Methods for screening compounds that modulate lipid metabolism

• US 20040096435A1
• Filed: 11/24/2003
• Published: 05/20/2004
• Est. Priority Date: 10/26/2000
• Status: Active Grant

• Current Assignee: Xenon Pharmaceuticals Inc.
• Original Assignee: Xenon Pharmaceuticals Inc.
• Inventors: Knickle, Leah Christine, Haardt, Martin, Ponton, Andre, Winther, Michael David, De Antueno, Roberto Justo, Allen, Stephen John, Jenkins, D Kenneth, Nwaka, Solomon O
• Granted Patent: US 7,256,028 B2
• Time in Patent Office: Days
• US Class Current: 424/94.4
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)

Abstract

Drug screening assays useful in the discovery of pharmaceutically active compounds for use in the treatment of diseases involving abnormal lipid metabolism including diabetic neuropathy are taught. In particular, the control region of delta-5-desaturase gene is taught as a target for the drug screening methods, which serve to identify nucleotides, proteins, compounds and/or other pharmacological agents, which modulate the activity of desaturase enzymes or regulate the level of expression of the desaturase genes. Cell-based and cell lysate assays are taught for detecting components that interact with the desaturase enzymes and modify fatty acid profiles. In addition, cell-based and cell lysate assays are used to identify functional and regulatory elements controlling expression of the desaturase genes as well as to screen for components that modulate the transcriptional activity of the desaturase genes. Also taught is the gene for rat delta-5-desaturase.

Claims

1. An isolated nucleic acid comprising a DNA sequence selected from the group consisting of:

(a) SEQ ID NO;

1

(b) a fragment of (a) having at least 15 nucleotides;

(c) a sequence which is at least 90% homologous with (a), and;

(d) a sequence which hybridizes to (a), (b) or (c) under stringent hybridization conditions.

4. The isolated nucleic acid of claim 1, wherein the DNA sequence includes a control region of a human delta-5-desaturase gene.

8. A recombinant nucleic acid molecule comprising a control region of a mammalian delta-5-desaturase gene according to claims 1 and 4 and a reporter gene, wherein the control region is transcriptionally linked to the reporter gene so as to effectively initiate, terminate or regulate transcription of the reporter gene.

2. An isolated nucleic acid comprising a DNA sequence selected from the group consisting of:

(a) SEQ ID NO;

2

(b) a fragment of (a) having at least 15 nucleotides;

(c) a sequence which is at least 90% homologous with (a), and;

(d) a sequence which hybridizes to (a), (b) or (c) under stringent hybridization conditions.

5. The isolated nucleic acid of claim 2, wherein the DNA sequence encodes a rat delta-5-desaturase gene.

3. An isolated polypeptide comprising a polypeptide sequence selected from the group consisting of:

(a) SEQ ID NO;

3

(b) a fragment of (a) having at least 15 amino acids;

(c) a sequence which is at least 90% homologous with (a) or its salt.

6. The isolated polypeptide of claim 3, wherein the polypeptide sequence denotes a rat delta-5-desaturase protein.

32. An antibody immunoreactive with a polypeptide of claim 3 or an immunogenic portion thereof.

7. A recombinant nucleic acid molecule comprising a control region of a mammalian delta-5-desaturase gene and a reporter gene, wherein the control region is transcriptionally linked to the reporter gene so as to effectively initiate, terminate or regulate transcription of the reporter gene.

9. The recombinant nucleic acid molecule according to claim 7 or 8, wherein the reporter gene is selected from the group consisting of luciferase, chloramphenicol acetyl transferase (CAT), beta-galactosidase, alkaline phosphatase, placental alkaline phosphatase, glucuronide synthetase, and green fluorescence protein (GFP).

10. The recombinant nucleic acid molecule according to claim 9, wherein the reporter gene is chloramphenicol acetyl transferase (CAT) or luciferase.

11. A vector construct containing the recombinant nucleic acid molecule defined in any one of claims 7 to 10, wherein the vector construct is capable of expressing the reporter gene to generate a reporter gene product that can be detected upon introduction of the vector construct into an appropriate host cell or host system.

12. A host cell or host system transformed or transfected with the vector construct defined in claim 11.

13. The host cell according to claim 12, wherein the host cell is a mammalian cell.

14. The host cell according to claim 13, wherein the mammalian cell is a human cell.

15. The host cell according to claim 14, wherein the mammalian cell is from the human carcinoma cell lines, ZR-75-1 or HepG2.

16. A method of screening for a modulator which is capable of modulating or regulating the transcriptional expression of a mammalian delta-5-desaturase enzyme gene, comprising the steps of:

(a) providing a host system containing a control region of mammalian delta-5-desaturase gene and a reporter gene operably associated with the control region, wherein the control region is effective to initiate, terminate or regulate a level of transcription of the reporter gene;

(b) contacting the host system with a test component;

(c) evaluating the level of transcription of the reporter gene, wherein a measurable difference in the level of transcription of the reporter gene in the presence of the test component compared to a control under identical conditions but in the absence of the test component is an indicator of an ability of the test component to transcriptionally modulate or regulate expression of the delta-5-desaturase gene; and

(d) selecting as said modulator the test component which exhibits said ability.

17. The method according to claim 16, wherein the control region has a DNA sequence represented by SEQ ID NO:

1, or a fragment thereof.

