# US 7,070,947 B2 · Human protein kinase, phosphatase, and protease family members and uses thereof

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- Number: US 7,070,947 B2
- Document ID: US7070947B2
- Status: Granted
- Kind: B2
- Application number: 10/170,789
- Filed: 2002-06-13
- Published: 2003-09-25
- Granted: 2006-07-04
- Earliest claimed: 2000-02-29
- Assignees: Millennium Pharmaceuticals, Inc.
- Inventors: Rachel E. Meyers; Peter J. Olandt; Rosana Kapeller-Libermann; Rory A. J. Curtis; Mark Williamson; Nadine Weich
- Web page: https://patentgrep.com/patent/US7070947B2

## CPC

- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- C07K 16/40: against enzymes
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12N 9/14: Hydrolases (3)
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/6424: {Serine endopeptidases (3.4.21)}
- C12N 9/6427: {Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)}
- C12N 9/6437: {Coagulation factor VIIa (3.4.21.21)}
- C12Q 1/34: involving hydrolase
- C12Q 1/44: involving esterase
- C12Y 207/01001: Hexokinase (2.7.1.1)
- C12Y 301/02015: Ubiquitin thiolesterase (3.1.2.15)
- C12Y 304/21004: Trypsin (3.4.21.4)
- C12Y 304/21021: Coagulation factor VIIa (3.4.21.21)
- G01N 33/5011: {for testing antineoplastic activity}
- G01N 33/502: {for testing non-proliferative effects}
- G01N 33/5041: {involving analysis of members of signalling pathways}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2039/505: {comprising antibodies}
- C07K 2319/00: Fusion polypeptide
- G01N 2500/00: Screening for compounds of potential therapeutic value
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The invention provides isolated nucleic acids molecules, designated 2504, 15977, 14760, 53070, 15985, 50365, 26583, 21953, m32404, 14089, and 23436 nucleic acid molecules, which encode novel human protein kinase family members, serine/threonine protein kinase family members, hexokinase family members, serine/threonine phosphatase family members, prolyl oligopeptidase family members, trypsin family members, trypsin serine protease family members, and ubiquitin protease family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 2504, 15977, 14760, 53070, 15985, 50365, 26583, 21953, m32404, 14089, or 23436 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 2504, 15977, 14760, 53070, 15985, 50365, 26583, 21953, m32404, 14089, or 23436 gene has been introduced or disrupted. The invention still further provides isolated 2504, 15977, 14760, 53070, 15985, 50365, 26583, 21953, m32404, 14089, or 23436 proteins, fusion proteins, antigenic peptides and anti-2504, 15977, 14760, 53070, 15985, 50365, 26583, 21953, m32404, 14089, or 23436 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

## Claims

### Claim 1

A method for identifying a candidate compound which modulates the ubiquitin hydrolase activity of a polypeptide selected from the group consisting of:
a) a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:58, 60, or a complement thereof,
b) a polypeptide comprising the amino acid sequence of SEQ ID NO:59, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:58, 60, or a complement thereof under conditions of hybridization in 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes in 0.2×SSC, 1% SDS at 65° C.;
c) a polypeptide comprising amino acids 89 to 420 of SEQ ID NO:59; and
d) a polypeptide comprising the amino acid sequence of SEQ ID NO:59, the method comprising the steps of:
i) contacting the polypeptide, or a cell expressing the polypeptide with a test compound;
ii) determining the effect of the test compound on ubiquitin hydrolase activity of the polypeptide, to thereby identify a candidate compound which modulates ubiquitin hydrolase activity of the polypeptide; and
iii) determining the effect of the candidate compound identified in ii) on a cellular activity selected from the group consisting of cell proliferation, cell signaling, cell death, cell motility, receptor-mediated endocytosis, organelle biogenesis, hematopoietic cell proliferation or differentiation, and cytokine-mediated signaling events.

### Claim 2 (depends on 1)

The method of claim 1, wherein the test compound is labeled.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polypeptide is in liquid phase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the polypeptide is on a solid support.

### Claim 5 (depends on 1)

The method of claim 1, wherein the test compound contacts a polypeptide expressed by a cell.

### Claim 6 (depends on 5)

The method of claim 5, wherein the cell is selected from the group consisting of an erythroid cell, an erythroid progenitor cell, a liver cell, a prostate cell, a hypothalamus cell, a bone marrow cell, a brain cell, a kidney cell, an ovary cell, a human vascular endothelial cell, and a hematopoietic progenitor cell.

### Claim 7 (depends on 5)

The method of claim 5, wherein the cell is selected from the group consisting of an erythroid cell, an erythroid progenitor cell, a liver cell, a prostate cell, and a hypothalamus cell.

### Claim 8 (depends on 1)

The method of claim 1, wherein the cellular activity modulated by the test compound is cell signaling that is mediated by the 23436 protein or a protein de-ubiquitinated by 23436.

### Claim 9 (depends on 1)

The method of claim 1, wherein the polypeptide is a fusion protein further comprising a polypeptide of the group consisting of glutathione-S-transferase (GST) and all or part of a serum protein.

### Claim 10 (depends on 5)

The method of claim 5, wherein the cellular activity modulated by the test compound is cell proliferation or cell differentiation.

### Claim 11 (depends on 10)

The method of claim 10, wherein the cell proliferation modulated by the test compound is growth factor mediated cell proliferation.

### Claim 12 (depends on 1)

The method of claim 1, further comprising the step:
(iv) determining the effect of the candidate compound identified in ii) on a hematopoietic disorder, an erythroid disorder, or a neoplastic disorder.

### Claim 13

A method for identifying a candidate compound which modulates ubiquitin hydrolase activity of a polypeptide comprising the amino acid sequence of SEQ ID NO:59 the method comprising the steps of:
a) contacting the polypeptide, or a cell expressing the polypeptide with a test compound;
b) determining the effect of the test compound on ubiquitin hydrolase activity of the polypeptide, to thereby identify a candidate compound which modulates ubiquitin hydrolase activity of the polypeptide; and
c) determining the effect of the candidate compound identified in b) on a cellular activity selected from the group consisting of cell proliferation, cell signaling, cell death, cell motility, receptor-mediated endocytosis, organelle biogenesis, hematopoietic cell proliferation or differentiation, and cytokine-mediated signaling events.