucleic acid is a conserved or unique region, for example, designing hybridization probes to facilitate identification and cloning of additional polypeptides, facilitating cloning of additional polypeptides. This is a useful region for designing a PCR probe to obtain an antibody, obtaining an antibody against the polypeptide region, and designing an antisense oligonucleotide.

A “conserved nucleic acid region” refers to a kinase polypeptide-encoding 2 polypeptide that allows a particular nucleic acid sequence to hybridize under lower stringency conditions.
A region present on one or more nucleic acids. Examples of lower stringency conditions suitable for screening for nucleic acids encoding kinase polypeptides are described in Abe et al. (J. Biol. Chem. 19: 13361-13368, 1992). Preferably, the conserved regions differ by only 5 out of 20 nucleotides, more preferably by 2 out of 20 nucleotides, or most preferably by 1 out of 20 nucleotides.

By “unique nucleic acid region” is meant a sequence that is not present in the sequence encoding another naturally occurring polypeptide, but is present in the nucleic acid encoding the kinase polypeptide. Such regions are preferably 32 (preferably 40, more preferably 45, most preferably 55, as shown in the full length amino acid sequence of SEQ ID NO: 2.
Or more contiguous amino acids, 33 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids shown in the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 4 8 (preferably 15, more preferably 20, most preferably 30) or more consecutive amino acids shown in the amino acid sequence of 6, 33 (shown in the amino acid sequence of SEQ ID NO: 8) Is 40
, More preferably 45, most preferably 55) or more contiguous amino acids, 33 (preferably 40, more preferably 45, most preferably 55) shown in the amino acid sequence of SEQ ID NO: 10. ) Or more consecutive amino acids. In particular, it is preferred that the unique nucleic acid region be of mammalian origin.

In the second aspect of the present invention, a BUB1 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 2 contained in a sample or a nucleic acid encoding an NEK kinase polypeptide contained in a sample is detected. And a nucleic acid probe.
In a preferred embodiment, the nucleic acid probe encodes a kinase polypeptide that is a protein fragment encoded by the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 6. Preferably, the NEK kinase polypeptide comprises NEK4, NEK5, and N
Selected from the group consisting of EK6; preferably, NEK4 comprises NEK4a having the amino acid sequence shown in SEQ ID NO: 8 and NEK4b having the amino acid sequence shown in SEQ ID NO: 10; more preferably, the kinase polypeptide is Is selected from the group consisting of NEK5 and NEK6 having the amino acid sequence shown in SEQ ID NO: 4; and most preferably, the kinase polypeptide is NEK having the amino acid sequence shown in SEQ ID NO: 6.
6 The nucleic acid probe is capable of hybridizing to a sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a functional derivative thereof. Contains an array.

In a preferred embodiment, the nucleic acid probe comprises at least 12, 32, 75, 90, 105, 120, 200, 250, 300 or 300 of the full-length sequence shown in SEQ ID NO: 2. 350 contiguous amino acids; at least the full-length sequence represented by SEQ ID NO: 4
35, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids; at least 12, 32 of the full length sequence shown in SEQ ID NO: 6 , 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids; at least 35, 75 of the full-length sequence represented by SEQ ID NO: 8 , 90
, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids; at least 35, 75, 90, 105 of the full-length sequence shown in SEQ ID NO: 10 , 120, 150, 200, 250, 300 or 350 contiguous amino acids; or a nucleic acid encoding a functional derivative thereof.

The method using a probe includes contacting a nucleic acid probe with a sample under conditions that allow hybridization to occur, and detecting the presence or absence or amount of the probe bound to the kinase RNA. Detecting the presence or absence or the amount of the The nucleic acid duplex formed between the probe and the nucleic acid sequence encoding the kinase polypeptide may be used to identify the sequence of the detected nucleic acid (see drawings, figures or tables herein for reference). Nelson et al., "Nonisotopic DNA Probe Technology (Nonisotopic DNA P
robe Techniques), Academic Press, San Diego, Kricka
(Eds., P. 275, 1992). A kit for performing such a method may be constructed to include a container means having a nucleic acid probe disposed therein.

In a third aspect, the present invention provides a BU having the amino acid sequence set forth in SEQ ID NO: 2
Recombinant cells or tissues that contain a nucleic acid molecule that encodes a B1 kinase polypeptide or that encodes a NEK kinase polypeptide have been described. Preferably, the NEK kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6; preferably, NEK4 has NEK4a having the amino acid sequence of SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO: 10. More preferably, the kinase polypeptide is selected from the group consisting of NEK5 having the amino acid sequence shown in SEQ ID NO: 4 and NEK6 having the amino acid sequence shown in SEQ ID NO: 6;
And most preferably, the kinase polypeptide is NEK6. In such cells, the nucleic acid may be under the control of a genomic regulatory element or under the control of an exogenous regulatory element, including an exogenous promoter. "Exogenous" refers to a promoter that is not normally transcriptionally coupled in vivo to the coding sequence of a kinase polypeptide.

[0051] The polypeptide is preferably a fragment of the protein encoded by the full-length amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. "Fragment" refers to the amino acid sequence present in a kinase polypeptide. Preferably, such a sequence comprises at least 32, 45, 50, 60, 100, 200 or 300 contiguous amino acids of the full length sequence shown in SEQ ID NO: 2, : At least 35, 45, 5 of the sequence shown in 4
Contains 0, 60, 100, 200, or 300 contiguous amino acids, SEQ ID NO: 6
At least 10, 15, 20, 30, 45, 50, 60
Contains at least 45, 50, 60, 100, 200, or 300 contiguous amino acids of the full-length sequence shown in SEQ ID NO: 8, containing 100, 200, or 300 contiguous amino acids At least the full-length sequence comprising amino acids and represented by SEQ ID NO: 10
Contains 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids.

In a fourth aspect, the present invention provides an isolated, enriched, or purified BUB1 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, or an isolated, enriched, or purified BUB1 kinase polypeptide. Features a NEK kinase polypeptide. Preferably, the NEK kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6; preferably, NEK4 has NEK4a having the amino acid sequence of SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO: 10. More preferably the kinase polypeptide is selected from the group consisting of NEK5 having the amino acid sequence shown in SEQ ID NO: 4 and NEK6 having the amino acid sequence shown in SEQ ID NO: 6; and most preferably This kinase polypeptide is NEK6.

“Isolated” with respect to a polypeptide refers to six (preferably twelve, more preferably eighteen, and most preferably sixteen) attached to each other, including polypeptides isolated or synthesized from natural sources. (Preferably 25, 32, 40, or 50) or more amino acids. In certain aspects, long polypeptides are preferred, eg, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8
Or 100, 200, 300, 400, or more contiguous amino acids of the full-length sequence set forth in SEQ ID NO: 10.

An isolated polypeptide of the present invention is unique in that it is not found in pure or isolated form in nature. Use of the term "isolated" indicates that the naturally occurring sequence has been separated from its normal cellular environment. Thus, the sequences may be in a cell-free solution or may be in a different cellular environment. The term does not mean that the sequence is the only amino acid chain present, but that it is essentially free of at least about 90-95% pure naturally related non-amino acid material. are doing.

The term “enrichment” as used in reference to polypeptides refers to the total amount of a particular amino acid sequence present in the cell or solution concerned, relative to the amount present in normal or diseased cells or the cell from which the sequence was taken. A significantly higher percentage of amino acid sequences (2
５5 times). This can be done artificially by selectively reducing the other amino acid sequences present, or by selectively increasing the amount of the particular amino acid sequence involved, or by a combination of both. Note, however, that the term enriched does not mean that no other amino acid sequence is present, but merely that the relative amount of the relevant sequence is significantly increased. The term "significant", as used herein, is used to indicate that the level of increase is useful to the person making such an increase, and generally is at least as compared to other amino acid sequences. It means an increase of about 2 times, more preferably at least 5 to 10 times or more. The term also does not mean that amino acid sequences from other sources are not present.
Amino acid sequences of other origins may include, for example, those encoded by yeast or bacterial genomes, or cloning vectors such as pUC19. The term is meant to apply only to situations where a person intervenes to increase the proportion of the desired amino acid.

[0056] It is also advantageous for some purposes that the amino acid sequence is in purified form. The term "purified" with respect to a polypeptide does not require absolute purity (as in a homogeneous preparation). Instead, the sequences are meant to be relatively purer than in their natural environment. This level should be at least 2-5 times greater (eg, in terms of mg / ml) compared to the native level.
A purification of at least one order, preferably two or three orders, more preferably four or five orders, is clearly envisaged. The material is preferably free of contamination at functionally significant levels, for example 90%, 95% or 99% pure.

[0057] In a preferred embodiment, the kinase polypeptide is encoded by the full length amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. It is a protein fragment. Preferably, the kinase polypeptide comprises at least 32, 45, 50, 60 of the full-length sequence set forth in SEQ ID NO: 2.
Contains at least 35, 45, 50, 60, 100, 200 or 300 contiguous amino acids comprising 100, 200, or 300 contiguous amino acids Including at least contiguous amino acids or at least 10, 15, 25, 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence shown in SEQ ID NO: 6. Or at least 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ ID NO: 8, or SEQ ID NO: 10
At least 35, 45, 50, 60, 100, 200, or
Contains 300 contiguous amino acids or includes functional derivatives thereof.

In a preferred embodiment, the kinase polypeptide comprises (a) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, (b) the sequence Positions 1 to 785, 786 to 1028, or 1029 to 1085 of No .: 2, Positions 1 to 31, 32 to 283, or 284 to 302 of SEQ ID NO: 4, or SEQ ID NO: 6
The amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, unless one or more segments of amino acid residues consisting of positions -22 or 23-302 are missing, (c) 1 to 785, 786 to 1028, or 1029-1 of SEQ ID NO: 2
085, 1-31, 32-283, or 284-302 of SEQ ID NO: 4, or 1 of SEQ ID NO: 6
Or SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 from the amino acid residues consisting of 2222 or 23-302, or (d) a C-terminal catalytic domain, an N-terminal domain, and Unless one or more domains selected from the group consisting of the C-terminal tail are missing, the amino acid sequence represented by SEQ ID NO: 2, or from the N-terminal domain, the catalytic domain, the C-terminal catalytic domain, and the C-terminal domain Not including all but one or more of the domains selected from the group consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 .

[0059] Polypeptides can be isolated from natural sources by methods well known in the art. Natural sources may be mammals, preferably humans, blood, sperm, or tissue, and polypeptides may be synthesized using an automated polypeptide synthesizer.

In some embodiments, the invention includes a recombinant kinase polypeptide selected from the group consisting of BUB1, NEK4a, NEK4b, NEK5, and NEK6 kinase polypeptides. A "recombinant kinase polypeptide" is one which differs from a naturally occurring polypeptide in either its location (eg, in a different cell or tissue than is found in nature), purity or structure. And polypeptides produced by recombinant DNA technology. Generally, such recombinant polypeptides will be present in cells in amounts that are different from those normally found in nature.

In a fifth aspect, the present invention provides a BUB1 kinase polypeptide, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 2, a NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 8, : N having the amino acid described in 10
A kinase polypeptide or kinase selected from the group consisting of EK4b kinase polypeptide, NEK5 kinase polypeptide having the amino acid sequence of SEQ ID NO: 4, and NEK6 kinase polypeptide having the amino acid sequence of SEQ ID NO: 6 An antibody having specific binding affinity for a polypeptide domain or fragment (
For example, a monoclonal antibody or a polyclonal antibody). By "specific binding affinity" is meant that the antibody binds to the target kinase polypeptide with greater affinity than it would under certain conditions to bind another polypeptide. An antibody or antibody fragment is a polypeptide that contains a region that can bind to another polypeptide. The term "specific binding affinity" refers to an antibody that binds to a kinase polypeptide with greater affinity than it would when binding to another polypeptide under certain conditions. Antibodies can be used to identify endogenous sources of BUB1 or NEK kinase polypeptide, to monitor cell cycle regulation, and for immunolocalization of NEK kinase polypeptide to the centrosome. .

[0062] The term "polyclonal" refers to a heterogeneous population of antibodies consisting of antibody molecules derived from the sera of animals immunized with the antigen or a derivative thereof having antigenic function. For production of polyclonal antibodies, immunization may be performed by injecting an antigen into various host animals. Various adjuvants can be used to boost the immune response, depending on the type of host.

A “monoclonal antibody” is a substantially homogeneous population of antibodies to a particular antigen. They can be obtained by any method used to produce antibody molecules in continuous subculture cell lines. Monoclonal antibodies may be obtained by methods known to those of skill in the art (Kohler et al., Nature 256: 495-497, 1975 and U.S. Pat. Which are incorporated by reference.)

The term “antibody fragment” refers to a portion of an antibody, often a hypersensitive region or a portion of the surrounding heavy and light chains that exhibit specific binding affinity for a particular molecule. The hypersensitive variable region is the portion of the antibody that physically binds to the polypeptide target.

An antibody or antibody fragment having specific binding affinity for a kinase polypeptide of the present invention can be obtained by searching for a sample using the antibody under conditions suitable for forming a kinase-antibody immune complex. Thus, the present invention can be used in a method for detecting the presence of a kinase polypeptide and the amount of a kinase polypeptide in the sample, and in the method for detecting the presence and amount of an antibody binding to the kinase polypeptide. Diagnostic kits for performing such methods may include antibodies or antibody fragments specific to the kinase as well as complexes of the binding partner of the antibody specific to the kinase or the antibody itself. It is good to build.

[0066] Antibodies or antibody fragments that have specific binding affinity for a kinase polypeptide of the present invention can be isolated, enriched, or purified from prokaryotes or eukaryotes. Routine methods known to those of skill in the art allow for the production of antibodies or antibody fragments in either prokaryotes or eukaryotes. Purification, enrichment, and isolation of antibodies comprising polypeptide molecules are described above.

An antibody having specific binding affinity for a kinase polypeptide of the present invention can be obtained by contacting the sample with the antibody under conditions that allow the formation of an immune complex, followed by antibody complexation with the kinase polypeptide. It can be used in a method for examining the presence or amount of a kinase polypeptide contained in the sample by detecting the presence or amount of the kinase polypeptide. A diagnostic kit for performing such a method is constructed to comprise a first container containing an antibody and a complex of a binding partner of the antibody and a second container containing a label, eg, a radioisotope. Good to do. The diagnostic kit should also include instructions and instructions for optimal use of the FDA.

In a sixth aspect, the invention features a hybridoma that produces an antibody that exhibits specific binding affinity for a kinase polypeptide or a kinase polypeptide domain, wherein the polypeptide has SEQ ID NO: BUB1 kinase polypeptide having the amino acid sequence of SEQ ID NO: 2, NEK4a kinase polypeptide having the amino acid sequence of SEQ ID NO: 8, NEK4b kinase polypeptide having the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 4 It is selected from the group consisting of a NEK5 kinase polypeptide having the amino acid sequence described, and a NEK6 kinase polypeptide having the amino acid sequence described in SEQ ID NO: 6. By "hybridoma" is meant an immortalized cell line that can secrete antibodies, such as, for example, antibodies to the kinases of the invention. In a preferred embodiment, an antibody against this kinase comprises an amino acid sequence capable of specifically binding a kinase polypeptide of the present invention.

[0069] In a seventh aspect, the present invention provides a BU having the amino acid sequence of SEQ ID NO: 2.
B1 kinase polypeptide, NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 8, NEK4b kinase polypeptide having the amino acid set forth in SEQ ID NO: 10, NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 4 A kinase polypeptide binding reagent capable of binding to a peptide and a kinase polypeptide selected from the group consisting of an NEK6 kinase polypeptide having the amino acid sequence of SEQ ID NO: 6. The binding reagent is preferably a purified antibody that recognizes an epitope present on the kinase polypeptide of the present invention. Other binding reagents include molecules that bind to kinase polypeptides and similar molecules that bind to one kinase polypeptide. Such binding reagents can be identified using assays that measure kinase binding partner activity, such as assays that measure PDGFR activity.

The invention also features a method of screening for human cells containing a kinase polypeptide of the invention or an equivalent sequence. This method includes methods such as those described herein for identifying the kinases of the invention (eg, cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.). Identifying novel polypeptides in human cells using standard methods routine in the art.

In an eighth aspect, the present invention provides (a) a BUB1 kinase polypeptide having the amino acid sequence of SEQ ID NO: 2, and an NE having the amino acid sequence of SEQ ID NO: 8.
K4a kinase polypeptide, NEK4b kinase polypeptide having the amino acid set forth in SEQ ID NO: 10, NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and NEK6 kinase having the amino acid sequence set forth in SEQ ID NO: 6 Contacting a kinase polypeptide selected from the group consisting of polypeptides with a test substance; (b) measuring the activity of the polypeptide; and (c) determining whether the substance modulates the activity of the polypeptide. And a method for identifying a substance that regulates kinase activity, which comprises the steps of detecting

The term “modulate” refers to the ability of a compound to alter the function of a kinase of the present invention. The modulator preferably activates or suppresses the activity of the kinase according to the concentration of the compound contacted with the kinase of the invention.

The term “activate” means to increase the intracellular activity of the kinase. The term inhibition means reducing the intracellular activity of the kinase. The kinase activity is preferably an interaction with the original binding partner.

The term “modulates” also refers to increasing or decreasing the probability of forming a complex between a kinase of the present invention and its native binding partner, thereby altering the function of the kinase. . Modulators preferably increase the probability of forming such a complex between the kinase and the native binding partner, and more preferably increase the probability of forming a complex between the kinase and the native binding partner. It is increased or decreased depending on the concentration of the compound contacted with, and most preferably reduces the probability of forming a complex between the kinase and its native binding partner.

The term “complex” refers to an assembly of at least two molecules bound together. Signaling complexes often include at least two protein molecules bound together. For example, the protein tyrosine receptor protein kinases GRB2, SOS, RAF, and RAS assemble to form signaling complexes in response to mitogen ligands.

The term “native binding partner” refers to a polypeptide, lipid, small molecule, or nucleic acid that binds a kinase in a cell. A change in the interaction between the kinase and the native binding partner manifests itself as an increase or decrease in the probability of forming an interaction, ie, an increase or decrease in the concentration of the kinase / intrinsic binding partner complex. it can.

The term “contacting” as used herein refers to a solution containing a test compound,
This means mixing with a solution of a liquid medium containing the cells of the present method. The solution containing the compound may also include another component such as, for example, dimethyl sulfoxide (DMSO) to facilitate uptake of one or more test compounds into the cells of the method. The solution containing the test compound may be added to the cell-soaked medium using a supply device such as a pipette-based device or a cylinder-based device.

In a ninth aspect, the present invention relates to (a) a BUB1 kinase polypeptide having the amino acid sequence of SEQ ID NO: 2, and an NE having the amino acid sequence of SEQ ID NO: 8.
K4a kinase polypeptide, NEK4b kinase polypeptide having the amino acid set forth in SEQ ID NO: 10, NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and NEK6 kinase having the amino acid sequence set forth in SEQ ID NO: 6 Expressing in a cell a kinase polypeptide selected from the group consisting of polypeptides; (b) adding a test substance to said cell; and (c) cell phenotype or native binding to said polypeptide. A method for identifying a substance that modulates kinase activity in a cell, comprising the steps of monitoring changes in interaction with a partner, is characterized.

As used herein, the term “express” means that the kinase of the present invention is produced in a cell from a nucleic acid vector containing the kinase gene. The nucleic acid vector is transfected into the nucleic acid using methods well known in the art as described herein.

In a tenth aspect, the present invention relates to a method for treating a disease by administering a substance that regulates the activity of a BUB1 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 2 to a patient in need of treatment. A method of treating is provided. Preferably the disease is cancer, more preferably the cancer is leukemia, cervical cancer, lymphoma, colon cancer,
It is selected from the group consisting of lung cancer, melanoma, ovarian cancer, CNS cancer, prostate cancer, kidney cancer, and breast cancer. The disease or disorder is CREST syndrome.

CREST syndrome is caused by cutaneous calcinosis, Raynaud's phenomenon, esophageal imvo
lvement), hyperdactyly, and the first stage of telangiectasia.
Patients with CREST syndrome have progressive systemic sclerosis, diffuse systemic sclerosis, primary pulmonary hypertension, and idiopathic portal hypertension with increased incidence of malignant cancer .

An agent useful for treating a kinase-related disease or disorder is preferably in one or more in vitro assays for activity corresponding to the treatment of the disease or disorder in question. A positive result is shown (examples of such assays are provided below in section VIII). Examples of substances that can be screened for advantageous activity are shown and referenced in Section VIII below.
Agents that modulate the activity of the kinase preferably include, but are not limited to, antisense oligonucleotides and inhibitors of protein kinases, as determined by the methods and screens referenced in Section VIII below.

The term “suppress” refers to reducing the probability that an organism will change or develop into an abnormal state.

The term “treating” means having a therapeutic effect and at least partially alleviating or eliminating the abnormal condition of the organism.

The term “therapeutic effect” refers to an inhibitor or activator that causes or is associated with an abnormal condition. Depending on the therapeutic effect, one or more of the symptoms of the abnormal condition may be relieved to some extent. With respect to the treatment of an abnormal condition, a therapeutic effect means one or more of the following. (A) increased cell proliferation, growth, or differentiation; (b) suppressed cell death (ie, slowed or stopped); (c) suppressed degeneration; (d) abnormal One or more of the symptoms associated with the condition are to some extent relieved, and (e) the function of the affected cell population is enhanced. Compounds that exhibit an effect on abnormal conditions can be identified as described herein.

The term “abnormal condition” with respect to human BUB1 kinase refers to the function of an organism that deviates from the function of the cells or normal tissues of the organism. The abnormal condition may be related to cell proliferation, cell differentiation, or cell viability. Abnormal conditions include leukemia,
Includes cervical, lymphoma, colon, lung, melanoma, ovarian, CNS, prostate, kidney, and breast cancer, and CREST syndrome.

[0087] Aberrant cell proliferative conditions include cancers such as fibrotic and vascular diseases, abnormal angiogenesis and angiogenesis, wound adhesions, psoriasis, diabetes, and inflammation, and cancers of the invention. It is.

An abnormal differentiation state includes, but is not limited to, a neurodegenerative disease, a delayed rate of wound healing, and a delayed rate of tissue transplant healing.

The condition in which abnormal cells survive involves a condition in which the programmed cell death (apoptosis) pathway is activated or eliminated. The number of protein kinases is associated with the apoptotic pathway. Aberrant function of any one of the protein kinases may result in cell immortality or cell death before maturation.

The term “aberrant” with respect to the function of a kinase in the signal transduction process
Is overexpressed or underexpressed in the organism, mutated so that its catalytic activity is lower or higher than wild-type protein kinase activity, or suddenly becomes incapable of further interacting with its native binding partner A kinase that is mutated, is not further modified by another protein kinase or protein phosphatase, or does not further interact with its native binding partner.

The term “administering” refers to a method of introducing a compound into cells or tissues of an organism. An abnormal condition can be suppressed or treated when cells or tissues of the organism are present within the organism or outside the organism. Cells that are present in vitro can be maintained in a cell culture dish, ie, can grow. For cells that remain in the organism, there are many methods in the art for administering the compounds, including (but not limited to) oral application, parenteral application, , Mucosal application, infusion application, and aerosol application. For cells in vitro, there are many methods in the art for administering this compound, including (but not limited to) cell microinjection, transformation, and Includes carrier method.

[0092] An abnormal condition can also be suppressed or treated by administering to a group of cells having an abnormal form by one route of transduction of an organism. Subsequently, the effect of the compound administration on the biological function may be monitored. The organism is preferably a mouse,
Rats, rabbits, guinea pigs or goats, more preferably monkeys or apes, most preferably humans.

[0093] In the eleventh aspect of the present invention, there is provided a method for detecting a nucleic acid encoding a BUB1 kinase polypeptide having the amino acid sequence of SEQ ID NO: 2 contained in a sample as a tool for diagnosing a disease or disorder, The method comprises the steps of: (a) contacting a nucleic acid probe that hybridizes with a sample under conditions for performing a hybridization assay on a nucleic acid target region of bub1, wherein the probe comprises a nucleic acid sequence encoding a polypeptide; A step comprising a fragment and a probe comprising the sequence and the complement of the fragment; and (b) detecting the presence or amount of a hybrid of the probe and target region, which is indicative of a disease or disorder. including.

In a preferred aspect of the invention, the disease or disorder is cancer. More preferably, the cancer is leukemia, cervical cancer, lymphoma, colon cancer, lung cancer, melanoma, ovarian cancer, CNS cancer,
It is selected from the group consisting of prostate cancer, kidney cancer, and breast cancer. Alternatively, the disease or disorder is CREST syndrome.

The nucleic acid “target region” is a full-length nucleobase sequence described in SEQ ID NO: 1, a functional derivative thereof or a fragment thereof, which is a sequence to which a nucleic acid probe specifically hybridizes. Specific hybridization indicates that only the probe, in the presence of other nucleic acids, will detectably hybridize to the kinase of the target region of the invention. Putative target regions can be identified by methods well known in the art, consisting of alignment and comparison of the most closely related sequences present in the database.

