WO2003066811A2 - Genes msrebp utilises en tant que modificateurs de la voie des proteines srebp, et procedes d'utilisation associes - Google Patents

Genes msrebp utilises en tant que modificateurs de la voie des proteines srebp, et procedes d'utilisation associes Download PDF

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WO2003066811A2
WO2003066811A2 PCT/US2003/003384 US0303384W WO03066811A2 WO 2003066811 A2 WO2003066811 A2 WO 2003066811A2 US 0303384 W US0303384 W US 0303384W WO 03066811 A2 WO03066811 A2 WO 03066811A2
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msrebp
srebp
assay
agent
activity
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WO2003066811A3 (fr
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Arthur Brace
Agnes V. Eliares
Kimberly Carr Ferguson
Cynthia Seidel-Dugen
Felipa A. Mapa
Donald Ruhrmund
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Exelixis, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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

Definitions

  • SREBP sterol regulatory element binding protein
  • SREBPs are also implicated in the process of fat cell differentiation and adipose cell gene expression, particularly as transcription factors that can promote adipogenesis in a dominant fashion (reviewed by Spiegelman et al, Cell (1996) 87:377-389). SREBP function is regulated by intracellular levels of sterols or polyunsaturated fatty acids (PUFAs) (Xu J. et al, J. Biol. Chem. (1999) 274:23577- 23583).
  • PUFAs polyunsaturated fatty acids
  • SREBPs are retained as membrane-bound protein precursors that are kept inactive by virtue of being attached to the nuclear envelope and endoplasmic reticulum (ER) and therefore, excluded from the nucleus.
  • An SREBP in its membrane-bound form has large N-terminal and C-terminal segments facing the cytoplasm and a short loop projecting into the lumen of the organelle.
  • the N-terminal domain is a transcription factor of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) family, and contains an "acid blob" typical of many transcriptional activators (Brown and Goldstein, Cell (1997) 89:331-340).
  • the N-terminal acid blob is followed by a basic helix-loop-helix/leucine zipper domain (bHLH-Zip) similar to those found in many other DNA-binding transcriptional regulators.
  • SIP site 1 protease
  • S2P site 2 protease
  • SCAP SREBP Cleavage Activating Protein
  • NOT2 and S. Cerevisiae ortholog CDC36 have been shown to be part of a complex of proteins that interact with the Polymerase II holoenzyme to regulate gene expression.
  • the complex contains CCR4, CAF and NOT family proteins, among others.
  • the NOT proteins likely restrict access of TATA box proteins to noncanonical TATAAs. Loss of NOT2 can result in the derepression of genes (Benson et al. 1998, EMBO 17:6714-6722; Collart et al. 1994, Genes Dev. 8:525-537; Liu, et. al.
  • Rga (regena) gene was originally identified in a Drosophila screen for genes modifying the expression of the white eye color gene. Regena was shown to affect the expression of four of seven genes tested, which suggested that it is involved in general regulation of gene expression. Expression of the RP49 ribosomal gene was unaffected by mutations in Rga. Based on sequence similarity and functional similarity, Rga was shown to be the homolog of the yeast gene CDC36/NOT2 (Frolov et al, 1998, Genetics 148: 317-329).
  • Tuberous sclerosis (TCS) complex in humans is a disease that results in the formation of benign tumors in many tissues (Cheadle et al 2000, Hum. Genet. 107:97- 114). These tumors contain differentiated cells, but these cells are much larger than normal. This disorder manifests itself most severely in the central nervous system, which can result in epilepsy, retardation and autism, and is caused by mutations in either the TSC1 or TSC2 genes (Consortium T.E.C.T.S., 1993, Cell 75:1305-1315; van Sightenhorst et al. 1997, Science 277:805-808).
  • TSC1 encodes hamartin
  • TSC2 encodes tuberin
  • the human proteins interact in vitro (Plank et al 1998, Cancer Res. 58: 4766-4770; van S lightsenhorst et al 1998, Hum. Mol. Genet. 7:1053-1057).
