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Methods for Screening Compounds That Modulate Lipid Metabolism

a technology of lipid metabolism and compounds, applied in the direction of metabolism disorders, immunological disorders, drug compositions, etc., can solve the problems of affecting the balance of eicosanoids produced, affecting the biosynthesis of polyunsaturated fatty acids (pufas) by the desaturation and elongation system, and prone to certain long-term complications for diabetics

Inactive Publication Date: 2009-03-05
XENON PHARMACEUTICALS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Diabetics are prone to certain long-term complications.
Evidence from experimental diabetes studies in animals indicates that the biosynthesis of polyunsaturated fatty acids (PUFAs) by the desaturation and elongation systems is impaired.
This changes the amount and alters the balance of eicosanoids produced.
However, these models which use different species are limited by the fact that they are not close enough to the desired target, i.e. the human delta-5-desaturase, for drug screening.
This is particularly so in recognition of the fact that the relationship between a protein's unique role in a metabolic pathway and the expression of the gene encoding that protein is normally a well coordinated event such that subtle deviations can often lead to abnormal physiological processes.

Method used

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  • Methods for Screening Compounds That Modulate Lipid Metabolism
  • Methods for Screening Compounds That Modulate Lipid Metabolism
  • Methods for Screening Compounds That Modulate Lipid Metabolism

Examples

Experimental program
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Effect test

example 1

Cloning of Rat and Human Desaturase Genes

[0167]RNA Extraction: Total RNA was extracted from rat liver or the human cell line Chang (ATCC No. CCL-13), using TRIzol Reagent solution (Gibco BRL) as described by the manufacturer.

[0168]Reverse Transcription About 1 μg of each RNA sample was reverse-transcribed in 3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl (pH 8.3), 2 ng / μl of random primers (Perkin-Elmer), 1.0 mM each dNTP, 2.0 U / μl of RNase inhibitor (Perkin-Elmer) and 10 U / μl of MuLV reverse transcriptase (Perkin-Elmer). The reactions were carried out at 42° C. for 30 min in a final volume of 20 μl. The enzyme was then inactivated at 94° C. for 5 min.

[0169]Amplification of Desaturase Genes by PCR and Cloning in a Yeast Vector: Aliquots (5 μl) of the reverse transcription reactions were amplified by polymerase chain reaction (PCR), using primers designed to generate cDNA corresponding to the coding sequence for the human desaturase gene.

[0170]The hD5D gene was amplified in a two step process...

example 2

Cloning and Identification of the Human Desaturase Control Region

[0176]The hD5D control region was cloned from human leukocyte genomic DNA by two rounds of PCR reactions. The first PCR reaction used the following primers: a reverse primer starting at position +127 of the hD5D coding sequence (5′-CACCTTACGGTCGATCACTA-3′; SEQ ID NO. 12) and a forward primer starting at position −1357 upstream from the translation initiation codon, ATG (5′-CTCAGTGCTTGGGACAGTTA-3′; SEQ ID NO. 13).

[0177]The PCR amplification was conducted in a Perkin-Elmer GeneAMP PCR system 9700 instrument in a 50 μl reaction volume containing: 0.5 μg of genomic DNA, 0.4 μM of each primer, 1× dNTP mix (Clontech, CA), 1×PCR reaction buffer (Clontech) and 1× Advantage-polymerase mix (Clontech).

[0178]The conditions for the PCR reaction were:

7 cycles at 94° C. for 2 seconds, 72° C. for 3 minutes

32 cycles at 94° C. for 2 seconds, 67° C. for 3 minutes

67° C. for 4 minutes

[0179]The PCR products were gel-purified using QIAquick ...

example 3

Cell Transfection

[0181]The cell lines ZR-75-1 and HepG2 are transfected with 5 μg of the plasmids pBh4013.1 and pGh4014.1 using 5 μl of Lipofectamine 2000 Reagent (Gibco BRL) in a 6-well plate as described by the manufacturer. The control plasmids pCAT-3-CTL and pCAT3-Basic or pGL3-CTL and pGL3-Basic are also transfected as positive and negative controls, respectively. In all cases, 5 μg of the plasmid pCMV-β-Gal (pCMV-β-Galactosidase control vector; Clontech) is co-transfected and used as an internal control to standardize the transfection efficiency between each transfection.

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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.

Description

[0001]This application is a divisional of U.S. application Ser. No. 10 / 415,232, filed Nov. 24, 2003, which is the U.S. national stage of PCT / CA01 / 01520, filed Nov. 29, 2001, which claims priority to U.S. Provisional Application No. 60 / 243,009, filed Oct. 26, 2000. All applications are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]This invention relates to the identification of nucleotides, proteins, compounds and / or pharmacological agents that either inhibit or enhance the activity of fatty acid desaturase enzymes involved in lipid metabolism and / or effectively regulate the level of expression of the desaturase genes.BACKGROUND OF THE INVENTION[0003]Diabetes mellitus represents a collection of genetically determined disorders one of which is the altered metabolism of lipids associated with a deficiency of insulin or insulin resistance. Diabetics are prone to certain long-term complications. The range of medical problems includes cardiovascular diseas...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C12Q1/26C07K16/00G01N33/50A61K45/00A61K48/00A61P3/06A61P3/10A61P9/10A61P11/00A61P17/06A61P19/02A61P25/00A61P29/00A61P35/00A61P37/00A61P37/08A61P43/00C07K16/40C12N1/15C12N1/19C12N1/21C12N5/10C12N9/02C12N15/09G01N33/15
CPCC12N9/0083A61P11/00A61P17/06A61P19/02A61P25/00A61P29/00A61P3/10A61P35/00A61P3/06A61P37/00A61P37/08A61P43/00A61P9/10
Inventor WINTHER, MICHAEL DAVIDKNICKLE, LEAH CHRISTINEHAARDT, MARTINALLEN, STEPHEN JOHNPONTON, ANDREDE ANTUENO, ROBERTO JUSTOJENKINS, D. KENNETHNWAKA, SOLOMON O.
Owner XENON PHARMACEUTICALS INC
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