In a preferred embodiment, the nucleic acid probe comprises at least 12, 32, 50, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full length sequence as set forth in SEQ ID NO: 2. Encodes or hybridizes to a nucleic acid target region encoding a functional derivative thereof. Hybridization conditions should be such that they hybridize only to the kinase gene, even if other nucleic acid molecules are present. Under stringent hybridization conditions, only highly complementary nucleic acid sequences will hybridize. Preferably, such conditions prevent hybridization of nucleic acids with one or two mismatches from the 20 adjacent nucleotides. Such conditions are defined above.

[0097] Diseases that can be diagnosed by detecting a kinase gene contained in a sample include diseases in which a nucleic acid (DNA and / or RNA) encoding BUB1 kinase is amplified compared to normal cells. By "amplification" is meant that the number of kinase DNA or RNA in the cell is greater than in a normal cell. In the selected disease, chromosomal localization of the kinase gene may be amplified, resulting in many copies of the gene, ie, amplification. Gene amplification may cause kinase RNA amplification, i.e., kinase RNA may be amplified where kinase DNA amplification has not occurred.

[0098] In some normal cells, kinase RNA is essentially not expressed, but "amplification" makes the kinase RNA in the cell detectable. Other normal cells show basal levels of kinase expression, so that in these cases amplification can be detected at least 1-2 times, preferably more, kinase RNA compared to basal levels.

Diseases that can be diagnosed by detecting a nucleic acid encoding a BUB1 kinase polypeptide contained in a sample preferably include cancer and CREST syndrome. Test samples suitable for the nucleic acid probe method of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The samples used in the methods described above will vary based on the format of the assay, the method of detection and the nature of the tissue, cell or extract on which the assay is to be performed. Methods for preparing nucleic acid extracts of cells are well known in the art,
It can be easily adapted to obtain samples that do not affect the method used.

The invention in a twelfth aspect provides a method of treating a disease by administering a substance that modulates the activity of a NEK kinase polypeptide to a patient in need of such treatment. Preferably, the kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6. Preferably, NEK4 comprises an NEK4a kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 8 and an NEK4b kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 10, and more preferably the kinase polypeptide Is selected from the group consisting of an NEK5 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 4 and an NEK6 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 6, and most preferably, the kinase polypeptide is NEK6.

An agent useful for treating a kinase-related disease or disorder preferably has a positive result in one or more in vitro assays for an activity corresponding to the treatment of the disease or disorder in question. (Examples of such assays are provided below with reference to Section IX, and Examples 22-24). Examples of substances that can be screened for preferred activity are shown and referenced in Section IX below. Substances that modulate the activity of this kinase are preferably
Includes, but is not limited to, protein kinase antibodies, antisense oligonucleotides and inhibitors as determined by the methods and screens referenced in Section IX and Examples 19 and 22-24 below. . In a preferred embodiment, the agent is an antisense oligonucleotide that inhibits expression of the NEK kinase polypeptide and fragments thereof. Preferably, the kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6. Preferably NEK4
Contains NEK4a and NEK4b, the antisense oligonucleotides of which are synthesized as phosphorothioates.

The term “abnormal condition” with respect to NEK kinase refers to the function of an organism's cells or tissues that deviates from normal function in the organism. The abnormal condition may be involved in cell proliferation, cell differentiation, or cell survival. Abnormal conditions include cancer, neurodegenerative disorders and immune disorders.

In the thirteenth aspect of the present invention, as a diagnostic tool for a disease or disorder,
A method for detecting a nucleic acid encoding a NEK kinase polypeptide contained in a sample, comprising: (a) combining the sample with a nucleic acid probe that hybridizes under hybridization assay conditions to a nucleic acid target region of the kinase polypeptide. Contacting, wherein the probe is a probe comprising the kinase polypeptide, a fragment thereof, and a sequence complementary to the sequence and the fragment; and (b)
Detecting the presence or amount of a probe: target region hybrid as an indicator of the disease or disorder. Preferably, the kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6. Preferably, NEK4 comprises an NEK4a kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 8 and an NEK4b kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 10, and more preferably, the kinase polypeptide thereof. Is NEK having the amino acid sequence represented by SEQ ID NO: 4.
5 is selected from the group consisting of a kinase polypeptide and a NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 6; and most preferably, the kinase polypeptide is NEK6.

The disease or disorder according to a preferred embodiment of the present invention is selected from the group consisting of cancer and neurodegenerative disorders and immune disorders. More preferably, the disease or disorder is cancer.

A “target region” of a nucleic acid is a full-length nucleotide base sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, a functional derivative thereof, or a fragment thereof. To which the nucleic acid probe hybridizes specifically. Specific hybridization indicates that only the probe, even in the presence of other nucleic acids, will detectably hybridize to the kinase of the target region of the invention. Multiple target regions can be identified by methods well known in the art consisting of alignment and comparison of the most closely related sequences in the database.

In a preferred embodiment, the nucleic acid probe comprises at least 6, 12, or at least a sequence represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or a functional derivative thereof. , 32, 50, 75, 90, 105, 120, 150, 20
It hybridizes to a nucleic acid target region encoding 0, 250, 300 or 350 contiguous amino acids. Hybridization conditions should be such that hybridization occurs only with the kinase gene, even in the presence of other nucleic acid molecules. Under stringent hybridization conditions, only highly complementary nucleic acid sequences will hybridize. Preferably, such conditions inhibit the hybridization of nucleic acids having one or two mismatches other than 20 contiguous nucleotides. Such conditions have been clarified in the literature.

[0107] Diseases that can be diagnosed by detecting a kinase gene contained in a sample include diseases in which NEK kinase polypeptide nucleic acid (DNA or RNA) is amplified compared to normal cells.

[0108] Diseases that can be diagnosed by detecting a nucleic acid encoding a NEK kinase polypeptide contained in a sample preferably include cancer, neurolytic disorders and immune disorders. Test samples suitable for the nucleic acid probe method of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The sample used in the above methods will vary based on the morphology of the assay, the method of detection, and the characteristics of the tissue or cell or extract to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted to obtain samples that are consistent with the methods used.

[0109] In the fourteenth aspect of the present invention, B having the amino acid sequence represented by SEQ ID NO: 2
It features an antisense oligonucleotide for inhibiting expression of a UB1 kinase polypeptide or a NEK kinase polypeptide. Preferably, the kinase polypeptide is selected from the group consisting of BUB1, NEK4, NEK5, and NEK6, and fragments thereof. Preferably, NEK4 comprises NEK4a having an amino acid sequence represented by SEQ ID NO: 8 and a NEK4b kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 10, and more preferably, the kinase polypeptide has SEQ ID NO: The kinase polypeptide is selected from the group consisting of an NEK5 kinase polypeptide having the amino acid sequence represented by 4 and an NEK6 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 6, and most preferably, the kinase polypeptide is NEK6. In preferred embodiments, the oligonucleotides are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8
Or a sequence complementary to a sequence encoding a fragment of the protein encoded by the amino acid sequence represented by SEQ ID NO: 10. Preferably, the antisense oligonucleotide is synthesized as a phosphothioate.

The antisense oligonucleotides are preferably designed to specifically inhibit BUB1 or NEK kinase expression. Preferably, this can be done by identifying a sequence (one or more) that is unique to the kinase polypeptide of the present invention. Preferably, these sequences are not present when other non-BUB1 or non-NEK kinase polypeptides are present in the sample or in the patient.
Methods for identifying such sequences are well known in the art and therefore will only be briefly described. Preferably, the antisense oligonucleotide is between 10 and 100 oligonucleotides, more preferably between 15 and 30 nucleotides, and most preferably between 18 and 25 nucleotides in length.

The antisense oligonucleotides of the present invention are preferably used to inhibit BUB1 or NEK protein expression in normal and tumor cells in vivo. Antisense oligonucleotides can be used alone or in combination. Preferably, the expression of BUB1 kinase or NEK kinase is significantly reduced, more preferably below the limit of detection. By performing the treatment using the antisense oligonucleotide of the present invention in a preferred embodiment,
Cell growth is inhibited and / or apoptosis is induced.
Antisense oligonucleotides can also be used to inhibit BUB1 or NEK protein expression in xenografts of human tumor cells in nude mice. In a preferred embodiment, the antisense oligonucleotides are used in treating various human diseases and disorders in which a BUB1 kinase polypeptide or NEK kinase polypeptide is over-expressed or abnormally expressed.

The addition or effective combination of another antisense oligonucleotide can be confirmed by methods well known in the art.
Briefly, cells or tissues expressing a BUB1 or NEK kinase polypeptide can be contacted with any of the antisense oligonucleotides, alone or in combination, to express bub1 or nekRNA and / or BUB1 kinase or NEK kinase. Polypeptide expression can be determined by the methods described herein. Preferably, treatment with an antisense oligonucleotide to BUB1 kinase or NEK kinase reduces bub1 or nekRNA, and / or NEK polypeptide expression, and more preferably significantly reduces expression (1-2 fold). )
, Most preferably reducing expression to undetectable levels.

The summary of the invention described above is not limiting, and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.

[0114] The nucleic acid of the Detailed Description of the Invention The present invention A. Mammalian BUB1 full-length human bub1 cDNA (SEQ ID NO: 1) is 3408 bp long and 44 bp (1-44 bp).
5'UTR and a 3255 bp ORF (positions 45 to 3300) adjacent to the 108 bp 3'UTR.
The sequence adjacent to the first ATG is the Kozak consensus sequence for the starting methionine (Kozak,
M., Nucleic Acids Res. 15, 8125-8148 (1987)), which is considered to be the translation initiation site for human BUB1.

The two human bub1 isoforms were PCR fragments, SuSTK34a (R51-40-18, 238 bp) and SuSTK
First identified as 34b (R51-40-4, 246 bp). SuSTK34b is identical to SuSTK34a except that there is a 15 nucleotide deletion at the kinase subdomain VII, the very N-terminus that results in a typically conserved "DFG" motif in the activation loop. This isoform predicts an in-frame deletion of the five amino acid sequence, “GLALI”.

Eight human EST fragments are present in the dbEST database, ie R94348 spans the human bub1 cDNA consisting of nucleotide positions 898-1259, and the other seven are human bub1
From nucleotides 2049 to 3422.

B. Mammalian NEK4a full length human NEK4a cDNA (SEQ ID NO: 7) is 1,449 bp long and 121 bp 5 '.
It consists of a UTR and a 1,107 bp open reading frame (ORF) flanked by a 21 nucleotide polyadenylation region followed by a 200 bp 3 ′ UTR. No polyadenylation signal (AATAAA) was found before the polyadenylation region. First ATG
The sequence adjacent to is the Kozak consensus sequence for the starting methionine (Kozak, M. (1987)
Nucleic Acids Res. 15, 8125-8148), which is considered to be the translation initiation site for NEK4a. The NEK4a isoform (NEK4b) was also identified as having a sequence that differs from NEK4a starting at position 1005, as a result of replacing the terminal 75 amino acids of NEK4a with a unique 19 amino acid sequence in the C-terminal sequence. Was.

Some EST fragments span the complete NEK4a sequence with AA214523 at the 5 ′ end and AA147800 at the 3 ′ end.

C. The mammalian NEK4b partial human NEK4b cDNA (SEQ ID NO: 9) is 1,346 bp long and consists of a 41 nucleotide polyadenylation region followed by a 804 bp ORF flanked by a 499 bp 3 ′ UTR. No polyadenylation signal (AATAAA) was found before the polyadenylation region. The first 750 nucleotides of NEK4b are identical to NEK4a, indicating that these genes alternatively splice one NEK4 gene isoform.

Several EST fragments span the NEK4b sequence with N30324 at the 5 ′ end and AA461376 at the 3 ′ end.

D. Mammalian NEK5 full-length human NEK5 cDNA (SEQ ID NO: 3) is 3,854 bp long and has 31 bp of 5'UT.
Flanked by R and a 2,897 bp 3 'UTR following the 20 nucleotide polyadenylation region
Consists of a 906 bp ORF. The polyadenylation signal (AATAAA) is found at positions 3817-3822. The sequence adjacent to the first ATG corresponds to the Kozak consensus sequence for the starting methionine, which is thought to be the translation initiation site for NEK5.

At least one EST fragment, AA181165, spans the complete NEK5 sequence.

E. The mammalian NEK6 partial mouse NEK6 cDNA (SEQ ID NO: 5) is 1310 bp long and 403 bp.
Consists of a 907 bp ORF before the 5 'UTR. The sequence adjacent to the initial ATG only slightly matches the Kozak consensus sequence for the starting methionine, but is thought to be the translation initiation site for NEK5. There is adenine at position -3 before the start codon, but no pyrimidine at position +4. Multiple stop codons 5 'for multiple start sites are present in frame and out frame, but there are no other possible start codons in any frame. The sequence before this start site is GC-rich (57.6
%), So it is characterized in the 5'UTR region.

One EST fragment, AA030068, shows the partial NEK6 sequence.

[0125] proteins of the present invention A. The mammalian BUB1 3408 bp human bub1 nucleotide sequence encodes a polypeptide of 1085 amino acids (SEQ ID NO: 2). Analysis of the deduced amino acid sequence predicts that BUB1 protein is an intracellular serine / threonine kinase that lacks both the signal sequence and the transmembrane domain. The BUB1 protein has a large N-terminal region (amino acids 1-785).
Position), followed by the C-terminal kinase domain (amino acids 786-1028),
Includes the C-terminal tail (amino acids 1029-1085) (FIG. 3). 785 amino acid N of human BUB1
The terminal domain has 68% amino acid identity with mouse BUB1. The 140 amino acid subdomain in this N-terminal region of human BUB1 contains the mouse BUB1 (AF002823), Saccharomyces cerevisiae BUB1 (L32027), and the non-kinase S. cerevisiae MAD3 protein (P47074). ) Has 81%, 31%, and 29% amino acid sequence identity to the N-terminal domain, respectively. The putative 243 amino acids of the catalytic domain of human BUB1 consist of mouse BUB1 and S.
It has 83% and 36% amino acid identity to S. cerevisiae BUB1, respectively. Human
The 57 amino acid C-terminal tail of BUB1 has 72% and 24% amino acid identity with mouse BUB1 and S. cerevisiae BUB1, respectively.

B. Mammalian NEK4a (Long C-Terminal Isoform ) The 1,449 bp human NEK4a nucleotide sequence encodes a 369 amino acid (SEQ ID NO: 8) polypeptide with a predicted molecular weight of 41,825 daltons. Analysis of the deduced amino acid sequence suggests that NEK4a is an intracellular serine / threonine kinase lacking both a signal sequence and a transmembrane domain. NEK4a contains a 42 amino acid N-terminal domain, a 252 amino acid catalytic domain with all the motifs characteristic of serine / threonine kinase, and a 75 amino acid C-terminal domain.

NEK4a is human NEK5 (SEQ ID NO: 4), C. elegans kinase F1
9H6.1 (GB: Z68115), mouse NEK1 (GB: S45828), human NEK3 (GB: Z29067),
Very closely related to human NEK2 (GB: U11050) and Emericella nidulans NIMA (GB: M20249), 82.6%, 78.8%, 41.2%, 40.2%, 44.2%, 42
.6% and 30.4% amino acid identity. NEK4a is also international publication 9
It has 97.2% identity with human HPK1, a partial protein kinase disclosed in 8/11234.

NEK4a (369 amino acids) and NEK4b (268 amino acids) have identical amino acids (and nucleotides) up to amino acids 304 and 249 in NEK4a and NEK4b, respectively.
It is likely that these two proteins may be the result of different, alternative splicing (FIG. 10).

The 42 amino acid N-terminal domain of human-NEK4a does not show any significant homology in a Smith-Waterman search of a non-redundant protein database. Human NEK4a lacks a glycine residue at position 2 and therefore does not appear to undergo myristylation.

The 252 amino acid catalytic domain of human NEK4a includes human NEK5 (SEQ ID NO: 4), C. elegans kinase F19H6.1 (GB: Z68115), mouse NEK1 (GB: S45828), human NEK3
(GB: Z29067), human NEK2 (GB: U11050), and E. nidulans NIMA (GB: M20249)
) Are very closely related to 88.5%, 78.8%, 41.6%, 40.6%, 44
It has .2%, 42.6%, and 30.2% amino acid identity. NEK4a contains a potential "TPY" motif-regulated phosphorylation site in the activation loop. This "TPY
The motif is also present in NEK5. NEK4a and NEK5 are also STK's NIMA
It also contains a catalytic motif HPN (I / V) that maps the C-terminus to a conserved catalytic lysine characteristic of the / NEK-subfamily (positions 102-105 in NEK4a in Figure 9). The kinase domain of NEK4a also has 97.2% identity with human HPK1, a partial protein kinase disclosed in WO 98/11234.

The 75 amino acid C-terminal domain of human NEK4a is most closely related to human NEK5 (SEQ ID NO: 4) with 61.5% amino acid identity. The C-terminus of NEK4a is human HP, a partial protein kinase disclosed in WO 98/11234.
It does not show any detectable homology with K1.

Apart from the potential phosphorylation sites in the “TPY] activation loop, the following additional potential phosphorylation sites were placed at the position indicated by NEK4a (FIG. 9), with only the ortholog of C. elegans. But not in NEK4a and NEK5: protein kinase C [ST] -X- [RK] (PROSITE: PD0C00005) at position 165 and casein kinase II at position 111
[ST-X (2)-[DE] (PROSITE: PD0C00006), tyrosine kinase [RK]-at position 224
X- (2)-[DE] -X- (2/3) -Y (PROSITE: PD0C00007) and Cdc2 kinase [S / T] -P at position 245 (Nigg, E. (1993) Trends in Cell Biol 3: 296-301). The 75 amino acid C-terminal region unique to NEK4a contains additional potential phosphoryl sites at 308 (Cdc2) and 344 (casein kinase II).

C. Mammalian NEK4b (short C-terminal isoform) NEK4a (369 amino acids) and NEK4b (268 amino acids) have identical amino acids (and nucleotides) up to amino acids 304 and 249 in NEK4a and NEK4b, respectively.
It is therefore possible that these two proteins are the result of different, alternative splicing (Figure 4). This diversity immediately precedes the putative kinase domain, making these two proteins distinct
It has a C-terminal tail.

The 19 amino acid C-terminal domain of human-NEK4b is most closely related to human NEK5 (SEQ ID NO: 4), with 61.5% amino acid identity. The C-terminal domain of NEK4b also has 89.5% identity to human HPK1, a partial protein kinase disclosed in WO 98/11234.

D. Mammalian NEK5 The 3,854 bp human NEK5 nucleotide sequence encodes a 302 amino acid (SEQ ID NO: 4) polypeptide with a predicted molecular weight of 34,550 daltons. Analysis of the deduced amino acid sequence indicated that NEK5 was found to have a serine / cell sequence in cells lacking both signal sequences and transmembrane domains.
Expected to be a threonine kinase. NEK5 contains a 31 amino acid N-terminal domain, a 252 amino acid catalytic domain with all the motifs characteristic of serine / threonine kinases, and a 19 amino acid C-terminal domain.

NEK5 is human NEK4a (SEQ ID NO: 8), C. elegans kinase F19H6.1 (GB: Z6
8115), mouse NEK1 (GB: S45828), human NRK2 (GB: P51957), human NEK3 (GB:
Z299067), and most closely related to E. nidulans NIMA (GB: M20249)
They have 82.6%, 79.8%, 38.3%, 40.2%, 44.0% and 32.2% amino acid identity, respectively. NEK5 also has 86.1% identity to human HPK1, a partial protein kinase disclosed in WO 98/11234.

The 31 amino acid N-terminal domain of NEK5 is a C. elegans kinase F19H6.1 (GB: Z681
It has 34.6% identity with the N-terminus of 15). Human NEK5 lacks a glycine residue at position 2 and therefore does not appear to undergo myristylation. A Smith-Waterman search of the non-redundant protein database did not reveal any significant homology to this domain suggesting a potential function.

The 252 amino acid catalytic domain of human NEK5 includes human NEK4a (SEQ ID NO: 8), C. elegans kinase F19H6.1 (GB: Z68115), mouse NEK1 (GB: S45828), human NEK2
(GB: U11050), human NRK2 (GB: 51957), human NEK3 (GB: Z299067), and E. n
most closely related to idulans NIMA (GB: M20249), 88.5% and 79.8 respectively
%, 39.0%, 42.3%, 40.2%, 44.1%, and 32.2% amino acid identity.
NEK5 contains a potential "TPY" motif-regulated phosphorylation site in the activation loop. This "TPY" motif is also present in NEK4a. Also NEK4
a and NEK5 are NIMA, as well as other NEK-subfamily kinases, and a catalytic motif HPN (I / V) that maps the C-terminus to a conserved catalytic lysine (positions 102-105 in NEK4a in FIG. 9) have. The kinase domain of NEK5 is described in WO 9
It has 88.1% identity with human HPK1, a partial protein kinase disclosed in 8/11234.

The 19 amino acid C-terminal domain of human NEK5 is most closely related to human NEK4a (SEQ ID NO: 8) with 61.5% amino acid identity.

Apart from potential phosphorylation sites within the “TPY] activation loop, the following potential phosphorylation sites were located at the positions indicated by NEK4a (FIG. 9), as well as the C. elegans ortholog as well as NEK4a. And NEK5: PKC at position 165, casein kinase II at position 111, tyrosine kinase at position 224, and Cdc2 kinase at position 245.

E. Mammalian NEK6 The 1,310 bp partial cDNA nucleotide sequence of mouse NEK6 encodes a 302 amino acid (SEQ ID NO: 6) polypeptide with a predicted molecular weight of 33,590 daltons. Analysis of the deduced amino acid sequence indicates that NEK6 is an intracellular serine / threonine kinase that lacks both the signal sequence and the transmembrane domain. NEK6 is a 22 amino acid N
It contains a terminal domain and a catalytic domain of 280 amino acids with all the motifs characteristic of serine / threonine kinases. The NEK6 sequence is a partial sequence and thus does not include the C-terminus of the catalytic domain or the carboxyl terminus encoded by the gene.

NEK6 is a Drosophila heteroneura kinase (GB: AF05229).
6), C. elegans kinase (GB: Z46242), Saccharomyces cerevisiae kinase (GB: YILO95W), human GAK (GB
: D88435), human NEK1 (GB: S45828), human NEK3 (GB: Z299067), and human NRK
(GB: L20321), which are 67.8%, 43.7%, 37.0%,
It has 39.5%, 30.5%, 33.9%, and 28.4% amino acid identity.

The 22 amino acid N-terminal domain of mouse NEK6 lacked significant homology when a Smith-Waterman search of the non-redundant protein database was performed.

The 281 amino acids in the catalytic domain of mouse NEK6 are D. heteroneura kinase (GB: A
F052296), C. elegans kinase (GB: Z46242), S. cerevisiae kinase (GB
: YILO95W), human GAK (GB: D88435), human NEK1 (GB: S45828), human NEK3 (GB
: Z299067) and human NRK (GB: L20321), each of which is 67.8%
, 43.7%, 37.0%, 39.5%, 30.5%, 33.9%, and 28.4% amino acid identity.

NEK6 has three features that distinguish it from other NEK-subfamily members.
Provided with EK6-subfamily kinases: (1) an insert between the catalytic glycine-rich loop and catalytic lysine, (2) a three amino acid insert within the activation loop, and (3) an " Lack of TPY "motif-regulated phosphorylation motif.

NEK6 contains, in defined positions, the following potential nucleotide sequence phosphorylation sites. That is, cAMP-dependent kinase [RK] (2) -X- [ST] (PROSITE
: PS00004), PKC at positions 9, 199, 226, 235, and 242, and 7, 47, 115, 147.
, 241 and 278 contain casein kinase II. Of these, P in the 199th place
The KC site (the C-terminus of the two amino acids attached to the “DFG” catalytic motif) extends across species (
From plants to mammals) NEK6-related kinase (NEK from mouse and Drosophila)
Conserved in A. thaliana and rat and human GAK, and Saccharomyces NEK-related kinases such as YILO95W. Low but significant conservation is seen for PKC at position 235.

Much attention has now been paid to the relevance between the biological significance of the serine / threonine protein kinase of human BUB1 and the development of the cell cycle and the development of cancer. Only a few genes involved in cell cycle regulation have been found in human cells. Examples include p53, a tumor suppressor gene, ATM, a gene involved in an autoimmune disease that causes a high incidence of cancer, and the SDI1 gene, which causes cell senescence. All three genes are cell cycle checkpoint genes that function during the G1 / S transition.

The group of yeast cell cycle checkpoint genes includes G2 to M transition, BUB
1, also demonstrated in mitosis, including BUB2, BUB3, MAD1, MAD2, MAD3, and MPS1.

Little is known about mammalian proteins that regulate the transition from G2 to M phase or monitor the spindle or centromere. However, evidence suggests that deficits in controlling these processes are important in tumorigenesis.