  • Tuberin the TSC2 protein product contains coiled-coil domains, as well as a predicted GTPase activating protein (GAP) domain, and has GAP activity in vitro (Wienecke et al 1995, J. Biol. Chem. 270:16409-16414).
  • GAP GTPase activating protein
  • GAP GTPase activating protein
  • Rapl nuclear Ras-related regulatory proteins
  • Rsrl nuclear Ras-related regulatory proteins
  • Ran nuclear Ras-related regulatory proteins
  • GIG GTPase activating protein
  • GIG loss-of-function mutants display a range of phenotypes, depending on the strength of the mutant allele, including larval lethality and various neuroanatomical and behavioral defects (Meinertzhagen, 1994, J. Neurogenet 9:157-176; Canal et al. 1998, J. Neurosci 18:999-1008; Acebes and Ferrus 2001, J. Neurosci 21:6264-6273).
  • TSC1 and TSC2 are Drosophila TSC1 and TSC2 (GIG) genes that lead to a reduction in cell size, number and organ size (Potter et al. 2001, Cell 105:357-368; Tapon et al. 2001). Genetic experiments in the fly have demonstrated that the TSC1 and TSC2 GIG genes act together to antagonize insulin receptor signaling (Gao et al. 2001, Genes and Dev. 15:1383-1392; Potter et al. 2001; Tapon et al. 2001, Cell 105:345-355). One copy of a GIG loss of function allele is sufficient to rescue the lethality associated with fly insulin receptor mutants. Genetic data indicate that TSC1 and TSC2 (GIG) likely function downstream of Akt, and upstream of S6 kinase in the same pathway as these genes, or in a parallel pathway.
  • the EIF2B3 protein is a heteromeric guaninine nucleotide exchange factor involved in protein synthesis initiation.
  • the eukaryotic translation initiation factor EIF-2B is a complex made up of five different subunits, alpha, beta, gamma, delta and epsilon, and catalyzes the exchange of EIF-2-bound GDP for GTP, thus regenerating an active complex required for peptide-chain initiation.
  • Initiation factor 2 binds to Met-tRNA, GTP and the small ribosomal subunit (Webb and Proud, 1997, Int J Biochem Cell Biol 10:1127-1131). It is regulated by phosphorylation and by allosteric effectors.
  • EEF2B The activity of EEF2B is a key control point for protein synthesis and is altered in response to a variety of signals including hormones, nutrients, growth factors and stress (Campell et al., 1999, Biochem J. 344:433-441). Insulin, amino acids and glucose have been shown to increase the activity of EIF2. Exercise also increases rates of protein synthesis by modulating activity of EIF2B, and severe diabetes has been shown to inhibit this increase (Kostyak et al., 2001, J. Appl. Physiol. 91:79-84).
  • CAF-1 also known as a CCR4-NOT transcription complex subunit 7
  • CAF-1 is a component of a complex of proteins that interact with the RNA polymerase U holoenzyme to regulate gene expression (Albert et al., 2000, Nucleic Acids Res. 28:809-817).
  • the complex also contains CCR4 and NOT proteins, among others.
  • CAF-1 may also regulate gene expression by regulating early ribosome assembly (Schaper et al., 2001, Curr. Biol. 11:1885-1890).
  • CCR4 and CAF-1 are also components of the major cytoplasmic mRNA deadenylase in S.
  • BAP B-cell associated protein belongs to a family of Prohibitin genes found in many species including human, mouse, C. elegans, Arabidopsis, and yeast.
  • HSP70s Mitochondrial heat shock proteins
  • PP2 Protein Phosphatase 2, also called PP2A
  • PP2A is a serine/threonine protein phosphatase that has been implicated in dephosphorylation of the proteins Akt and Gsk3- beta (Ivaska et al. 2002, Mol Cell Biol 22:1352-1359); dephophorylation of Gsk leads to increased glycogen synthase activity. Additional reports show that the insulin resistance mediated by ceramide induce a PP2 activity and can be relieved by treatment with a PP2 inhibitor okadaic acid (Teruel et al. 2001, Diabetes 50:2563-2571).
