Human diacylglycerol acyltransferase 2 (DGAT2) family members and uses therefor

• US 20060183210A1
• Filed: 12/07/2005
• Published: 08/17/2006
• Est. Priority Date: 12/19/2001
• Status: Active Grant

• Current Assignee: Millennium Pharmaceuticals Incorporated (Takeda Pharmaceutical Company Limited)
• Original Assignee: Millennium Pharmaceuticals Incorporated (Takeda Pharmaceutical Company Limited)
• Inventors: Kapeller-Libermann, Rosana, Hubbard, Brian K., Wu, Zhidan, Gimeno, Ruth E.
• Granted Patent: US 7,527,962 B2
• Time in Patent Office: Days
• US Class Current: 435/193
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Y 203/0102 Diacylglycerol O-acyltransferase (2.3.1.20)
• CPC Class Codes: G01N 2333/91051 Acyltransferases other than aminoacyltransferases (general) (2.3.1)
• CPC Class Codes: G01N 2800/044 Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

The present invention relates to compositions and methods for the diagnosis and treatment of obesity and related metabolic disorders. The invention provides isolated nucleic acids molecules, designated DGAT2 family member nucleic acid molecules, which encode diacylglycerol acyltransferase family members. The invention also provides recombinant expression vectors containing DGAT2 family member nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a DGAT2 family member gene has been introduced or disrupted. The invention still further provides isolated DGAT2 family member proteins, fusion proteins, antigenic peptides and anti-DGAT2 family member antibodies. Methods of use of the provided DGAT2 family member compositions for screening, diagnostic and therapeutic methods in connection with obesity disorders are also disclosed.

Claims

1. An isolated nucleic acid molecule selected from the group consisting of:

a) a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to the nucleotide sequence of SEQ ID NO;

7, SEQ ID NO;

19, or SEQ ID NO;

61;

b) a nucleic acid molecule comprising a fragment of at least 300 nucleotides of the nucleotide sequence of SEQ ID NO;

7, SEQ ID NO;

19 or SEQ ID NO;

61;

c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20 and SEQ ID NO;

62;

d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO;

8, SEQ ID NO;

20, or SEQ ID NO;

62, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO;

8, SEQ ID NO;

20 or SEQ ID NO;

62;

e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO;

8, SEQ ID NO;

20 or SEQ ID NO;

62, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising a sequence consisting of SEQ ID NO;

7, SEQ ID NO;

19, or SEQ ID NO;

61 or a complement thereof, under stringent conditions; and

f) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

11, SEQ ID NO;

13, SEQ ID NO;

15, SEQ ID NO;

17, SEQ ID NO;

19, and SEQ ID NO;

61.

2. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences or nucleic acid sequences encoding a heterologous polypeptide.

3. A host cell which contains the nucleic acid molecule of claim 1.

7. A method for producing a polypeptide-selected from the group consisting of:

a) a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

18, SEQ ID NO;

20, and SEQ ID NO;

62;

b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO;

8 or SEQ ID NO;

20, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO;

8, SEQ ID NO;

20, or SEQ ID NO;

62;

c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO;

7, SEQ ID NO;

19, or SEQ ID NO;

61, or a complement thereof; and

d) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO;

8, SEQ ID NO;

20 or SEQ ID NO;

62, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO;

7, SEQ ID NO;

19, SEQ ID NO;

61, or a complement thereof under stringent conditions;

comprising culturing the host cell of claim 3 under conditions in which the nucleic acid molecule is expressed.

16. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising:

a) contacting the sample with a compound which selectively hybridizes to a nucleic acid molecule of claim 1;

and

b) determining whether the compound hybridizes to the nucleic acid or binds to the polypeptide in the sample.

18. A method of identifying a subject having a disorder or at risk for developing a disorder comprising:

a) contacting a sample obtained from the subject comprising nucleic acid molecules with a hybridization probe comprising at least 25 contiguous nucleotides of a nucleic acid molecule of claim 1;

and

b) detecting the presence of a nucleic acid molecule in the sample that hybridizes to the probe, thereby identifying a subject having a disorder or at risk for developing a disorder.

