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Title:
REGULATION OF HUMAN TYROSINE PHOSPHATASE-LIKE ENZYME
Document Type and Number:
WIPO Patent Application WO/2002/020747
Kind Code:
A2
Abstract:
Reagents which regulate human tyrosine phosphatase-like enzyme and reagents which bind to human tyrosine phosphatase-like enzyme gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, obesity, diabetes, CNS disorders, chronic obstructive pulmonary disease, cardiovascular diseases, and cancer.

Inventors:
KOSSIDA SOPHIA (US)
Application Number:
PCT/EP2001/010205
Publication Date:
March 14, 2002
Filing Date:
September 05, 2001
Export Citation:
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Assignee:
BAYER AG (DE)
KOSSIDA SOPHIA (US)
International Classes:
C12N9/16; C12Q1/68; A61K48/00; (IPC1-7): C12N15/00
Domestic Patent References:
WO2001012819A22001-02-22
WO2001009345A12001-02-08
WO2001009316A12001-02-08
WO2002057460A22002-07-25
Other References:
DATABASE EMBL [Online] RNA,746 bp, 22 February 2000 (2000-02-22) "Homo sapiens cDNA FLJ20442 fis, clone KAT04828" retrieved from EBI Database accession no. AK000449 XP002211889
DATABASE EMBL [Online] 190 aa, tyrosine phosphatase, 1 May 1997 (1997-05-01) retrieved from EBI Database accession no. P91585 XP002211892
DATABASE EMBL [Online] 150 aa, 1 October 2000 (2000-10-01) WATANABE K ET AL.: "Hypothetical protein FLJ20442" retrieved from EBI Database accession no. Q9NX48 XP002211926
FAUMAN E B ET AL: "Structure and function of the protein tyrosine phosphatases" TIBS TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER PUBLICATION, CAMBRIDGE, EN, vol. 21, no. 11, 1 November 1996 (1996-11-01), pages 413-417, XP004071016 ISSN: 0968-0004
Attorney, Agent or Firm:
BAYER AKTIENGESELLSCHAFT (Leverkusen, DE)
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Claims:
CLAIMS
1. An isolated polynucleotide encoding a tyrosine phosphataselike enzyme polypeptide and being selected from the group consisting of : a) a polynucleotide encoding a tyrosine phosphataselike enzyme poly peptide comprising an amino acid sequence selected form the group consisting of : amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO:.
2. b) a polynucleotide comprising the sequence of SEQ ID NO: 1 ; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleo tide sequences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a to (d).
3. 2 An expression vector containing any polynucleotide of claim 1.
4. A host cell containing the expression vector of claim 2.
5. A substantially purified tyrosine phosphataselike enzyme polypeptide en coded by a polynucleotide of claim 1.
6. A method for producing a tyrosine phosphataselike enzyme polypeptide, wherein the method comprises the following steps: a) culturing the host cell of claim 3 under conditions suitable for the ex pression of the tyrosine phosphataselike enzyme polypeptide; and b) recovering the tyrosine phosphataselike enzyme polypeptide from the host cell culture.
7. A method for detection of a polynucleotide encoding a tyrosine phosphatase like enzyme polypeptide in a biological sample comprising the following steps: a) hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting said hybridization complex.
8. The method of claim 6, wherein before hybridization, the nucleic acid mate rial of the biological sample is amplified.
9. A method for the detection of a polynucleotide of claim 1 or a tyrosine phos phataselike enzyme polypeptide of claim 4 comprising the steps of : contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the tyrosine phosphataselike enzyme polypeptide.
10. A diagnostic kit for conducting the method of any one of claims 6 to 8.
11. A method of screening for agents which decrease the activity of a tyrosine phosphataselike enzyme, comprising the steps of : contacting a test compound with any tyrosine phosphataselike enzyme poly peptide encoded by any polynucleotide of claiml ; detecting binding of the test compound to the tyrosine phosphataselike en zyme polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a tyro sine phosphataselike enzyme.
12. A method of screening for agents which regulate the activity of a tyrosine phosphataselike enzyme, comprising the steps of : contacting a test compound with a tyrosine phosphataselike enzyme poly peptide encoded by any polynucleotide of claim 1; and detecting a tyrosine phosphataselike enzyme activity of the polypeptide, wherein a test compound which increases the tyrosine phosphataselike en zyme activity is identified as a potential therapeutic agent for increasing the activity of the tyrosine phosphataselike enzyme, and wherein a test com pound which decreases the tyrosine phosphataselike enzyme activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the tyrosine phosphataselike enzyme.
13. A method of screening for agents which decrease the activity of a tyrosine phosphataselike enzyme, comprising the steps of : contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of tyrosine phosphataselike enzyme.
14. A method of reducing the activity of tyrosine phosphataselike enzyme, com prising the steps of : contacting a cell with a reagent which specifically binds to any polynucleo tide of claim 1 or any tyrosine phosphataselike enzyme polypeptide of claim 4, whereby the activity of tyrosine phosphataselike enzyme is reduced.
15. A reagent that modulates the activity of a tyrosine phosphataselike enzyme polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.
16. A pharmaceutical composition, comprising: the expression vector of claim 2 or the reagent of claim 14 and a pharmaceu tically acceptable carrier.
17. Use of the pharmaceutical composition of claim 15 for modulating the activ ity of a tyrosine phosphataselike enzyme in a disease.
18. Use of claim 16 wherein the disease is obesity, diabetes, CNS disorder, chronic obstructive pulmonary disease, cardiovascular disease and cancer.
19. A cDNA encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO : 2.
20. The cDNA of claim 18 which comprises SEQ ID NO : 1 or SEQ ID NO : 14.
21. The cDNA of claim 18 which consists of SEQ ID NO : 1 or SEQ ID NO : 14.
22. An expression vector comprising a polynucleotide which encodes a polypep tide comprising the amino acid sequence shown in SEQ ID NO : 2.
23. The expression vector of claim 21 wherein the polynucleotide consists of SEQ ID NO : 1 or SEQ ID NO : 14.
24. A host cell comprising an expression vector which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO : 2.
25. The host cell of claim 23 wherein the polynucleotide consists of SEQ ID NO: 1 or SEQ ID NO : 14.
26. A purified polypeptide comprising the amino acid sequence shown in SEQ ID NO : 2.
27. The purified polypeptide of claim 25 which consists of the amino acid se quence shown in SEQ ID N0 : 2.
28. A fusion protein comprising a polypeptide having the amino acid sequence shown in SEQ ID N0 : 2.
29. A method of producing a polypeptide comprising the amino acid sequence shown in SEQ ID N0 : 2, comprising the steps of : culturing a host cell comprising an expression vector which encodes the poly peptide under conditions whereby the polypeptide is expressed; and isolating the polypeptide.
30. The method of claim 28 wherein the expression vector comprises SEQ ID NO : 1 or SEQ ID N0 : 14.
31. A method of detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID N0 : 2, comprising the steps of : hybridizing a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NO : 1 or SEQ ID N0 : 14 to nucleic acid material of a biological sample, thereby forming a hybridization complex ; and detecting the hybridization complex.
32. The method of claim 30 further comprising the step of amplifying the nucleic acid material before the step of hybridizing.
33. A kit for detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID N0 : 2, comprising: a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NO : 1 or SEQ ID NO : 14; and instructions for the method of claim 30.
34. A method of detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO : 2, comprising the steps of : contacting a biological sample with a reagent that specifically binds to the polypeptide to form a reagentpolypeptide complex; and detecting the reagentpolypeptide complex.
35. The method of claim 33 wherein the reagent is an antibody.
36. A kit for detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO : 2, comprising: an antibody which specifically binds to the polypeptide; and instructions for the method of claim 33.
37. A method of screening for agents which can modulate the activity of a human tyrosine phosphataselike enzyme, comprising the steps of : contacting a test compound with a polypeptide comprising an amino acid se quence selected from the group consisting of : (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO : 2 and (2) the amino acid sequence shown in SEQ ID NO : 2; and detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential agent for regulating activity of the human tyrosine phosphataselike enzyme.
38. The method of claim 36 wherein the step of contacting is in a cell.
39. The method of claim 36 wherein the cell is in vitro.
40. The method of claim 36 wherein the step of contacting is in a cellfree sys tem.
41. The method of claim 36 wherein the polypeptide comprises a detectable label.
42. The method of claim 36 wherein the test compound comprises a detectable label.
43. The method of claim 36 wherein the test compound displaces a labeled ligand which is bound to the polypeptide.
44. The method of claim 36 wherein the polypeptide is bound to a solid support.
45. The method of claim 36 wherein the test compound is bound to a solid sup port.
46. A method of screening for agents which modulate an activity of a human tyrosine phosphataselike enzyme, comprising the steps of : contacting a test compound with a polypeptide comprising an amino acid se quence selected from the group consisting of : (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO : 2 and (2) the amino acid sequence shown in SEQ ID NO : 2; and detecting an activity of the polypeptide, wherein a test compound which in creases the activity of the polypeptide is identified as a potential agent for in creasing the activity of the human tyrosine phosphataselike enzyme, and wherein a test compound which decreases the activity of the polypeptide is identified as a potential agent for decreasing the activity of the human tyro sine phosphataselike enzyme.
47. The method of claim 45 wherein the step of contacting is in a cell.
48. The method of claim 45 wherein the cell is in vitro.
49. The method of claim 45 wherein the step of contacting is in a cellfree sys tem.
50. A method of screening for agents which modulate an activity of a human tyrosine phosphataselike enzyme, comprising the steps of : contacting a test compound with a product encoded by a polynucleotide which comprises the nucleotide sequence shown in SEQ ID NO : 1 or SEQ ID NO : 14; and detecting binding of the test compound to the product, wherein a test compound which binds to the product is identified as a potential agent for regulating the activity of the human tyrosine phosphataselike enzyme.
51. The method of claim 49 wherein the product is a polypeptide.
52. The method of claim 49 wherein the product is RNA.
53. A method of reducing activity of a human tyrosine phosphataselike enzyme, comprising the step of : contacting a cell with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO : 1 or SEQ ID NO : 14, whereby the activity of a human tyrosine phosphataselike enzyme is reduced.
54. The method of claim 52 wherein the product is a polypeptide.
55. The method of claim 53 wherein the reagent is an antibody.
56. The method of claim 52 wherein the product is RNA.
57. The method of claim 55 wherein the reagent is an antisense oligonucleotide.
58. The method of claim 56 wherein the reagent is a ribozyme.
59. The method of claim 52 wherein the cell is in vitro.
60. The method of claim 52 wherein the cell is in vivo.
61. A pharmaceutical composition, comprising: a reagent which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ID NO : 2; and a pharmaceutically acceptable carrier.
62. The pharmaceutical composition of claim 60 wherein the reagent is an anti body.
63. A pharmaceutical composition, comprising: a reagent which specifically binds to a product of a polynucleotide compris ing the nucleotide sequence shown in SEQ ID NO : 1 or SEQ ID NO : 14; and a pharmaceutically acceptable carrier.
64. The pharmaceutical composition of claim 62 wherein the reagent is a ribo zyme.
65. The pharmaceutical composition of claim 62 wherein the reagent is an an tisense oligonucleotide.
66. The pharmaceutical composition of claim 62 wherein the reagent is an anti body.
67. A pharmaceutical composition, comprising: an expression vector encoding a polypeptide comprising the amino acid se quence shown in SEQ ID NO : 2; and a pharmaceutically acceptable carrier.
68. The pharmaceutical composition of claim 66 wherein the expression vector comprises SEQ ID NO : 1 or SEQ ID NO : 14.
69. A method of treating a tyrosine phosphataselike enzyme disfunction related disease, wherein the disease is selected from obesity, diabetes, CNS disorder, chronic obstructive pulmonary disease, cardiovascular disease and cancer, comprising the step of : administering to a patient in need thereof a therapeutically effective dose of a reagent that modulates a function of a human tyrosine phosphataselike en zyme, whereby symptoms of the tyrosine phosphataselike enzyme disfunc tion related disease are ameliorated.
70. The method of claim 68 wherein the reagent is identified by the method of claim 36.
71. The method of claim 68 wherein the reagent is identified by the method of claim 45.
72. The method of claim 68 wherein the reagent is identified by the method of claim 49.
Description:
REGULATION OF HUMAN TYROSINE PHOSPHATASE-LIKE ENZYME TECHNICAL FIELD OF THE INVENTION The invention relates to the area of enzyme regulation. More particularly, the inven- tion relates to the regulation of human tyrosine phosphatase-like enzyme and its regulation.