Human BUB1 is a novel gene encoding a serine / threonine kinase that clearly functions as a mitotic checkpoint. Human BUB1 is a centromere protein that interacts with microtubules during mitosis.

If the metaphase mitotic spindle does not function properly, cell cycle progression can be arrested. For example, meiosis of a metaphase-late transition chromosome is suppressed if one or more chromosomes are not yet associated in the metaphase plate. Furthermore, the onset of a new cell cycle is suppressed if mitosis was not completed in the previous cell cycle due to inhibition of microtubule assembly. Defective mitosis can cause genomic alterations (aneuploid or tetraploid) and chromosomal abnormalities. Human BUB1
Studying the biological function of genes may help to control mitotic checkpoints and understand the development of cancer.

[0152] biological significance of the novel NEK- sub-family kinase A. NEK4 and NEK5 Human NEK4 and NEK5 are most closely related to C. elegans kinase F19H6.1 and also to four mammalian NEK-subfamily kinases: mouse NEK1 and human NRK2.
, NEK2 and NEK3. They are also E. nidulans NIMA
It is clear that the homology is far apart for the kinase.

[0153] Mouse NEK1 (N IMA R e lated K inase) is 77 with predicted molecular weight of 88.4kDa
It is a four amino acid STK. NEK1 has an extracatalytic C-terminal domain of 526 amino acids containing four putative nuclear-localization signals. For the catalytic and extra-catalytic regions, the percent amino acid sequence identity between NEK1 and NIMA is 42% and 14%, respectively.

NEK1 is prominently expressed in male and female germ cells, suggesting a role for this kinase in meiosis. In addition, NEK1 is enriched in post-mitotic neurons from peripheral ganglia, indicating a role for differentiated cells in this STK.

NEK1 functions as a bispecific kinase of phosphorylated serine / threonine and phosphorylated tyrosine residues in vitro. Double phosphorylation in vivo remains to be elucidated (Arama, E. et al. (1998) On
cogene 16: 1813-1823).

[0156] Human NRK2 (N IMA- r elated k inase ) is a 841 amino acid STK with a predicted molecular weight of 94.6kDa. NRK2 contains a 580 amino acid extracatalytic C-terminal domain with unrelated C-terminal sequences found in NIMA and other NEK-subfamily kinases. The percent amino acid sequence identity between NRK2 and NIMA for the catalytic region is 39%.

NRK2 is prominently expressed in liver, skeletal muscle, kidney, and pancreas. In contrast, the transcription levels of NIMA and NRK2 do not change during the cell cycle. The NRK2 gene is
Localizes to a region of chromosome 3p21.1 that is commonly altered in kidney cancer (Levedakou, E
et al. (1994) Oncogene 9: 1977-1988).

Human NEK2 is a 445 amino acid STK with a predicted molecular weight of 51.7 kDa. For the catalytic region and the C-terminal extracatalytic region, the percent amino acid sequence identity between NEK2 and NIMA is 47% and 19%, respectively.

NEK2 expression is highest in germ cells found in meiotic thickening spermatocytes and oocytes. NEK2 levels are elevated during the G2 / M transition of the meiotic cell cycle (Rhe
e, K and Wolgemuth, D. (1997) Development 124: 2167-2177). NEK2 also
Since the level and activity of this kinase is greatest during the S and G2 phases of the cell cycle, it also appears to play a role in meiosis. The role of centrosome segregation during mitosis was a requirement for NEK2 (Fry, A. et al. (1998)
EMBO J. 17: 470-481). This NEK2 gene is located on human chromosome 14q12 (
Schultz, S. et al. (1994) Cell Growth and Diff. 5: 625-635).

Human NEK3 is a 459 amino acid STK with a predicted molecular weight of 52.3 kDa. NEK3 (G
B: Z29067) shows that the protein lacks the N-terminal catalytic region and the starting methionine. For the catalytic region and the C-terminal extracatalytic region, the percent amino acid sequence identity between NEK3 and NIMA was 43% and 43%, respectively.
19% (Schultz, S. et al. (1994) Cell Growth and Diff. 5: 625-635)
.

NIMA, a STK found in the filamentous fungi Aspergillus and E. nidulans, is a 699 amino acid protein with a predicted molecular weight of 78.9 kDa. Mitosis in A. nidulans requires both Cdc2 and NIMA.

NIMA expression and kinase activity are coordinately regulated throughout the cell cycle, with the lowest in G1 and the highest in S and G2 phases. A number of phosphorylation events are required for sufficient NIMA activation, some of which appear to be performed by Cdc2. When transitioning from mitosis to G1, it is necessary to inactivate both NIMA and Cdc2, which is possible by ubiquinone-mediated proteolysis. The C-terminus of NIMA displays a common Cdc2 phosphorylation site, as well as a PEST motif that mediates the function of this protein by phosphorylation and proteolysis.

Evidence for a NIMA-like pathway during mitosis in higher eukaryotic cells is based on the observation that NIMA is expressed in a non-dominant form in Healer cells and arrests G2.
As in higher eukaryotic cells, Aspergillus also results from the observation that NIMA is overexpressed and causes chromatin contraction (Fry, A. and Nigg, EA (1
995) Current Biology, 5: 1122-1125).

NEK4 and NEK5 are in sharp contrast to all other NEK-subfamily kinases that lack a C-terminus outside the long catalytic domain. The function outside of these catalytic domains in NIMA and NEK2 is to regulate their catalytic activity and rotation in a cell cycle-dependent manner. NEK4 and NEK5 are predicted to be half as long in duration as other NEK-subfamily kinases due to their lack of proteolytic targeting motifs as found in NIMA and NEK2.

The short C-terminal extra-catalytic tail found in NEK4a can play a regulatory role in kinase activation and / or substrate recognition.

Modulation of NEK4 function can be achieved in part by alternative splicing of the C-terminus resulting in NEK4a and NEK4b isoforms. NEK4 and NEK5 can also be regulated by phosphorylation when these proteins have multiple conserved consensus sites that are phosphorylated by various kinases.

NEK4 and NEK5 are predicted to be small, compact kinases similar to MAPK's and CDK's. MAPK's are regulated by bispecific MAPK kinases (MEK's) by phosphorylating "TXY" residues in the activation loop. These phosphorylations redirect the activation loop in a new direction, resulting in over 1000-fold enzyme activation. The mechanism of this rearrangement involves the interaction between the 50 amino acid C-terminus of MAPK (α-helix L16) and the phosphorylated residue of the activation loop. Rearrangement of the activation loop shows that proline is followed by phosphorylated serine /
As a proline-directed kinase capable of phosphorylating only a substrate having a threonine residue, it forms a tight pocket that defines the substrate specificity of MAPK's (
(P + 1 loop) (Canagarajah BJ et al. (1997) Cell, 90: 859-869).

The three characteristics of NEK4 and NEK5 suggest that they can behave similarly to MAPK's. The three features are: (1) their expected small and compact nature; (2) the presence of a potential double phosphorylation "TPY" site in the activation loop;
And (3) 50 amino acid C-terminal α-helix of MAPK when generating an active site
The presence of a short C-terminus (19 amino acids in NEK4b and NEK5, and 75 amino acids in NEK4a) that can play a role similar to L16.

NEK4 and NEK5, similar to MAPK, can also be amenable to activation by MEK's, which performs double phosphorylation in their activation loop. NEK4 and NEK5 are MAP
Like K's and CDK's, they appear to be proline-directed kinases and are often observed specifically in kinases that phosphorylate transcriptional sequences and cell cycle-regulatory molecules.

NEK4 and NEK5 can potentially transduce signals generated at the level of activated membrane receptors to the nucleus, where their targets become transcriptional or cell cycle regulatory proteins. Clarify its position as a new subclass of possible NEK-subfamily kinases.

B. NEK6 mouse NEK6 contains D. heteroneura kinase AF052296, S. cerevisiae kinase (GB
: YIL095W) and the mammalian kinases GAK, NEK1, NEK3, and NRK2, and are distantly homologous to E. nidulans NIMA.

Human GAK is a 1,311 amino acid STK with a predicted molecular weight of 141.3 kDa. GAK contains an extracatalytic C-terminal domain of 998 amino acids with sequences unrelated to the C-terminus found in NIMA and other NEK-subfamily kinases. The C-terminus of GAK contains a tensin / auxilin-like domain. GAK expression and kinase activity fluctuate during the cell cycle with the highest levels found in G1. The GAK gene has a map on human chromosome 4p16 (Kimura, SH et al.
(1997) Genomics, 44: 179-187).

Cyclin G is a member of the cyclin family consisting of regulatory subunits of the cyclin-dependent family of STK's. Unlike cyclins A and B, which regulate the transition of G2 / M, cyclin G regulates the transition from the G1 phase to the S phase of the cell cycle (Tamura, K. et al. (1993) Oncogene, 8: 2113 -2118). Cyclin G is p5
3 It is a transcription target of tumor suppressor protein. Overexpression of it overturns p53-mediated cell cycle checkpoints, resulting in a hypersensitivity cytotoxic effect of cancer chemotherapy (Smith, ML et al. (1997) Exp. Cell Res. 230: 61-68).

Based on the sequence homology between NEK6 and GAK, NEK6 also has a long C-terminal region and is likely to contain a tensin / auxin-like domain similar to GAK. At least three regions unique to the kinase domain of NEK6 appear to have a strong effect on substrate specificity. In these three regions, (1) NEK6 and human GAK-like related kinases, S. cerevisiae kinases YNL020C, YIL095W, and TBR059C, and C. elegans Z46242 are conserved with a glycine-rich loop (subdomain I). Compared to other STK's in that there is a variable length insert located between the designated nucleotide-binding lysine residues (subdomain II) (FIG. 3 shows NEK6 and / or
Or only for GAK. ), (2) NEK6 and related kinases are "DFG" (subdomain VII) and "TPF" activation loop (subdomain VIII)
It shows a six amino acid insert between the catalytic motifs, and (3) the activation loop of NEK6 and its related kinases lack the "TPY" found in NEK4 and NEK5, with two Contains consecutive, equally conserved threonine residues.

NEK6 is clearly another subclass of GAK-like NEK-subfamily kinases, and is therefore regulated by regulatory subunits such as cyclins,
It can also be controlled by domains found in the extra-catalytic region.

Nucleic acid probes, detection methods and methods for detecting BUB1-related kinase and NEK kinase
In order to obtain another nucleic acid molecule of the present invention, an appropriate chromosomal DNA or cDNA library can be screened by a usual hybridization technique using the nucleic acid probe of the present invention. Libraries of chromosomal DNA or cDNA can be prepared using appropriate cells by methods known in the art (eg, Mole
cular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor L
aboratory, Sambrook, Fritsch, & Maniatis eds. 1989)).

Chemical synthesis can also be performed to obtain a nucleic acid probe having a base sequence corresponding to the amino acid sequence at the N-terminal or C-terminal portion of the polypeptide of interest. The synthesized nucleic acid probe may be used as a primer in a polymerase chain reaction (PCR). In order to obtain the fragment of the present invention, a polymerase chain reaction is carried out using an appropriate chromosomal DNA or cDNA library according to a recognized PCR method. Specifically, “A Guide to Metho
ds and Applications), Academic Press, Michael et al., 1990, 5).

Based on the sequences disclosed in the present application, computer-based sequence parallelization and sequence analysis techniques known in the art ("Molecular Cloning: A Laborator")
y Manual ", 1989, supra), those skilled in the art can easily design such a probe. The hybridization probe of the present invention can be used for isotopic labeling, enzyme labeling, fluorescent labeling, biotin-avidin labeling, chemical labeling, or the like. The probes can be labeled by luminescent labels or other standard labeling techniques .. After hybridization, the probes are visualized by known methods.

[0179] The nucleic acid probes of the present invention include not only DNA probes, but also RNA probes produced by methods known in the art. The nucleic acid probe may be immobilized on a supporting solid. Examples of the supporting solid include the following, but are not limited to these examples. Plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins such as polyacrylamide and latex particles. Techniques for immobilizing a nucleic acid probe on such a support solid are known in the art.

A test sample suitable for the method of the present invention using a nucleic acid probe is, for example, a nucleic acid extracted from a cell, a cell or a biological fluid sample. The sample used in the above method varies depending on the type of measurement, the detection method, and the properties of the tissue, cells, and extract as the specimen. Techniques for preparing nucleic acid extracts from cells are known in the art. Using these techniques, samples suitable for the method used can be easily prepared.

The method for examining the presence or absence of the nucleic acid of the present invention in a sample includes the following (a) and (b)
Including the stage. (A) contacting the sample with the nucleic acid probe under conditions that allow hybridization to occur, and (b) detecting the presence or absence of the probe bound to the nucleic acid molecule. By procedures known in the art as described above,
Those skilled in the art can select and use a nucleic acid probe. Test samples include, but are not necessarily limited to, RNA samples derived from human tissues.

A kit for detecting the presence or absence of a nucleic acid of the present invention in a sample comprises at least one
Means as individual containers, wherein said nucleic acid probes are held in said containers.
The kit may further include another container containing one or more of the following reagents. The reagent is a washing reagent and a detection reagent used for detecting the presence or absence of a bound nucleic acid probe. Examples of the detection reagent include an isotope-labeled probe, an enzyme-labeled probe (horseradish peroxidase, alkaline phosphatase), an affinity-labeled probe (biotin, avidin or streptavidin), and the like. However, the present invention is not limited to this. Preferably, the kit further comprises instructions for use.

Specifically, kits having containers divided into compartments include:
All kits containing reagents in different containers are included. Such containers include small glass containers, plastic containers, plate-like plastics and paper, and the like. Such containers allow efficient transfer of reagents from one compartment to another, prevent sample and reagents from mixing with each other, and quantitatively remove drugs or solutions from each container from one compartment. , Allows you to add to another compartment. Such containers include a container for a test sample, a container for a probe or a primer used for measurement, a container for a washing reagent (a phosphate buffer solution, a Tris buffer solution, etc.), a hybridized probe, a bound antibody, Includes containers and the like that contain reagents used to detect amplification products and the like. One skilled in the art will readily appreciate that the nucleic acid probes of the present invention can be readily incorporated into practical kit forms well known in the art.

DNA constructs containing BUB1-related kinase or NEK kinase nucleic acid molecules and their construction
The present invention also relates to a recombinant DNA molecule comprising a promoter and a nucleic acid molecule for efficiently performing transcription in the direction from the 5 'end to the 3' end in a host cell. Furthermore, the present invention relates to a recombinant DNA molecule comprising a vector and the above nucleic acid molecule. The present invention also relates to a nucleic acid molecule comprising a transcription region that functions in a cell, a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the above polypeptide, and a transcription termination region that functions in the cell. Said molecule may be an isolated and / or purified DNA molecule.

The present invention further relates to a cell or organism comprising the above-described nucleic acid molecule and capable of expressing a polypeptide. The polypeptide can be purified from cells that have been modified to express the polypeptide. A cell is "modified to express a desired polypeptide" when the cell does not normally produce the protein or produces only a small amount, but the cell is produced by genetic engineering techniques. It means that it has been manipulated to produce a protein. Those skilled in the art can easily understand genomic DNA, cDN
Either A or the synthetic DNA sequence can be introduced and expressed in either eukaryotic or prokaryotic cells.

A nucleic acid molecule such as DNA is “expressible” for a polypeptide when the molecule has a base sequence that retains information about the control of transcription and translation, and the sequence encodes the polypeptide. It is "operably linked" to the sequence of the nucleic acid. Functionally operable linkage means that the regulatory DNA sequence and the DNA sequence to be expressed are combined in such a way as to allow expression of the gene sequence. The nature of the regulatory regions required for expression of the gene sequence will vary depending on the exact type of organism, but will generally include a promoter region. In prokaryotes, this promoter region contains both the promoter (which controls the initiation of RNA transcription) and the DNA sequence which, when transcribed into RNA, directs the start of synthesis. Such regions usually include the 5'-noncoding regions involved in initiating transcription and translation. For example, TATA box,
The sequence of the cap site, the CAAT sequence and the like.

If necessary, the non-coding region 3 ′ to the sequence encoding the kinase of the present invention can also be obtained by the above method. Such regions are known to be transcription termination or poly
It is considered that it contains a transcription termination control sequence related to A addition and the like. therefore,
By retaining the 3'-region that is naturally contiguous to the DNA sequence encoding the kinase of the present invention, a transcription termination signal is incorporated. If the transcription termination signal does not function sufficiently when expressed in the host cell, it may be replaced with a 3 ′ region that functions in the host cell.

The nature of the binding between two DNA sequences (eg, the sequence of the promoter region and the sequence encoding the kinase of the invention) does not (1) result in the introduction of a frameshift mutation, but ( 2) Transcription activity of the kinase gene sequence of the present invention which does not interfere with the activity of the promoter region sequence that directs transcription of the gene sequence encoding the kinase of the present invention, and (3) is transcribed by the promoter region sequence When conditions such as not hindering the DNA sequence are satisfied, it can be said that the two DNA sequences are "operably linked". Thus, if a promoter region is capable of activating transcription of a DNA sequence, the promoter region is operably linked to the DNA sequence. Therefore, in order to express the gene encoding the kinase of the present invention, a transcription / translation signal recognized by an appropriate host is required.

The present invention includes expression in eukaryotic or prokaryotic cells of a gene encoding a kinase of the present invention (or a functional analog thereof). In general, a prokaryotic host can produce a recombinant protein very easily and efficiently, and is therefore considered to be one of the preferred expression systems for the kinase of the present invention. The most frequently used prokaryote is E. coli and includes various strains, but other microbial strains, including bacterial strains other than E. coli, can also be used.

In prokaryotic expression systems, plasmid vectors containing the replication site and control sequences obtained from a species compatible with the host are used. Examples of preferred plasmid vectors include pBR322, pUC118, pUC119 and the like. Preferred phage vectors or bacteriophage vectors include λgt10, λgt11 and the like. Suitable viral vectors include pMAM-neo, pKRC, and the like. The vector used in the present invention is preferably a vector that can be replicated in the host used.

Recognized prokaryotic hosts include E. coli, Bacillus subtilis (Bacillu
s), Streptomyces, Pseudomonas,
Salmonella, Serratia and the like are included. However, under such conditions (in prokaryotic cells), glycosylation of the polypeptide does not occur. Prokaryotic hosts must be compatible with respect to the replication unit and control sequences in the expression plasmid.

When expressing the kinase of the present invention (or a functional analog thereof) in a prokaryotic host, the sequence encoding the kinase of the present invention may function as a prokaryotic promoter capable of functioning. Must be combined. Such promoters may be constitutive, but are more preferably controllable (eg, inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of β-lactamase gene of pBR322, and the CAT promoter of chloramphenicol acetyltransferase gene of pPR325. Examples of inducible prokaryotic promoters include:
Bacteriophage lambda major right-left promoter, E. coli trp, r
ecA, lacZ, lacI, gal, etc. promoters, Bacillus subtilis α-amylase promoter
(Ulmanen et al., J. Bacteriol. 162: 176-182, 1985) and a ζ-28 specific promoter
(Oilman et al., Gene Sequence 32: 11-20, 1984), Bacteriophage bacteriophage promoter (Gryczan, In: The Molecular Biology of the Bacilli, Academic).
Press, Inc., NY, 1982), Streptomyces promoter (Ward et al., Mol. G).
en. Genet. 203: 468-478, 1986). For a review on prokaryotic promoters, see Glick (Ind. Microbiot. 1: 277-282. 1987), Centatiempo.
(Biochimie 68: 505-516, 1986), and Gottesman (Ann. Rev. Genet. 18: 415-442).
, 1984).

[0193] Proper expression in prokaryotic cells also requires a ribosome binding site upstream of the coding region of the gene. Regarding the ribosome binding site, for example, Gold et al. (Ann.
Rev. Microbiol. 35: 365-404, 1981). Control array,
The choice of an expression vector, a transformation method, and the like depends on the host cell used to express the gene. As used herein, the terms “cell,” “cell line,” and “cultured cell” are interchangeable. All such names also include their descendants. Therefore, the words "transformants" or "transformed cells" refer to both the very first, base cells and the cultured cells derived therefrom. Such names are independent of the cell's passage number. In addition, since there are intentionally introduced mutations and naturally-occurring mutations, etc., all of the passaged cells have the same DN.
It is not considered to have A. However, even progeny cells with mutations, by definition, have the same function as the cells originally transformed.

The host cell used in the expression system of the present invention is not strictly limited, as long as it can be suitably used for expressing the target kinase polypeptide. Preferred hosts also include, at least, eukaryotic cells. Suitable eukaryotic host cells include, for example, yeast, fungi, insect cells, mammalian cells in vivo and in tissue culture, and the like. Mammalian cells useful as hosts include HeLa cells, cells derived from fibroblasts such as VERO and CHO-K1, or cells derived from the lymphoid system and derivatives thereof. Suitable mammalian cells include cells such as SP2 / 0 and J558L, as well as cells derived from neuroblastomas, such as IMR332, which are believed to have a better ability to perform post-translational modifications of the protein. Can be

Further, plant cells can also be used as hosts. It is also possible to use control sequences compatible with plant cells, such as cauliflower mosaic virus 35S and 19S, a nopaline synthase promoter and a polyA addition signal sequence. Another suitable host is an insect cell. For example, Drosophila larvae.
If an insect cell is used as a host, a Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240: 1453-1459, 1988). Alternatively, using a silkworm polyhedrovirus vector, the kinase of the present invention can be used in large amounts in insect cells (Jasny, Science 238: 1653, 1987; Miller et al., In: Genetic Engi.
neering, Vol. 8, Plenum, Setlow et al., eds., 277-297, 1986).

When a yeast grows in a medium rich in glucose, a large number of enzymes involved in sugar degradation are produced. Any yeast expression system that incorporates a promoter encoding such a glycolytic enzyme and a promoter and a transcription termination sequence element derived from a gene that is expressed at a high level can be used. Sequences derived from known glycolytic genes are very efficient as transcription control signals. Yeasts have a great advantage in that post-translational modifications also occur. There are several types of recombinant DNA techniques using strong promoter sequences and high copy number plasmids, which can be used to produce the desired protein. Yeast recognizes a leader sequence on a cloned gene of a mammal and secretes a peptide having the leader sequence (ie, a pre-peptide). When it is desired to express the kinase of the present invention using a mammalian cell as a host, several types of vector systems can be applied.

Various transcription / translation control sequences are used depending on the nature of the host. Sequences that play such transcriptional and translational control signals include adenovirus, bovine papilloma virus, cytomegalovirus (cy
Such genes may be derived from viruses such as tomegalovirus and simian virus, and may be sequences involved in the control of specific genes expressed at high levels. Alternatively, a promoter derived from a mammalian gene such as actin, collagen, myosin and the like can be used. A signal sequence related to the control of transcription initiation activates or suppresses transcription, and the use of such a sequence can control the level of expression. What appears to be advantageous are temperature sensitive control sequences. It can suppress and activate gene expression by changing the temperature. Others are controlled by chemicals (eg, metabolites).

Expression of the kinase of the present invention in eukaryotic cells requires control regions from eukaryotes. Such regions typically include a promoter region that directs initiation of RNA synthesis. Preferred eukaryotic cell promoters include, for example, the promoter of the mouse metallothionein I gene (Hamer et al., J. Mol.
ppi. Gen. 1: 273-288, 1982), herpesvirus TK promoter (McKnight, C
ell 31: 3.55-365, 1982), the early promoter of SCV40 (Benoist et al., Nature (Londo
n) 290: 304-31, 1981), and the promoter of the yeast gal4 gene (Johnston et al.,
Proc. Nati. Acad. Sci. (USA) 79: 6971-6975, 1982; Silver et al., Proc. Natl. A.
cad. Sci. (USA) 81: 5951-5955, 1984).

Translation of eukaryotic mRNA begins at the first methionine-encoding codon. Therefore, it encodes the eukaryotic promoter sequence and the kinase of the present invention.
Care should be taken that the sequence of the binding moiety connecting the DNA sequences (or their functional analogs) does not contain any methionine codons (ie, AUG). If such a codon is present, a fusion protein is produced (if the AUG is in the same reading frame as the sequence encoding the kinase of the invention) or (if the AUG encodes the kinase of the invention) Frameshift mutations (if they are in a different reading frame from the sequence in which they are present).

A nucleic acid molecule comprising a sequence encoding the kinase of the present invention and a promoter operably linked thereto can be introduced into prokaryotic or eukaryotic cells as non-replicating DNA or RNA. Such a nucleic acid molecule may be a linear molecule, but is more preferably a closed circular molecule. Since such a molecule cannot self-replicate, the gene is expressed by transient expression of the introduced sequence. Alternatively, if the introduced sequence is integrated into the host chromosome, gene expression will be permanent.