  • PP2 stimulates Acetyl CoA Carboxylase, an enzyme that catalyzes the production of long chain fatty acids, which may regulate insulin secretion (Kowluru et al. 2001, Diabetes 50:1580-1587). PP2 also appears to inhibit Acyl CoA: cholesterol acyltransferase (ACAT) and cholesterol ester synthesis (Hernandez et al. 1997, Biochim Biophys Acta 1349:233-41). Drosophila MTS (microtubule star) is ortholog of human PP2, and plays an essential role in spindle formation, where it is critical for the attachment of microtubules to the kinetochore during mitosis (Snaith et al. 1996, J. Cell Sci.
  • mouse PP2 is necessary for meiosis (Lu et al 2002, Biol Reprod. 66(l):29-37). It has been speculated that the MTS/PP2 requirement is due to the hyperphosphorylation and inactivation of the Tau protein, which associates with and promotes stabilization of microtubules (Brandt and Lee 1993, J Neurochem. 61:997-1005; Planel et al. 2001, J. Biol. Chem. 276(36):34298-34306).
  • DYRK1A is a dual specificity protein kinase that is presumably involved in brain development and that belongs to a family of DYRK protein kinases comprising at least seven mammalian isoforms, as well as yeast and Drosophila orthologs (Becker et al. 1998 J Biol Chem 273:25893-902).
  • the human DYRK1A gene maps to the Down Syndrome critical region of human chromosome 21; the mouse ortholog has been implicated in neurodevelopmental delay, motor abnormalities and cognitive deficits (Becker et al. 1998, supra; Altafaj et al. 2001, Hum Mol Genet 10:1915-23).
  • DYRK kinases While the exact cellular function of the DYRK kinases is unknown, a common enzymatic property is their ability to autophosphorylate on tyrosine residues as well as phophorylate serine/threonine residues of exogenous substrates. Except for their catalytic domain, they differ in substrate specificity, tissue distribution and sub-cellular localization (Guimera et al. 1999, Genomics 57:407-18).
  • the Drosophila MNB minibrain is ortholog of human DYRK1A, and is expressed during neuroblast proliferation.
  • MNB-mutant flies show abnormalities in visual and olfactory behavior due to a reduction of the optic lobes and central brain hemispheres (Tejedor et al. 1995, Neuron 14:287-301).
  • YAK-1 has the highest sequence similarity to MNB and DYRK 1 A (Becker et al., 1998, supra).
  • YAK-1 was identified as a functional agonist of the RAS/protein kinase A pathway and has been characterized as a negative growth regulator (Garrett and Broach, 1989 Genes Dev 3:1336-1348; Garrett et al. 1991, Mol Cell Biol 11:4045-4052).
  • CSNK1 (Casein kinase 1, gamma 3), a serine/threonine protein kinase, belongs to a family of mammalian casein kinase I genes, producing multiple isoforms. Family members contain a highly conserved ⁇ 290-residue N-terminal catalytic domain coupled to a variable C-terminal region. The C-terminal region serves to promote differential subcellular localization of individual isoforms and to modulate enzyme activity (Mashhoon, et al. 2000, J Biol Chem 275: 20052-20060).
  • CSNK1 appears to play a role in the regulation of circadian rhythms, intracellular trafficking, DNA repair, cellular morphology, and protein stabilization (Liu et al. 2001, Proc Natl Acad Sci 98:11062- 11068). CSNK1 also has been shown to be involved in the regulation of eIF2B in coordination with GSK3 as part of an insulin signaling response (Wang et al. 2001, EMBO 20:4349-4359). Drosophila GISH (gilgamesh) is ortholog of CSNK1 and has been characterized as being part of a repulsive signaling mechanism that coordinates glial migration and neuronal development in the eye (Hummel, et al.
  • ERFl is responsible for terminating protein biosynthesis. Termination of protein biosynthesis and release of the nascent polypeptide chain are signaled by the presence of an in-frame stop codon at the aminoacyl site of the ribosome. ERFl recognizes the stop codon and promotes the hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA (Frolova et al. 1994, Nature 372: 701-703).
  • the crystal structure of the release factor has been determined, the overall shape and dimensions of ERFl resemble a tRNA molecule, with domains designated 1, 2, and 3 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively (Song et al. 2000, Cell 100: 311-321).