BACKGROUND OF THE INVENTION Phosphorylation of proteins is a fundamental mechanism for regulating diverse cel- lular processes. U. S. Patent 5,952,212. While the majority of protein phosphoryla- tion occurs at serine and threonine residues, phosphorylation at tyrosine residues is attracting a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established. Hunter et al., Ann. Rev. Biochem. 54, 987-30,1985 ; Ullrich et al., Cell 61,203-12,1990; Nurse, Nature 344, 503-08,1990; Cantley et al., Cell 64, 281-302,1991.

Biochemical studies have shown that phosphorylation on tyrosine residues of a vari- ety of cellular proteins is a dynamic process involving competing phosphorylation and dephosphorylation reactions. The regulation of protein tyrosine phosphorylation is mediated by the reciprocal actions of protein tyrosine kinases (PTKases or PTKS) and protein tyrosine phosphatases (PTPs). The tyrosine phosphorylation reactions are catalyzed by PTKs. Tyrosine phosphorylated proteins can be specifically dephosphorylated through the action of PTPs. The level of protein tyrosine phos- phorylation of intracellular substances is determined by the balance of PTK and PTP activities. Hunter, Cell 58, 1013-16,1989.

Protein Tyrosine Kinases The protein tyrosine kinases (PTKS; ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) are a large family of proteins that includes many growth factor receptors and potential oncogenes. Hanks et al., Science 241, 42-52,1988. Many PTKs have been linked to initial signals required for induction of the cell cycle. Weaver et al., Mol. Cell. Biol. 11 (9), 4415-22,1991. PTKs comprise a discrete family of enzymes having common ancestry with, but major differences from, serine/threonine-specific protein kinases. Hanks et al., supra. The mechanisms leading to changes in activity of PTKs are best understood in the case of receptor-type PTKs having a transmem- brane topology. Ullrich et al., 1990, supra. The binding of specific ligands to the extracellular domain of members of receptor-type PTKs is thought to induce their oligomerization, leading to an increase in tyrosine kinase activity and activation of the signal transduction pathways. Ullrich et al., 1990, supra. Deregulation of kinase activity through mutation or overexpression is a well-established mechanism for cell transformation. Hunter et al., 1985; Ullrich et al., 1990.

Protein Tyrosine Phosphatases The protein phosphatases are composed of at least two separate and distinct families: the protein serine/threonine phosphatases and the protein tyrosine phosphatases (PTPs ; protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48. Hunter, 1989.

The PTPs are a family of proteins that have been classified into two subgroups. The first subgroup is made up of the low molecular weight, intracellular enzymes that contain a single conserved catalytic phosphatase domain. All known intracellular type PTPs contain a single conserved catalytic phosphatase domain. Examples of the first group of PTPs include (1) placental PTP 1B (Charbonneau et al., Proc. Natl.

Acad. Sci. USA 86, 5252-56,1989; Chernoff et al., Proc. Natl. Acad. Sci USA 87, 2735-89, 1990, (2) T-cell PTP (Cool et al., Proc. Natl. Acad. Sci. USA 86, 5257-61, 1989, (3) rat brain PTP (Guan et al., Proc. Natl. Acad. Sci. USA 87, 1501-02,1990,

(4) neuronal phosphatase (STEP) (Lombroso et al., Proc. Natl. Acad. Sci. USA 88, 7242-46,1991, and (5) cytoplasmic phosphatases that contain a region of homology to cytoskeletal proteins (Gu et al., Proc. Natl. Acad Sci. USA 88, 5867-871,1991; Yang et al., Proc. Natl. Acad. Sci. USA 88, 5949-53,1991.

The second subgroup is made up of the high molecular weight, receptor-linked PTPs, termed RPTPs. RPTPs consist of (a) an intracellular catalytic region, (b) a single transmembrane segment, and (c) a putative ligand-binding extracellular domain. The structures and sizes of the putative ligand-binding extracellular"receptor"domains of RPTPs are quite divergent. In contrast, the intracellular catalytic regions of RPTPs are highly homologous. All RPTPs have two tandemly duplicated catalytic phosphatase homology domains, with the prominent exception of an RPTP termed HPTPß, which has only one catalytic phosphatase domain. Tsai et al., J : Biol. Chem.

266, 0534-43,1991.

Since the initial purification, sequencing, and cloning of a PTP, additional potential PTPs have been identified at a rapid pace. The number of different PTPs that have been identified is increasing steadily, leading to speculations that this family may be as large as the PTK family. Hunter, 1989.

Conserved amino acid sequences designated"consensus sequences"have been iden- tified in the catalytic domains of known PTPs. Krueger et al., EMBO J 9, 3241-52, 1990; Yi et al., Mol. Cell. Biol. 12, 836-46,1992. Yi et al. aligned the catalytic phosphatase domain sequences of the following PTPs : LCA, PTP1B, TCPTP, LAR, DLAR, and HPTPa, HPTPß, and HPTPy. This alignment includes the following "consensus sequences" (Yi et al., supra) : 1. K C XX Y W P (SEQ ID N0 : 12) 2. HCSXGXGRXG (SEQIDNO : 13).

Krueger et al., aligned the catalytic phosphatase domain sequences of PTP1B, TCPTP, LAR, LCA, HPTPa, p, 8, c and , and DLAR and DPTP. This alignment includes the following"consensus sequences" (Krueger et al., supra) : 1. K C X X Y W P (SEQ ID NO : 12) 2. HCSXGXGRXG (SEQIDNO : 13).

It is becoming clear that dephosphorylation of tyrosine residues can by itself function as an important regulatory mechanism. Dephosphorylation of a C-terminal tyrosine residue has been shown to activate tyrosine kinase activity in the src family of tyro- sine kinases. Hunter, Cell 49, 1-4,1987. Tyrosine dephosphorylation has been sug- gested to be an obligatory step in the mitotic activation of the maturation-promoting factor (MPF) kinase. Morla et al., Cell 58, 193-203,1989. These observations point out the need in the art for understanding the mechanisms that regulate tyrosine phos- phatase activity.

Because of the importance of these enzymes in a variety of cellular processes, there is a continuing need in the art to identify additional tyrosine phosphatases, which can be regulated to provide therapeutic effects.

SUMMARY OF THE INVENTION It is an object of the invention to provide reagents and methods of regulating a hu- man tyrosine phosphatase-like enzyme. This and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a tyrosine phosphatase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of :

amino acid sequences which are at least about 50% identical to the amino acid se- quence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.

Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a tyro- sine phosphatase-like enzyme polypeptide comprising an amino acid sequence se- lected from the group consisting of : amino acid sequences which are at least about 50% identical to the amino acid se- quence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.

Binding between the test compound and the tyrosine phosphatase-like enzyme poly- peptide is detected. A test compound which binds to the tyrosine phosphatase-like enzyme polypeptide is thereby identified as a potential agent for decreasing extra- cellular matrix degradation. The agent can work by decreasing the activity of the tyrosine phosphatase-like enzyme.

Another embodiment of the invention is a method of screening for agents which de- crease extracellular matrix degradation. A test compound is contacted with a polynu- cleotide encoding a tyrosine phosphatase-like enzyme polypeptide, wherein the poly- nucleotide comprises a nucleotide sequence selected from the group consisting of : nucleotide sequences which are at least about 50% identical to the nucleotide se- quence shown in SEQ ID NO: 1 ; and the nucleotide sequence shown in SEQ ID NO: 1.

Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the tyrosine phosphatase-like enzyme through interacting with the tyrosine phosphatase- like enzyme mRNA.

Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a tyro- sine phosphatase-like enzyme polypeptide comprising an amino acid sequence se- lected from the group consisting of : amino acid sequences which are at least about 50% identical to the amino acid se- quence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2.

A tyrosine phosphatase-like enzyme activity of the polypeptide is detected. A test compound which increases tyrosine phosphatase-like enzyme activity of the poly- peptide relative to tyrosine phosphatase-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation. A test compound which decreases tyrosine phosphatase-like en- zyme activity of the polypeptide relative to tyrosine phosphatase-like enzyme activity in the absence of the test compound is thereby identified as a potential agent for de- creasing extracellular matrix degradation.

Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a tyro- sine phosphatase-like enzyme product of a polynucleotide which comprises a nu- cleotide sequence selected from the group consisting of :

nucleotide sequences which are at least about 50% identical to the nucleotide se- quence shown in SEQ ID NO: 1; and the nucleotide sequence shown in SEQ ID NO: 1.