4. An isolated polypeptide selected from the group consisting of:

a) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO;

7, SEQ ID NO;

19 or SEQ ID NO;

61, or a complement thereof;

b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO;

8, SEQ ID NO;

20 or SEQ ID NO;

62, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO;

7, SEQ ID NO;

19 or SEQ ID NO;

61 or a complement thereof under stringent conditions;

c) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO;

8, SEQ ID NO;

20 or SEQ ID NO;

62, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO;

8, SEQ ID NO;

20 or SEQ ID NO;

62; and

d) a polypeptide comprising the amino acid sequence selelcted from the group consisting of SEQ ID NO;

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

18, SEQ ID NO;

20 and SEQ ID NO;

62.

5. The polypeptide of claim 4 further comprising heterologous amino acid sequences.

6. An antibody which selectively binds to a polypeptide of claim 4.

8. A method for identifying a compound which binds to a polypeptide of claim 4 comprising the steps of:

a) contacting a polypeptide, or a cell expressing a polypeptide of claim 4 with a test compound; and

b) determining whether the polypeptide binds to the test compound;

wherein binding of the test compound to the polypeptide is detected by a method selected from the group consisting of;

i) detection of binding by direct detecting of test compound/polypeptide binding;

ii) detection of binding using a competition binding assay; and

iii) detection of binding using an assay for diacylglycerol acyltransferase activity.

9. A method for identifying a compound which modulates the activity of a polypeptide of claim 4, comprising:

a) contacting a polypeptide of claim 4 with a test compound; and

b) determining the effect of the test compound on the diacylglycerol acyltransferase activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.

12. A method of identifying a compound which modulates the activity of a polypeptide of claim 4, comprising:

a) combining a sample comprising the polypeptide with a first fatty acyl coA substrate and a second acylglyceride substrate under conditions suitable for enzyme activity;

b) contacting the sample comprising the acyltransferase and substrates with a test compound;

c) determining the amount of acylglycerol product formation, thereby determining acyltransferase activity; and

d) comparing the acyltransferase activity in the presence of compound with acyltransferase activity in the absence of compound;

wherein a difference between the acyltransferase activity in the presence of compound with acyltransferase activity in the absence of compound indicates that the compound modulates the activity of the polypeptide,

thereby identifying a compound which modulates activity of the acyltransferase.

13. The method of claim 12, wherein the method detects diacylglycerol acyltransferase activity or monoacylglycerol acyltransferase activity.

14. The method of claim 12, wherein one substrate is biotinylated and the other substrate is radiolabeled.

15. A method for treating a subject having an obesity disorder characterized by expression or activity of a polypeptide of claim 4, comprising administering to the subject a DGAT2 family member modulator, thereby treating said subject having an obesity disorder.

17. A method for detecting the presence of a polypeptide of claim 4 in a sample, comprising:

a) contacting the sample with a compound which binds to the polypeptide; and

b) determining whether the compound hybridizes to the nucleic acid or binds to the polypeptide in the sample.

19. A method of identifying a subject having a disorder or at risk for developing a disorder comprising:

a) contacting a sample obtained from the subject comprising polypeptides with a compound which binds a polypeptide of claim 4;

and

b) detecting the presence of a polypeptide in the sample that binds to the compound, thereby identifying a subject having a disorder or at risk for developing a disorder.

10. A method for identifying a compound capable of treating a disorder comprising:

a) contacting an adipocyte with a test compound; and

b) assaying the ability of the test compound to modulate the expression of a DGAT2 family member nucleic acid or the activity of a DGAT2 family member polypeptide; and

c) identifying a compound capable of modulating an adipocyte activity when a test compound modulates the expression of a DGAT2 family member nucleic acid or the activity of a DGAT2 family member polypeptide.

11. The method of claim 10, wherein said adipocyte activity comprises any one of DGAT2 family member nucleic acid expression, diacylglyceroltransferase activity, hyperplastic growth, hypertrophic growth, or lipogenesis.