  • model organisms such as Drosophila and C. elegans
  • Drosophila and C. elegans provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example Dulubova I, et al, J Neurochem 2001 Apr;77(l):229-38; Cai T, et al., Diabetologia 2001 Jan;44(l):81-8; Pasquinelli AE, et al., Nature.
  • a genetic screen is performed in an invertebrate model organism displaying a mutant (generally visible or selectable) phenotype due to misexpression - generally reduced, enhanced or ectopic expression - of a known gene (the "genetic entry point"). Additional genes are mutated in a random or targeted manner.
  • a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a "modifier" involved in the same or overlapping pathway as the genetic entry point.
  • modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
  • RNA interference RNA interference
  • ds double stranded
  • Suitable methods for introduction of dsRNA into an animal include injection, feeding, and bathing (Tabara et al, 1998, Science 282:430-431).
  • RNAi has further been shown to produce specific gene disruptions in cultured Drosophila and mammalian cells (Paddison et al., Proc Natl Acad Sci U S A published Jan 29, 2002 as 10.1073/pnas.032652399; Clemens et al., 2000, Proc Natl Acad Sci U S A 97:6499- 503; Wojcik and DeMartino, J Biol Chem, published Dec 5, 2001 as 10.1074/jbc.M109996200; Goto et al., 2001, Biochem J 360:167-72; Elbashir et al., 2001, Nature 411:494-8). All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.
  • MSREBP Modifiers of SREBP
  • the invention provides methods for utilizing these SREBP modifier genes and polypeptides to identify MSREBP-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired SREBP function and/or MSREBP function.
  • MSREBP-modulating agents specifically bind to MSREBP polypeptides and restore SREBP function.
  • MSREBP modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress MSREBP gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
  • MSREBP modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with an MSREBP polypeptide or nucleic acid.
  • candidate MSREBP modulating agents are tested with an assay system comprising a MSREBP polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate SREBP modulating agents.
  • the assay system may be cell-based or cell-free.
  • MSREBP- modulating agents include MSREBP related proteins (e.g. dominant negative mutants, and biotherapeutics); MSREBP -specific antibodies; MSREBP -specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with MSREBP or compete with MSREBP binding partner (e.g. by binding to an MSREBP binding partner).
  • a small molecule modulator is identified using a binding assay.
  • the assay system comprises cultured cells or a non-human animal expressing SREBP, and the assay system detects an agent-biased change in the SREBP pathway, lipid metabolism, and/or adipogenesis.
  • candidate SREBP pathway modulating agents are further tested using a second assay system that detects an agent-biased change in an activity associated with the SREBP pathway, lipid metabolism, and/or adipogenesis to confirm the SREBP pathway modulating activity of the candidate agent.
  • the second assay detects an agent-biased change in an activity associated with SREBP pathway.
  • Preferred second assay systems are carried out in cultured cells.
  • the invention further provides methods for modulating the MSREBP function and/or the SREBP pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a MSREBP polypeptide or nucleic acid.
  • the agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be administered to a mammalian animal predetermined to have a pathology associated the SREBP pathway.
  • RNAi Drosophila cells, where various specific genes were silenced by RNA inhibition (RNAi).
  • Methods for using RNAi to silence genes in cells are known in the art (Adams et al., 2000, Science 287:2185-95).
  • Genes affecting SREBP pathway activity were identified as modifiers of the SREBP pathway. Accordingly, vertebrate orthologs of these modifiers, and preferably the human orthologs, MSREBP genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of disorders related to lipid (e.g., fatty acid and cholesterol) metabolism, adipogenesis, and/or other pathologies associated with the SREBP signaling pathway. In one example, treatment involves decreasing signaling through the SREBP pathway in order to treat pathologies related to metabolic syndrome. Table 1 (example 1) lists the modifiers and their orthologs.
  • MSREBP modulating agents are useful in diagnosis, therapy and pharmaceutical development.
  • Genbank referenced by Genbank identifier (Gl) or RefSeq number
  • Table 1 Sequences related to MSREBP nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (Gl) or RefSeq number), and shown in Table 1.
