WO2003033656A2 - Proteines msrebp utilisees comme modificateurs de la voie srebp et leurs methodes d'utilisation - Google Patents

Proteines msrebp utilisees comme modificateurs de la voie srebp et leurs methodes d'utilisation Download PDF

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Publication number
WO2003033656A2
WO2003033656A2 PCT/US2002/032807 US0232807W WO03033656A2 WO 2003033656 A2 WO2003033656 A2 WO 2003033656A2 US 0232807 W US0232807 W US 0232807W WO 03033656 A2 WO03033656 A2 WO 03033656A2
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WIPO (PCT)
Prior art keywords
srebp
msrebp
assay
agent
assay system
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PCT/US2002/032807
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English (en)
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WO2003033656A3 (fr
Inventor
Lisa C. Kadyk
Carol L. O'brien
Eric C. Kong
Glenn R. Hammonds, Jr.
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Exelixis, Inc.
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Application filed by Exelixis, Inc. filed Critical Exelixis, Inc.
Priority to AU2002332130A priority Critical patent/AU2002332130A1/en
Publication of WO2003033656A2 publication Critical patent/WO2003033656A2/fr
Publication of WO2003033656A3 publication Critical patent/WO2003033656A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • SREBP sterol regulatory element binding protein
  • RNA interference RNA interference
  • ds double stranded
  • Suitable methods for introduction of dsRNA include injection, feeding, and bathing (Tabara et al, 1998, Science 282:430-431).
  • the human enzyme INPP5A is a 43 kD membrane-associated protein expressed in heart, skeletal muscle, brain and platelets.
  • INPP5A is classified as a Type 15-phosphatase based on its elution properties on anion exchange columns and its affinity for the second messengers Ins (1,4,5)P 3 and
  • DAB 1 binds to the intracellular domains of APP, LDLR family members, the apoE2 receptor-2 and the cytoplasmic signaling protein SHIP (Howell et al., 1999, Nature 389: 733-7; Hussain, 2001, Front. Biosci. 6: D417-428).
  • the interaction with the LDLR occurs through an NPXY motif required for receptor intemalization and degradation. This motif has been implicated not only in LDL receptor turnover, but also in signaling pathways crucial for neuronal migration in brain development (Howell, 1999, supra).
  • the Yptlp GTPase is an essential protein that functions in the exocytic pathway, playing important roles in ER-to-Golgi transport and intra-Golgi transport (Jedd, et al, 1995, J. Cell Biol. 131:583-590).
  • moderately stringent hybridization conditions comprise: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS.
  • the MSREBP is an ortholog of human MSREBP.
  • Methods of identifying the human orthologs of these genes are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al, Genome Research (2000) 10:1204-1210).
  • the antisense oligomers interfere with the function of MSREBP nucleic acids, such as DNA replication, transcription, MSREBP RNA translocation, translation of protein from the MSREBP RNA, RNA splicing, and any catalytic activity in which the MSREBP RNA participates.
  • the antisense oligomer is an oligonucleotide that is sufficiently complementary to an MSREBP mRNA to bind to and prevent translation from the MSREBP mRNA, preferably by binding to the 5' untranslated region.
  • MSREBP-specific antisense oligonucleotides preferably range from at least 6 to about 200 nucleotides.
  • a protease substrate contains the amino acid proteolysis recognition sequences separating two different fluorescent tags; fluorescence resonance energy transfer detects the proximity of these fluorophores, which indicates whether the substrate is cleaved (Mahajan NP et al, Chem Biol (1999) 6:401-409).
  • appropriate primary assays are binding assays that test the antibody's affinity to and specificity for the MSREBP protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra).
  • the enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting MSREBP-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.
  • Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express MSREBP) in the presence and absence of the candidate modulator.
  • such assays test whether treatment of cells or animals with a candidate MSREBP-modulating agent results in changes in the SREBP pathway, lipid metabolism, and/or adipogenesis, in comparison to untreated (or mock- or placebo-treated) cells or animals.
  • Certain assays use sensitized genetic backgrounds, used herein to describe cells or animals engineered for altered expression of genes in the SREBP or interacting pathways, or other pathways associated with lipid metabolism and/or adipogenesis.
  • SREBP pathway activity is assessed by measuring expression of SREBP transcriptional targets.
