Methods for Screening Compounds That Modulate Lipid Metabolism

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.

1 . 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.   
     
     
         2 . The method according to  claim 1 , wherein the mammalian delta-5-desaturase enzyme is rat or human delta-5-desaturase. 
     
     
         3 . A method of screening for a modulator according to  claim 1 , wherein the screening method is an assay for identifying modulators that modulate lipid metabolism. 
     
     
         4 . A method of screening for a modulator according to  claim 1 , wherein the screening method is an assay for identifying modulators that modulate diabetic neuropathy. 
     
     
         5 . A modulator identified by a screening method according to  claim 1 . 
     
     
         6 . A modulator according to  claim 5 , wherein said modulator is in a purified form. 
     
     
         7 . A pharmaceutical composition comprising a modulator according to  claim 5  and a pharmaceutically acceptable carrier or diluent. 
     
     
         8 . The method according to  claim 1 , wherein the mammalian delta-5-desaturase enzyme is in a purified form. 
     
     
         9 . 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.   
     
     
         10 . A method for the treatment of a patient with a disease selected from the group consisting of diabetes; arterial hypertension; hypercholesterolemia; atherosclerotic heart disease; chronic inflammatory disorders; autoimmune disorders; allergic eczema; atopic disorders; 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, comprising administering to said patient a pharmaceutical composition according to  claim 7 .