  • MSREBP polypeptide refers to a full-length MSREBP protein or a functionally active fragment or derivative thereof.
  • a “functionally active” MSREBP fragment or derivative exhibits one or more functional activities associated with a full- length, wild-type MSREBP protein, such as antigenic or immunogenic activity, ability to bind natural cellular substrates, etc.
  • the functional activity of MSREBP proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds., John Wiley & Sons, Inc., Somerset, New Jersey) and as further discussed below.
  • a functionally active MSREBP polypeptide is a MSREBP derivative capable of rescuing defective endogenous MSREBP activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species.
  • functionally active fragments also include those fragments that comprise one or more structural domains of an MSREBP, such as a binding domain. Protein domains can be identified using the PFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260- 2). Methods for obtaining MSREBP polypeptides are also further described below.
  • preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of an MSREBP.
  • the fragment comprises the entire functionally active domain.
  • MSREBP nucleic acid refers to a DNA or RNA molecule that encodes a MSREBP polypeptide.
  • the MSREBP polypeptide or nucleic acid or fragment thereof is from a human, but can also be an ortholog, or derivative thereof with at least 70% sequence identity, preferably at least 80%, more preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with human MSREBP.
  • 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).
  • Programs for multiple sequence alignment may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees.
  • CLUSTAL Thimpson JD et al, 1994, Nucleic Acids Res 22:4673-4680
  • orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species.
  • Structural threading or other analysis of protein folding may also identify potential orthologs.
  • a single gene in one species may correspond to multiple genes (paralogs) in another, such as human.
  • paralogs encompasses paralogs.
  • percent (%) sequence identity with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0al9 (Altschul et al., J. Mol.
  • HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched.
  • a % identity value is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. "Percent (%) amino acid sequence similarity" is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
  • Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
  • an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489; database: European Bioinformatics Institute; Smith and Waterman, 1981, J. of Molec.Biol., 147:195-197; Nicholas et al., 1998, "A tutorial on Searching Sequence Databases and Sequence Scoring Methods” (www.psc.edu) and references cited therein.; W.R. Pearson, 1991, Genomics 11:635-650).
  • This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.
  • Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of an MSREBP.
  • the stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing.
  • a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of an MSREBP under high stringency hybridization conditions that are: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C in a solution comprising 6X single strength citrate (SSC) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 ⁇ g/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 ⁇ g/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for lh in a solution containing 0.1X SSC and 0.1% SDS (sodium dodecyl sulfate).
  • SSC single strength citrate
  • moderately stringent hybridization conditions are used that are: 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 18-20h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH7.5), 5mM 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.
  • low stringency conditions can be used that are: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.
  • MSREBP nucleic acids and polypeptides useful for identifying and testing agents that modulate MSREBP function and for other applications related to the involvement of MSREBP in the SREBP pathway.
  • MSREBP nucleic acids and derivatives and orthologs thereof may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art.
  • PCR polymerase chain reaction
  • the particular use for the protein will dictate the particulars of expression, production, and purification methods. For instance, production of proteins for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of proteins for antibody generation may require structural integrity of particular epitopes.
  • Proteins to be purified for screening or antibody production may require the addition of specific tags (e.g., generation of fusion proteins).
  • Overexpression of an MSREBP protein for assays used to assess MSREBP function, such as involvement in lipid metabolism, may require expression in eukaryotic cell lines capable of these cellular activities.
  • the necessary transcriptional and translational signals can derive from the native MSREBP gene and/or its flanking regions or can be heterologous.
  • a variety of host- vector expression systems may be utilized, such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA.
  • virus e.g. vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g. baculovirus
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA.
  • An isolated host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.
  • the expression vector can comprise a promoter operably linked to an MSREBP gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.).
  • selectable markers e.g. thymidine kinase activity, resistance to antibiotics, etc.
  • recombinant expression vectors can be identified by assaying for the expression of the MSREBP gene product based on the physical or functional properties of the MSREBP protein in in vitro assay systems (e.g. immunoassays).