[0201] Vectors capable of integrating the desired gene sequence into the host chromosome can also be used. Selection of cells in which the introduced sequence has been stably integrated into the chromosome can be achieved by simultaneously introducing one or more types of markers such that the host cell containing the vector can be selected into the host. Such markers may confer prototrophy to an auxotrophic host or may be resistant to drugs (eg, antibiotics,
Resistance to heavy metals such as copper). The sequence of the selectable marker gene may be directly linked to the gene DNA to be expressed, or may be co-transfected into the same cell. Other sequence elements are also required for optimization of mRNA synthesis. Such elements include splicing signal sequences, transcription promoters, enhancers, transcription termination signals, and the like. Okayama (M
ol. Cell. Biol. 3: 280-289, 1983).

[0202] The nucleic acid molecule to be introduced can also be inserted into a plasmid or viral vector that is replicable in the recipient host. A variety of vectors are available for this purpose. Important conditions for actually selecting a plasmid or viral vector include the following. Receptor cells containing a vector and those not containing a vector can be easily distinguished and selected. The desired number of copies of the vector in a particular host. It is necessary to consider whether it is necessary to use a so-called “shuttle-type” vector, which can introduce the vector into both the host cell and cells of a different species.

Preferred prokaryotic vectors include plasmids that are replicable in E. coli (eg, pBR322, ColE1, pSClOl, pACYC184, πVX: "Molecular Cloning: A La
boratory Manual ", 1989, supra). Bacillus subtilis plasmids include pC194, pC2
21, pT127, etc. (Gryczan, In: The Molecular Biology of the Bacilli, Ac
ademic Press, NY, pp. 307-329, 1982). Suitable Streptomyces plasmids are p1J101 (Kendall et al., J. Bacteriol. 169: 4177-4183, 1987), and φ1
Streptomyces bacteriophage such as C31 (Chater et al., In: Sixth
International Symposium on Actinomycetales Biology, Akademiai Kaido, Bud
apest, Hungary, pp. 45-54, 1986). Pseudomonas plasmids are described in John et al. (Rev. Infect. Dis. 8: 693-704, 1986) and Izaki (Jpn.
Bacteriol. 33: 729-742, 1978).

[0204] Preferred eukaryotic plasmids are, for example, bovine papillomavirus (BPV),
There are vaccinia virus, viral vectors such as SV40, and circular plasmids having a 2-μ origin of replication of yeast, and analogs derived therefrom. Such plasmids are well known in the art (Botstein
Symp. 19: 265-274, 1982; Broach, In: "The Molecular Biol.
ogy of the Yeast Saccharomyces: Life Cycle and Inheritance ", Cold Sprin
g Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, C
ell 28: 203-204, 1982; Bollon et a1., J. Clin. Hematol. Oncol. 10: 39-48,
1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene
Sequence Expression, Academic Press, NY, pp. 563-608, 1980).

After preparing a vector or nucleic acid molecule containing the construct of interest for expression, the DNA construct is introduced into a suitable host cell by any suitable method. There are various introduction methods, examples of which include, for example, a transformation method, a transfection method, a conjugation method, and a protoplast fusion method (protopl fusion method).
ast fusion), electroporation, particle gun method (particl
e gun technology), calcium phosphate-precipit
ation), direct microinjection and the like.
After the introduction of the vector, recipient cells are grown in a selective medium. In this medium, only the cells having the vector grow selectively. Expression of the cloned gene produces the kinase of the present invention or a fragment thereof. Protein expression thus occurs in the transformed cells or by inducing differentiation of the cells (eg, administration of bromodeoxyuridine to neuroblasts). In order to produce the peptide of the present invention, culture can be performed under various conditions. Most preferred conditions are conditions similar to physiological conditions.

Antibodies, hybridomas, methods using the same, and hBUB1-related kinases and NEK
The present invention relates to an antibody having a binding affinity for the kinase of the present invention. This polypeptide has SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
The amino acid sequence set forth in SEQ ID NO: 10, or a functional derivative thereof, or at least 9 consecutive amino acids (preferably at least 20, 30,
35 or 40 consecutive amino acids).

The present invention also relates to antibodies having specific affinity for the kinase of the present invention. Such antibodies can be isolated by comparing the affinity for the kinase of the invention with the affinity for other polypeptides. Antibodies that selectively bind to the kinase of the present invention will be selected for use in a method for distinguishing the kinase of the present invention from other polypeptides. Such a method is, for example, a method of analyzing a change in the expression level of a kinase expressed in a tissue in which another polypeptide is mixed, but is not limited to this exemplified method.

The BUB1 kinase or NEK kinase of the present invention can be used in various procedures and methods for producing antibodies, identifying pharmaceutical compositions, and studying DNA-protein interactions, for example.

[0209] The kinase of the present invention can be used for the production of antibodies or hybridomas. It is easily understood by those skilled in the art that when an antibody is required, a peptide can be prepared by the method described in the present application and used as an antigen. The antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, fragmented versions of these antibodies, and humanized antibodies. Humanized versions of the antibodies of the present invention can be created using methods known in the art, such as chimerization or immunoglobulin complementarity determining region transfer (CDR grafting).

The present invention also relates to a hybridoma that produces the above-described monoclonal antibody or a fragment of a portion related to the binding of the antibody. A hybridoma is an immortalized cell line that can secrete a specific monoclonal antibody.

In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, "Monoclonal Antibody Technology: L
aboratory Techniques in Biochemistry and Molecular Biology, "Elsevier Sc
ience Publishers, Amsterdam, The Netherlands, 1984; St. Groth et al., J. Immu
nol. Methods 35: 1-21, 1980). Animals producing antibodies (mouse, rabbit, etc.) can be immunized with the polypeptide used. Immunization techniques are well known in the art. Such methods include subcutaneous and intraperitoneal administration of the polypeptide. It is understood by those skilled in the art that the amount of polypeptide required for immunization varies depending on the type of animal to be immunized, the antigenicity of the polypeptide, the site of administration, and the like.

To enhance antigenicity, the polypeptide may be modified or administered in admixture with an adjuvant. Methods for enhancing the antigenicity of a polypeptide are known in the art. Such methods include a method in which an antigen is bound to a heterologous protein (globulin or β-galactosidase or the like), a method in which an antigen is mixed with an adjuvant at the time of immunization, and the like.

For preparation of monoclonal antibodies, splenocytes were excised from immunized animals and SP2 / 0-Agl4
, And established as a hybridoma cell producing a monoclonal antibody. As a method for identifying whether a hybridoma cell produces an antibody having desired properties, a plurality of methods are known in the art, and any method may be used. Examples of such a method include enzyme immunoassay (ELISA) used for hybridoma screening.
assay), western blot analysis, radioimmunoassay
) (Lutz et al., Exp. Cell Res. 175: 109-124, 1988). Cloning of a hybridoma secreting a desired antibody, determination of the class and subclass of the antibody, and the like can be performed by methods known in the art (Campbell, "Monoclo
nal Antibody Technology: Laboratory Techniques in Biochemistry and Molec
ular Biology ", supra, 1984).

[0214] The production of polyclonal antibodies is carried out by a procedure in which an antiserum containing an antibody is removed from an immunized animal, and the presence or absence of an antibody having a desired specificity is screened by any of the above methods. The above antibodies are labeled to allow for detection. The detectable labeling of the antibody can be performed using the following. Radioisotopes, affinity labels (such as biotin, avidin), enzyme labels (such as horseradish peroxidase, alkaline phosphatase), (FI
Fluorescent labels (TC, rhodamine, etc.), and paramagnetic atoms. Labeling can be performed by methods known in the art (for example, references include: Stemberger et al., J Histochem. Cytochem. 18: 315, 1970; Ba.
yer et al., Meth. Enzym. 62: 308, 1979; Engval et al., Immunol. 109: 129, 1972; Godi.
ng, J. Immunol. Meth. 13: 215, 1976). The labeled antibody of the present invention can be used for any of in vitro measurement, in vivo measurement, and in situ measurement for identifying tissues and cells expressing a specific peptide.

[0215] The antibodies described above can also be immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins such as polyacrylamide and latex particles. Techniques for immobilizing antibodies on such supporting solids are known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Bla.
ckwell Scientific Publications, Oxford, England, Chapter 10, 1986; Jacob
y et al., Meth. Enzym. 34, Academic Press, NY, 1974). The immobilized antibody of the present invention can be used for any of in vitro measurement, in vivo measurement, and in situ measurement in addition to immunochromatography and the like.

Furthermore, in order to rationally design and produce peptides, anti-peptide peptides, that specifically bind to a particular peptide sequence, one of skill in the art would have to use only the techniques, methods, and kits disclosed herein for antibodies. Instead, one can use currently available techniques (Hurby et al., "Application of Synthetic Peptides: Antisense Peptides".
In Synthetic Peptides, A User's Guide, WH Freeman, NY, pp. 289-307, 19
92; Kaspczak et al., Biochemistry 28: 9230-9238, 1989).

Anti-peptide peptides can be created by exchanging basic amino acids with acidic amino acids while leaving the hydrophobic amino acids and uncharged polar amino acids present in the peptide sequence of the kinase of the present invention. . For example, a lysine, arginine, and / or histidine residue is replaced with aspartic acid or glutamic acid, and a glutamic acid residue is replaced with a lysine, arginine, or histidine residue.

The present invention further relates to a method comprising the following two steps of detecting a BUB1-related kinase or NEK kinase polypeptide in a sample. That is, (1) a step of bringing a sample into contact with the above-mentioned antibody under conditions such that an immune complex is formed; In detail, the method includes a step of mixing and reacting a test sample with one or more kinds of the antibodies of the present invention, and a step of measuring whether or not the antibody binds to the test sample. A change in the kinase of the present invention relative to normal expression levels is indicative of a possible disease.

[0219] Conditions for incubating the antibody with the test sample vary. Reaction conditions vary depending on the type of measurement, the detection method, the type and properties of the antibody used for measurement, and the like. When using the antibody of the present invention, any of the commonly used immunological measurement methods (radioimmunoassay, enzyme-labeled immunoassay, diffusion octalony, rocket fluorescence immunoassay, etc.) can be used. Is known to those skilled in the art. Examples of such measurement methods are described in books written by the following authors. That is, Chard ("An Introduction to Radioimmunoassay and Related Techniques
"Elsevier Science Publishers, Amsterdam, The Netherlands, 1986), Bulloc
k et al. ("Techniques in Immunocytochemistry," Academic Press, Orlando, FL Vo
l. 1, 1982; Vol. 2, 1983; Vol. 3, 1985), Tijssen ("Practice and Theory o
f Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecul
ar Biology, "Elsevier Science Publishers, Amsterdam, The Netherlands, 19
85).

The test samples in the immunoassay of the present invention include protein extracts and membrane extracts of cells, and biological fluid samples such as blood, plasma, and urine. The test sample used in the above method varies depending on the type of measurement, the nature of the detection method, and the types of tissues, cells, and extracts used as the test sample. Methods for preparing a protein extract / membrane extract from cells are known in the art and can be used to obtain a sample that can be measured in a measurement system to be used.

The kit contains all the reagents necessary for measurement by the above-described detection method. The kit includes: That is. (I) a means such as a first container for holding the above-mentioned antibody, and (ii) a second container for holding a complex containing a molecule with which the antibody and the labeling substance react or bind. Means. In another preferred embodiment, the kit further comprises one or more containers including one or more of the following: That is, a washing reagent and a reagent capable of detecting the presence or absence of a bound antibody.

Examples of the detection reagent include a labeled secondary antibody, or, if the primary antibody is labeled, a coloring reagent, an enzyme reagent, and an antibody-binding reagent capable of reacting with the labeled antibody. However, the present invention is not necessarily limited to these. For kits with compartmented containers, already for nucleic acid probe kits,
It is described in the present application. One of ordinary skill in the art will readily appreciate that the antibodies described in the present invention can be readily incorporated into practical kit forms well known in the art.

Isolation of Compounds Interacting with BUB1-Related Kinase or NEK Kinase The invention further comprises incubating the kinase of the invention with a compound and detecting the presence or absence of a compound bound to the kinase. The present invention relates to a method for detecting a compound capable of binding to the BUB1-related kinase or NEK kinase of the present invention. This compound
It may be a mixture of various substances, such as those contained in serum, body fluids or cell extracts.

The invention further comprises incubating cells producing the kinase of the invention in the presence of a compound, and detecting a change in the level of kinase activity or activity of the molecule to which the kinase binds. , Kinase activity or the activity of the molecule to which the kinase binds. The compound identified in this way causes a change in activity that is an indicator of the presence or absence of the compound. The compound may be contained in a mixture of various substances, such as serum, body fluids or cell extracts. Once this compound has been identified, it can be isolated by techniques well known in the art.

The invention further provides a method for agonizing (enhancing) a kinase-related activity in a mammal, comprising administering to the mammal an agonist or antagonist in an amount sufficient to elicit an agonistic or antagonistic effect. ) Or a method of producing an antagonistic action. Administering to a mammal an agonist or antagonist in an amount sufficient to agonize or antagonize a function associated with a BUB1-related kinase or NEK kinase, the agonist or antagonist of the activity of the BUB1-related kinase or NEK kinase. Methods for treating mammalian diseases using are also included in this application.

To find new treatments for disease, biochemists and chemists have designed, synthesized, and tested molecules that inhibit the function of protein kinases. Certain low molecular weight organic compound molecules have been classified as compounds that modulate the function of protein kinases. Examples of molecules that have been reported to inhibit the function of protein kinases include bis-monocyclic, bicyclic, or heterocyclic aryl compounds (M
International Patent Application WO 92/2064, issued November 26, 1992 by Aguire et al.
2), vinylene-azaindole derivatives (International Patent Application WO 94/14808 issued July 7, 1994 by Ballinari et al.), 1-cyclopropyl-4-pyridyl-quinolones (US Pat. No. 5,330,992), Styryl compounds (US Patent No. 5,217,999)
Issued on February 17, 1994 by Styryl group-substituted pyridyl compounds (US Pat. No. 5,302,606), certain quinazoline derivatives (European Patent Application No. 0 566 266 Al), selenindoles and selenides (Denny et al.). International Patent Application WO 94/0
3427), tricyclic polyhydric compounds (International Patent Application WO 92/21660 filed by Dow on December 10, 1992), and benzyl phosphonate compounds (filed by Dow et al. On October 17, 1991). International Patent Application WO 91/15495) and the like, but are not limited thereto.

Compounds that are cell permeable and resistant to acid hydrolysis have high bioavailability after oral administration to patients and can be said to have potential benefits as therapeutic agents.
However, many of these protein kinase inhibitors do not significantly inhibit the function of protein kinases. In addition, many inhibit various types of protein kinases, and therefore have multiple side effects when used as therapeutics for diseases.

However, certain indolinones have been classified as acid-resistant, membrane-permeable organic molecules. WO 96/22976 (issued August 1, 1996 by Ballinari et al.) Describes water-soluble indolinone compounds in which substituents such as tetralin, naphthalene, quinoline, and indole are attached to the oxidol ring. These bicyclic substituents are sequentially substituted with polar groups such as hydroxylated alkyl groups, phosphate groups, and ethers. "Indolinone Combinatorial Libraries and Related Products and Methods for Treating Diseases," filed Aug. 23, 1996 by Tang et al.
No. 08 / 702,232 entitled "thods for the Treatment of Disease"
(Lyon & Lyon Docket No. 221/187) and Tang et al. Filed June 7, 1995, entitled "Benzylide-Z-indoline Compounds for Treating Diseases.
ne-Z-Indoline Compounds for the Treatment of Disease), U.S. Patent Application No. 08 / 485,323 (Lyon & Lyon Docket No. 223/298);
International Patent Application WO 96/40116 published December 19, 996, WO 96/22976 published August 1, 1996 by Ballinari et al. These are all
The entire text, including the figures or tables, is incorporated herein by reference and describes the indolinone chemical library for indolinone compounds having monocyclic and other bicyclic substituents attached to the oxidol ring. . "The Indolinone Combinatorial Library and Related Products and Methods for Treating Diseases, filed August 23, 1996 by Tang et al.
al Libraries and Related Products and Methods for the Treatment of Disea
patent application Ser. No. 08 / 702,232 (Lyon & Lyon Docket No. 221/187).
And Tang et al., Filed June 7, 1995, entitled "Benzylidene-Z-Indoline Compounds for the Treatment of Diseases".
No. 08 / 485,323 entitled Treatment of Disease) (Lyon & Lyon D
ocket No. 223/298), and International Patent Application WO 96/22976, filed August 1, 1996 by Ballinari et al., describe a method for synthesizing indolinone, a method for testing the biological activity of indolinone compounds in cells, Describes the mode of inhibition of indolinone derivatives.

Examples of other substances that modulate kinase activity include, but are not limited to, tyrphostins, quinazolines, quinoxalines, and quinolines. The above-mentioned quinazolines, tyrphostins, quinolines, and quinoxalines also include known compounds described in the literature. For example, representative publications on quinazolines include Barker et al., European Patent Application Publication No. 0 520 722 Al, Jones et al., U.S. Pat.No. 4,447,608, Kabbe et al., U.S. Pat.
No. 5,757,072; U.S. Pat.No. 5,316,553 to Kaul and Vougioukas; Kreighbaum and C.
omer U.S. Pat.No. 4,343,940; Pegg and Wardleworth European Patent Application 0 562
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Orq. Chem. 54: 3511-3518 (1989); Ley and Seng, Synthesis 1975: 415-522 (197
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99 (1977), Sculier et al. Cancer Immunol. And Immunother. 23: A65 (1986), Si
Kora et al. Cancer Letters 23: 289-295 (1984), and Sikora et al. Analytical Bio
chem. 172: 344-355 (1988). All of which are cited in this application by reference to the full text including the figures.

For quinoxalines, see US Pat. No. 5,316,553 to Kaul and Vougioukas.
This reference is incorporated herein by reference in its entirety, including the figures.

For quinolines, see Dolle et al., J. Med. Chem. 37: 2627-2629 (1994); MaG
uire, J. Med. Chem. 37: 2129-2131 (1994), Burke et al., J. Med. Chem. 36: 425-.
432 (1993), and Burke et al., BioOrqanic Med. Chem. Letters 2: 1771-1774 (199
All of these references are cited in the present application by reference to the entire text including the figures.

For tilphostines, see Alien et al., Clin. Exp. Immunol. 91: 141-156 (
1993), Anafi et al., Blood 82: 12: 3524-3529 (1993), Baker et al., J. Cell Sci. 10
2: 543-555 (1992), Amer. Phvsiol. Soc. Pp. 6363-6143-.C721-C by Bilder et al.
730 (1991), Brunton et al., Proceedings of Amer. Assoc. Cancer Rsch. 33: 558.
(1992), Bryckaert et al., Exp. Cell Research 199: 255-261 (1992), Dong et al., J.
Leukocyte Bioloov 53: 53-60 (1993); Dong et al., J. Immunol. 151 (5): 2717-2.
724 (1993), Gazit et al., J. Med. Chem. 32: 2344-2352 (1989), Gazit et al., J. Me.
d. Chem. 36: 3556-3564 (1993), Kaur et al., Anti-Cancer Drugs 5: 213-222 (1994).
), King et al., Biochem. J. 275: 413-418 (1991), Kuo et al., Cancer Letters 74:
197-202 (1993); Levitzki's The FASEB J. 6: 3275-3282 (1992); Lyall et al.
Biol. Chem. 264: 14503-14509 (1989); Peterson et al., The Prostate 22: 335-34.
5 (1993), Pillemer et al., Int.J. Cancer 50: 80-85 (1992), Posner et al., Molec.
ular Pharmacology 45: 673-683 (1993), Rendu et al., Biol. Pharmacology 44 (5),
881-888 (1992), Life Sciences of Sauro and Thomas 53: 371-376 (1993), Sa
uro and Thomas, J. Pharm. And Experimental Therapeutics 267 (3): 119-1125.
(1993), Wolbring et al., J. Biol. Chem. 269 (36): 22470-22472 (1994), and
Yoneda et al. In Cancer Research 51: 4430-4435 (1991) and other documents,
All of these references are cited in the present application as full text including drawings.

Other compounds that can be used as substances that affect the activity of protein kinases include oxyindolines as described in US patent application Ser. No. 08/702/232, filed Aug. 23, 1996. And the like. This reference is incorporated herein by reference in its entirety, including figures.

[0234] can utilize various methods for producing transgenic animals associated with the transgenic animal present invention. DNA is injected into the pronucleus of a fertilized egg, and then the male and female pronuclei are fused, or DN is transferred to the nucleus of embryonic cells (eg, a 2-cell goblet nucleus) after cell division has begun.
A may be injected (Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442,
1985). Embryos may be infected with a virus, particularly a retrovirus, and modified to carry the inorganic-ion acceptor nucleotides of the invention.

Pluripotent stem cells, derived from the inner cell mass of the embryo and stabilized in culture, can be manipulated in culture to incorporate the nucleotide sequences of the present invention. Transgenic animals can be produced from such cells by implantation into blastocysts implanted in foster parents and reach maturity. Animals suitable for transgenic experiments include:
Charles River (Wilmington, MA),
It can be obtained from standard sources such as Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN) and the like.

Techniques for engineering rodent embryos and microinjecting DNA into the pronuclei of zygotes are well known to those skilled in the art (Hogan et al., Supra). Microinjection techniques for fish, amphibian eggs and birds are described in detail in Houdebine and Chourrout (Experimentia 47: 897-905, 1991).
Another technique for introducing DNA into animal tissues is described in US Pat. No. 4,945,050 (Stanford).
Et al., July 30, 1990).

As one example for generating transgenic mice, female mice are induced to be superovulated. Bred female with males, the mated females were sacrificed by CO ₂ asphyxiation or decapitation, recovering embryos from dissected oviducts. The surrounding cumulus cells are removed. The pronuclear embryo is then washed and stored until injection. Adult females with random cycles were paired with vasectomized males. Recipient females were mated simultaneously with donor females. The embryos were then surgically transferred. The procedure for producing transgenic rats is similar to that for mice (Hammer et al., Cell 63: 1099-1112, 1990).

The production of transgenic animals by introducing DNA into ES cells using methods such as culturing embryonic stem (ES) cells and using methods such as electroreporation, calcium phosphate / DNA precipitation and direct injection, is invaluable. (Teratocarcin
omas and Embryonic Stem Cells, A Practical Approach, EJ Robertson, ed
., IRL Press, 1987).

In the case of random gene transfer, a clone containing a sequence of the invention is co-transfected with a gene encoding resistance. Alternatively, a gene encoding neomycin resistance is physically linked to a sequence of the invention. Transgenic and isolation of the desired clones is performed by any one of several methods well known to those skilled in the art (EJ Robertoson, supra).

[0240] DNA molecules introduced into ES cells can also be integrated into the chromosome by homologous recombination methods (Capecchi, Science 244: 1288-1292, 1989). Methods for positive selection of recombination events (ie, neo resistance) and dual positive-negative selection (ie, neo resistance and ganciclovir resistance) and identification of the desired clones by PCR are described in The teachings are described by Capecchi, supra, and Joyner et al. (Nature 338: 153-156, 1989), which are incorporated herein by reference. The final step in the procedure is to inject targeted ES cells into blastocysts and transfer the blastocysts to pseudopregnant females. The resulting chimeric animals are bred and analyzed by Southern blot to identify individuals carrying the transgene. Techniques for producing non-rodent mammals and other animals are discussed by another author (Houdebine and Chourrout, supra; Pursel et al., Science 244: 1281-1288, 1989;
and Simms et al., Bio / Technology 6: 179-183, 1988).

Thus, the present invention provides a transgenic non-human mammal containing a transgene encoding a kinase of the invention or a gene that affects expression of the kinase. Such transgenic non-human mammals are particularly useful as in vivo test systems to study the effects of introducing a kinase or to regulate the expression of a kinase (ie, to introduce additional genes, antisense nucleic acids or ribozymes). By).

A “transgenic animal” is an animal having cells into which certain DNA has been artificially inserted into cells, wherein the DNA becomes part of the genome generated from the cells. Preferred transgenic animals are primates, mice, rats, cows, pigs, horses,
Goats, sheep, dogs and cats. The transgenic DNA can encode human STS20-related kinase. Native expression in an animal can be reduced by providing an effective amount of antisense RNA or DNA to reduce receptor expression.

Gene Therapy BUB1-related kinases or NEK kinases or their gene sequences are also useful for gene therapy (discussed in Miller, Nature 357: 455-460, 1992). Miller notes that advances demonstrating positive initial results will be a viable approach to human gene therapy. The basic science of gene therapy is Mulligan (Sc
ience 260: 926-931, 1993).

In a preferred embodiment, an expression vector containing a coding sequence for a BUB1-related kinase or NEK kinase is inserted into cells, the cells are grown in vitro, and then injected into a patient in large quantities. In another preferred embodiment, the promoter segment enhances expression of the endogenous kinase gene in a cell containing an endogenous gene encoding a kinase of the present invention, with the DNA segment containing the superior promoter (eg, a strong promoter) in the cell. (Eg, transferring the promoter segment to the cell to link directly to the endogenous kinase gene).