Binding of the test compound to the tyrosine phosphatase-like enzyme product is detected. A test compound which binds to the tyrosine phosphatase-like enzyme product is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a tyrosine phosphatase-like enzyme polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of : nucleotide sequences which are at least about 50% identical to the nucleotide se- quence shown in SEQ ID NO: 1 ; and the nucleotide sequence shown in SEQ ID NO: 1.

Tyrosine phosphatase-like enzyme activity in the cell is thereby decreased.

The invention thus provides a human tyrosine phosphatase-like enzyme which can be used to identify test compounds which may act, for example, as agonists or antago- nists at the enzyme's active site. Human tyrosine phosphatase-like enzyme and fragments thereof also are useful in raising specific antibodies which can block the enzyme and effectively reduce its activity.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : 1).

Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig. l (SEQ ID NO : 2).

Fig. 3 shows the amino acid sequence of the C. Elegans protein identified by EMBL Accession No. AF000363 (SEQ ID NO : 3).

Fig. 4 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : 4).

Fig. 5 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : 5).

Fig. 6 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : 6).

Fig. 7 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : 7).

Fig. 8 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : 8).

Fig. 9 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : 9).

Fig. 10 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : 10).

Fig. 11 shows the DNA-sequence encoding a tyrosine phosphatase-like enzyme poly- peptide (SEQ ID NO : lu).

Fig. 12 shows the BLASTP alignment of SEQ ID NO : 2 with SEQ ID NO : 3.

Fig. 13 shows the HMMPFAM-alignment of 218 (SEQ ID NO : 2) against pfamlhmmlDSPc.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to an isolated polynucleotide encoding a tyrosine phosphatase- like enzyme polypeptide and being selected from the group consisting of : a) a polynucleotide encoding a tyrosine phosphatase-like enzyme polypeptide comprising an amino acid sequence selected from the group consisting of : amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 2. b) a polynucleotide comprising the sequence of SEQ ID NO: 1; c) a polynucleotide which hybridizes under stringent conditions to a poly- nucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleotide se- quences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).

Furthermore, it has been discovered by the present applicant that a novel tyrosine phosphatase-like enzyme, particularlay a human tyrosine phosphatase-like enzyme, is a discovery of the present invention. Human tyrosine phosphatase-like enzyme com-

prises the amino acid sequence shown in SEQ ID NO : 2. Human tyrosine phos- phatase-like enzyme was identified by searching human sequences with the C. ele- gans protein having the sequence shown in SEQ ID NO : 3 and identified with EMBL Accession No. AF000363.

Human tyrosine phosphatase-like enzyme is 30% identical over 159 amino acids to the C. elegans protein identified with EMBL Accession No. AF000363 and anno- tated as"protein phosphatase CDC14" (FIG. 12). A dual specificity phosphatase catalytic region was detected from a pfam search and is shown in bold in FIG. 12. A TYRPHOSPHATASE region was detected from both Prosite and BLOCKS searches and is underlined in FIG. 12.

A coding sequence for SEQ ID NO : 2 is shown in SEQ ID NO : 1 or SEQ ID NO : 14.

This sequence is contained within the longer sequence shown in SEQ ID NO : 13.

Related ESTs (SEQ ID NOS: 4-10) are expressed in fetal brain, kidney (mouse), mammary gland (mouse), placenta (mouse), endocrine tumor, lung, epithelium, immune privileged tissue, tumor tissue, colon tumor, carcinoid, uterus, pooled germ cell tumors, pooled human melanocytes, fetal heart, and pregnant uterus.

Human tyrosine phosphatase-like enzyme is expected to be useful for the same pur- poses as previously identified phosphatases. Thus, human tyrosine phosphatase-like enzyme can be used in therapeutic methods to treat disorders such as obesity, diabe- tes, CNS disorders, chronic obstructive pulmonary disease, cardiovascular diseases, and cancer. Human tyrosine phosphatase-like enzyme also can be used to screen for human tyrosine phosphatase-like enzyme agonists and antagonists.

Polypeptides Human tyrosine phosphatase-like enzyme polypeptides according to the invention comprise at least 6,10,15,20,25,50,75,100,125, or 150 contiguous amino acids

selected from the amino acid sequence shown in SEQ ID NO : 2 or a biologically ac- tive variant thereof, as defined below. A human tyrosine phosphatase-like enzyme polypeptide of the invention therefore can be a portion of a human tyrosine phos- phatase-like enzyme, a full-length human tyrosine phosphatase-like enzyme, or a fusion protein comprising all or a portion of a human tyrosine phosphatase-like en- zyme.

Biologically Active Variants Human tyrosine phosphatase-like enzyme polypeptide variants which are biologi- cally active, e. g., retain the ability to protein tyrosine phosphate to tyrosine and phosphate, also are human tyrosine phosphatase-like enzyme polypeptides. Prefer- ably, naturally or non-naturally occurring tyrosine phosphatase-like enzyme poly- peptide variants have amino acid sequences which are at least about 50,55,60,65, or 70, preferably about 75,80,85,90,96,96, or 98% identical to the amino acid se- quence shown in SEQ ID NO : 2 or a fragment thereof. Percent identity between a putative polypeptide variant and an amino acid sequence of SEQ ID NO : 2 is deter- mined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replace- ments are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence.

They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing

biological or immunological activity of an tyrosine phosphatase-like enzyme poly- peptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active polypeptide can readily be determined by assaying for tyrosine phosphatase activity, as described, for example, in Calvert-Evers & Hammond, Cell Biol. Int. 24, 559-68, 2000.

Fusion Proteins Fusion proteins are useful for generating antibodies against tyrosine phosphatase-like enzyme amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of an tyrosine phosphatase-like enzyme polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

An tyrosine phosphatase-like enzyme fusion protein comprises two polypeptide seg- ments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6,10,15,20,25,50,75,100,125, or 150 contiguous amino acids of SEQ ID NO : 2 or of a biologically active variant, such as those described above.

The first polypeptide segment also can comprise full-length tyrosine phosphatase- like enzyme.

The second polypeptide segment can be a full-length protein or a protein fragment.

Proteins commonly used in fusion protein construction include p-galactosidase, ß- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horse- radish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Addition- ally, epitope tags are used in fusion protein constructions, including histidine (His)

tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding pro- tein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the tyrosine phosphatase-like enzyme polypeptide-encoding sequence and the heterologous pro- tein sequence, so that the desired polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from the complement of SEQ ID NO : 1 or SEQ ID NO : 14 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of Species Homologs Species homologs of human tyrosine phosphatase-like enzyme polypeptide can be obtained using tyrosine phosphatase-like enzyme polypeptide polynucleotides (de- scribed below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of tyrosine phosphatase-like enzyme polypeptide, and ex- pressing the cDNAs as is known in the art.

Polynucleotides An tyrosine phosphatase-like enzyme polynucleotide can be single-or double- stranded and comprises a coding sequence or the complement of a coding sequence for an tyrosine phosphatase-like enzyme polypeptide. A coding sequence for tyro- sine phosphatase-like enzyme shown in SEQ ID NO : 2 is shown in SEQ ID NO : 1 or SEQ ID NO : 14.

Degenerate nucleotide sequences encoding human tyrosine phosphatase-like enzyme polypeptides, as well as homologous nucleotide sequences which are at least about 50,55,60,65,70, preferably about 75,90,96, or 98% identical to the nucleotide sequence shown in SEQ ID NO : 1 or SEQ ID NO : 14 or its complement also are tyrosine phosphatase-like enzyme polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of-12 and a gap extension penalty of-2.

Complementary DNA (cDNA) molecules, species homologs, and variants of tyrosine phosphatase-like enzyme polynucleotides which encode biologically active tyrosine phosphatase-like enzyme polypeptides also are tyrosine phosphatase-like enzyme polynucleotides. Polynucleotides comprising at least 6,7,8,10,12,15,18, 20, or 25 contiguous nucleotides of SEQ ID NO : 1 or SEQ ID NO : 14 or its complement also are tyrosine phosphatase-likeenzyme polynucleotides. Such polynucleotides can be used, for example, as oligonucleotide probes or as antisense oligonucleotides.

Identification ofPolynucleotide Variants and Homologs Variants and homologs of the polynucleotides described above also are tyrosine phosphatase-like enzyme polynucleotides. Typically, homologous polynucleotide

sequences can be identified by hybridization of candidate polynucleotides to known tyrosine phosphatase-like enzyme polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions--2X SSC (0. 3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 min- utes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each--homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the tyrosine phosphatase-like enzyme polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.

Human variants of tyrosine phosphatase-like enzyme polynucleotides can be identi- fied, for example, by screening human cDNA expression libraries. It is well known that the Tm of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al., J Mol. Bill 81, 123 (1973). Variants of human tyro- sine phosphatase-like enzyme polynucleotides or tyrosine phosphatase-like enzyme polynucleotides of other species can therefore be identified by hybridizing a putative homologous polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO : 1 or SEQ ID NO : 14 or the complement thereof to form a test hybrid.

The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to tyrosine phosphatase-like enzyme poly- nucleotides or their complements following stringent hybridization and/or wash con- ditions also are tyrosine phosphatase-like enzyme polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example,

in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9. 50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calcu- lated Tm of the hybrid under study. The Tm of a hybrid between a polynucleotide having a nucleotide sequence shown in SEQ ID NO : 1 or SEQ ID NO : 14 or the complement thereof and a polynucleotide sequence which is at least about 50,55,60, 65,70, preferably about 75,90,96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad Sci. US. A. 48, 1390 (1962): Tm = 81.5 °C-16.6 (loglo [Na+]) + 0.41 (% G + C)-0.63 (% formamide)-600/1), where I = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% form- amide, 4X SSC at 42 °C, or 0. 5X SSC, 0.1% SDS at 65 °C. Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.

Preparation of Polynucleotides An tyrosine phosphatase-like enzyme polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Poly- nucleotides can be made by a cell and isolated using standard nucleic acid purifica- tion techniques, or synthesized using an amplification technique, such as the poly- merase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated tyrosine phosphatase- like enzyme polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprises tyrosine phosphatase-like

nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70,80, or 90% free of other molecules.