20. A method of screening for a test component according to any one of the claims 16 to 19, wherein the screening method is an assay for identifying modulators that modulate lipid metabolism.

21. A method of screening for a test component according to any one of the claims 16 to 19, wherein the screening method is an assay for identifying modulators that modulate diabetic neuropathy.

22. A modulator identified by a screening method according to any one of claims 16 to 21.

23. A modulator according to claim 22, wherein said modulator is in a purified form.

24. A pharmaceutical composition comprising a modulator according to claims 22 or 23 and a pharmaceutically acceptable carrier or diluent.

25. The use of a modulator or composition according to any one of claims 22 to 24, for the treatment of lipid metabolism abnormalities.

26. The use of a modulator or composition according to any one of the claims 22 to 24, for the treatment of diabetic neuropathy.

27. The use of a modulator or composition according to any one of the claims 22 to 24, for the treatment of human diabetic neuropathy.

35. The use of a modulator or pharmaceutical composition according to any one of the claims 22 to 24 for the treatment of a disease selected from the group consisting of diabetes;

arterial hypertension;

hypercholesterolemia;

atherosclerotic heart disease;

chronic inflammatory disorders;

autoimmune disorders;

allergic eczema and other atopic disorders;

inflammatory processes such as rheumatoid arthritis;

diminished lymphocyte proliferation, T-cell-mediated cytotoxicity, natural killer cell activity, macrophage-mediated cytotoxicity, monocyte and neutrophil chemotaxis, major histocompatibility class II expression and antigen presentation, production of pro-inflammatory cytokines (interleukins I and 6, tumour necrosis factor) and adhesion molecule expression;

eczema;

psoriasis;

acute respiratory distress syndrome (ARDS);

articular cartilage degradation (ACD); and

cancer.

37. A method as claimed in claim 20, wherein the screening method is an assay for identifying modulators that modulate the n-3 lipid metabolic pathway, conversion of 18:

3n3 to 22;

6n3.

38. A method as claimed in claim 20, wherein the screening method is an assay for identifying modulators that modulate the n-9 lipid metabolic pathway, conversion of 16:

0 to 22;

4n9.

39. A method as claimed in claim 20, wherein the screening method is an assay for identifying modulators that modulate the n-6 lipid metabolic pathway, conversion of 8:

2n6 to 22;

5n6.

18. A method of screening for a modulator which is capable of modulating the enzymatic activity of a functional mammalian delta-5-desaturase enzyme, comprising the steps of:

(a) providing a host system containing a nucleic acid sequence which encodes a mammalian delta-5-desaturase enzyme operably associated with a promoter region, wherein the promoter region is effective to initiate, terminate or regulate the level of expression of the nucleic acid sequence;

(b) contacting the host system with a test component,

(c) evaluating the enzymatic activity of the delta-5-desaturase, wherein a measurable difference in a level of lipid metabolite or associated cofactors in the presence of the test component compared to a control under identical conditions but in the absence of the test component is an indicator of the ability of the test component to modulate delta-5-desaturase enzyme activity; and

(d) selecting as said modulator a test component which exhibits said ability.

19. The method according to claim 18, wherein the mammalian delta-5-desaturase enzyme is rat or human delta-5-desaturase.

31. The method according to claim 18, wherein the mammalian delta-5-desaturase enzyme is in a purified form.

28. An isolated rat delta-5-desaturase.

29. A delta-5-desaturase protein having a linked tag sequence.

30. A delta-5-desaturase protein according to claim 29, wherein the tag sequence is a V5 epitope or a 6×

Histidine tag.

33. An antibody immunoreactive with a polypeptide of a human delta-5-desaturase or an immunogenic portion thereof, wherein the antigen is produced in a host system.

34. A method of screening for a modulator which is capable of modulating the enzymatic activities of functional mammalian delta-5- and/or delta-6-desaturase enzymes within the same host system, comprising the steps of:

(a) providing a host system containing nucleic acid sequences, which encode both mammalian delta-5- and delta-6-desaturase enzymes operably associated with promoter regions, wherein the promoter regions are effective to initiate, terminate or regulate the level of expression of the nucleic acid sequence;

(b) contacting the host system with a test component;

(c) simultaneously evaluating the enzymatic activities of the delta-5- and delta-6-desaturases, wherein a measurable difference in a level of lipid metabolites or associated cofactors in the presence of the test component compared to a control under identical conditions but in the absence of the test component is an indicator of the ability of the test component to modulate delta-5- and/or delta-6-desaturase enzyme activity; and

(d) selecting as said modulator a test component which exhibits said ability.

36. The use of n-propyl gallate for the treatment of a disease selected from the group consisting of arterial hypertension;

hypercholesterolemia;

atherosclerotic heart disease;

chronic inflammatory disorders;

autoimmune disorders;

allergic eczema and other atopic disorders;

inflammatory processes such as rheumatoid arthritis;

diminished lymphocyte proliferation, T-cell-mediated cytotoxicity, natural killer cell activity, macrophage-mediated cytotoxicity, monocyte and neutrophil chemotaxis, major histocompatibility class II expression and antigen presentation, production of pro-inflammatory cytokines (interleukins 1 and 6, tumour necrosis factor) and adhesion molecule expression;

eczema;

psoriasis;

acute respiratory distress syndrome (ARDS);

articular cartilage degradation (ACD); and

cancer.