Gene therapy involves the use of adenoviruses containing tumor-targeted kinase cDNA, systemic increase of kinases by implantation of engineered cells, injection of the kinase-encoding virus or naked injection into appropriate tissues. It may include injection of (naked) kinase DNA.

To regulate the activity of such a protein complex, one of the protein complexes
Target cell populations can be modulated by introducing altered forms of one or more components. For example, reducing or inhibiting the activity of a complex component in a target cell can reduce, inhibit or ameliorate a signaling event beyond a certain state. Prevent abnormal, deleterious signaling events using deletions or missense variants of components that retain the ability to interact with other components of the protein complex but cannot affect signaling. Can be.

Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes viruses, some RNA viruses or bovine papilloma virus are used to encode the recombinant kinase of the protein of the invention. The sequence (eg, cDNA) can be used for delivery to a target cell population (eg, tumor cells). Recombinant viral vectors containing the coding sequence can be constructed using methods well known to those of skill in the art (Maniatis
Et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laborato
ry, NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and Wiley Interscience, NY, 1989). Alternatively, a recombinant nucleic acid molecule encoding a protein sequence may be used as naked DNA, or in a reconstituted system such as a liposome or other lipid system for delivery to target cells. (Eg, Felgner et al., Nature 337: 387-8, 1989). There are several other ways to introduce plasmid DNA directly into cells for use in human gene therapy, targeting DNA to receptors on cells by complexing the plasmid DNA to proteins (Miller, supra).

In the simplest form, gene transfer can be performed by simple injection of small amounts of DNA into the nucleus of cells by the microinjection method (Cape
cchi, Cell 22: 479-88, 1980). When recombinant DNA is introduced into cells, they
It can be recognized by the normal mechanisms of the cell for transcription and translation, and the genetic product is expressed. Other methods for introducing DNA into larger cells have been attempted. These methods include transfection in which DNA is precipitated with calcium phosphate and taken up by cells by phagocytosis (Chen et al., Mol. Cell. Biol. 7:
2745-52, 1987), electroporation in which cells are exposed to high-voltage pulses to make holes in the membrane (Chu et al., Nucleic Acids Res. 15: 1311-26, 1987), lipophilic vesicles that fuse with target cells Liposomes encapsulating DNA in DNA (Felgner et al., Pr
oc. Natl. Acad. Sci. USA 84: 7413-7417, 1987), and particle guns using DNA attached to small projectiles (Yang et al., Proc. Natl. Acad. Sci. USA 87: 9568-9572). ,
1990). Another method for introducing DNA into cells is to attach the DNA to a chemically modified protein.

It has also been reported that adenovirus proteins can destabilize endosomes and increase uptake of DNA into cells. Mixing the adenovirus in a solution containing the DNA complex, or binding the DNA to polylysine covalently linked to the adenovirus using a protein crosslinker, substantially improves uptake and expression of the recombinant gene ( Curiel et al., Am. J. Respir. Cell. Mol. Biol., 6: 247-
52,1992).

[0250] As used herein, "gene transfer" refers to a method for introducing a foreign nucleic acid molecule into a cell. Gene transfer is routinely performed so that the particular product encoded by the gene can be expressed. Products may include proteins, polypeptides, antisense DNA or RNA, enzymatically active RNA. Gene transfer may be performed in cultured cells or by direct administration to animals. In general, gene transfer involves methods of contacting a nucleic acid with a target cell by non-specific or receptor-mediated interactions, incorporation of the nucleic acid into cells through a membrane or by phagocytosis, and into the cytoplasm from the plasma membrane or endosomes. Release of the nucleic acid. Expression also requires that the nucleic acid migrate to the nucleus of the cell and bind to an appropriate nuclear factor for transcription.

“Gene therapy” as used herein is a form of gene transfer.
Included within the definition of gene transfer as used herein, specifically refers to gene transfer for expressing a therapeutic product from a cell in vivo or in vitro. Gene transfer may be performed ex vivo on the cells and then implanted into the patient, or may be performed by directly administering the nucleic acid or nucleic acid-protein complex to the patient.

In another preferred embodiment, the BU wherein the nucleic acid sequence is expressed only in certain tissues
A vector having a nucleic acid sequence encoding a B1-related kinase or NEK kinase polypeptide is provided. How to Perform Tissue-Specific Gene Expression November 3, 1992
No. 93/09236, filed on May 13, 1993 and published on May 13, 1993.

In all of the above vectors, yet another aspect of the present invention is that the nucleic acid sequence contained in the vector may be added, deleted or modified to part or all of the nucleic acid sequence, as described above. That is, it may be included.

In another preferred embodiment, a method for gene replacement is described. As used herein, "gene replacement" refers to providing a nucleic acid sequence that is expressed in vivo in an animal and thereby can provide or enhance the function of an endogenous gene that has been deleted or deleted in the animal. Means

Pharmaceutical Formulations and Routes of Administration The compounds described herein may be administered to the patient as such, or as a combination therapy, in a pharmaceutical composition mixed with other active ingredients, or with a suitable carrier or excipient. Can be administered as a pharmaceutical composition. The compounding method and administration method of the compound of this application are described in “Remington's Phamaceutical Sciences,” Mack Publishing Co.,
Easton, PA, can be found in the latest edition.

A. Routes of administration Preferred routes of administration include, for example, oral, rectal, transmucosal or intestinal administration; intramuscular, subcutaneous, intravenous and intraspinal injection, as well as
It includes parenteral administration, such as intrathecal injection, direct intraventricular injection, intraperitoneal injection, intranasal injection, or intraocular injection.

Alternatively, rather than administering the compound systemically, it may be administered topically, for example by injection of the compound directly into a solid tumor, often in a depot or in a sustained release dosage form.

In some cases, the drug can also be administered in a targeted drug delivery system, for example, in liposomes coated with a tumor-specific antibody. The liposome is targeted and will be selectively harvested by the tumor.

B. Compositions / Formulations The pharmaceutical compositions of the present invention can be prepared in a manner known per se, for example, by means of simple mixing, dissolving, granulating, dragee-forming, wet-milling, emulsifying, encapsulating, entrapping steps. Or a freeze-drying process.

The pharmaceutical compositions used according to the present invention therefore comprise one or more physiologically acceptable preparations which contain excipients and auxiliaries which facilitate the processing of the active compounds into pharmaceutically usable preparations. Can be compounded by a conventional method using a carrier which is acceptable. Proper formulation is dependent upon the route of administration chosen.

In the case of injection, the agents according to the invention may be administered in aqueous solution, preferably in Hank's solution.
solution), Ringer's solution, or physiologically compatible buffers such as saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers include tablets, suitable for ingestion by the patient to be treated,
It allows the compounds of the present invention to be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like. Suitable carriers include excipients, for example, fillers such as lactose, sucrose, mannitol, or sugars such as sorbitol, and, for example, corn starch, wheat starch, rice starch,
Cellulose preparations such as potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP) are included. If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or a salt of alginic acid such as alginic acid or sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, a lacquer solution, and a concentrated sugar solution optionally containing a suitable organic solvent or solvent mixture are prepared. Can be used. Dyestuffs or pigments may be added to the tablets or dragee coatings to identify themselves or to characterize different combinations of the active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of a plasticizer, such as gelatin or glycerol or sorbitol. included. Fillers like lactose, binders like starch,
It may contain the active ingredient in admixture with lubricants such as talc and magnesium stearate, and optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable solutions, for example in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Further, a stabilizer may be added. All formulations for oral administration should be in dosage forms suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges which can be formulated in a conventional manner.

For administration by inhalation, the compounds which can be used according to the invention can be prepared by means of a pressurized container or insufflator, for example a suitable gas such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It is easily delivered in the form of an aerosol spray propellant using a suitable propellant. In the case of a compressed aerosol, the dosage unit can be determined by using a valve to eject a measured amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration by injection, eg, as a single or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may be in such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may be incorporated with, for example, suspending, stabilizing or dispersing agents. Things may be included.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or excipients include fatty oils such as sesame oil, esters of synthetic fatty acids such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl, cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or reagents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

The active ingredient can also be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

The compounds can also be formulated in rectal compositions such as suppositories and retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound may be formulated with a suitable polymeric or hydrophobic material (eg, as an emulsion in an acceptable oil) or an ion exchange resin, or may not be sufficient, such as a poorly soluble salt. You may mix | blend as a soluble derivative.

The pharmaceutical carrier for the hydrophobic compounds of the present invention is a co-solvent system such as benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is a solution made up of 3% w / v benzyl alcohol, 8% w / v nonpolar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300 based on the volume of absolute ethanol. VPD co-solvent system (VPD: D5W) diluted 1: 1 with 5% dextrose in water
Consists of VPD. This co-solvent system dissolves hydrophobic compounds well and as such has low toxicity even for systemic administration. In general, the proportions of a co-solvent system can be varied in order to avoid destroying the solubility and toxicity characteristics. In addition, the components of the co-solvent component may be themselves replaced.For example, other low-toxic nonpolar surfactants may be used in place of polysorbate 80, the size of the polyethylene glycol fraction may be changed, or other biocompatible polymers may be used. For example, polyvinylpyrrolidone can be replaced with polyethylene glycol, and other sugars and polysaccharides can be replaced with dextrose.

[0273] Other delivery systems suitable for hydrophobic pharmaceutical compounds can also be used. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents, such as dimethyl sulfoxide, can also be used, but will usually result in greater toxicity. Further, the compounds can be delivered using a sustained release system, such as a semipermeable matrix of solid hydrophobic polymer containing the therapeutic agent. Various sustained-release materials are well established and are well known by those skilled in the art. Sustained-release capsules can, depending on their chemical nature, release the compound for a period of weeks to more than 100 days. Other strategies for stabilizing proteins depending on the chemical and biological stability of the therapeutic agent may be used.

The pharmaceutical compositions may also include suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. .

Many of the tyrosine or serine / threonine kinases that modulate compounds of the present invention can be provided as salts with counterions that do not adversely affect pharmaceutically. Salts that do not have pharmaceutically adverse effects can be formed using many acids such as, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents, which are the corresponding free salt form.

C. Effective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions where the active ingredients are contained in an effective amount to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to suppress, reduce or alleviate the symptoms of the disease, or to prolong the life of the patient. Determining a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, doses can be adjusted in animal models to achieve a circulating concentration range that includes the IC ₅₀ (ie, the concentration of the test compound that can achieve half-maximal inhibition of tyrosine or serine / threonine kinase activity) as measured in cell culture. Can be devised. Such information can be used to more accurately determine useful doses in humans.

The toxicity and therapeutic efficacy of the compounds described herein can be demonstrated, for example, in cell cultures or experimental animals, such as LD ₅₀ (the dose that kills 50% of the population) and ED ₅₀ (50% of the population).
Therapeutically effective dose) can be determined by standard pharmaceutical techniques. The dose ratio between therapeutic effect and toxicity is the therapeutic index and it can be expressed as the ratio between LD ₅₀ and ED _50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture measurements and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds, little or is preferably to fall within a range of circulating concentrations that include the ED ₅₀ with no toxicity. This dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, eg, Fingl et al., 1975, in “The P
harmacological Basis of Terapeutics ", Ch.1 p.1).

Dosage amount and interval may be adjusted individually to provide the kinase modulating effect, ie, sufficient plasma levels of the active moiety to maintain a minimum effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. That is, it is the concentration required to achieve 50-90% inhibition of the kinase using, for example, the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays are available for measuring plasma concentrations.

[0280] The dose interval can also be determined using the value of MEC. The compound maintains a plasma level above the MEC for a period of 10-90%, preferably for a period within the range of 30-90%,
Most preferably it should be administered under a regimen which maintains for a time in the range of 50-90%.

When administered topically and selectively taken up, the effective local concentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

D. The composition as long as the packaging desired, may be accommodated in a pack or dispenser device could contain one or more units of the dosage form containing the active ingredient. The pack can include, for example, a metal or plastic foil, such as a blister pack. The pack or dispenser device will be accompanied by instructions for administration. The pack or dispenser will also accompany the container with related instructions in a form prescribed by a government agency governing the manufacture, use, or sale of the drug. The notice reflects government approval for forms of the polynucleotide for administration to humans and animals. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for pharmaceutical regulations, or the product insert. Compositions containing a compound of the present invention formulated in a non-toxic pharmaceutical carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Appropriate symptoms indicated on the label may include treatment of tumors, inhibition of angiogenesis, treatment of fibrosis, diabetes and the like.

Functional Derivatives Functional derivatives of the polypeptides or nucleic acids of the invention are also provided herein. "Functional derivative" means a "chemical derivative", "fragment", or "variant" of a polypeptide or nucleic acid of the invention, which is defined as follows. A functional derivative retains the function of at least a portion of a protein, such as reactivity with an antibody specific for the protein, enzymatic action or binding action mediated by a non-catalytic domain, and is thereby utilized in the present invention. It is possible to do. It is well known in the art that many different nucleic acid sequences can encode the same amino acid sequence due to the degeneracy of the genetic code. Similarly, it is well known in the art that conservative changes to amino acids can still result in a protein or polypeptide that retains its original function. In both cases, all changes are
It is intended to be included by this disclosure.

[0285] Functional equivalents of the isolated nucleic acid molecules described herein are included within the scope of the invention. The degeneracy of the genetic code allows one codon to be replaced with another codon specific for the same amino acid, thus giving rise to the same protein. Nucleic acid sequences can vary substantially because known amino acids other than methionine and tryptophan can be encoded by one or more codons. Therefore, some or all of the BUB1 and NEK genes have a nucleic acid sequence significantly different from the nucleic acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. It may be synthesized to have. However, their encoded amino acid sequences are conserved.

In addition, the nucleic acid sequence may comprise the nucleic acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or a derivative thereof at the 5 ′ end and / or Alternatively, a nucleic acid sequence obtained by adding, deleting or substituting at least one nucleotide at the 3 ′ end may be contained. Any nucleotide or polynucleotide can be used in this regard, including adding SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 6, wherein the addition, deletion or substitution is encoded by the nucleotide sequence. The amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 10 is not changed. For example, the present invention is intended to include a nucleic acid sequence obtained by adding ATG as an initiation codon at the 5 'end of the nucleic acid sequence of the present invention or a derivative thereof, or 3' of the nucleic acid sequence of the present invention or a derivative thereof. It is intended to include a nucleic acid sequence obtained by adding TTA, TAG or TGA as a termination codon at the terminus. Further, the nucleic acid molecule of the present invention has a restriction endonuclease recognition site optionally added to the 5 'end and / or 3' end.

[0287] Such functional alterations in the subject nucleic acid sequences provide the opportunity to facilitate secretion and / or cleavage of the atypical protein encoded by the foreign nucleic acid sequence fused thereto. Accordingly, all variants of the nucleic acid sequences of the BUB1 and NEK genes of the present invention permitted by this genetic code, and fragments thereof, are included in the present invention.

In addition, a polypeptide is produced that is a structurally modified polypeptide, but that has substantially the same utility or effect as the polypeptide produced by the unmodified nucleic acid molecule. To this end, it is possible to delete codons or replace one or more codons with codons other than degenerate codons. As is recognized in the art, if there are two nucleic acid molecules that cause the production of those polypeptides, the two polypeptides will be separated even if the difference between the nucleic acid molecules is not related to the degeneracy of the genetic code. Functionally equivalent.

“Chemical derivatives” of this complex generally contain additional chemical moieties that are not part of the protein. Covalent modifications of the protein or peptide are included within the scope of the invention. Such modifications are made by reacting the targeted amino acid residue in the peptide with an organic derivatizing reagent capable of reacting with the selected side chain or terminal residue as described below. Can be introduced into the molecule.

Most commonly, cysteine residues form carboxymethyl or carboxamidomethyl derivatives by reacting with α-haloacetates (and the corresponding amines), such as chloroacetic acid or chloroacetamide. . Cysteine residues can also include bromotrifluoroacetone, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2
-Pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-
It can also be derived by reacting with a diazole.

Histidine residues can be derived by reacting with diethylprocarbonate at pH 5.5-7.0, since the reagent has considerable specificity for the histidine side chain. Parabromophenacyl bromide is also useful,
The reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

The lysine and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Induction with these reagents affects or reverses changes in lysine residues. Other suitable reagents for deriving residues including primary amines include imide esters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzene sulfonic acid, O-methyl Includes transaminase-catalyzed reactions with isourea, 2,4-pentanedione, and glyoxylate.

The arginine residue can be modified by reacting with one or several conventional reagents, ie, phenylglyoxal, 2,3-butanedione, 1,2-chlorohexanedione, and ninhydrin. . Induction of arginine residues, because of the high pK _a of _the guanidine functional group, it is necessary to proceed the reaction under alkaline conditions. Moreover, these reagents may react not only with the α-amino group of arginine, but also with the functional groups of lysine.

Tyrosine residues are well-known targets for modification to introduce spectral labels by reacting with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form the O-acetyltyrosyl moiety and the 3-nitro derivative, respectively.

The carboxyl group of the side chain (of aspartic acid or of glutamic acid) is 1-cyclohexyl-3- (2-morpholinyl (4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4- It can be selectively modified by a reaction using a carbodiimide (R'-N-C-N-R ') such as dimethylpentyl) carbodiimide, and the aspartic acid residue and the glutamic acid residue can be reacted with an ammonium ion. , To asparagine and glutamine residues.

Glutamine and asparagine residues are often deaminated to the corresponding glutamic and aspartic acid residues. These residues are also deaminated under mildly acidic conditions. Either form of these residues falls within the scope of the invention.

Derivation with bifunctional reagents can be achieved, for example, by cross-linking the peptide components of the protein or cross-linking with other proteins to form a complex with a water-insoluble support matrix or other macromolecular carrier. This is useful when converting Commonly used crosslinking reagents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, Homobifunctional imide esters, including disuccinimidyl esters such as 3'-dithiobis (succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane Is included. Derivative reagents such as methyl-3- [p-azidophenyl) dithiolpropioimidate produce photoactive intermediates capable of forming crosslinks in the presence of light. Also, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates, and U.S. Patent Nos. 3,969,287, 3,691,016, 4,195,128
Nos. 4,247,642, 4,229,537, and 4,330,440 are used to immobilize proteins.

Other modification reactions include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of serine or threonine residues, α-linkage of lysine, arginine, and histidine side chains.
-Methylation of amino group (Creighton, TE, Proteins: Structure and Mole
cular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983))
, N-terminal amine acetylation reactions, and, to name but a few examples, C-terminal carboxyl group amidation reactions.

By deriving the moiety in this way, stability, solubility, absorbability, biological half-life and the like can be improved. This portion can also reduce or mitigate unwanted side effects such as protein complexes. Portions capable of mediating such effects are described, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing.
Co., Easton, PA (1990).

The term “fragment” is used to indicate a polypeptide derived from the amino acid sequence of a protein, which is a complex that is smaller in length than the full-length polypeptide from which it is derived. Used. Such fragments may be produced, for example, by proteolysis of a full-length protein. Preferably, the fragment will have one or more amino acids at the C-terminus, N-terminus, and / or at one or more sites within the original sequence, by appropriately modifying the DNA sequence encoding the protein. It is obtained by recombination by deletion. Protein fragments are
It is used to screen for substances that act to modulate signal transduction as described herein. It will be appreciated that such fragments will retain one or more characteristic portions of the original complex. Examples of such retained features include catalytic activity, substrate specificity, interaction with other molecules in intact cells, regulatory functions, or antibodies specific for the original complex, or epitopes thereof. And the binding property.

[0301] Another functional derivative allegedly included within the scope of the present invention is one that lacks or has one or more amino acids, or has been added or substituted, relative to the original polypeptide. A "variant" polypeptide comprising amino acids or any of them. This variant adds codons for one or more amino acids from the naturally occurring complex component at the C-terminus, N-terminus, and / or at one or more sites in the original sequence, It can be induced by removing and / or modifying the DNA coding sequence of the protein appropriately. Such variants having amino acids that are added, substituted and / or additional, can be used to remove one or more characteristic portions of the original protein, as described above. We understand that we hold.

[0302] Functional derivatives of proteins with deleted, inserted and / or substituted amino acid residues can be prepared using standard methods well known to those skilled in the art. For example, the modified component of the functional derivative may be subjected to a modification reaction of the nucleotides of the DNA encoding the modified coding sequence such that the modified coding sequence is modified, and then the prokaryote or Site-directed mutagenesis to express this recombinant DNA in eukaryotic host cells (Adelman et al., 1983, DNA 2
: 183). Amino acid deletion,
Proteins with insertions and / or substitutions can be readily synthesized by direct chemical synthesis using methods well known in the art. A functional derivative of this protein usually exhibits the same quality of biological action as the original protein.

[0303] The following examples are not intended to limit the various aspects and features of the present invention is intended to be merely illustrative. The following example illustrates the isolation and characterization of a serine / threonine kinase of the present invention.

Example 1 Isolation of cDNA Encoding Human BUB1 Protein Kinase A. Materials and Methods Identification of New Clones A guanidine salt / phenol extraction method from Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987)
) Was used to isolate total RNA from primary human colon cancer cells HCC2998. This RNA is used in the Superscript Preamplification System (Superscript Preamp
lification System) (GIBCO BRL, Gaithersburg, MD; Gerard, GF
(1989) FOCUS 11, 66) was used under the conditions recommended by the manufacturer to produce single-stranded cDNA. Typical reaction is 1.5μ with 60μl reaction volume
10 μg of total RNA was used with g of oligo (dT) 12-18. This product was treated with RNaseH and diluted with 100 μl of H2O. 3 μl of this single-stranded cDNA was used in each reaction for subsequent PCR amplification.

The degenerate oligonucleotides were synthesized on an Applied biosystems 3948 DNA synthesizer using established phosphoramidite chemistry,
Ethanol precipitated and used for PCR without purification. The sequence of the degenerate oligonucleotide primer is as follows: These primers were derived from the sense and antisense strands of the amino acid motifs BUB HRD = EVGIIHG (SEQ ID NO: 16) and BUB DVW = GDXKPDNS (SEQ ID NO: 17), respectively. Nomenclature of degenerate nucleotide residues is: N = A, C, G or T; R = A or G
Y = C or T; H = A, C or T but not G; D = A, G or T but not C;

A PCR reaction was performed using degenerate primers applied to NCI colon HCC2998 single stranded cDNA. Primers are 10 mM TrisHCl (pH 8.3) to a final concentration of 25 μM each.
, 50 mM KCl, 1.5 mM MgCl 2, 200 μM deoxynucleoside triphosphate, 0.001
% Gelatin, 1.5 U AmpliTaq DNA polymerase (Perkin-Elmer / Cetus) and 3μ
l Added to the mixture containing cDNA. Following denaturation at 95 ° C for 3 minutes, cycle conditions were 9
Perform 3 cycles of 4 ° C for 30 seconds, 37 ° C for 1 minute, 72 ° C for 1 minute and 45 seconds, followed by 94 ° C for 30 seconds and 50 ° C for 1 minute.
And 35 cycles of 1 minute and 45 seconds at 72 ° C. PCR migrated between 220-250 bp
Fragments were isolated from a 2% agarose gel using the GeneClean Kit (Bio101) and TA cloned into pCRII vector (Invitrogen Corp. USA) according to the manufacturer's protocol.

Colonies were selected for plasmid DNA mini preparation, plasmid DNA was purified using Qiagen columns and cycle sequencing dye terminator kit FS (ABI, Foster City, CA) using AmpliTaq DNA polymerase. )
Was used for sequencing. ABI Pris for sequencing reaction products
run on a m377 DNA sequencer and run the BLAST alignment algorithm (Altsc
hul, SF) et al. (1990) J. Mol. Biol. 215: 403-10).

B. Isolation of cDNA clones A human leukemia 5'-stretch plus λgt11 cDNA library (Clontech) was probed with a 238 bp PCR fragment of SuSTK34a (R51-40-18) corresponding to human BUB1. Probes are ³² P-labeled by random priming and 2x10 ⁶ cp
used at m / ml, followed by standard procedures for library screening. Prehybridization (3 hours) and hybridization (overnight) were performed using 5X SSC, 5X Denhart containing 100 mg / ml denatured salmon sperm DNA.
) Solution, 2.5% dextran sulfate, 50 mM Na2PO4 / NaHPO4, pH 7.0, 50% formamide at 42 ° C. Stringent washes 65 in 0.1X SSC and 0.1% SDS
C. was performed. DNA sequencing was performed on both strands using the cycle sequencing dye terminator kit FS (ABI, Foster City, CA) using AmpliTaq DNA polymerase. The sequencing reaction products were run on an ABI Prism 377 DNA sequencer.

Results SuSTK34a (R51-40-18) and SuSTK34b (R51
Two fragments corresponding to the two isoforms of human BUB1 designated -40-4) are isolated. SusTK34a and SusTK34b have a 1 in kinase subdomain VII of SusTK34b.
Identical except that 5 nucleotides have been deleted, resulting in a predicted deletion of 5 amino acids.