  • Many transcriptional targets are known (e.g., Osborne TF, 2001, J Biol Chem 275:32379-32382; Horton JD et al, 1998, J Clin Invest 101:2331- 2339; Shimano H et al, 1997, J Clin Invest 100:2115-2124; Shimomura I et al, 1999, J Biol Chem 274: 30028-30032). Any available means for expression analysis, as previously described, may be used. Typically, mRNA expression is detected. In a preferred application, Taqman analysis is used to directly measure mRNA expression.
  • assays monitor SREBP processing events, such as cleavage of the membrane-bound form of SREBP, or nuclear translocation or nuclear accumulation of the activated form of SREBP. These events can be monitored directly by monitoring levels of membrane bound and cleaved forms of the protein. Typically, cells are fractionated, and protein levels in nuclear and membrane fractions are measured using immunohistochemistry. Alternatively, SREBP cleavage can be monitored indirectly using specific reporters for SREBP cleavage. In one example, a fusion construct comprising sequences encoding the signal peptide and soluble catalytic domain of alkaline phosphatase (AP) linked to the C-terminal (regulatory) domain of SREBP is introduced into cells.
  • AP alkaline phosphatase
  • mice with knockouts in both leptin and LDL receptor genes display hypercholesterolemia, hypertriglyceridemia and arterial lesions and provide a model for the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis (Hasty AH et al, 2001, supra.).
  • INPP5A C. elegans gene C09B8.1 (GI#868274), which is an ortholog of the human INPP5A (SEQ ID NOs:l and 14) gene, is an enhancer of the SREBP phenotype.
  • INPP5A negatively regulates inositol polyphosphate signaling, it is predicted that knockdown of the function of the INPP5A phosphatase would result in increased inositol polyphosphate signaling. Because such increased signaling appears to enhance the phenotype of an SREBP loss-of- function mutation, it is predicted that signaling via these second messengers works antagonistically to SREBP function at some level.
  • one or more of the defects resulting from loss of worm Sacl (F30A10.6) function could enhance an SREBP mutant.
  • a defect in this process would be expected to enhance the phenotype of a weak SREBP mutant.
  • other aspects of the defects resulting from loss of this phosphatase function might have effects on SREBP pathway function.
  • Candidate suppressors gave a similar phenotype in at least one re- test, and the clone that was used to generate the dsRNA was sequenced to confirm the identity of the gene. Soaking and injection of the two SREBP mutant strains with dsRNA corresponding to M60.2 resulted in strong suppression (robust growth for several generations) of Strain 1 but not Strain 2.
  • DAB genes SEQ DO NOS: 5, 6, 18, 19 is an enhancer of the SREBP phenotype.
  • GYPl. C. elegans F32B6.8 (Gl 3876566) gene, ortholog of the human GYP1 genes (SEQ ID NOs:7, 8, 20, 21), is an enhancer of SREBP function.
  • RNAi treatment with a known component of the SREBP pathway (site 2 protease, or S2P) causes enhancement to larval lethality in the SREBP mutant but in neither of the control strains. Sequence analysis of C47D12.2 was performed using Smith - Waterman, SignalP, PSORT, PFAM, and TM-HMM.
  • ARFGEF. C. elegans Y6B3A.1 (Gl 6425428 ortholog of the human guanine nucleotide exchange proteins for ADP-ribosylation factors (ARFGEF2/BIG2, SEQ DO NOs 10, 11, 23, 24), is an enhancer of SREBP function.
  • elegans proteins C10C5.6a b, domains A, B, C, and D are located at approximately amino acid positions 86-250, 299-445, 499-604, and 756-892, respectively, of Gl 15718133 and 15718132. Because the KIAA1303 human clone was annotated as incomplete at the 5' end and lacked the region encoding domain A, we used the program GeneWise (Guigo R et al., 2000, Genome Res 10:1631-42; Birney E and Durbin R, 2000, Genome Res 10:547-8) to identify the 5' region of this gene. GeneWise combines gene prediction with homology searching to identify potential coding sequences.
  • the S. cerevisiae protein ortholog Yhrl86C (Gl 6321980) is an essential gene (Winzeler EA et al., 1999, Science 285:901-906) and has a yeast two hybrid interaction with DCP2 (Fromont-Racine M, et al., 2000, Yeast 17:95).
  • DCP2 itself interacts with multiple yeast proteins, including two involved in mRNA turnover or nonsense-mediated decay, one involved in splicing, one involved in export of proteins from the nucleus (exportin), one that's part of cytochrome be complex DI (ubiquinol cytochrome C reductase), and one that is related to dynamin and is involved in cortical localization of mitochondria.
  • DCP2 acts as a transcriptional co-activator for ectopically expressed nuclear hormone receptors in yeast (Gaudon, et al., 1999, EMBO J. 18:2229).