Human tyrosine phosphatase-like enzyme cDNA molecules can be made with stan- dard molecular biology techniques, using human tyrosine phosphatase-like enzyme mRNA as a template. Human tyrosine phosphatase-like enzyme cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the in- vention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesizes tyrosine phosphatase-like enzyme polynucleotides. The degeneracy of the genetic code al- lows alternate nucleotide sequences to be synthesized which will encode a polypep- tide having, for example, an amino acid sequence shown in SEQ ID NO : 2 or a bio- logically active variant thereof.

Extending Polynucleotides Various PCR-based methods can be used to extend the nucleic acid sequences dis- closed herein to detect upstream sequences such as promoters and regulatory ele- ments. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2,318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker se- quence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., Nucleic Acids Res. 16, 8186,1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleo- tides in length, to have a GC content of 50% or more, and to anneal to the target se- quence at temperatures about 68-72 °C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplifica- tion of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1, 111-119,1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al., Nucleic Acids Res. 19, 3055-3060,1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5'regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d (T) library does not yield a full-length cDNA. Genomic libraries can be use- ful for extension of sequence into 5'non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For ex- ample, capillary sequencing can employ flowable polymers for electrophoretic sepa- ration, four different fluorescent dyes (one for each nucleotide) which are laser acti- vated, and detection of the emitted wavelengths by a charge coupled device camera.

Output/light intensity can be converted to electrical signal using appropriate software (e. g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire pro- cess from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the se- quencing of small pieces of DNA which might be present in limited amounts in a particular sample.

Obtaining Polypeptides Human tyrosine phosphatase-like enzyme polypeptides can be obtained, for example, by purification from human cells, by expression of tyrosine phosphatase-like enzyme polynucleotides, or by direct chemical synthesis.

Protein Purification Human tyrosine phosphatase-like enzyme polypeptides can be purified from any cell which expresses the enzyme, including host cells which have been transfected with tyrosine phosphatase-like enzyme expression constructs. A purified tyrosine phos- phatase-like enzyme polypeptide is separated from other compounds which normally associate with the tyrosine phosphatase-like enzyme polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified tyrosine phosphatase-like enzyme polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%,

or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.

Expression of Polynucleotides To express a human tyrosine phosphatase-like enzyme polynucleotide, the poly- nucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Meth- ods which are well known to those skilled in the art can be used to construct expres- sion vectors containing sequences encoding tyrosine phosphatase-like enzyme poly- peptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sam- brook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N. Y., 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding an tyrosine phosphatase-like enzyme polypeptide. These in- clude, but are not limited to, microorganisms, such as bacteria transformed with re- combinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast trans- formed with yeast expression vectors, insect cell systems infected with virus expres- sion vectors (e. g., baculovirus), plant cell systems transformed with virus expression vectors (e. g., cauliflower mosaic virus, CaMV ; tobacco mosaic virus, TMV) or with bacterial expression vectors (e. g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector--enhancers, promoters, 5'and 3'untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host util- ized, any number of suitable transcription and translation elements, including con-

stitutive and inducible promoters, can be used. For example, when cloning in bacte- rial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTI plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e. g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e. g., viral promoters or leader sequences) can be cloned into the vector. In mam- malian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding an tyrosine phosphatase-like enzyme polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the tyrosine phosphatase-like enzyme polypeptide. For ex- ample, when a large quantity of a polypeptide is needed for the induction of anti- bodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding the polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of p-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster, J Biol. Chem. 264, 5503-5509,1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage

sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.

For reviews, see Ausubel et al. (1989) and Grant et al., Methods Enzymol. 153, 516-544,1987.

Plant and Insect Expression Systems If plant expression vectors are used, the expression of sequences encoding tyrosine phosphatase-like enzyme polypeptides can be driven by any of a number of pro- moters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO R 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3,1671-1680,1984; Broglie et al., Science 224, 838-843,1984; Winter et al., Results Probl. Cell Differ. 17, 85-105,1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.

Such techniques are described in a number of generally available reviews (e. g., Hobbs or Murray, in McGRAw HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N. Y., pp. 191-196,1992).

An insect system also can be used to express an tyrosine phosphatase-like enzyme polypeptide. For example, in one such system Autographa californica nuclear poly- hedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding tyrosine phospha- tase-like enzyme polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.

Successful insertion of tyrosine phosphatase-like enzyme polypeptides will render

the polyhedrin gene inactive and produce recombinant virus lacking coat protein.

The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplu- sia larvae in which tyrosine phosphatase-like enzyme polypeptides can be expressed (Engelhard etal., Proc. Nat. Acad. Sci. 91, 3224-3227,1994).

Mammalian Expression Systems A number of viral-based expression systems can be used to express tyrosine phos- phatase-like enzyme polypeptides in mammalian host cells. For example, if an ad- enovirus is used as an expression vector, sequences encoding tyrosine phosphatase- like enzyme polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing an tyrosine phosphatase-like enzyme polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659,1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 1 OM are constructed and delivered to cells via conventional delivery methods (e. g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding tyrosine phosphatase-like enzyme polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding an tyrosine phosphatase-like enzyme polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous transla-

tional control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of vari- ous origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl. Cell Differ. 20, 125-162,1994).

Host Cells A host cell strain can be chosen for its ability to modulate the expression of the in- serted sequences or to process the expressed tyrosine phosphatase-like enzyme poly- peptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipida- tion, and acylation. Post-translational processing which cleaves a"prepro"form of the polypeptide also can be used to facilitate correct insertion, folding and/or func- tion. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e. g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express tyrosine phosphatase-like en- zyme polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced tyrosine phosphatase-like enzyme sequences.

Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R. I. Freshney, ed., 1986.

Any number of selection systems can be used to recover transformed cell lines.

These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11, 223-32,1977) and adenine phosphoribosyltransferase (Lowy et al., Cell 22, 817-23,1980) genes which can be employed in tk-or aprt-cells, re- spectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77,3567-70,1980), npt confers resistance to the amino- glycosides, neomycin and G-418 (Colbere-Garapin et al., J : Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyl- transferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of trypto- phan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51,1988). Visible markers such as antho- cyanins, p-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of tran- sient or stable protein expression attributable to a specific vector system (Rhodes et al., MethodsMol. Biol. 55, 121-131,1995).

Detecting Expression Although the presence of marker gene expression suggests that the tyrosine phos- phatase-like enzyme polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding an tyrosine phosphatase- like enzyme polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode an tyrosine phosphatase-like enzyme polypeptide can be identified by the absence of marker gene function. Alternatively, a marker

gene can be placed in tandem with a sequence encoding an tyrosine phosphatase-like enzyme polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tyrosine phosphatase-like enzyme polynucleotide.

Alternatively, host cells which contain an tyrosine phosphatase-like enzyme poly- nucleotide and which express an tyrosine phosphatase-like enzyme polypeptide can be identified by a variety of procedures known to those of skill in the art. These pro- cedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding an tyro- sine phosphatase-like enzyme polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding an tyrosine phosphatase-like enzyme polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding an tyrosine phosphatase-like enzyme polypeptide to detect trans- formants which contain an tyrosine phosphatase-like enzyme polynucleotide.

A variety of protocols for detecting and measuring the expression of an tyrosine phosphatase-like enzyme polypeptide, using either polyclonal or monoclonal anti- bodies specific for the polypeptide, are known in the art. Examples include en- zyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluores- cence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on an tyrosine phosphatase-like enzyme polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al., SEROLOGICAL METHODS : A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al., J. Exp. Med. 158, 1211-1216,1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for pro- ducing labeled hybridization or PCR probes for detecting sequences related to poly- nucleotides encoding tyrosine phosphatase-like enzyme polypeptides include oligo- labeling, nick translation, end-labeling, or PCR amplification using a labeled nucleo- tide. Alternatively, sequences encoding an tyrosine phosphatase-like enzyme poly- peptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthe- size RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a vari- ety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides Host cells transformed with nucleotide sequences encoding an tyrosine phosphatase- like enzyme polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a trans- formed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode tyrosine phosphatase-like enzyme polypeptides can be designed to contain signal sequences which direct secretion of soluble tyrosine phosphatase-like enzyme polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane- bound tyrosine phosphatase-like enzyme polypeptide.

As discussed above, other constructions can be used to join a sequence encoding an tyrosine phosphatase-like enzyme polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such puri- fication facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the tyrosine phosphatase-like enzyme polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fu- sion protein containing an tyrosine phosphatase-like enzyme polypeptide and 6 his- tidine residues preceding a thioredoxin or an enterokinase cleavage site. The his- tidine residues facilitate purification by IMAC (immobilized metal ion affinity chro- matography, as described in Porath et al., Prot. Exp. Purif 3,263-281,1992), while the enterokinase cleavage site provides a means for purifying the tyrosine phospha- tase-like enzyme polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al., DNA Cell Biol. 12, 441-453,1993.

Chemical Synthesis Sequences encoding an tyrosine phosphatase-like enzyme polypeptide can be synthe- sized, in whole or in part, using chemical methods well known in the art (see Caruth- ers et al., Nucl. Acids Res. Sy np. Ser. 215-223,1980; Horn et al. NucL Acids Res.

Symp. Ser. 225-232,1980). Alternatively, an tyrosine phosphatase-like enzyme polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154,1963; Roberge et al., Science 269, 202-204,1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Bio-

systems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of tyrosine phosphatase-like enzyme polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified by preparative high per- formance liquid chromatography (e. g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N. Y., 1983). The com- position of a synthetic tyrosine phosphatase-like enzyme polypeptide can be con- firmed by amino acid analysis or sequencing (e. g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the tyrosine phosphatase-like enzyme polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to pro- duce a variant polypeptide or a fusion protein.

Production of Altered Polypeptides As will be understood by those of skill in the art, it may be advantageous to produce tyrosine phosphatase-like enzyme polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of pro- tein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occur- ring sequence.

The nucleotide sequences disclosed herein can be engineered using methods gener- ally known in the art to alter tyrosine phosphatase-like enzyme polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide se-

quences. For example, site-directed mutagenesis can be used to insert new restric- tion sites, alter glycosylation patterns, change codon preference, produce splice vari- ants, introduce mutations, and so forth.

Antibodies Any type of antibody known in the art can be generated to bind specifically to an epitope of an tyrosine phosphatase-like enzyme polypeptide."Antibody"as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F (ab') 2, and Fv, which are capable of binding an epitope of an tyrosine phos- phatase-like enzyme polypeptide. Typically, at least 6,8,10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-con- tiguous amino acids may require more, e. g., at least 15,25, or 50 amino acids.