The full-length human bub1 clones are four EcoRI cDNA fragments, J6E0.45 # 9, J7E1
.3 # 3, J6E1.3 # 5, and J2E0.9 # 8. 3 of a single lambda clone
The 436 bp NotI fragment was subcloned into the pBluescript (SKII +) vector and
Yielded 10. The full-length human bub1 cDNA (SEQ ID NO: 1) is 3408 bp long and 3255 bp ORF (45-3300) adjacent to the 44 bp (1-44) 5 'UTR and the 108 bp 3' UTR.
Consists of A potential polyadenylation signal begins at position 3404. The sequence adjacent to ATG is the same as the Kozak consensus sequence for the starting methionine, which is thought to be the translation start site for human BUB1.

Example 2: Mammalian BUB1 Protein Kinase Expression Assay Materials and Methods Northern Blot Analysis Bub1 expression was determined in 22 adult tissues (thymus, lung, duodenum, colon, testis, cerebrum,
Cerebellum, cortex, salivary gland, liver, pancreas, kidney, spleen, stomach, uterus, prostate, skeletal muscle, placenta, mammary gland, bladder, lymph nodes, adipose tissue), two human fetal normal tissues (fetal liver, fetus) Brain) and 60 human tumor cell lines (HOP-92, EKVX, NCI-H23, NCI-
H226, NCI-H322M, NCI-H460, NCI-H522, A549, HOP-62, OVCAR-3, OVCAR-
4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, SNB-19, SNB-75, U251, SF-26
8, SF-295, SF-539, CCRF-CEM, K-562, MOLT-4, HL-60, RPMI8226, SR, D
U-145, PC-3, HT-29, HCC-2998, HCT-116, SW620, Colo-205, HTC15, KM
-12, UO-31, SN12C, A498, CaKi1, RXF-393, ACHN, 786-0, TK-10, LOX IM
VI, Malme-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257
, M14, MCF-7, MCF-7 / ADR RES, Hs578T, MDA-MB-231, MDA-MB-435, MDA
-N, BT-549, T47D) were analyzed by Northern blot prepared for analysis.

[0312] Total RNA samples were analyzed on a denaturing formaldehyde 1% agarose gel and transferred to nitrocellulose membranes (BioRad, CA). Thirteen different on charged nylon membranes (human blot # 7760-1, human immune system blot # 7754-1, and human cancer cell line blot # 7757-1, Clontech, Palo Alto, CA) Human normal tissues (heart, cerebrum, placenta, lung, liver, kidney, pancreas, spleen, lymph nodes, thymus, peripheral blood leukocytes, bone marrow, fetal liver) and eight human tumor cell lines (HL-60, Hela Cell S3, K-562, M
OLT-4, Raji, SW480, A549, G361) 2 µg per lane of polyA + mRNA
Northern blots of two other human normal tissues containing were also hybridized. The filters were hybridized using a randomly primed [α ³² P] dCTP-labeled probe synthesized from EcoRI / PstI, a 809 bp human bub1 fragment. Hybridization overnight at 42 ° C, 6x SSC, 0.1% SDS, 1x Denhardt's solution, 100x
In semen in [mu] g / mL denatured herring was performed with 1~2 × 10 ⁶ cpm / mL of ³² P- labeled DNA probe. The filter was washed with 0.1 × SSC / 0.1SDS at 65 ° C. and applied to a Molecular Dynamics phosphor imager.

[0313] STK plasmid sequence blots show that many S, such as bub1 (pSG110),
Prepared by loading 0.5 μg of the denatured plasmid encoding TK. [Α ³² P
The dCTP-labeled single-stranded DNA probe was synthesized from RNA isolated from immune cells or tissues. Hybridization for 16 hours at 42 ℃, 6 × SSC, 0.1 % of SDS, 1 × Denhardt's solution, ^[32 P] in the DNA of the semen of 100 [mu] g / mL denatured herring dCTP labeled - single-stranded probe, 10 ⁶ cpm / ML. 0.1 × SSC / 0.1% SDS
At 65 ° C. and run on a Molecular Dynamics phosphor imager for quantitative analysis.

Results Expression of human BUB1 RNA in normal tissues and tumor cell lines A single human bub1 mRNA transcript of approximately 4.2 kb was confirmed by Northern analysis,
It was found to be expressed in nine normal human tissues (thymus, testis, heart, placenta, lung, lymph nodes, thymus, bone marrow, and fetal liver). The remaining 19 normal human tissues (duodenum,
Colon, cerebrum, cerebellum, cortex, salivary glands, liver, pancreas, kidney, spleen, stomach, uterus, prostate, skeletal muscle, fetal brain, mammary gland, bladder, adipose tissue, peripheral blood leukocytes) were negative. Of the human tumor cell lines, 60 out of 64 were positive for expression of human bub1. The very strong expression of bub1 is shown in the following cell lines: K-562, SW-
480, NCI-H23, NCI-H226, NCI-H522, OVCAR-4, CCRF-CEM, RPMI8226, HTC1
5, CaKi1, RXF-393, 786-0, TK-10, SK-MEL-28, UACC-62, M14, MCF-7,
Detected in MCF-7 / ADR RES, MDA-MB-231, MDA-N. BUB1 expression was negative in four cell lines (SK-OV-3, PC-3, HT-29, A498) and moderate in the remaining cell lines (Table 1).

[0315]

[Table 1] Analysis of bub1 RNA expression in human normal tissues and cancer cells Scale: 0 = negative, 1 = weak, 2 = medium, 3 = strong, 4 = very strong * mRNA Northern blot

Expression of human bub1 in immune tissues and cells Expression of human bub1 was determined by [α ³² P] dCTP-labeled single-stranded DNA obtained from human immune cells.
Was analyzed by hybridizing to a nylon membrane containing plasmid DNA encoding bub1 and other STKs. Expression analysis based on this DNA sequence revealed 14 human immune tissues and cell lines: thymus, dendritic cells, mast cells, monocytes, B cells (primitive, Jurkat, RPMI8226, SR), T cells ( CD8 / CD4 +, TH1, TH2, CEM, MOLT4),
And K562 (megakaryocytes). bub1 expression is expressed in four of these immunogens,
That is, it was restricted to RPMI (B cells), MOLT4 (T cells), CEM (T cells), and primitive monocytes, and was not expressed from the other 10 samples (data not shown) .
This expression profile indicates that human bub1 is expressed in several hematopoietic cell types.

[0317] Real 施例3: construct expression construct expression results expression plasmid of human bub1 gene expression plasmid construction and recombinant bub1 were formed for human bub1, include: a) to c) to it . That is, a) a full-length bub1 nucleotide base sequence with or without an HA label, H
Nucleotide base sequence encoding the N-terminal domain of BUB1 with or without the A label (amino acids 1-332), nucleotide base encoding the central-domain of BUB1 with or without the HA label (amino acids 333-757) Sequence, nucleotide sequence encoding the C-terminal kinase domain of BUB1 with or without HA tag (amino acids 775-1085), and amino acid 821 of BUB1 with or without HA tag
Encodes a pcDNA3.1 expression plasmid containing the nucleotide sequence of full-length bub1 in which Lys is mutated to Arg, and b) encodes the full-length nucleotide sequence of bub1 and the N-terminal domain of BUB1 (amino acids 1-32) A pGEX 4T-3 fusion plasmid comprising a nucleotide base sequence, a nucleotide base sequence encoding the central domain of BUB1 (amino acids 333-757), and a nucleotide base sequence encoding the C-terminal kinase domain of BUB1 (amino acids 775-1085) C) pEGFP fusion plasmid containing the full-length nucleotide sequence encoding bub1. The N-terminal domain of BUB1 and the K to R mutation are predicted to function as dominant negative constructs, which may help elucidate the function of BUB1.

Example 4: Recombinant Expression of BUB1 Serine / Threonine Protein Kinase Materials and Methods Transient Expression of BUB1 in Mammalian Cells Encoding full length, truncated, or dead form of BUB1 The pcDNA expression plasmid (10 μg DNA / 100 mm plate) containing the sequence was introduced into human 293 cells with Lipofectamine (Gibco BRL). After 72 hours, cells were diluted with 0.5 mL of lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 μg / mL aprotinin). Aliquot 7.5% acrylamide / 0.5% bis-
The sample was analyzed by SDS polyacrylamide gel electrophoresis on an acrylamide gel, and transferred to nitrocellulose by electrophoresis. Non-specific binding was blocked by pre-incubating the blot in Blotto (phosphate buffered saline containing 5% w / v skim milk and 0.2% v / v nonidet p40 (Sigma)). Recombinant proteins include various
Detection was performed by Western analysis using BUB1 antiserum or anti-HA specific antiserum. BUB1-antiserum was diluted 1: 1,000 fold and the anti-HA monoclonal antibody (
(Babco, Berkeley, Califoynia) diluted 1: 10,000-fold and used for the primary antibody reaction of Western blot analysis. Goat anti-rabbit HRP (BioRad, Richmond, Calif
ornia) as a secondary antibody to BUB1-antiserum, and goat anti-mouse HRP
(BioRad, Richmond, California) was used as a secondary antibody to the anti-HA antibody. ECL reagent (Amersham Life Science,) was used for detection.

Results The cell lysates (30 μg each) were subjected to 7.5% SDS PAGE followed by Western blot analysis. The BUB1 protein has a predicted molecular weight of 120 kDa. Recombinant human BUB1 migrates as a single polypeptide band of 150 kDa. The higher molecular weight than expected is likely due to a post-translational modification reaction, probably a cetine / threonine phosphorylation reaction. The cut form moved as expected. That is, the N-terminal domain is 45 kDa (with HA tag) and the central-domain is 56 kD
moved to a.

Example 5: BUB1 Serine / Threonine Protein Kinase Specific Immunoreagent
Production Materials and Methods Specific immunoreagents were raised in rabbits against a MAP-conjugated synthetic peptide corresponding to human BUB1. Each peptide was injected into two rabbits. The peptide number, amino acid sequence, and the region of BUB1 from which the peptide was derived are as follows. 505A: QKYNQRRKHEQWVNE (SEQ ID NO: 18, amino acids 57-271, N-terminal), 506A: D
DKDEWQSLDQNED (SEQ ID NO: 19, amino acids 485-498, central domain), 507A: KNI
QKFVLKVQK (SEQ ID NO: 20, amino acids 813-824, catalytic domain), and 508A: RQK
LKKVFQQHYTNK (SEQ ID NO: 21, amino acids 1051-1965, catalytic domain).

Another immunoreagent is generated by immunizing a rabbit with a bacterially expressed GST-fusion protein containing the N-terminal or C-terminal domain of BUB1.

Affinity Purification of Anti-BUB1 Peptide Antibody 10 mg of BUB1 peptide was added to 2 mL of Immunolink (Pierce AminoLin).
k Plus). This link consists of an aldehyde-supporting arm that forms a covalent bond to a primary amine via a Schiff base intermediate. The BUB1 peptide (ligand) was immobilized on an aminolink plus coupling gel. The remaining active sites were blocked by adding a weakening reagent. The column was subsequently washed before use.

20 mL of the filtered anti-BUB1 antiserum was applied to the immobilized antigen column. Purified anti-BUB1 IgG was eluted with 1 M propionic acid while neutralizing with 3 M Tris pH 7.4 during elution and buffered with 100 mM TEA buffered with 1 M Tris pH 6.8. did.
The purified antibody was dialyzed against phosphate buffer pH 7.4 and stored in a solution containing 20% glycerol and 0.03% NaN ₃ in PBS.

Results The antiserum raised from the anti-BUB1 peptides 506A, 507A and 508A was
-Tested by Western analysis using BUB1 fusion protein. Although binding was clearly detected in blood collected from two rabbits, 505A failed to elicit a specific immunogenic response.

The affinity-purified anti-BUB1 antibodies were tested by Western analysis using a GST-BUB1 fusion protein or a mammalian cell lysate obtained from transiently transfected and expressed BUB1 protein. Purified anti-506A is very antigenic and has a 1: 20,000 ratio for testing against recombinant BUB1 protein.
It should have been diluted by a factor of two. Anti-507A and anti-508A can be purified similarly.

Example 6 Endogenous Expression of BUB1 Serine / Threonine Protein Kinase Materials and Methods A number of human tumor cell lines (HT-29, Calu-6, HCT15, MCF7, A549, C33a, Hela,
HT-29, H1299, SH422C, COLO205, SW480, SW837, SW948, and SNB19) were grown to confluence. Transfer the protein lysate to HNTG buffer (20 mM HEPES, pH7
.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 2 mM
Phenylmethylsulfonyl fluoride, 1 μ / mL aprotinin). Use this protein concentrate as a BioRad assay (BioRad, Richmond, California)
Was measured by The protein obtained from each sample (30 μg each) was tested by Western analysis using purified anti-BUB1 506A antibody.

Results Fifteen human tumor cell lines expressed detectable levels of endogenous BUB1 protein. However, very low RNA and protein levels of BUB1 were detected in MCF7 and SNB19 cell lines.

Example 7: Cell cycle preparation of BUB1 Materials and methods Hera cells were synchronized by adding 0.5 M thymidine and 250 μg / mL aphidicolin. 0, 2 hours, 4 hours, 6 hours after removing thymidine and aphidicolin
Sampled after hours, 8, 9, 10 and 12 hours. In other experiments, thymidine (
G1 / S / G2) or nocodazole (M) was used to block Hela cells at different stages of the cell cycle by starving serum. Total RNA was isolated from synchronized cells and cell cycle blocked cells. Total RNA (10μg)
Was placed on a nylon membrane. RNA blots were hybridized using the 809 bp probe for bub1 EcoRI / PstI.

Results Northern blot analysis involving RNA derived from Hela cells either at an exponential growth stage or arrested at various stages of the cell cycle showed that bub1 expression Probe operation. Although detectable bub1 signal was observed in cells has increased exponentially, cells growing in serum-starved contrast, i.e. G ₁ / S / G ₂ stage block by thymidine / aphidicolin No signal was present in the samples obtained from the cells being tested. However, high levels of bub1 expression were observed in cells arrested at the mitotic stage by nocodazole. Bub1 RNA was expressed at very low levels immediately upon release from the synchronized state, peaking 8-10 hours (during mitosis) after drug release. This expression level decreased 11-12 hours after the drug release (terminal phase, ie, exiting the mitotic phase). We believe that bub1 is regulated at the mitotic stage,
And they came to the conclusion that it is greatly expressed in mitosis.

Example 8: Protein localization of serine / threonine protein kinase of BUB1
Materials and Methods The subcellular localization of the BUB1 protein was determined by immunofluorescence using a BUB1-specific antibody. Hela and H1299 cells were grown to confluence. Cells were trypsinized, then seeded onto 60 mm plates with 1 cm poly-lysine pretreated coverslips and cultured at 37 ° C. for 1 day. After removing the medium, the cells were washed with 5 mL of ice-cold PBS, followed by fixation with -20 ° C cooled 100% methanol. Methanol-fixed cells can be used for about a week. Cells were blocked with 30 μL of immunofluorescence blocking buffer (3% BSA, PBS solution containing 0.01% Triton X100) for 30 minutes and washed with PBS containing 0.01% Triton X100. The purified anti-BUB1 peptide 506 antibody was diluted 1: 100 in blocking buffer. Goat anti-rabbit Ig conjugated with rhodamine or fluorescein (FITC)
The AffinPure F (ab) 2 fragment of G was diluted 1: 400 and used as a secondary antibody. Dapi (62.5 μg / mL) was used for nuclear staining.

Results Immunofluorescence experiments to examine the intracellular localization of endogenous BUB1 were performed using anti-BUB1 antibodies affinity purified on both Hela and H1299 cells. BUB1 protein was localized exclusively to the centromere during mitosis. The BUB1 antibody stained all chromosome pairs in prophase and early metaphase, proving that BUB1 is a centromere protein.

The immunofluorescence experiments also showed that 293T transiently transfected with pcDNA3.1.BUB1 was used.
It was also performed on cells. The results showed that BUB1 protein did not co-localize with Crest, but appeared to bind to microtubules when BUB1 protein was overexpressed after transient transfection. Cells overexpressing BUB1 divide normally and normally, but the BUB1 protein is not localized to the centromere.

Example 9 In Vivo and In Vitro Kinase Assay for BUB1 Human 293T cells were transfected with the pcDNA3.1.BUB1.HA expression plasmid. Crude protein lysates were extracted from the transfected cells and used in the BUB1 kinase activity assay. Four separate assays were performed: (1) BUB1 kinase autophosphorylation in vitro, (2) BUB1 phosphorylation of exogenous substrate,
(3) BUB1 kinase activity in vivo, and (4) BUB1 phosphoamino acid analysis.

In Vitro Assay for BUB1 Kinase Autophosphorylation Reaction Materials and Methods HA-labeled wild type (BUB1.HA), N-terminal truncated (BUB1.N.HA), or kinase inactivated form of BUB1 (BUB1.N.HA) PcDNA3.1BUB1 expression plasmid containing BUB1.KR.HA) was introduced into human 293T cells together with lipofectamine (Gibco BRL, Gaithersburg, MD). 48
After time, the cells are washed with 0.4 mL of kinase lysis buffer (50 mM HEPES, pH 7.5, 100 mM
KCl, 25 mM NaF, 1 mM sodium ortho-vanadate, 0.5 mM Nonidet P-40
(Shigma, St. Louis, MO), 1 mM DTT, 170 μg / mL PMSF, 25 μg / mL leupeptin, 20 μg / mL trypsin inhibitor, 25 μg / mL aprotinin). Anti-HA antibody (Babco, Berkeley,
CA) and protein G beads (Boehringer Mannheim, Indianapolis, IN), followed by three washes with kinase lysis buffer, followed by kinase buffer (20 mM HEPES, pH 7.5, 150 mM KCl). , 10 mM MgCl ₂ ) twice. 20 μL of kinase reaction mixture (1 × kinase buffer, 1 mM DTT, 0.1 mM ATP, 10 μCi ³² P
-R-ATP) is added to the washed beads and the mixture is stirred with constant stirring.
Incubated at 37 ° C for minutes. The reaction was stopped by adding 8 μL of 5 × PAGE buffer and boiling at 100 ° C. for 5 minutes.

Results Kinase assay using BUB1.HA, BUB1.KR.HA (kinase is inactivated), and BUB1.N.HA (dominant / negative) contained in the transient cell lysate of 293T. Was done. Radiolabeled-protein was immunoprecipitated with anti-HA antibody, 10%
On a polyacrylamide SDS gel. 150kDa protein is wild type BUB1
It was detected by autoradiography from the sample containing the gene, but not from the kinase-inactivated BUB1 and the dominant / negative BUB1, that is, the vector-only sample. BUB1 protein has kinase activity and can autophosphorylate.

In Vitro BUB1 Phosphorylation of Exogenous Substrates Materials and Methods In vitro kinase assays using exogenous substrates indicate that the kinase reaction mixture contains 1 mg / mL of histone H1, myelin basic protein, or alpha casein protein. (Boehringer Mannheim) but identical to the in vitro autophosphorylation assay.

Results According to the autoradiography using BUB1 obtained from the immunoprecipitated transiently transfected cell extract, the phosphorylation of histone H1 by BUB1 was strong and the phosphorylation of myelin basic protein was weak. And no signal was detected for alpha casein. Histone H1 is a suitable substrate for BUB1 protein kinase in vitro.

The in vitro kinase assay also uses radiolabeled-BUB1.N protein (50k
Da) is detected in an in vivo kinase assay (herein), so that BUB1
N-terminal, i.e., truncated form, can also be used to test whether it can be a substrate for BUB1 kinase, which means that the BUB1.N protein
It suggests that it is a substrate for an unidentified kinase or a BUB1 kinase. Immunoprecipitated BUB1.N as substrate for immunoprecipitated BUB1
Autoradiography of this assay with, showed that the BUB1.N protein could be detected (results not shown). Therefore, BUB1.N protein is BUB1
It turns out to be a weak substrate for the kinase.

In Vivo BUB1 Kinase Assay Materials and Methods pcDN containing HA-labeled full-length, truncated, or kinase inactivated forms of BUB1
A3.1. BUB1 expression plasmid (10 μg DNA / 100 mM plate) was introduced into human 293T cells together with lipofectamine (Gibco BRL). Forty-eight hours later, the cell culture medium was dialyzed with 10% fetal bovine serum (Gibco BRL) and contained phosphate-free medium (Gibco BRL).
). Twenty hours after the medium was changed, inorganic ³² P-phosphate was added at 2 mCi / 100.
The plate was added to the medium in a mm plate and the cells were then incubated for 3 hours. After addition of 12 μL pervanadate solution (50 mM sodium ortho-vanadate, 15% hydrogen peroxide), the cells were incubated for another 10 minutes. The cell culture dish was placed on ice and rinsed with 4 ° C. phosphate-buffered saline. The crude protein lysate was mixed with 450 μL of kinase lysis buffer (50 mM HEPES, pH 7.5, 100 mM
KCl, 25 mM NaF, 1 mM sodium ortho-vanadate, 0.5 mM Nonidet P-
40 (Shigma), 1 mM DTT, 170 μg / mL PMSF, 25 μg / mL leupeptin, 20 μg / mL
mL trypsin inhibitor, 25 μg / mL aprotinin) and immunoprecipitated with anti-HA antibody.

Results The assay was transiently transfected with BUB1 or BUB1.N.
This was done by adding inorganic ³² P to 93T cells. Crude protein was extracted from the radiolabeled cells. Immunoprecipitating the cell lysate with an anti-HA antibody,
This was followed by running on a 10% polyacrylamide SDS gel. Phosphorylated band is BUB
It was observed at the position of the molecular weight of 150 kDa corresponding to 1. No signal was detected in the vector only control. A weak signal was observed from the BUB1.N transfected sample. This was not seen in an in vitro kinase assay.

BUB1 Kinase Phosphoamino Acid Analysis Materials and Methods The Hunter protocol was used for BUB1 kinase phosphoamino acid analysis. After completing the in vitro kinase assay, radiolabeled-BUB1
Bands were cut out of the dried polyacrylamide gel. A piece of this gel
It was rehydrated by placing it in 0.5 mL of 50 mM ammonium bicarbonate, pH 7.3-7.6 for 5 minutes and then homogenized into a fine suspension. The volume of the suspension was adjusted to 1 mL using 50 mM ammonium bicarbonate with a final concentration of 5% beta-mercaptoethanol (v / v) and 0.1% SDS (w / v). The mixture was incubated at 100 ° C. for 5 minutes, followed by incubation at 37 ° C. for 12 hours with agitation. The particulate fraction was collected by centrifugation, extracted once more with 50 mM ammonium bicarbonate, pH 7.3-7.6, 5% beta-mercaptoethanol (v / v), 0.1% SDS (w / v) Using 100
Incubated for 5 minutes at 37 ° C, followed by 60 minutes at 37 ° C. After centrifugation, the liquid extract was combined with the previous extract to make a total volume of 1.2 mL. The extract was centrifuged again to remove particulate matter, and the supernatant was isolated and refrigerated at 4 ° C. A total of 20 μg of bovine serum albumin was added to the extract, and the protein was precipitated by adding 250 μL of 100% trichloroacetic acid followed by 4 μl.
Incubated at 60 ° C for 60 minutes. The precipitated protein was isolated by centrifugation, and the resulting pellet was washed at 4 ° C. with 500 μL of 100% acetone. The protein pellet was air dried and then dissolved in 5.7 M HCl, 100 μL. The solution was heated at 100 ° C. for 60 minutes in a tube with a screw cap, followed by vacuum drying.
The pellet of the residue was treated with 67 μg / mL phosphoserine, 67 μg / mL phosphothreonine,
PH1.9 formic acid (2% v / v) and glacial acetic acid (7.3% v / v) containing 67 μg / mL phosphotyrosine
/ V). A sample containing a total of 600 cpm was transferred to a cellulose TLC plate (VWR sc
ientific, San Francisco, CA). Two-dimensional chromatography was performed. Gel in one dimension is formic acid (2% v / v) and glacial acetic acid (7.3% v / v), p
It was developed in a buffer consisting of H1.9 at 1.0 kV for 25 minutes. The plate was air dried followed by glacial acetic acid (5% v / v), pyridine (0.5% v / v), 0.5 mM EDTA, pH 3.5.
The gel was subjected to two-dimensional gel electrophoresis at 1.3 kV for 16 minutes in a buffer consisting of
The plate was air dried again, followed by spraying with a solution containing 0.25% ninhydrin in acetone and coloring at 65 ° C for 15 minutes. The position and identity of the labeled amino acids were detected by autoradiography.

Results A hunter protocol was performed to examine the phosphorylation site (amino acid) for BUB1 protein kinase. Radiolabeled-amino acids were detected at the phosphoserine and phosphothreonine positions, but no signal was detected at the phosphotyrosine position. Thus, BUB1 kinase is a serine / threonine protein kinase.