  • the MSREBP protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i.e. it is joined via a peptide bond to a heterologous protein sequence of a different protein), for example to facilitate purification or detection.
  • a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other using standard methods and expressing the chimeric product.
  • a chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer (Hunkapiller et al., Nature (1984) 310:105-111).
  • the gene product can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis).
  • native MSREBP proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification). Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.
  • the methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of MSREBP or other genes associated with the
  • mis-expression encompasses ectopic expression, over- expression, under-expression, and non-expression (e.g. by gene knock-out or blocking expression that would otherwise normally occur).
  • Animal models that have been genetically modified to alter MSREBP expression may be used in in vivo assays to test for activity of a candidate SREBP modulating agent, or to further assess the role of MSREBP in SREBP pathway and/or adipogenesis, and lipid metabolism.
  • the altered MSREBP expression results in a detectable phenotype, such as modified lipid profile compared to control animals having normal MSREBP expression.
  • the genetically modified animal may additionally have altered SREBP expression (e.g. SREBP knockout).
  • Preferred genetically modified animals are mammals such as primates, rodents (preferably mice or rats), among others.
  • Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila.
  • Preferred genetically modified animals are transgenic animals having a heterologous nucleic acid sequence present as an extrachromosomal element in a portion of its cells, i.e. mosaic animals (see, for example, techniques described by Jakobovits, 1994, Curr. Biol. 4:761- 763.) or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells).
  • Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal.
  • transgenic mice see Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat.
  • Clones of the nonhuman transgenic animals can be produced according to available methods (see Wilmut, I. et al. (1997) Nature 385:810-813; and PCT International Publication Nos. WO 97/07668 and WO 97/07669).
  • the transgenic animal is a "knock-out" animal having a heterozygous or homozygous alteration in the sequence of an endogenous MSREBP gene that results in a decrease of MSREBP function, preferably such that MSREBP expression is undetectable or insignificant.
  • Knock-out animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it.
  • the transgene can be a human gene (e.g., from a human genomic clone) but more preferably is an ortholog of the human gene derived from the transgenic host species.
  • a mouse MSREBP gene is used to construct a homologous recombination vector suitable for altering an endogenous MSREBP gene in the mouse genome.
  • homologous recombination vector suitable for altering an endogenous MSREBP gene in the mouse genome.
  • Detailed methodologies for homologous recombination in mice are available (see Capecchi, Science (1989) 244:1288-1292; Joyner et al., Nature (1989) 338: 153-156). Procedures for the production of non-rodent transgenic mammals and other animals are also available (Houdebine and Chourrout, supra; Pursel et al, Science (1989) 244:1281-1288; Sirnms et al., Bio/Technology (1988) 6:179-183).
  • knock-out animals such as mice harboring a knockout of a specific gene, may be used to produce antibodies against the human counterpart of the gene that has been knocked out (Claesson MH et al., (1994) Scan J Immunol 40:257-264; Declerck PJ et al., (1995) J Biol Chem. 270:8397-400).
  • the transgenic animal is a "knock-in" animal having an alteration in its genome that results in altered expression (e.g., increased (including ectopic) or decreased expression) of the MSREBP gene, e.g., by introduction of additional copies of MSREBP, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the MSREBP gene.
  • a regulatory sequence include inducible, tissue-specific, and constitutive promoters and enhancer elements.
  • the knock-in can be homozygous or heterozygous.
  • Transgenic nonhuman animals can also be produced that contain selected systems allowing for regulated expression of the transgene.
  • a system that may be produced is the cre/loxP recombinase system of bacteriophage PI (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182).
  • both Cre-LoxP and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun X et al (2000) Nat Genet 25:83-6).
  • the genetically modified animals can be used in genetic studies to further elucidate the SREBP pathway, as animal models of disease and disorders implicating defective SREBP function, and for in vivo testing of candidate therapeutic agents, such as those identified in screens described below.
  • the candidate therapeutic agents are administered to a genetically modified animal having altered MSREBP function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered MSREBP expression that receive candidate therapeutic agent.
  • animal models having defective SREBP function can be used in the methods of the present invention.