An antibody which specifically binds to an epitope of an tyrosine phosphatase-like enzyme polypeptide can be used therapeutically, as well as in immunochemical as- says, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.

Various immunoassays can be used to identify antibodies having the desired speci- ficity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.

Typically, an antibody which specifically binds to an tyrosine phosphatase-like en- zyme polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical as- say. Preferably, antibodies which specifically bind to tyrosine phosphatase-like en- zyme polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate an tyrosine phosphatase-like enzyme polypeptide from solution.

Human tyrosine phosphatase-like enzyme polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, an tyrosine phosphatase-like enzyme polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e. g., aluminum hydroxide), and sur- face active substances (e. g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are espe- cially useful.

Monoclonal antibodies which specifically bind to an tyrosine phosphatase-like en- zyme polypeptide can be prepared using any technique which provides for the pro- duction of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al., Nature 256, 495-497, 1985; Kozbor et al., J : Immunol. Methods 81, 31-42,1985; Cote et al., Proc. Natl.

Acad. Sci. 80, 2026-2030,1983; Cole et al., Mol. Cell Biol. 62, 109-120,1984).

In addition, techniques developed for the production of"chimeric antibodies,"the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al., Proc. Natl. Acad. Sci. 81, 6851-6855,1984; Neuberger et al., Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454,1985). Monoclonal and other antibodies also can be"humanized"to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be suffi- ciently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent anti-

bodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual resi- dues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to an tyrosine phosphatase-like enzyme polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U. S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to tyrosine phosphatase-like enzyme polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl.

Acad. Sci 88, 11120-23,1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., 1996, Eur. R Cancer Prev. 5, 507-11). Single-chain antibodies can be mono-or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. I5, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, R Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be pro- duced directly using, for example, filamentous phage technology (Verhaar et al., 1995, Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J : Immunol. Meth. 165, 81- 91).

Antibodies which specifically bind to tyrosine phosphatase-like enzyme polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989 ; Winter et al., Nature 349, 293-299,1991).

Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the"diabodies"described in WO 94/13804, also can be prepared.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which an tyrosine phosphatase-like enzyme polypeptide is bound. The bound anti- bodies can then be eluted from the column using a buffer with a high salt concentra- tion.

Antisense Oligonucleotides Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12,15,20,25,30,35,40,45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oli- gonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of tyrosine phosphatase-like enzyme gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a com- bination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5'end of one nucleotide with the 3'end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphos- phonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkyl- phosphonates, phosphoramidates, phosphate esters, carbarnates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol.

Biol. 20, 1-8, 1994 ; Sonveaux, Meth. Mol. Biol. 26, 1-72,1994; Uhlmann et al., Chem. Rev. 90, 543-583,1990.

Modifications of tyrosine phosphatase-like enzyme gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the tyrosine phosphatase-like enzyme gene. Oligonucleo- tides derived from the transcription initiation site, e. g., between positions-10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix"base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e. g., Gee et al., in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N. Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of an tyrosine phos- phatase-like enzyme polynucleotide. Antisense oligonucleotides which comprise, for example, 2,3,4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to an tyrosine phosphatase-like enzyme polynucleotide, each sepa- rated by a stretch of contiguous nucleotides which are not complementary to adjacent

tyrosine phosphatase-like enzyme nucleotides, can provide sufficient targeting speci- ficity for tyrosine phosphatase-like enzyme mRNA. Preferably, each stretch of com- plementary contiguous nucleotides is at least 4,5,6,7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1,2,3, or 4 nu- cleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tol- erated between a particular antisense oligonucleotide and a particular tyrosine phos- phatase-like enzyme polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybrid- ize to an tyrosine phosphatase-like enzyme polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, inter- nucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3'hydroxyl group or the 5'phos- phate group are substituted, also can be employed in a modified antisense oligo- nucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e. g., Agrawal et al., Trends Biotechnol. 10, 152-158,1992; Uhlmann et al., Chem. Rev. 90, 543-584,1990; Uhlmann et al., Tetrahedron. Lett.

215, 3539-3542,1987.

Ribozymes Ribozymes are RNA molecules with catalytic activity. See, e. g., Cech, Science 236, 1532-1539; 1987; Cech, Anfz. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin.

Struct. Biol. 2,605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e. g., Haseloff et al., U. S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme mole-

cule to complementary target RNA, followed by endonucleolytic cleavage. Ex- amples include engineered hammerhead motif ribozyme molecules that can specifi- cally and efficiently catalyze endonucleolytic cleavage of specific nucleotide se- quences.

The coding sequence of an tyrosine phosphatase-like enzyme polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the tyrosine phosphatase-like enzyme polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete"hybridization"region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321, 201).

Specific ribozyme cleavage sites within an tyrosine phosphatase-like enzyme RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate tyrosine phosphatase-like enzyme RNA targets also can be evaluated by testing ac- cessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the af- finity of the hybridization sequence for the target. The hybridizing and cleavage re- gions of the ribozyme can be integrally related such that upon hybridizing to the tar- get RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease tyrosine phosphatase-like enzyme expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribo- zyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al., U. S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Diffef°entially Expressed Genes Described herein are methods for the identification of genes whose products interact with human tyrosine phosphatase-like enzyme. Such genes may represent genes which are differentially expressed in disorders including, but not limited to, obesity, diabetes, CNS disorders, chronic obstructive pulmonary disease, and cardiovascular diseases. Further, such genes may represent genes which are differentially regulated in response to manipulations relevant to the progression or treatment of such dis- eases. Additionally, such genes may have a temporally modulated expression, in- creased or decreased at different stages of tissue or organism development. A differ- entially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human tyrosine phosphatase-like enzyme gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quan- titative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes To identify differentially expressed genes total RNA or, preferably, mRNA is iso- lated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique which does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al., ed."CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc.

New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U. S. Patent 4,843,155.

Transcripts within the collected RNA samples which represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al., Proc.

Natl. Acad. Sci. U. S. A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al., Nature 308, 149-53; Lee et al., Proc. Natl. Acad. Sci. U. S. A. 88, 2825,1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71,1992 ; U. S.

Patent 5,262,311).

The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human tyrosine phosphatase-like enzyme. For example, treatment may include a modulation of expression of the differentially ex-

pressed genes and/or the gene encoding the human tyrosine phosphatase-like en- zyme. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human tyrosine phosphatase-like enzyme gene or gene product are up-regulated or down-regulated.

Screening Methods The invention provides assays for screening test compounds which bind to or modu- late the activity of an tyrosine phosphatase-like enzyme polypeptide or an tyrosine phosphatase-like enzyme polynucleotide. A test compound preferably binds to an tyrosine phosphatase-like enzyme polypeptide or polynucleotide. More preferably, a test compound decreases or increases tyrosine phosphatase-like enzyme by at least about 10, preferably about 50, more preferably about 75,90, or 100% relative to the absence of the test compound.

Test Compounds Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The com- pounds can be naturally occurring or designed in the laboratory. They can be iso- lated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvo- lution, the"one-bead one-compound"library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypep- tide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145,1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al., Proc. Natl. Acad Sci. US. A. 90, 6909,1993; Erb et al. Proc.

Natl. Acad. Sci. U. S. A. 91, 11422,1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303,1993; Carell et al., Angew. Chem. Int. Ed. Efzgl.

33,2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33,2061 ; Gallop et al., J.

Med. Chem. 37,1233,1994). Libraries of compounds can be presented in solution (see, e. g., Houghten, BioTechniques 13, 412-421,1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556,1993), bacteria or spores (Ladner, U. S. Patent 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. U. S. A.

89, 1865-1869,1992), or phage (Scott & Smith, Science 249, 386-390,1990; Devlin, Science 249, 404-406,1990); Cwirla et al., Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. MoL Biol. 222,301-310, 1991; and Ladner, U. S. Patent 5,223,409).

High Throughput Screening Test compounds can be screened for the ability to bind to tyrosine phosphatase-like enzyme polypeptides or polynucleotides or to affect tyrosine phosphatase-like en- zyme activity or tyrosine phosphatase-like enzyme gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates.

The wells of the microtiter plates typically require assay volumes that range from 50 to 500 u. l. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively,"free format assays,"or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al., Proc. Natl. Acad. Sci. U. S. A. 19, 1614-18 (1994). The cells are

placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky,"Strategies for Screening Combinatorial Libraries : Novel and Traditional Approaches,"reported at the First Annual Conference of The Society for Biomolecular Screening in Philadel- phia, Pa. (Nov. 7-10,1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial com- pounds via a photolinker were placed inside the gel and the compounds were par- tially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Yet another example is described by Salmon et al., Molecular Diversity 2,57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al., U. S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.

When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

Binding Assays

For binding assays, the test compound is preferably a small molecule which binds to and occupies, for example, the ATP/GTP binding site of the enzyme or the active site of the tyrosine phosphatase-like enzyme polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the tyrosine phosphatase-like enzyme polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the tyro- sine phosphatase-like enzyme polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining con- version of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to an tyrosine phosphatase-like enzyme polypeptide can be determined without labeling either of the interactants. For ex- ample, a microphysiometer can be used to detect binding of a test compound with an tyrosine phosphatase-like enzyme polypeptide. A microphysiometer (e. g., Cytosen- sorTM) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and an tyrosine phosphatase-like enzyme polypeptide (McConnell et al., Science 257, 1906-1912,1992).

Determining the ability of a test compound to bind to an tyrosine phosphatase-like enzyme polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Cliem. 63, 2338-2345,1991, and Szabo et al., Curr. Opin. Struct. Biol. 5, 699-705,1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e. g., BIAcore). Changes in the optical phenomenon surface

plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, an tyrosine phosphatase-like enzyme polypep- tide can be used as a"bait protein"in a two-hybrid assay or three-hybrid assay (see, e. g., U. S. Patent 5,283,317; Zervos et al., Cell 72,223-232,1993; Madura et al., J : Biol. Chem. 268, 12046-12054, 1993; Bartel et al., BioTechniques 14, 920-924, 1993; Iwabuchi et al., Oncogene 8, 1693-1696,1993; and Brent W094/10300), to identify other proteins which bind to or interact with the tyrosine phosphatase-like enzyme polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding an tyrosine phosphatase-like enzyme polypeptide can be fused to a poly- nucleotide encoding the DNA binding domain of a known transcription factor (e. g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey"or"sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the"bait"and the"prey"proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e. g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Ex- pression of the reporter gene can be detected, and cell colonies containing the func- tional transcription factor can be isolated and used to obtain the DNA sequence en- coding the protein which interacts with the tyrosine phosphatase-like enzyme poly- peptide.