Example 10: High throughput screen for BUB1 protein kinase inhibitor
Developing a Protocol to Perform BUNing Because BUB1 RNA is overexpressed in many cancer cells, BUB1 kinase activity is probably up-regulated in many cancer cells, causing loss of the mitotic checkpoint. Inhibition of BUB1 kinase activity could be a way to suppress uncontrolled cell division and thus inhibit tumor cell growth. By utilizing the overexpression of the recombinant BUB1 protein, in vitro kinase inhibitor screening can be performed.

Recombinant baculoviruses have become widely used as vectors for expressing atypical genes in cultured insect cells. Atypical genes under the control of the strong polyhedrin promoter of Autograhpa california nucleopolyhedrovirus (AcNPV) are frequently expressed in late stages of infection. In most cases, the recombinant protein is cleaved, modified and then directed to the appropriate cell location, where it is functionally equivalent to its actual counterpart.

Expression of Recombinant BUB1 in Baculovirus System Materials and Materials The baculovirus expression system was purchased from Gibco BRL, Gaithersburg, MD. BUB1.
The HA wild type and BUB1.KR.HA (kinase was inactivated) were subcloned into pFastBacHtb (having a histidine label at the N-terminus) and pFastBacG2T (a glutathione transferase gene with the histidine label replaced). Recombinant plasmids were constructed with the normal E. coli host strain, XL2-blue. Then this bacmid (b
acmid) were grown in E. coli DH10Bac cells. Recombinant bacmid DNA was isolated and transfected into insect cells. The recombinant baculovirus particles were then used to infect insect cells to express the recombinant protein.

Results Insect cells were harvested 72 hours after baculovirus infection and crude protein lysates were extracted. Expression of the recombinant BUB1 protein was measured by Western analysis using an anti-HA antibody. The expected BUB1 band was detected at a molecular weight of 150 kDa. Both wild-type and kinase-inactivated recombinant proteins of BUB1 fused to HISx6 and GST were expressed in the baculovirus system.

In vitro Baculovirus-Expressed BUB1 Kinase Activity Assay Materials and Methods Protein extracts obtained from insect cells overexpressing recombinant BUB1 protein were used in an in vitro BUB1 kinase activity assay. The protocol for this kinase assay was the same as described in Example 9.

Results Protein extracts from BUB1 and BUB1.KR fused to the HIS label were isolated from insect cells and subjected to an in vitro kinase activity assay. Radiolabeled-protein was immunoprecipitated with anti-HA antibody before running on a 10% polyacrylamide SDS gel. The 150 kDa BUB1 protein was observed only in samples containing recombinant wild-type BUB1. This recombinant BUB1 can autophosphorylate and can also phosphorylate the exogenous substrate histone H1.

BUB1 overexpressed in the baculovirus system provides a powerful tool in performing high-throughput screening. Small chemical molecules are an example of a compound to be screened for use as a therapeutic.

One of ordinary skill in the art can readily appreciate that the present invention is well-adapted, not only to its original nature, but also to achieve its objectives and to obtain the above-described results and advantages. The molecular complexes and methods, treatments, therapies, molecules, specific compounds described herein are presently representative of preferred embodiments, which are exemplary and within the scope of the present invention. There is no intention to limit. Variations and other uses made by those skilled in the art are within the spirit of the invention and are defined by the appended claims.

It will be readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the level of those skilled in the art to which the invention pertains.

The invention exemplarily described herein may be embodied in a form in which there are no one or more elements or one or more restrictions not specifically disclosed herein. Can be. Thus, for example, in each instance herein, the terms "comprising", "consisting essentially of" and "consisting of" can be replaced with either of the other two terms. The terms and phrases used are intended to be illustrative, not limiting, and the use of such terms and phrases is not intended to imply equivalents or parts of the features shown and described. It is recognized that various modifications may be made without departing from the scope of the present invention as set forth in the claims.

In particular, although some of the formulations described herein are identified by excipients added to the formulation, the present invention also relates to final formulations formed by combining these excipients. Means included. Specifically, in the present invention, all of the added excipients react at the compounding stage and may no longer be present in the final formulation or may be present in a modified form. Preparations are included.

In addition, if a feature or aspect of the invention is described on the basis of a Markush group, the invention is thereby also described on the basis of any individual element or subgroup of elements of the Markush group. Those skilled in the art will recognize this. For example, if X is stated to be selected from the group consisting of bromine, chlorine and iodine, claims wherein X is bromine and claims wherein X is bromine and chlorine are fully described.

Example 11 Characterization of Antibodies Materials and Methods Rabbit polyclonal antibodies were generated against specific peptide sequences contained in the region encoding BUB1. This peptide (506A) has the amino acid sequence of BUB1
It consists of the following amino acid sequence corresponding to the residues at positions 385-498: DDKDEWQSLDQNED (SEQ ID NO: 19). The IgG fraction was purified by affinity chromatography using protein A agarose beads based on standard methodology. The IgG fraction was dialyzed against PBS, adjusted to 1 mg / ml BSA, and aliquots were frozen at -20 ° C.

Results The specificity of the antibody was confirmed using the recombinant BUB1 protein expressed in human 293T cells. This antibody detects a protein of about 150 kDa in both immunoprecipitation and immunoblotting experiments. This signal competed by including the immunizing peptide with the antibody. This BUB1 antibody immunoprecipitated a kinase activity capable of transphosphorylating histone H1. BUB1 also autophosphorylated in the BUB1 immune complex kinase assay.

Example 12 Cell Cycle Regulatory Materials and Methods Hela cells were synchronized by blocking with double thymidine / aphidicolin in the G1 / S translocation state. The cells were released from the block and collected as cells progressing through the cell division cycle over time.

[0359] Results Not only the activity of the associated kinase, but also the amount of BUB1 protein peaks in cell cycle regulated mitosis. This activity appears to be maximal during metaphase corresponding to maximal activation of p34 ^cdc2 (CDK1). Consistent with this observation, activity is also induced by nocodazole treatment, which arrests cells at G2 and mitosis, but not by serum starvation or thymidine treatment, which arrests at G1. .

Example 13 Antisense Oligonucleotide Materials and Methods Antisense oligonucleotides were identified to specifically down regulate BUB1 protein levels. The sequence of this oligonucleotide is GTGCCA
AGCCAGCAGATAAAT (SEQ ID NO: 24). This corresponds to nucleotides 2889-2909 of the human BUB1 cDNA sequence.

Results This oligonucleotide down-regulates BUB1 protein levels as detected by immunoblotting with a BUB1-specific antiserum. β-tubulin levels remain unchanged. Aurora 1, Aurora 2
Other mitotic proteins, such as cyclin A, cyclin B, and polo-like kinase 1 (PLK1), were upregulated in cells treated with BUB1 antisense oligonucleotide compared to control cells. To confirm the DNA content of cells treated with this BUB1 antisense oligonucleotide, a study by flow cytometry was performed. This analysis indicated that 4N DNA content stagnated in cells treated with the BUB1 antisense oligonucleotide, consistent with mitotic arrest. To better characterize this stop point, the treated cells were stained with DAPI and stained for DNA, but also stained for microtubules and BUB1 protein with α-tubulin and antibodies to BUB1, respectively. The same is true. BUB1
Cells that did not express mitosis were arrested, especially at metaphase where aggregated chromosomes on a metaphase plate and the normal bipolar mitotic spindle are present. In a small percentage of these cells, the chromosomes were randomly distributed, as if the chromosomes were no longer associated with the mitotic spindle. This is consistent with the BUB1 protein being involved in the microtubules associated with the chromosomal kinetochore. The cell number of cells treated with BUB1 antisense was significantly reduced 2 days after treatment, which means that the loss of BUB1 was fatal. To determine whether such a decrease in cell number was due to apoptosis, TUNEL
The assay was performed. This TUNEL assay measures the amount of DNA fragmentation in the resulting cell population that is indicative of apoptotic cell death. Cells treated with control antisense oligonucleotide had no increase in DNA fragmentation by day 3 post-treatment, whereas cells treated with BUB1 antisense oligonucleotide had 10 days of apoptosis 2 days post-treatment. Increased by a factor of two. These observations suggest that BUB1
Is essential.

Example 14 BUB1 Synergizes with Ras. Materials and Methods To investigate the effect of BUB1 overexpression on cell transformation, NIH3T3
Cells are transfected with activated H-ras (valine 12) alone and combined with wild-type BUB1 (BUB1WT) or an N-terminal deletion mutant corresponding to amino acids 1-332 (BUB1N). Transfected.

Results In three independent experiments, activated H-ras and BUB1WT were activated.
As many lesions as H-ras alone or twice as much as when BUB1N was added to activated H-ras occurred. These data suggest that BUB1 cooperates with oncogenes that transform cells.

Example 15: Isolation of cDNA Encoding Mammalian NEK-Related Plasmid Kinase Materials and Methods Makegene Bioinformation EST Assembler EST report was downloaded from ncbi (www.ncb.nim.gov.). After unzipping the file, use the program "report2est" to get the following information: 1) EST name,
2) Gene bank related number, 3) Gene bank gi number, 4) Clone Id
No., 5) EST nucleic acid sequence, 6) microorganism, 7) library name, 8) laboratory name, and 9) information of the facility were extracted. The output of "report2est" has all of the above listed information except the sequence on the first line of each entry, and the sequence is a file in FASTA format as listed on the secondary line of each entry. is there. The resulting file was formatted for BLAST using "pressdb" (available as part of the ncbi toolkit).

To create a gene or gene portion from an EST, the program “Makegene
Was used. The query sequences and genes for the microorganism / species were entered into this program so that they would be created. An initial search of the formatted database described above was performed using BLAST (blastn). Consequences that include indications such as poly-A tails or other repeating elements have reduced future queries. By using the program "blast_parse_reports" to extract the FASTA headline from the search results and then filtering its output, only the FASTA headline for the desired species can be extracted.

The first result, filtered for signs and species, is put into a loop where iteratively searches its database until no new ESTs are found. This loop consists of the following steps: 1) If possible, rename both ends of the EST to "Clone
Extracting from the database using the "Id" field or by searching in front of the postscript .r or .s using the "EST name"field; 2) The EST used as the query in the previous loop Step away from the current query by the program "subtract" 3) Use the list that is the result of the EST to extract sequences from the database by the program batch_parse_fasta 4) BLAST scans the database using each sequence 5) removing the output file from BLAST containing symptoms, 6) filtering the results by species, and 7) looping if there is a new EST found in the last pass through the loop. To re-enter.

The EST selected by “Makegene” is a program “mpd2_cluster” in which partially overlapping sequences are clustered (not disclosed, Hide, W., Bruke, J, and Davison, DU o
f Houston). The program "contig" (private Ker
lavage, T., TIGR), “gde2mult”, and “gde2sing” [Smith, SW, et al.
, CABIOS 10: 671-675 (1994)] were used to generate overlapping and consensus sequences for ESTs.

DNA sequencing Plasmid DNA was purified using a Quagen column and Taq, FS DNA polymerase (ABI
, Foster City, CA) using BIG-DYE
-Sequenced using terminator chemistry. The sequencing reaction was performed using ABI Prism 37
7 Proceed with the DNA sequencer and use the BLAST alignment algorithm (Altschul,
SF et al. (1990) J. Mol. Biol. 215: 403-410). Sequencing oligonucleotides to which primers migrate are based on Applied Biosystems utilizing established phosphoramidate chemistry (ABI, Foster, City, CA).
It was synthesized on a 3948 DNA synthesizer.

Results Cloning and Characterization of NEK4a cDNA The cDNA sequence of human nek4a is represented by AA214523, an EST fragment identified by a Smith-Waterman search of the EST database. The complete sequence of this clone was determined and used to generate the predicted full-length NEK4a amino acid sequence (SEQ ID NO: 8).

The full-length human NEK4a cDNA (SEQ ID NO: 7) is 1,449 bp long and has 121 bp of 5 ′.
It consists of a UTR and a 1,107 bp open reading frame (ORF) flanked by a 21 nucleotide polyadenylation region followed by a 200 bp 3 ′ UTR. No polyadenylation signal (AATAAA) was found before the polyadenylation region. First AT
The sequence adjacent to G is the Kozak consensus sequence for the starting methionine (Kozak, M. (1987)
Nucleic Acids Res. 15, 8125-8148), which is known to be the translation initiation site for NEK4a.

Several EST fragments have AA214523 at the 5 'end and span the complete nek4a sequence with AA147800 at the 3' end.

CDNA Cloning and Characterization of NEK4b The cDNA sequence of human nek4b is represented by AA147800, an EST fragment identified by a Smith-Waterman search of the EST database. The complete sequence of this clone was determined and used to generate the predicted full-length NEK4b amino acid sequence (SEQ ID NO: 10).

The partial human NEK4b cDNA (SEQ ID NO: 9) is 1,346 bp long and consists of a 41 nucleotide polyadenylation region followed by an 804 bp ORF flanked by a 499 bp 3′UTR. No polyadenylation signal (AATAAA) was found before the polyadenylation region. Since the coding region is open over the 5 'region of this sequence, it is clear that this is a partial cDNA lacking the N-terminal start methionine.

Several EST fragments have N30324 at the 5 ′ end and AA461376 at the 3 ′ end.
Spread in the ek4b sequence.

The cDNA cloning and characterization of NEK5, EST fragment AA181165, corresponds to the complete human nek5 gene, which is the EST Smith-
Identified by Waterman search. The complete sequence of this clone was determined and used to generate the predicted full-length NEK5 amino acid sequence (SEQ ID NO: 4).

The full length human NEK5 cDNA (SEQ ID NO: 3) is 3,854 bp long. This human NEK
The 5 cDNA contains a 906 bp ORF flanked by a 31 bp 5 'UTR (1-31) and a 2,897 bp 3' UTR (941--3,834) following a 20 nucleotide polyadenylation region. The polyadenylation signal (AATAAA) is found at positions 3817-3822. The sequence adjacent to the first ATG is similar to the Kozak consensus sequence for the starting methionine, which is thought to be the translation initiation site for NEK5.

At least one EST fragment, AA181165, spans the complete NEK5 sequence. AA18116
5 contains a 3,854 bp insert and a 906 bp ORF (302 amino acids).

CDNA Cloning and Characterization of NEK6 AA030068, an EST fragment corresponding to the partial human NEK6 gene, was identified by a Smith-Waterman search of the EST database. The complete sequence of this clone was determined and used to generate a partial human NEK6 sequence.

A partial mouse nek6 cDNA (SEQ ID NO: 5) is 1310 bp long. This mouse NEK6 contains a 907 bp ORF before a 403 bp 5 'UTR (positions 1-403). The sequence flanking the initial ATG fits slightly in the Kozak consensus sequence for the starting methionine.
However, it is thought to be the translation initiation site for NEK6 for four reasons: (1) there is adenine at position -3 before the start codon but no pyrimidine at position +4. (2) Multiple stop codons 5 'at multiple start sites are present in frame and out frame. (3) There are no other possible start codons in any frame 5 'of multiple start codons. And (4) the sequence before this start site is
57.7% Gs and Cs with regulatory features of the 5'UTR region (very GC-rich).

One EST fragment, AA030068, shows a partial NEK6 sequence. EST clone AA
030068 contains an insert of 1,310 bp and an ORF of 907 bp (302 amino acids).

Example 16: Expression analysis of mammalian Nek-related protein kinases Materials and methods Expression analysis using Dna arrays Plasmid DNA array blots were performed on nylon membranes with 0.5 μg of the denatured plasmid for each NEK-related kinase. Was prepared by loading
[Α ³² P] dCTP labeling-single-stranded DNA probe was used in the following cell lines: T cells (MOLT-4
, CEM / C1, jurkat), B cells (2PK3, WHHI-231, DAUDI, RAMOS, RAJI), TH2 (
D10.G4.1, pL104, pL3), TH1 (pGL10), CTL (L13), and basophil cells (RBL
-2H3), as well as RNA isolated from several human and rodent immunogens, including primary thymus (B6Thy), mast cells (MC / 9), and T cells (CD4 +) It was synthesized from total RNA. Hybridization was performed at 42 ° C for 16 hours, 6 x SSC, 0
0.1% SDS, 1 × Denhardt's solution, 100 μg / mL denatured herring in semen DNA [ ³² P
DCTP labeling—single-stranded probe, 10 ⁶ cpm / mL. 0.1 for this filter
Washed at 65 ° C. with × SSC / 0.1% SDS and run on a Molecular Dynamics phosphor imager for quantitative analysis.

Results Distribution of NEK-Related Gene Transcripts in RNA Panels of Immune Cells Expression of three NEK-related kinases was determined in human and rodent immune cell sources by three genes (NEK4 EST GB: H59937, NEK5 EST GB: AA102912 and NEK6 EST GB: A
A030253 was determined by hybridizing to a DNA-array blot containing the plasmids encoding each. (The NEK4 probe used in this experiment can detect the NEK4a and NEK4b isoforms with equal efficiency).

NEK4 was widely expressed in a panel of whole immune cells, with highest and lowest expression observed in mast cells and thymus, respectively. NEK5 was widely expressed in a panel of whole immune cells, with highest and lowest expression observed in TH1 cells and thymus, respectively. NEK
6 was widely expressed in a panel of whole immune cells, with highest and lowest expression observed in TH1 and basophils, respectively.

This analysis suggests that the novel NEK-subfamily kinases may be potential targets for various immune diseases. These broad expression patterns also suggest that they may mediate functions that are lethal to the basic biology of the most proliferating cells of the immune system.

[0385]

[Table 2] Expression of novel NEK-subfamily kinases in immune cell panel

Example 17: nek Gene Expression Plasmid: Construction and Expression of Recombinant NEK Expression plasmid construction Expression constructs can be made for NEK kinase, including, for example, a) to c) below. A) the full-length nek nucleotide sequence with and without the HA tag, the respective N-terminal domain of NEK4a, NEK5, or NEK6 with and without the HA tag (amino acids 1-42, 1-31, or 1 ~ 22), NEK4a, NEK4b, NEK5, or NEK6 with and without HA label
Each of the catalytic domains (amino acids 43-294, 1-259, 32-283, or 23-30
2) the nucleotide sequence coding for, and N with and without HA label
The respective C-terminal domain of EK4a, NEK4b, or NEK5 (amino acids 295-313, 260
-268, or 284-302), the pcDNA3.1 expression plasmid; b) the full-length nucleotide sequence of nek, the N-terminal domain of each of NEK4a, NEK5, or NEK6 (amino acids 1-42, 1-31, or NEK4a, nucleotide sequence encoding 1 to 22)
, NEK4b, NEK5, or NEK6 catalytic domain (amino acids 43-294, 1-2
59, 32-283, or 23-302), and NEK4a
, NEK4b, or NEK5 C-terminal domain (amino acids 295-313, 260-268
Or a pGEX 4T-3 fusion plasmid containing a nucleotide sequence encoding 284-302); and c) a pEGFP fusion plasmid containing NEK kinase. The N-terminal domain of NEK is predicted to function as a major negative construct, which may help elucidate the function of BUB1. This full-length gene is readily available by methods well known in the art for obtaining useful partial sequences.

Example 18 Recombinant Expression of NEK Plasmid Kinase Materials and Methods Transient Expression of NEK in Mammalian Cells A pcDNA expression plasmid (10 μg DNA) containing a sequence encoding the full-length or truncated form of NEK kinase / 100 mm plate) together with lipofectamine (Gibco BRL) into human 293 cells. After 72 hours, the cells were diluted with 0.5 mL of lysis buffer (20 mM HE).
PES PH7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EG
TA, 2 mM phenylmethylsulfonyl fluoride, 1 μg / mL aprotinin). Aliquots of samples are subjected to SDS polyacrylamide gel electrophoresis (PAGE)
On a 7.5% acrylamide / 0.5% bis-acrylamide gel and transfer to nitrocellulose by electrophoresis. Non-specific binding was determined by the Blotto method (5% w / v non-fat dry milk and 0.2% v / v Nonidet P-40).
(Sigma buffered phosphate buffer) by preincubation. Recombinant proteins are detected by performing Western analysis with various NEK antisera or anti-HA specific antisera. NEK-antiserum was diluted about 1: 1,000-fold, and anti-HA monoclonal antibody (Babco, Berkeley, Califoynia) was 1: 1.
: Dilute 10,000 times and use for primary antibody reaction in Western blot analysis. Goat anti-rabbit HRP (BioRad, Richmond, California) was used as a secondary antibody to NEK-antiserum, and goat anti-mouse HRP (BioRad, Richmond, California) was used as anti-H.
Used as a secondary antibody to antibody A. ECL reagent (Amersham Life Science,) is used for detection.

Cell lysates (30 μg each) are subjected to 7.5% SDS PAGE followed by Western blot analysis.

Example 19 Preparation of Immunoreagents Specific for NEK Protein Kinase Materials and Methods Specific immunoreagents are raised in rabbits against a MAP-conjugated synthetic peptide corresponding to NEK kinase. Each peptide is injected into two rabbits.

Another immunizing reagent is generated by immunizing rabbits with a bacterially expressed GST-fusion protein containing the terminal or C-terminal domain of NEK kinase.

Affinity Purification of Anti-NEK Peptide Antibody 10 mg of NEK peptide was combined with 2 mL of Immunolink (Pierce AminoLink).
Plus). This link consists of an aldehyde-supporting arm that forms a covalent bond to a primary amine via a Schiff base intermediate. NEK peptide (ligand) is immobilized on Aminolink Plus coupling gel. The remaining active sites are blocked by adding a reducing reagent. The column is subsequently washed before use.

[0392] 20 mL of the filtered anti-NEK antiserum is applied to the immobilized antigen column. Purified anti-NEK IgG was eluted with 1 M propionic acid neutralized with 3 M Tris to pH 7.4 during elution, and with 100 mM TEA buffered with 1 M Tris pH 6.8. Equilibrated.
Dialyzed The purified antibody against phosphate buffered saline pH 7.4, it is stored in a solution containing 20% glycerol and 0.03% NaN ₃ in PBS.

The anti-NEK antiserum is tested by Western analysis using a GST-NEK fusion protein.

The affinity-purified anti-NEK antibodies were tested by Western analysis using a GST-NEK fusion protein or a mammalian cell lysate obtained from transiently transfected and expressed NEK protein.

Example 20 Endogenous Expression of NEK Protein Kinase Materials and Methods Numerous human tumor cell lines (eg, HT-29, Calu-6, HCT15, MCF7, A549, C33a
, Hela, HT-29, H1299, SH422C, COLO205, SW480, SW837, SW948, and SNB19)
Is grown to confluence. Transfer the protein lysate to HNTG buffer (20 mM HE
PES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM E
GTA, 2 mM phenylmethylsulfonyl fluoride, 1 μ / mL aprotinin). This protein concentrate is used for the BioRad assay (BioRad, Richmond, California).
fornia). The proteins obtained from each sample (30 μg each) are tested by Western analysis using purified anti-NEK antibodies.

Example 21 NEK Cell Cycle Modulation Materials and Methods Hera cells are synchronized by adding 0.5 M thymidine and 250 μg / mL aphidicolin. 0, 2 hours, 4 hours, 6 hours after removing thymidine and aphidicolin
Cells are harvested after hours, 8 hours, 9 hours, 10 hours, 11 hours, and 12 hours. Other experiments are performed by blocking Hela cells at different stages of the cell cycle by starving serum with thymidine (G1 / S / G2) or nocodazole (M). Total RNA is isolated from synchronized cells and cells that have blocked the cell cycle. RNA
The whole amount (10 μg) is placed on a nylon membrane. The RNA blot is hybridized with the nek probe.

[0397] Northern blot analysis involving RNA derived from Hela cells, either in an exponential growth phase or arrested at various stages of the cell cycle, showed nek
Probe operation for the expression of

Example 22 Protein Localization of NEK Protein Kinase Materials and Methods The subcellular localization of NEK proteins is determined by immunofluorescence using NEK-specific antibodies. Hela and H1299 cells are grown to confluence.
Cells are trypsinized, then seeded on 60 mm plates in 1 cm poly-lysine pretreated coverslips and cultured at 37 ° C. for 1 day. After removing the medium, the cells are washed with 5 mL of ice-cold PBS, followed by fixation with -20 ° C cooled 100% methanol. Methanol-fixed cells can be used for about a week.

The cells were blocked with 30 μL of immunofluorescence blocking buffer (3% BSA, PBS solution containing 0.01% Triton X100) for 30 minutes, and 0.01% Triton X was added.
Wash with PBS containing 100. Purified anti-NEK antibody was added about 1:10 with blocking buffer.
Dilute 0-fold. Goat anti-conjugated with rhodamine or fluorescein (FITC)
AffinPure F (ab) 2 fragment of rabbit IgG is diluted 1: 400 and used as a secondary antibody. Dapi (62.5 μg / mL) is used for nuclear staining.

[0400] Immunofluorescence experiments to examine the subcellular localization of endogenous NEK include, for example, Hela cells and H1299
This is done using an affinity-purified anti-NEK antibody in both cells. Immunofluorescence experiments are also performed on 293T cells transiently transfected with pcDNA3.1.NEK.