Abstract

L'invention concerne des gènes MSREBP humains identifiés comme modulateurs de la voie SREBP et constituant des cibles thérapeutiques destinées aux troubles associés à la voie SREBP. L'invention concerne en outre des méthodes destinées à identifier des modulateurs de MSREBP et consistant à rechercher par criblage des agents modulant l'activité de MSREBP.
PCT/US2002/032807 2001-10-16 2002-10-15 Proteines msrebp utilisees comme modificateurs de la voie srebp et leurs methodes d'utilisation WO2003033656A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002332130A AU2002332130A1 (en) 2001-10-16 2002-10-15 Msrebps as modifiers of the srebp pathway and methods of use

Applications Claiming Priority (18)

Application Number Priority Date Filing Date Title
US32989001P 2001-10-16 2001-10-16
US60/329,890 2001-10-16
US34228801P 2001-12-21 2001-12-21
US34228701P 2001-12-21 2001-12-21
US60/342,287 2001-12-21
US60/342,288 2001-12-21
US34809602P 2002-01-14 2002-01-14
US60/348,096 2002-01-14
US35140102P 2002-01-25 2002-01-25
US35140302P 2002-01-25 2002-01-25
US35136102P 2002-01-25 2002-01-25
US60/351,401 2002-01-25
US60/351,403 2002-01-25
US60/351,361 2002-01-25
US35882602P 2002-02-21 2002-02-21
US60/358,826 2002-02-21
US36861502P 2002-03-27 2002-03-27
US60/368,615 2002-03-27

Publications (2)

Publication Number Publication Date
WO2003033656A2 true WO2003033656A2 (fr) 2003-04-24
WO2003033656A3 WO2003033656A3 (fr) 2003-11-06

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AU (1) AU2002332130A1 (fr)
WO (1) WO2003033656A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10364268B2 (en) 2011-12-22 2019-07-30 Genentech, Inc. Ion exchange membrane chromatography

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5891631A (en) * 1993-05-13 1999-04-06 Board Of Regents, The University Of Texas System Methods relating tosterol regulatory element binding proteins
US6322962B1 (en) * 1998-08-14 2001-11-27 Board Of Regents, The University Of Texas System Sterol-regulated Site-1 protease and assays of modulators thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5891631A (en) * 1993-05-13 1999-04-06 Board Of Regents, The University Of Texas System Methods relating tosterol regulatory element binding proteins
US6322962B1 (en) * 1998-08-14 2001-11-27 Board Of Regents, The University Of Texas System Sterol-regulated Site-1 protease and assays of modulators thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10364268B2 (en) 2011-12-22 2019-07-30 Genentech, Inc. Ion exchange membrane chromatography
US11945837B2 (en) 2011-12-22 2024-04-02 Genentech, Inc. Ion exchange membrane chromatography

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WO2003033656A3 (fr) 2003-11-06

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