It may be desirable to immobilize either the tyrosine phosphatase-like enzyme poly- peptide (or polynucleotide) or the test compound to facilitate separation of bound

from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the tyrosine phosphatase-like enzyme poly- peptide (or polynucleotide) or the test compound can be bound to a solid support.

Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (in- cluding, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test com- pound to a solid support, including use of covalent and non-covalent linkages, pas- sive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a v enzyme polypep- tide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro- centrifuge tubes.

In one embodiment, the tyrosine phosphatase-like enzyme polypeptide is a fusion protein comprising a domain that allows the tyrosine phosphatase-like enzyme poly- peptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.

Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed tyrosine phos- phatase-like enzyme polypeptide; the mixture is then incubated under conditions conducive to complex formation (e. g., at physiological conditions for salt and pH).

Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either an tyrosine phosphatase-like enzyme polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated tyrosine phosphatase-like enzyme polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e. g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, an- tibodies which specifically bind to an tyrosine phosphatase-like enzyme polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the tyrosine phosphatase-like enzyme polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addi- tion to those described above for the GST-immobilized complexes, include immuno- detection of complexes using antibodies which specifically bind to the tyrosine phos- phatase-like enzyme polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the tyrosine phosphatase-like enzyme polypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to an tyrosine phosphatase-like enzyme polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises an tyrosine phosphatase-like enzyme polypeptide or polynucleotide can be used in a cell-based assay system. An tyrosine phosphatase-like enzyme poly- nucleotide can be naturally occurring in the cell or can be introduced using tech- niques such as those described above. Binding of the test compound to an tyrosine phosphatase-like enzyme polypeptide or polynucleotide is determined as described above.

Enzyme Assays

Test compounds can be tested for the ability to increase or decrease the enzyme ac- tivity of a human tyrosine phosphatase-like enzyme polypeptide. Enzyme activity can be measured, for example, as described in Calvert-Evers & Hammond, Cell Biol.

Int. 24, 559-68,2000.

Enzyme assays can be carried out after contacting either a purified tyrosine phos- phatase-like enzyme polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which decreases activity of an tyrosine phospha- tase-like enzyme polypeptide by at least about 10, preferably about 50, more prefer- ably about 75,90, or 100% is identified as a potential therapeutic agent for decreas- ing tyrosine phosphatase-like enzyme activity. A test compound which increases activity of a human tyrosine phosphatase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75,90, or 100% is identified as a po- tential therapeutic agent for increasing human tyrosine phosphatase-like enzyme ac- tivity.

Gene Expression In another embodiment, test compounds which increase or decrease tyrosine phos- phatase-like enzyme gene expression are identified. An tyrosine phosphatase-like enzyme polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the v enzyme polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modu- lator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its ab- sence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide

is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of v enzyme mRNA or polypeptide expression in the cells can be deter- mined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide prod- ucts of an tyrosine phosphatase-like enzyme polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.

Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into an tyrosine phosphatase-like enzyme polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses an tyrosine phosphatase-like enzyme polynucleotide can be used in a cell-based assay system. The tyrosine phosphatase-like enzyme polynucleotide can be naturally occurring in the cell or can be introduced using tech- niques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions The invention also provides pharmaceutical compositions which can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the in- vention can comprise, for example, an tyrosine phosphatase-like enzyme polypep- tide, tyrosine phosphatase-like enzyme polynucleotide, ribozymes or antisense oligo- nucleotides, antibodies which specifically bind to an tyrosine phosphatase-like en- zyme polypeptide, or mimetics, agonists, antagonists, or inhibitors of an tyrosine phosphatase-like enzyme polypeptide activity. The compositions can be adminis- tered alone or in combination with at least one other agent, such as stabilizing com-

pound, which can be administered in any sterile, biocompatible pharmaceutical car- rier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be ad- ministered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be for- mulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol ; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxy- propylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i. e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid poly- ethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiologically buffered saline. Aqueous in- jection suspensions can contain substances which increase the viscosity of the sus- pension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubil- ity of the compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e. g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfu- ric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aque- ous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condi- tion. Such labeling would include amount, frequency, and method of administration.

Therapeutic Indications and Methods Human tyrosine phosphatase-like enzyme can be regulated to treat a variety of dis- eases and disorders, including cancer, obesity, diabetes, CNS disorders, chronic ob- structive pulmonary disease, and cardiovascular diseases.

Abnormal tyrosine phosphorylation has been postulated to be involved in the gen- erationofneoplasia. Zelivianskietal., Mol. Cell. biochem. 208, 1-18,2000. Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal coun- terparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortali- zation), increased cellular motility and invasiveness, increased ability to recruit blood

supply through induction of new blood vessel formation (angiogenesis), genetic in- stability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an in- tractable disease state in which organ failure and patient death ultimately ensue.

Most standard cancer therapies target cellular proliferation and rely on the differen- tial proliferative capacities between transformed and normal cells for their efficacy.

This approach is hindered by the facts that several important normal cell types are also highly proliferative and that cancer cells frequently become resistant to these agents. Thus, the therapeutic indices for traditional anti-cancer therapies rarely ex- ceed 2.0.

The advent of genomics-driven molecular target identification has opened up the possibility of identifying new cancer-specific targets for therapeutic intervention that will provide safer, more effective treatments for cancer patients. Thus, newly dis- covered tumor-associated genes and their products can be tested for their role (s) in disease and used as tools to discover and develop innovative therapies. Genes play- ing important roles in any of the physiological processes outlined above can be char- acterized as cancer targets.

Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins.

These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Agonists and/or antagonists of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxico- logical analyses form the basis for drug development and subsequent testing in hu- mans.

Obesity and overweight are defined as an excess of body fat relative to lean body mass. An increase in caloric intake or a decrease in energy expenditure or both can bring about this imbalance leading to surplus energy being stored as fat. Obesity is associated with important medical morbidities and an increase in mortality. The causes of obesity are poorly understood and may be due to genetic factors, environ- mental factors or a combination of the two to cause a positive energy balance. In contrast, anorexia and cachexia are characterized by an imbalance in energy intake versus energy expenditure leading to a negative energy balance and weight loss.

Agents that either increase energy expenditure and/or decrease energy intake, ab- sorption or storage would be useful for treating obesity, overweight, and associated comorbidities. Agents that either increase energy intake and/or decrease energy ex- penditure or increase the amount of lean tissue would be useful for treating cachexia, anorexia and wasting disorders.

This gene, translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity, overweight, anorexia, cachexia, wasting disorders, appetite suppression, appetite enhancement, increases or decreases in satiety, modulation of body weight, and/or other eating disorders such as bulimia.

Also this gene, translated proteins and agents which modulate this gene or portions of the gene or its products are useful for treating obesity/overweight-associated comor- bidities including hypertension, type 2 diabetes, coronary artery disease, hyperlipi- demia, stroke, gallbladder disease, gout, osteoarthritis, sleep apnea and respiratory problems, some types of cancer including endometrial, breast, prostate, and colon cancer, thrombolic disease, polycystic ovarian syndrome, reduced fertility, compli- cations of pregnancy, menstrual irregularities, hirsutism, stress incontinence, and depression.

CNS disorders which can be treated include brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron

disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease.

Dementias, such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corti- cobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld- Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoffs psy- chosis also can be treated. Similarly, it is possible to treat cognitive-related disor- ders, such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disor- ders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities, by regulating the activity of human tyrosine phosphatase- like enzyme.

Pain that is associated with CNS disorders also can be treated by regulating the ac- tivity of human tyrosine phosphatase-like enzyme. Pain which can be treated in- cludes that associated with central nervous system disorders, such as multiple sclero- sis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spi- nal cord (e. g., infarct, hemorrhage, vascular malformation). Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystro- phy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS re- lated pain, cancer pain, metabolic neuropathies (e. g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or car- cinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, ten- sion-type like headache, cluster headache, and chronic paroxysmal hemicrania.

Chronic obstructive pulmonary (or airways) disease (COPD) is a condition defined physiologically as airflow obstruction that generally results from a mixture of em- physema and peripheral airway obstruction due to chronic bronchitis (Senior & Sha- piro, Pulmonary Diseases and Disorders, 3d ed., New York, McGraw-Hill, 1998, pp.

659-681,1998; Barnes, Chest 117, 10S-14S, 2000). Emphysema is characterized by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung. Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years. In COPD, airflow obstruc- tion is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does occur in non-smokers.

Chronic inflammation of the airways is a key pathological feature of COPD (Senior & Shapiro, 1998). The inflammatory cell population comprises increased numbers of macrophages, neutrophils, and CD8+ lymphocytes. Inhaled irritants, such as ciga- rette smoke, activate macrophages which are resident in the respiratory tract, as well as epithelial cells leading to release of chemokines (e. g., interleukin-8) and other chemotactic factors. These chemotactic factors act to increase the neutrophil/mono- cyte trafficking from the blood into the lung tissue and airways. Neutrophils and monocytes recruited into the airways can release a variety of potentially damaging mediators such as proteolytic enzymes and reactive oxygen species. Matrix degra- dation and emphysema, along with airway wall thickening, surfactant dysfunction, and mucus hypersecretion, all are potential sequelae of this inflammatory response that lead to impaired airflow and gas exchange.

Cardiovascular diseases include the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, and peripheral vascular diseases.

Heart failure is defined as a pathophysiologic state in which an abnormality of car- diac function is responsible for the failure of the heart to pump blood at a rate com- mensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failure, such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (MI) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously nar- rowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included, as well as the acute treatment of MI and the prevention of complications.

Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.

This group of diseases includes stable angina, unstable angina, and asymptomatic ischemia.

Arrhythmias include all forms of atrial and ventricular tachyarrhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexcitation syndrome, ventricular tachycardia, ventricular flutter, and ventricular fibrillation), as well as bradycardic forms of arrhythmias.

Hypertensive vascular diseases include primary as well as all kinds of secondary arte- rial hypertension (renal, endocrine, neurogenic, others). The disclosed gene and its product may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications.

Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD),

acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon, and venous disorders.

This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e. g., a modulating agent, an an- tisense nucleic acid molecule, a specific antibody, ribozyme, or an tyrosine phos- phatase-like enzyme polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.

Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

A reagent which affects tyrosine phosphatase-like enzyme activity can be adminis- tered to a human cell, either in vitro or in vivo, to reduce tyrosine phosphatase-like enzyme activity. The reagent preferably binds to an expression product of a human tyrosine phosphatase-like enzyme gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells which have been removed from the body.

The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome. Preferably, the lipo- some is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of

targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its con- tents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 u. g of DNA per 16 nmole of liposome delivered to about 106 cells, more preferably about 1.0 Rg of DNA per 16 nmole of liposome delivered to about 106 cells, and even more preferably about 2.0 u. g of DNA per 16 nmol of liposome delivered to about 106 cells.

Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes stan- dardly used in, for example, gene delivery methods known to those of skill in the art.

More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.

Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.

Complexing a liposome with a reagent such as an antisense oligonucleotide or ribo- zyme can be achieved using methods which are standard in the art (see, for example, U. S. Patent 5,705,151). Preferably, from about 0.1 llg to about 10 u. g ofpoly- nucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 sug to about 5 u. g ofpolynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 u. g ofpolynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 26J, u21-24 (1988); Wu et al., J Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad. Sci.

U. S. A. 87, 3655-59 (1990); Wu et al., J : Biol. Chem. 266, 338-42 (1991).

Determination of a Therapeutically Effective Dose The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of ac- tive ingredient which increases or decreases enzyme activity relative to the tyrosine phosphatase-like enzyme activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e. g., ED5 (l (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.

Pharmaceutical compositions which exhibit large therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies is used in formulating a

range of dosage for human use. The dosage contained in such compositions is pref- erably within a range of circulating concentrations that include the EDso with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to pro- vide sufficient levels of the active ingredient or to maintain the desired effect. Fac- tors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination (s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clear- ance rate of the particular formulation. Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is pro- vided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-estab- lished techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation,"gene gun,"and DEAE-or calcium phos- phate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 ug to about 50 g/kg, about 50 ag to about 5 mg/kg, about 100 jig to about 500 ug/kg of patient body weight, and about 200 to about 250 ug/kg of patient body weight. For ad- ministration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ug to about 2 mg, about 5 ug to about 500 u. g, and about 20 u. g to about 100 llg of DNA.

If the expression product is mRNA, the reagent is preferably an antisense oligo- nucleotide or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of an tyrosine phosphatase-like enzyme gene or the activity of an tyrosine phosphatase-like enzyme polypeptide by at least about 10, preferably about 50, more preferably about 75,90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of an tyrosine phosphatase-like enzyme gene or the activity of an tyrosine phosphatase-like enzyme polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to tyrosine phosphatase- like enzyme-specific mRNA, quantitative RT-PCR, immunologic detection of an tyrosine phosphatase-like enzyme polypeptide, or measurement of tyrosine phos- phatase-like enzyme activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate thera- peutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this ap-

proach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic Methods Human tyrosine phosphatase-like enzyme also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme. For example, differences can be determined between the cDNA or geno- mic sequence encoding tyrosine phosphatase-like enzyme in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequenc- ing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for

example, by high resolution gel electrophoresis. DNA fragments of different se- quences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e. g., Myers et al., Science 230,1242,1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e. g., Cotton et al., Proc. Natl. Acad. Sci. USA 85, 4397-4401,1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.

In addition to direct methods such as gel-electrophoresis and DNA sequencing, mu- tations can also be detected by in situ analysis.

Altered levels of an tyrosine phosphatase-like enzyme also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention.

A more complete understanding can be obtained by reference to the following spe- cific examples which are provided for purposes of illustration only and are not in- tended to limit the scope of the invention.

EXAMPLE 1 Detection of tyrosine phosphatase-like enzyme activity The polynucleotide of SEQ ID NO: 1 is inserted into pGEX vector and expressed as a fusion protein with glutathione S-transferase. The fusion protein is purified from lysed cells by adsorption by glutathion-agarose-beads followed by elution in the presence of free glutathione. The activity of the fusion protein (tyrosine phosphatase- like enzyme polypeptide of SEQ ID NO: 2) is assessed according to the following procedures: The fusion protein is incubated at 37°C for 2h in 2501 of lOmM Tris HCL, pH 7,4, containing 7,5 nM tyrosine phosphopeptide (RRLIEDAEpYAARG), and the reaction is terminated by addition of Malachite green solution (UBI). Phosphate release is measured after 15 min by evaluating absorbance at 605 nm. By comparing the ab- sorbance of the fusion protein to the absorbance of a negative standard such as heat- inactivated enzyme and to a positive standard such as tyrosine phosphatase-like en- zyme the tyrosine phosphatase-like enzyme activity of the fusion protein (tyrosine phosphatase-like enzyme polypeptide of SEQ ID NO: 2 is demonstrated).

EXAMPLE 2 Expression of recombinant human tyrosine phosphatase-like enzyme The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human tyrosine phosphatase-like enzyme polypeptides in yeast. The tyrosine phosphatase-like enzyme-encoding DNA se- quence is derived from SEQ ID NO : 1 or SEQ ID NO : 14. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5'-end an initiation codon and at its 3'-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of

the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastors, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombi- nantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human tyrosine phos- phatase-like enzyme polypeptide is obtained.

EXAMPLE 3 Identification of test compounds that bind to tyrosine phosphatase-like enzyme poly- peptides Purified tyrosine phosphatase-like enzyme polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human tyrosine phosphatase-like enzyme polypeptides comprise the amino acid sequence shown in SEQ ID NO : 2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Bind- ing of a test compound to an tyrosine phosphatase-like enzyme polypeptide is de- tected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of

a well in which a test compound is not incubated is identified as a compound which binds to an tyrosine phosphatase-like enzyme polypeptide.

EXAMPLE 4 Identification of a test compound which decreases tyrosine phosphatase-like enzyme gene expression A test compound is administered to a culture of human cells transfected with an tyro- sine phosphatase-like enzyme expression construct and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells which have not been transfected is incubated for the same time without the test compound to provide a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al., Biochem. 18, 5294-99,1979). Northern blots are prepared using 20 to 30 ug total RNA and hybridized with a 32P-labeled tyrosine phosphatase-like enzyme-specific probe at 65 ° C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nu- cleotides selected from the complement of SEQ ID NO : 1 or SEQ ID NO : 14. A test compound which decreases the tyrosine phosphatase-like enzyme-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of tyrosine phosphatase-like enzyme gene expression.

EXAMPLE 5 Identification of a test compound which decreases tyrosine phosphatase-like enzyme activity A test compound is administered to a culture of human cells transfected with an tyro- sine phosphatase-like enzyme expression construct and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells which have not been transfected is

incubated for the same time without the test compound to provide a negative control.

Enzyme activity is measured using the method of Calvert-Evers & Hammond, 2000.

A test compound which decreases the activity of the tyrosine phosphatase-like en- zyme relative to the activity in the absence of the test compound is identified as an inhibitor of tyrosine phosphatase-like enzyme activity.

EXAMPLE 6 Tissue-specific expression of tyrosine phosphatase-like enzyme The qualitative expression pattern of tyrosine phosphatase-like enzyme in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT- PCR). To demonstrate that tyrosine phosphatase-like enzyme is involved in CNS disorders, the following tissues are screened: fetal and adult brain, muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothala- mus, cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord.

To demonstrate that tyrosine phosphatase-like enzyme is involved in the disease pro- cess of obesity, expression is determined in the following tissues: subcutaneous adi- pose tissue, mesenteric adipose tissue, adrenal gland, bone marrow, brain (cerebellum, spinal cord, cerebral cortex, caudate, medulla, substantia nigra, and pu- tamen), colon, fetal brain, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle small intestine, spleen, stomach, testes, thymus, thyroid trachea, and uterus. Neuroblastoma cell lines SK-Nr-Be (2), Hr, Sk-N-As, HTB-10, IMR-32, SNSY-5Y, T3, SK-N-D2, D283, DAOY, CHP-2, U87MG, BE (2) C, T986, KANTS, M059K, CHP234, C6 (rat), SK-N-F1, SK-PU- DW, PFSK-1, BE (2) M17, and MCIXC also are tested for tyrosine phosphatase-like enzyme expression. As a final step, the expression of tyrosine phosphatase-like en-

zyme in cells derived from normal individuals with the expression of cells derived from obese individuals is compared.

To demonstrate that tyrosine phosphatase-like enzyme is involved in the disease pro- cess of diabetes, the following whole body panel is screened to show predominant or relatively high expression: subcutaneous and mesenteric adipose tissue, adrenal gland, bone marrow, brain, colon, fetal brain, heart, hypothalamus, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, thyroid, trachea, and uterus. Human islet cells and an islet cell library also are tested. As a final step, the expression of tyrosine phosphatase-like enzyme in cells derived from normal individuals with the expression of cells derived from diabetic individuals is compared.

To demonstrate that tyrosine phosphatase-like enzyme is involved in the disease pro- cess of COPD, the initial expression panel consists of RNA samples from respiratory tissues and inflammatory cells relevant to COPD: lung (adult and fetal), trachea, freshly isolated alveolar type II cells, cultured human bronchial epithelial cells, cul- tured small airway epithelial cells, cultured bronchial sooth muscle cells, cultured H441 cells (Clara-like), freshly isolated neutrophils and monocytes, and cultured monocytes (macrophage-like). Body map profiling also is carried out, using total RNA panels purchased from Clontech. The tissues are adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, mammary gland, pancreas, prostate, salivary gland, skeletal muscle, small intesting, spleen, stomach, testis, thymus, trachea, thyroid, and uterus.

Quantitative expressio71 profili7lg. Quantitative expression profiling is performed by the form of quantitative PCR analysis called"kinetic analysis"firstly described in Higuchi et al., BioTechnology 10, 413-17,1992, and Higuchi et al., BioTechnology 11, 1026-30,1993. The principle is that at any given cycle within the exponential

phase of PCR, the amount of product is proportional to the initial number of template copies.

If the amplification is performed in the presence of an internally quenched fluores- cent oligonucleotide (TaqMan probe) complementary to the target sequence, the probe is cleaved by the 5'-3'endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al., Proc. Natl. Acad. Sci. U. S. A.

88, 7276-80,1991). Because the fluorescence emission will increase in direct pro- portion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentra- tion (Heid et al., Genome Res. 6, 986-94, 1996, and Gibson et al., Genome Res. 6, 995-1001,1996).

The amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction. In this kind of experiment, the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used.