Example 23 In Vivo and In Vitro NEK Kinase Assay The pcDNA3.1.NEK.HA expression plasmid is transfected into human 293T cells. The crude protein lysate is extracted from the transfected cells and used in a NEK kinase activity assay. Four separate assays are performed. (1) NEK kinase autophosphorylation in vitro, (2) NEK phosphorylation of exogenous substrate,
(3) NEK kinase activity in vivo, and (4) NEK phosphoamino acid analysis.

Assay for In Vitro NEK Kinase Autophosphorylation Materials and Methods pcDNA containing HA-labeled wild type (NEK.HA) or N-terminal truncated form (NEK.N.HA).
1NEK expression plasmid (10 μg DNA / 90 mm plate) was transferred to Lipofectamine (Gibco
(BRL, Gaithersburg, MD) into human 293T cells. After 48 hours, the cells were washed with 0.4 mL of kinase lysis buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF
, 1 mM sodium ortho-vanadate, 0.5 mM Nonidet P-40 (Shigma, St. L.
ouis, MO), 1 mM DTT, 170 μg / mL PMSF, 25 μg / mL leupeptin, 20 μg / m
L of trypsin inhibitor, 25 μg / mL aprotinin). The protein extract was immunoprecipitated using anti-HA antibody (Babco, Berkeley, CA) at a final dilution of 1: 750 and protein G beads (Boehringer Mannheim, Indianapolis, IN), followed by kinase lysis buffer. After washing three times, kinase buffer (20 mM HEPES,
Wash twice with pH 7.5, 150 mM KCl, 10 mM MgCl ₂ ). 20 μL of the kinase reaction mixture (1 × kinase buffer, 1 mM DTT, 0.1 mM ATP, 10 μCi of ³² P-r-ATP) is added to the washed beads and the mixture is stirred for 40 minutes with constant stirring. Incubate at 37 ° C. The reaction is stopped by adding 8 μL of 5 × PAGE buffer and boiling at 100 ° C. for 5 minutes.

A kinase assay is performed using NEK.HA and NEK.N.HA (dominant / negative) contained in the transient cell lysate of 293T. The radiolabeled-protein is immunoprecipitated with an anti-HA antibody and run on a 10% polyacrylamide SDS gel.

In Vitro BUB1 Phosphorylation of Exogenous Substrates Materials and Methods In vitro kinase assays using exogenous substrates indicate that the kinase reaction mixture contains 1 mg / mL of histone H1, myelin basic protein, or alpha casein protein. (Boehringer Mannheim) but identical to the in vitro autophosphorylation assay.

In Vivo NEK Kinase Assay Materials and Methods pcDNA3.1.NEK expression plasmid (10 μg DNA / 100 mM plate) containing HA-labeled full-length or truncated form of NEK was combined with Lipofectamine (Gibco BRL). Transfect into human 293T cells. After 48 hours, the cell medium is changed to phosphate-free medium (Gibco BRL) containing 10% dialyzed fetal bovine serum (Gibco BRL). Twenty hours after changing the medium, inorganic ³² P-phosphate is added to the medium in a 2 mCi / 100 mm plate, and the cells are incubated for three hours. Add 12 μL pervanadate solution (50 mM sodium ortho-vanadate, 15% hydrogen peroxide) and then incubate the cells for another 10 minutes. The cell culture dish is placed on ice and rinsed with 4 ° C. phosphate buffered saline. 450μ crude protein lysate
L kinase lysis buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium ortho-vanadate, 0.5 mM Nonidet P-40 (Shigma), 1 mM DTT, 1 mM
70 μg / mL PMSF, 25 μg / mL leupeptin, 20 μg / mL trypsin inhibitor, 2
5 μg / mL aprotinin) and immunoprecipitate with anti-HA antibody.

This assay was performed using transiently transfected 293 with NEK or NEK.N.
It is performed by adding inorganic ³² P to T cells. The crude protein is extracted from the radiolabeled cells. Cell lysates are immunoprecipitated with anti-HA antibody and subsequently run on a 10% polyacrylamide SDS gel.

NEK Kinase Phosphoamino Acid Analysis Materials and Methods The Hunter protocol is used for NEK kinase phosphoamino acid analysis.
After completing the in vitro kinase assay, the radiolabeled-NEK band was excised from a dried polyacrylamide gel. 0.5 mL of 50 mM
Rehydrate with 5 minutes in ammonium bicarbonate, pH 7.3-7.6, then homogenize to a fine suspension. The volume of the suspension was adjusted to 1 mL using 50 mM ammonium bicarbonate with a final concentration of 5% β-mercaptoethanol (v / v) and 0.1% SDS (w / v). This mixture is incubated at 100 ° C for 5 minutes, followed by 1 hour at 37 ° C.
Incubate for 2 hours with agitation. Collect the particulate fraction by centrifugation, 50m
Extract once more with M ammonium bicarbonate, pH 7.3-7.6, use 5% beta-mercaptoethanol (v / v), 0.1% SDS (w / v) at 100 ° C. for 5 minutes, followed by Incubate at 37 ° C for 60 minutes. After centrifugation, the liquid extract is combined with the previous extract to make a total volume of 1.2 mL. The extract is centrifuged again to remove particulate matter, the supernatant is isolated and refrigerated at 4 ° C. A total of 20 μg of bovine serum albumin was added to the extract, and 250 μL of protein was added.
And then incubated for 60 minutes at 4 ° C. The precipitated protein is isolated by centrifugation and the resulting pellet is washed at 4 ° C. with 500 μL of 100% acetone. The protein pellet is air dried and then dissolved in 100 μL of 5.7 M HCl. This solution is kept at 100 ° C for 60 minutes.
Heat in a tube with a screw cap, followed by vacuum drying. Residue pellets
Dissolve in formic acid (2% v / v) pH 1.9 and glacial acetic acid (7.3% v / v) containing 67 μg / mL phosphoserine, 67 μg / mL phosphothreonine, 67 μg / mL phosphotyrosine.
A sample containing a total of about 600 cpm was transferred to a cellulose TLC plate (VWR scientific, San F
rancisco, CA). Perform two-dimensional chromatography. 1
The dimensional gel is developed at 1.0 kV for 25 minutes in a buffer consisting of formic acid (2% v / v), glacial acetic acid (7.3% v / v) and pH 1.9. The plate is air dried, followed by 16 minutes at 1.3 kV in a buffer consisting of glacial acetic acid (5% v / v), pyridine (0.5% v / v), 0.5 mM EDTA, pH 3.5. Apply gel electrophoresis in the dimensional direction. The plate is air dried again, followed by spraying with a solution containing 0.25% ninhydrin in acetone and coloring at 65 ° C. for 15 minutes. The position and identity of the labeled amino acid is detected by autoradiography.

A hunter protocol is performed to examine phosphorylation sites (amino acids) for NEK protein kinase.

Example 24: High throughput screen for NEK protein kinase inhibitors
Development of Protocols in Necking NEK is involved in cell cycle regulation, so that over-expression or mutagenic events render these kinases unregulated and act as cancers. Inhibiting NEK kinase activity can suppress uncontrolled cell division, thereby inhibiting the growth of tumor cells. In vitro kinase inhibitor screening can be performed using overexpression of the recombinant NEK protein.

[0410] Recombinant baculoviruses have become widely used as vectors for expressing atypical genes in cultured insect cells. Atypical genes under the control of the strong polyhedrin promoter of Autograhpa california nucleopolyhedrovirus (AcNPV) are frequently expressed in late stages of infection. In most cases, the recombinant protein is cleaved, modified and then directed to the appropriate cell location, where it is functionally equivalent to its actual counterpart.

Materials for Expression of Recombinant NEK in Baculovirus System and Materials The baculovirus expression system is purchased from Gibco BRL, Gaithersburg, MD. NEK.H
The wild type of A and NEK.KR.HA (kinase is inactivated) are subcloned into pFastBacHtb (having a histidine label at the N-terminus) and pFastBacG2T (a glutathione transferase gene in which the histidine label has been replaced). This recombinant plasmid is constructed in a normal E. coli host strain, XL2-blue. The bacmid is then propagated in E. coli DH10Bac cells. Recombinant bacmid D
The NA is isolated and transfected into insect cells. The recombinant baculovirus particles are then used to infect insect cells to express the recombinant protein.

Constructs in which the kinase has been inactivated can be made by methods well known in the art, such as point mutations, insertions and deletions of the nucleic acid such that the kinase activity is lost.

[0413] Insect cells are harvested and the crude protein lysate is extracted 72 hours after infection with the baculovirus. Expression of the recombinant NEK protein is measured by Western analysis using an anti-HA antibody.

In Vitro Baculovirus-Expressed NEK Kinase Activity Assay Materials and Methods Protein extracts obtained from insect cells overexpressing recombinant NEK protein were used in an in vitro BUB1 kinase activity assay. The protocol for this kinase assay was the same as described in Example 9.

[0415] Protein extracts from NEK and NEK.KR fused to a HIS tag are isolated from insect cells and subjected to an in vitro kinase activity assay. Radiolabeled protein anti-H
After immunoprecipitation with the A antibody, it is run on a 10% polyacrylamide SDS gel.

NEK overexpressed in the baculovirus system provides a powerful tool in performing high-throughput screening. Small chemical molecules are an example of a compound to be screened for use as a therapeutic.

Example 25 NEK Antisense Oligonucleotides Antisense oligonucleotides are used to inhibit the expression of NEK in normal and tumor cells due to the potential effects of small molecule NEK expression inhibitors. Can be They can also be used to inhibit the expression of NEK in xenografts of human tumor cells in nude mice, thereby measuring the antitumor effect of NEK inhibitors. In addition, there is a use as a “drug” for inhibiting the expression of NEK in various human tumors characterized by overexpression or abnormal expression of the nek gene.

Materials and Methods Antisense oligonucleotides to be tested for inhibitory effects on nek expression include:
It can be generated by methods well known in the art. Usually this oligonucleotide is synthesized as a phosphorothioate and tested in the following manner.

[0419] Human H1299 cells are seeded in 6-well plates (Falcon) at approximately 40-50% confluence. The next day, lipofectin (Gibco) and the oligonucleotide are mixed with OptiMEM (Gibco) such that the final concentration of lipofectin is 100 μg / mL and the final concentration of each oligonucleotide is 1 μM per 200 μL volume.
Place the Lipofectin / Oligonucleotide / OptiMEM mixture at room temperature (20-25 ° C)
And add 800 μL of OptiMEM to the lipofectin / oligonucleotide / OptiMEM mixture and gently agitate. Growth medium, ~ 80%
From confluent H1299 cells. Wash cells once with OptiMEM. Aspirate the OptiMEM and remove it before lipofectin /
Add oligonucleotide / OptiMEM mixture and incubate at 37 ° C. for 4 hours. The lipofectin / oligonucleotide / OptiMEM mixture is removed and replaced with a common growth medium containing antisense oligonucleotide at a concentration of 200 nM. The plate is returned to the 37 ° C. incubator and incubated for 16-20 hours, after which time the growth medium is removed from the cells and subsequently washed once with OptiMEM. The OptiMEM is again aspirated and replaced with a general growth medium containing a 200 nM concentration of antisense oligonucleotide. Put this plate at 37 ° C
Return to the incubator and incubate for 16-20 hours. The cells are harvested (〜40 hours after the initial treatment) and analyzed for nek mRNA by Northern blot or for expression of NEK protein by immunoblotting.

One of ordinary skill in the art can readily appreciate that the present invention is well adapted to achieve the objects and obtain the results and advantages described above, as well as the original. The molecular conjugates and methods, steps, therapies, molecules, and specific compounds described herein are presently representative of preferred embodiments and are exemplary and are not limiting. There is no intention to add. Changes therein and other uses will occur to those skilled in the art, but they are encompassed within the spirit of the invention and are defined by the claims.

[0421] It will be readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned herein are indicative of the level of those skilled in the art to which the invention pertains.

The invention exemplarily described herein may be embodied in a form without one or more elements or one or more limitations not specifically disclosed herein. Can be. Thus, for example, in each instance herein, the terms "comprising", "consisting essentially of" and "consisting of" can be replaced with either of the other two terms. The terms and expressions used are intended to be illustrative and not restrictive, and in using such terms and expressions, any equivalents or parts of the shown or described feature may be used. It is recognized that various modifications may be made without departing from the scope of the present invention, which is set forth in the following claims.

In particular, although some of the formulations described herein are identified by excipients added to the formulation, the invention also relates to final formulations formed by combining these excipients. Means included. Specifically, in the present invention, all of the added excipients react at the compounding stage and may no longer be present in the final formulation or may be present in a modified form. Preparations are included.

Further, if a feature or aspect of the invention is described on the basis of a Markush group, the invention is thereby also described on the basis of any individual element or subgroup of multiple elements of the Markush group. Those skilled in the art will recognize this. For example, if X is stated to be selected from the group consisting of bromine, chlorine and iodine, claims wherein X is bromine and claims wherein X is bromine and chlorine are fully described.

Other embodiments are within the following claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the human bub1 nucleic acid sequence (SEQ ID NO: 1).

FIG. 2 shows the amino acid sequence of human BUB1 (SEQ ID NO: 2).

FIGS. 3A and 3B show multiple sequence alignments of human BUB1 (SEQ ID NO: 2), mouse BUB1 (SEQ ID NO: 22), and S. cerevisiae BUB1 (SEQ ID NO: 23). .

FIG. 4A, 4B, 4C, and 4D show human NEK4a (SEQ ID NO: 7), human NEK4
b (SEQ ID NO: 9), human NEK5 (SEQ ID NO: 3), and mouse NEK6 (SEQ ID NO: 5)
1 shows a partial or full length nucleic acid sequence of a kinase polypeptide.

FIG. 5: Human NEK4a (SEQ ID NO: 8), human NEK4b (SEQ ID NO: 10), human N
Figure 4 shows the partial or full length amino acid sequences of EK5 (SEQ ID NO: 4) and mouse NEK6 (SEQ ID NO: 6) kinase polypeptides.

FIG. 6: Human NEK4b (SEQ ID NO: 10), human NEK5 (SEQ ID NO: 4), mouse
NEK6 (SEQ ID NO: 6), Emericella nidulans NIMA (SEQ ID NO: 11), human NEK2
(SEQ ID NO: 12) and N containing human GAK (SEQ ID NO: 13) kinase polypeptide
1 shows a multi-sequence alignment of the amino acid sequences of EK-subfamily kinases.
Because NEK4, NEK5, and NEK6 lack a long C-terminal region, NIMA, NEK2 and G
The C-terminus of AK is not shown in this figure.

FIG. 7 shows a sequence alignment of the amino acid sequences of NEK4a (SEQ ID NO: 8) and NEK4b (SEQ ID NO: 10) kinase polypeptides.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 37/00 35/02 C07K 16/40 37/00 C12N 1/15 C07K 16/40 1 / 19 C12N 1/15 1/21 1/19 9/12 1/21 C12Q 1/48 Z 5/10 1/68 A 9/12 G01N 33/15 Z C12Q 1/48 33/50 Z 1/68 33 / 68 G01N 33/15 C12N 15/00 ZNAA 33/50 A61K 37/52 33/68 C12N 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD) , TG), AP (GH, GM, KE, LS, MW, S , SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant 230 East Grand Avenue, South San Francisco, California 94080, United States of America (72) Inventor Chu Infan, San Bruno, Oxford Lane, California, USA # 1 152

Claims (33)

[Claims] 1. An isolated, enriched or purified nucleic acid molecule encoding a BUB1 kinase polypeptide, NEK5 kinase polypeptide or NEK6 kinase polypeptide, wherein the nucleic acid molecule comprises the following nucleotide sequence: (a) SEQ ID NO: : 2, a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 4, or SEQ ID NO: 6; (b) a nucleotide sequence that is the complement of the nucleotide sequence (a); (c) a nucleotide molecule A nucleotide sequence that hybridizes with (a) under stringent conditions and encodes a native kinase polypeptide; (d) positions 1-785, 786-1028, or 1029-1085 of SEQ ID NO: 2; Positions 1-31, 32-283 or 284-302 of SEQ ID NO: 4, or 1-22 of SEQ ID NO: 6
A kinase kinase having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, except that one, but not all, of the segments of amino acid residues consisting of positions 23 or 302 are missing. (E) a nucleotide sequence that is the complement of the nucleotide sequence (d); (f) a nucleotide sequence of positions 1 to 785, 786 to 1028, or 1029 to 1085 of SEQ ID NO: 2, SEQ ID NO: Positions 1-31 of positions 4, 32-283, or 284-302 of 4, or 1-22 of SEQ ID NO: 6
SEQ ID NO: 2, SEQ ID NO: 4, derived from amino acid residues consisting of positions 23 or 302
Or a nucleotide sequence encoding a kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 6; (g) a nucleotide sequence that is the complement of the nucleotide sequence (f); (h) an N-terminal domain, a C-terminal catalytic domain, and a catalyst. A polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, except that one, but not all, of the domain, C-terminal domain, and C-terminal tail are missing. A coding nucleotide sequence; or (i) a nucleotide sequence that is the complement of the nucleotide sequence (h). 2. The nucleic acid molecule of claim 1, further comprising a vector or promoter effective to initiate transcription in a host cell. 3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is isolated, concentrated, or purified from a mammal. 4. The nucleic acid molecule according to claim 3, wherein the mammal is a human. 5. The nucleic acid probe according to claim 1, which is used for detecting a nucleic acid encoding a kinase polypeptide contained in a sample, wherein the kinase polypeptide has an amino acid sequence represented by SEQ ID NO: 2. A nucleic acid probe selected from the group consisting of a BUB1 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 4, an NEK5 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 4, and a NEK6 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 6. 6. A BUB1 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 2, an NEK5 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 4, or NEK6 having an amino acid sequence represented by SEQ ID NO: 6 A recombinant cell comprising the nucleic acid molecule of claim 1, which encodes a kinase polypeptide. 7. An isolated, concentrated, or purified BUB1 kinase polypeptide, NEK5 kinase polypeptide, or NEK6 kinase polypeptide, which comprises the following amino acid sequence: (a) SEQ ID NO: 2 , SEQ ID NO: 4, or the amino acid sequence represented by SEQ ID NO: 6; (b) positions 1 to 785, 786 to 1028, or 1029 to 1085 of SEQ ID NO: 2, 1-31 of SEQ ID NO: 4 Positions, 32-283, or 284-302, or 1-22 of SEQ ID NO: 6
An amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, respectively, except that one, but not all, of the segments of amino acid residues consisting of positions 23-302 are missing; (C) positions 1 to 785, 786 to 1028, or 1029 to 1085 of SEQ ID NO: 2, positions 1 to 31, 32 to 283, or 284 to 302 of SEQ ID NO: 4, or SEQ ID NO: 6 1 ~ 22
Or the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 from the amino acid residue consisting of positions 23 to 302; or (d) N-terminal domain, C-terminal catalytic domain, catalyst SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, respectively, except that one, but not all, of the domains selected from the group consisting of a domain, a C-terminal domain, and a C-terminal tail are missing. Amino acid sequence shown. 8. The kinase polypeptide according to claim 7, which is isolated, purified, or concentrated from a mammal. 9. The kinase polypeptide according to claim 8, wherein the mammal is a human. 10. The kinase polypeptide according to claim 7, which is the BUB1 kinase polypeptide represented by SEQ ID NO: 2. 11. The kinase polypeptide according to claim 7, which is the NEK5 kinase polypeptide represented by SEQ ID NO: 4 or the NEK6 kinase polypeptide represented by SEQ ID NO: 6. 12. An antibody or antibody fragment having specific binding affinity for a kinase polypeptide or for a domain of said polypeptide, wherein said polypeptide has the amino acid sequence set forth in SEQ ID NO: 2. Polypeptide, NEK4a kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 8, NEK4b kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 10, NEK5 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 4 Or an NEK6 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 6. 13. A BUB1 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 2, a NEK4a kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 8, and an NEK4b kinase having an amino acid sequence represented by SEQ ID NO: 10 A hybridoma that produces an antibody having specific binding affinity for the polypeptide, the NEK5 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 4, or the NEK6 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 6. 14. A method for identifying a substance that regulates the activity of a BUB1 kinase polypeptide, NEK4a kinase polypeptide, NEK4b kinase polypeptide, NEK5 kinase polypeptide, or NEK6 kinase polypeptide, comprising the steps of: : (A) a BUB1 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 2,
NEK4a kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 8, NEK4b kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 4
NEK5 kinase polypeptide having the amino acid sequence represented by: or SEQ ID NO: 6
Contacting a NEK6 kinase polypeptide having the amino acid sequence represented by with a test substance; (b) measuring the activity of the polypeptide; and (c) determining whether the substance regulates the activity of the polypeptide. Judgment stage. 15. A method for identifying a substance that regulates the activity of a BUB1 kinase polypeptide, NEK4a kinase polypeptide, NEK4b kinase polypeptide, NEK5 kinase polypeptide, or NEK6 kinase polypeptide in a cell, comprising:
A method comprising the following steps: (a) a BUB1 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 2,
NEK4a kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 8, NEK4b kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 4
NEK5 kinase polypeptide having the amino acid sequence represented by: or SEQ ID NO: 6
(B) adding a test substance to the cell; and (c) altering the cell phenotype or naturally binding the polypeptide to the NEK6 kinase polypeptide having the amino acid sequence of Measuring interactions with partners. 16. A method for treating a disease or disorder, which comprises the step of administering a substance that regulates the activity of a BUB1 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 2 to a patient in need of treatment. 17. The method of claim 18, wherein the disease is cancer. 18. The cancer is selected from the group consisting of leukemia, cervical cancer, lymphoma, colon cancer, lung cancer, melanoma, ovarian cancer, CNS cancer, prostate cancer, kidney cancer, and breast cancer.
The method according to claim 19. 19. The substance modulates kinase activity in vitro.
The described method. 20. A method for detecting a nucleic acid encoding a BUB1 kinase polypeptide as shown in SEQ ID NO: 2 contained in a sample as a diagnostic tool for a disease or disorder, comprising the following steps: (A) contacting the sample with a nucleic acid probe that hybridizes to a nucleic acid target region of bub1 having the amino acid sequence shown in SEQ ID NO: 1 under hybridization assay conditions, wherein the probe comprises the polypeptide A probe comprising a nucleic acid sequence encoding a fragment thereof, or a sequence complementary to the sequence and the fragment; and (b) detecting the presence or amount of a probe: target region hybrid as an indicator of the disease. 21. The method of claim 20, wherein the disease is cancer. 22. The cancer is selected from the group consisting of leukemia, cervical cancer, lymphoma, colon cancer, lung cancer, melanoma, ovarian cancer, CNS cancer, prostate cancer, kidney cancer, and breast cancer.
22. The method according to claim 21. 23. An NEK4a kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 8, an NEK4b kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 10, and an NEK5 kinase having an amino acid sequence represented by SEQ ID NO: 4. Administering a polypeptide or a substance that modulates the activity of a NEK6 kinase polypeptide having the amino acid sequence of SEQ ID NO: 6 to a patient in need of treatment,
A method for treating a disease or disorder. 24. The method of claim 23, wherein the disease or disorder is selected from the group consisting of a cancer, a neurodegenerative disorder, and an immune disorder. 25. The method of claim 24, wherein said disease or disorder is cancer. 26. The method wherein the substance is an in vitro NEK4a kinase polypeptide, NEK4b
24. The method of claim 23, wherein the method modulates the activity of a kinase polypeptide, a NEK5 kinase polypeptide, or a NEK6 kinase polypeptide. 27. The method of claim 26, wherein the modulation is an inhibitory effect. 28. The substance according to claim 27, wherein the substance is an antisense oligonucleotide.
The described method. 29. A method for detecting a nucleic acid encoding a kinase polypeptide contained in a sample as a diagnostic tool for a disease or disorder, comprising: (a) converting the sample to a kinase polypeptide. Contacting with a nucleic acid probe that hybridizes under hybridization assay conditions to a nucleic acid target region of the BUB1 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 2, represented by SEQ ID NO: 4. NEK5 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 6, NEK6 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 6,
NEK4a kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 8 or NEK4b kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 10, fragments thereof, and a nucleic acid sequence encoding a complementary sequence of the sequence and the fragment And (b) detecting the presence or amount of a probe: target region hybrid as an indicator of the disease. 30. The method of claim 29, wherein said disease is selected from the group consisting of cancer, neurodegenerative disorders, and immune disorders. 31. The method of claim 30, wherein the disease is cancer. 32. A BUB1 kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 2, an NEK4a kinase polypeptide having an amino acid sequence represented by SEQ ID NO: 8, and an NEK4b kinase having an amino acid sequence represented by SEQ ID NO: 10 An antisense oligonucleotide which inhibits the expression of a polypeptide, an NEK5 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 4, or an NEK6 kinase polypeptide having the amino acid sequence represented by SEQ ID NO: 6. 33. The oligonucleotide comprising a sequence encoding a fragment of the protein encoded by the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. 35. The antisense oligonucleotide according to claim 34, which is a complementary sequence.