All"real time PCR"measurements of fluorescence are made in the ABI Prism 7700.

RNA extraction and cDNA preparation. Total RNA from the tissues listed above are used for expression quantification. RNAs labeled"from autopsy"were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.

Fifty it of each RNA are treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/lll RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/lll RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; 10mM MgCl2 ; 50 mM NaCl ; and 1 mM DTT.

After incubation, RNA is extracted once with 1 volume of phenol: chloro- form : isoamyl alcohol (24: 24: 1) and once with chloroform, and precipitated with 1/10 volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol.

Fifty u. g of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectropho- tometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufac- turer's protocol. The final concentration of RNA in the reaction mix is 200ng/, uL.

Reverse transcription is carried out with 2.5uM of random hexamer primers.

TaqMan quantitative analysis. Specific primers and probe are designed according to the recommendations of PE Applied Biosystems and are listed below: forward primer: 5'- (gene specific sequence)-3' reverse primer: 5'- (gene specific sequence)-3' probe: 5'- (FAM)- (gene specific sequence) (TAMRA)-3' where FAM = 6-carboxy-fluorescein and TAMRA = 6-carboxy-tetramethyl-rhodamine.

The expected length of the PCR product is- (gene specific length) bp.

Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.

Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).

The assay reaction mix is as follows: 1X final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA) ; IX PDAR control-18S RNA (from 20X stock) ; 300 nM forward primer; 900 nM reverse primer ; 200 nM probe; 10 ng cDNA ; and water to 25 u. l.

Each of the following steps are carried out once: pre PCR, 2 minutes at 50° C, and 10 minutes at 95°C. The following steps are carried out 40 times: denaturation, 15 seconds at 95°C, annealing/extension, 1 minute at 60°C.

The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve bet- ter background subtraction as well as signal linearity with the starting target quantity.

EXAMPLE 7 In vivo testing of compoundsltarget validation 1. Acute Mechanistic Assays 1. 1. Reduction in Mitogenic Plasma Hormone Levels This non-tumor assay measures the ability of a compound to reduce either the en- dogenous level of a circulating hormone or the level of hormone produced in re- sponse to a biologic stimulus. Rodents are administered test compound (p. o., i. p., i. v., i. m., or s. c.). At a predetermined time after administration of test compound, blood plasma is collected. Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i. e., LHRH may be injected i. m. into mice at a dosage of 30 ng/mouse to induce a burst of testosterone synthesis). The timing of plasma collec- tion would be adjusted to coincide with the peak of the induced hormone response.

Compound effects are compared to a vehicle-treated control group. An F-test is pre-

formed to determine if the variance is equal or unequal followed by a Student's t-test.

Significance is p value < 0.05 compared to the vehicle control group.

1. 2. Hollow Fiber Mechanism of Action Assay Hollow fibers are prepared with desired cell line (s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p. o., i. p., i. v., i. m., or s. c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific bio- chemical activity (i. e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with sig- nificance at p < 0.05 as compared to the vehicle control group.

2. Subacute Functional In Vivo Assays 2.1. Reduction in Mass of Hormone Dependent Tissues This is another non-tumor assay that measures the ability of a compound to reduce the mass of a hormone dependent tissue (i. e., seminal vesicles in males and uteri in females). Rodents are administered test compound (p. o., i. p., i. v., i. m., or s. c.) ac- cording to a predetermined schedule and for a predetermined duration (i. e., 1 week).

At termination of the study, animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded. Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent. Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle- treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value < 0.05 compared to the vehicle control group.

2.2. Hollow Fiber Proliferation Assay

Hollow fibers are prepared with desired cell line (s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p. o., i. p., i. v., i. m., or s. c. Fibers are harvested in accordance with specific readout assay protocol. Cell proliferation is determined by measuring a marker of cell number (i. e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p < 0.05 as compared to the vehicle con- trol group.

2. 3. ABtti-angiogenesis Models 2.3.1. Corneal Angiogenesis Hydron pellets with or without growth factors or cells are implanted into a micro- pocket surgically created in the rodent cornea. Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet).

Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Rank Sum test.

Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p < 0.05 as compared to the growth factor or cells only group.

2.3.2. Matrigel Angiogenesis Matrigel, containing cells or growth factors, is injected subcutaneously. Compounds are administered p. o., i. p., i. v., i. m., or s. c. Matrigel plugs are harvested at predeter- mined time point (s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i. e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by

Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p < 0.05 as compared to the vehicle control group.

3. Primary Antitumor Efficacy 3. 1. Early TZlerapy Models 3.1.1. Subcutaneous Tumor Tumor cells or fragments are implanted subcutaneously on Day 0. Vehicle and/or compounds are administered p. o., i. p., i. v., i. m., or s. c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden. Body weights and tumor measurements are recorded 2-3 times weekly.

Mean net body and tumor weights are calculated for each data collection day. Anti- tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p < 0.05. The ex- periment may also be continued past the end of dosing in which case tumor meas- urements would continue to be recorded to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the con- trol group to attain that size. Growth delays are compared by generating Kaplan- Meier curves from the times for individual tumors to attain the evaluation size. Sig- nificance is p < 0.05.

3.1.2. IsztraperitoneallIntracranial Tumor Models Tumor cells are injected intraperitoneally or intracranially on Day 0. Compounds are administered p. o., i. p., i. v., i. m., or s. c. according to a predetermined schedule start- ing on Day 1. Observations of morbidity and/or mortality are recorded twice daily.

Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long-term survi-

vors is indicated separately. Survival times are used to generate Kaplan-Meier curves. Significance is p < 0.05 by a log-rank test compared to the control group in the experiment.

3.2. Established Disease Model Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p. o., i. p., i. v., i. m., or s. c. ac- cording to a predetermined schedule. Tumor and body weights are measured and re- corded 2-3 times weekly. Mean tumor weights of all groups over days post inocula- tion are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as com- pared to the control group. Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predeter- mined size divided by the median time for the control group to attain that size.

Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value< 0.05 com- pared to the vehicle control group.

3. 3. Ortliotopic Disease Models 3. 3. 1. Mammazy Fat Pad Assay Tumor cells or fragments, of mammary adenocarcinoma origin, are implanted di- rectly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Com- pounds are administered p. o., i. p., i. v., i. m., or s. c. according to a predetermined

schedule. Tumor and body weights are measured and recorded 2-3 times weekly.

Mean tumor weights of all groups over days post inoculation are graphed for com- parison. An F-test is preformed to determine if the variance is equal or unequal fol- lowed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as compared to the control group.

Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value< 0.05 compared to the vehicle control group.

In addition, this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p < 0.05 compared to the con- trol group in the experiment.

3.3.2. Intraprostatic Assay Tumor cells or fragments, of prostatic adenocarcinoma origin, are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents. The prostate is exter- nalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles.

The successfully inoculated prostate is replaced in the abdomen and the incisions throught e abdomen and skin are closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p. o., i. p., i. v., i. m., or s. c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is termi-

nated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal fol- lowed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as compared to the control group.

This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by count- ing the number of visible foci per target organ (i. e., the lungs), or measuring the tar- get organ weight (i. e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance deter- mined at p < 0.05 compared to the control group in the experiment.

3. 3. 3. Intrabronchial Assay Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea. The trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend. Compounds are ad- ministered p. o., i. p., i. v., i. m., or s. c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer at- tached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p < 0.05 as compared to the control group. This model provides an opportunity to increase the rate of sponta- neous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i. e., the contralat- eral lung), or measuring the target organ weight. The means of these endpoints are

compared by Student's t-test after conducting an F-test, with significance deter- mined at p < 0.05 compared to the control group in the experiment.

3. 3. 4. Intracecal Assay Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizing the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p. o., i. p., i. v., i. m., or s. c. ac- cording to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t- test to compare tumor sizes in the treated and control groups at the end of treatment.

Significance is p < 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor.

Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i. e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p < 0.05 compared to the control group in the ex- periment.

4. Secondary (Metastatic) Antitumor Efficacy 4. 1. Spontaneous Metastasis Tumor cells are inoculated s. c. and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p. o., i. p., i. v., i. m., or s. c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of

tumor metastasis. Observations of morbidity and/or mortality are recorded daily.

Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p < 0. 05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with signifi- cance determined at p < 0.05 compared to the control group in the experiment for both of these endpoints.

4. 2. Forced Metastasis Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively. Com- pounds are administered p. o., i. p., i. v., i. m., or s. c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include sur- vival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p < 0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance at p < 0.05 compared to the vehicle control group in the experiment for both end- points.

EXAMPLE 8 Diabetes : In vivo testing of compoundsltarget validation 1. Glucose Production:

Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes. Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injec- tion of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i. p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then com- pounds are administered via different routes (p. o., i. p., i. v., s. c.). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are ad- ministered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will de- crease plasma glucose levels compared to the vehicle-treated control group.

2. Insulin Sensitivity: Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant. The animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group. Compounds are administered daily either q. d. or b. i. d. by different routes (p. o., i. p., s. c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group.

3. Insulin Secretion: Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the ad- ministration of a glucose load. When measuring insulin levels, compounds are ad- ministered by different routes (p. o., i. p., s. c. or i. v.) to overnight fasted normal rats or

mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intra- peritoneal glucose load (Ig/kg), bled again after 15,30,60 and 90 minutes and plasma glucose levels determined. Compounds that increase insulin levels will de- crease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.

Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the ad- ministration of a glucose load. When measuring insulin levels, test compounds which regulate pristanoyl-CoA oxidase-like enzyme are administered by different routes (p. o., i. p., s. c., or i. v.) to overnight fasted normal rats or mice. At the appro- priate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Test compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glu- cose load (lg/kg), bled again after 15,30,60, and 90 minutes and plasma glucose levels determined. Test compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.

4. Glucose Production: Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes. Overnight fasted normal rats

or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injec- tion of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i. p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then com- pounds are administered via different routes (p. o., i. p., i. v., s. c.). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are ad- ministered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will de- crease plasma glucose levels compared to the vehicle-treated control group.

5. Insulin Sensitivity: Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant. The animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group. Compounds are administered daily either q. d. or b. i. d. by different routes (p. o., i. p., s. c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group.

6. Insulin Secretion: Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the ad- ministration of a glucose load. When measuring insulin levels, compounds are ad- ministered by different routes (p. o., i. p., s. c. or i. v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals

given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intra- peritoneal glucose load (lglkg), bled again after 15,30,60 and 90 minutes and plasma glucose levels determined. Compounds that increase insulin levels will de- crease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.