  • a SREBP knockout mouse can be used to assess, in vivo, the activity of a candidate SREBP modulating agent identified in one of the in vitro assays described below.
  • the candidate SREBP modulating agent when administered to a model system with cells defective in SREBP function, produces a detectable phenotypic change in the model system indicating that the SREBP function is restored, i.e., the cells exhibit normal adipogenesis, or lipid metabolism.
  • the invention provides methods to identify agents that interact with and/or modulate the function of MSREBP and/or the SREBP pathway. Modulating agents identified by the methods are also part of the invention. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the SREBP pathway, as well as in further analysis of the MSREBP protein and its contribution to the SREBP pathway. Accordingly, the invention also provides methods for modulating the SREBP pathway comprising the step of specifically modulating MSREBP activity by administering a MSREBP-interacting or -modulating agent.
  • an "MSREBP-modulating agent" is any agent that modulates
  • MSREBP function for example, an agent that interacts with MSREBP to inhibit or enhance MSREBP activity or otherwise affect normal MSREBP function.
  • MSREBP function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity.
  • the MSREBP - modulating agent specifically modulates the function of the MSREBP.
  • the phrases "specific modulating agent”, “specifically modulates”, etc., are used herein to refer to modulating agents that directly bind to the MSREBP polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the MSREBP. These phrases also encompasses modulating agents that alter the interaction of the MSREBP with a binding partner, substrate, or cofactor (e.g.
  • the MSREBP-modulating agent is a modulator of the SREBP pathway (e.g. it restores and/or upregulates SREBP function) and thus is also a SREBP-modulating agent.
  • MSREBP-modulating agents include small molecule compounds; MSREBP-interacting proteins, including antibodies and other biotherapeutics; and nucleic acid modulators such as antisense and RNA inhibitors.
  • the modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in "Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, PA, 19 th edition.
  • Small molecules are often preferred to modulate function of proteins with enzymatic function, and/or containing protein interaction domains.
  • Chemical agents referred to in the art as "small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500.
  • This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the MSREBP protein or may be identified by screening compound libraries.
  • Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for MSREBP-modulating activity.
  • Small molecule modulators identified from screening assays can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in treating pathologies associated with the SREBP pathway.
  • the activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing.
  • candidate clinical compounds are generated with specific regard to clinical and pharmacological properties.
  • the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
  • MSREBP-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the SREBP pathway and related disorders, as well as in validation assays for other MSREBP-modulating agents.
  • MSREBP-interacting proteins affect normal MSREBP function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity.
  • MSREBP-interacting proteins are useful in detecting and providing information about the function of MSREBP proteins, as is relevant to SREBP related disorders (e.g., for diagnostic means).
  • An MSREBP-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with an MSREBP, such as a member of the MSREBP pathway that modulates MSREBP expression, localization, and/or activity.
  • MSREBP-modulators include dominant negative forms of MSREBP-interacting proteins and of MSREBP proteins themselves.
  • Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous MSREBP-interacting proteins (Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach, eds. Glover D. & Hames B. D (Oxford University Press, Oxford, England), pp.
  • Mass spectrometry is an alternative preferred method for the elucidation of protein complexes (reviewed in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates JR 3 rd , Trends Genet (2000) 16:5-8).
  • An MSREBP-interacting protein may be an exogenous protein, such as an MSREBP-specific antibody or a T-cell antigen receptor (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using antibodies: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). MSREBP antibodies are further discussed below.
  • an MSREBP-interacting protein specifically binds an MSREBP protein.
  • an MSREBP-modulating agent binds an MSREBP substrate, binding partner, or cofactor.
  • the protein modulator is an MSREBP specific antibody agonist or antagonist.
  • the antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify MSREBP modulators.
  • the antibodies can also be used in dissecting the portions of the MSREBP pathway responsible for various cellular responses and in the general processing and maturation of the MSREBP.
  • Antibodies that specifically bind MSREBP polypeptides can be generated using known methods.
  • the antibody is specific to a mammalian ortholog of MSREBP polypeptide, and more preferably, to human MSREBP.
  • Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab').sub.2 fragments, fragments produced by a FAb expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • Epitopes of MSREBP which are particularly antigenic can be selected, for example, by routine screening of MSREBP polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci. U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89;
  • MSREBP fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an MSREBP protein.
  • MSREBP-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response.
  • the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response.
  • KLH keyhole limpet hemocyanin
  • An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.
  • MSREBP-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MSREBP polypeptides.
  • an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MSREBP polypeptides.
  • Other assays such as radioimmunoassays or fluorescent assays might also be used.
  • Chimeric antibodies specific to MSREBP polypeptides can be made that contain different portions from different animal species. For instance, a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment. Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al.,
  • Humanized antibodies which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann LM, et al., 1988 Nature 323: 323-327).
  • CDRs complementary-determining regions
  • Humanized antibodies contain -10% murine sequences and ⁇ 90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co MS, and Queen C. 1991 Nature 351: 501-501; Morrison SL. 1992 Ann. Rev. Immun. 10:239-265). Humanized antibodies and methods of their production are well-known in the art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).
  • MSREBP-specific single chain antibodies which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via an amino acid bridge, can be produced by methods known in the art (U.S. Pat. No. 4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; and Ward et al, Nature (1989) 334:544-546).
  • T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).
  • polypeptides and antibodies of the present invention may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells that express the targeted protein (Menard S, et al., Int J. Biol Markers (1989) 4:131-134).
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U.S. Pat. Nos.
  • the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously.
  • the therapeutically effective dose and dosage regimen is determined by clinical studies.
  • the amount of antibody administered is in the range of about 0.1 mg/kg -to about 10 mg/kg of patient weight.
  • the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle.
  • a pharmaceutically acceptable vehicle are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used.
  • the vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential.
  • the antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml to aboutlO mg ml. Immunotherapeutic methods are further described in the literature (US Pat. No. 5,859,206; WO0073469).
  • an MSREBP-interacting protein may have biotherapeutic applications.
  • Biotherapeutic agents formulated in pharmaceutically acceptable carriers and dosages may be used to activate or inhibit signal transduction pathways. This modulation may be accomplished by binding a ligand, thus inhibiting the activity of the pathway; or by binding a receptor, either to inhibit activation of, or to activate, the receptor.
  • the biotherapeutic may itself be a ligand capable of activating or inhibiting a receptor. Biotherapeutic agents and methods of producing them are described in detail in U.S. Pat. No. 6,146,628.
  • MSREBP When the MSREBP is a ligand, it may be used as a biotherapeutic agent to activate or inhibit its natural receptor. Alternatively, antibodies against MSREBP, as described in the previous section, may be used as biotherapeutic agents.
  • MSREBP When the MSREBP is a receptor, its ligand(s), antibodies to the ligand(s) or the MSREBP itself may be used as biotherapeutics to modulate the activity of MSREBP in the SREBP pathway.
  • MSREBP-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit MSREBP activity.
  • Preferred nucleic acid modulators interfere with the function of the MSREBP nucleic acid such as DNA replication, transcription, translocation of the MSREBP RNA to the site of protein translation, translation of protein from the MSREBP RNA, splicing of the MSREBP RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the MSREBP RNA.
  • the antisense oligomer is an oligonucleotide that is sufficiently complementary to an MSREBP mRNA to bind to and prevent translation, preferably by binding to the 5' untranslated region.
  • MSREBP-specific antisense oligonucleotides preferably range from at least 6 to about 200 nucleotides. In some embodiments the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length.
  • the oligonucleotide can be DNA or RNA or a chimeric mixture or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • the oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.
  • the antisense oligomer is a phosphothioate morpholino oligomer (PMO).
  • PMOs are assembled from four different morpholino subunits, each of which contain one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate intersubunit linkages. Details of how to make and use PMOs and other antisense oligomers are well known in the art (e.g. see WO99/18193; Probst JC, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev. :7: 187-95; US Pat. No.
  • RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene.
  • dsRNA double-stranded RNA
  • Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and humans are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev.
  • Nucleic acid modulators are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to inhibit gene expression with extraordinar specificity, are often used to elucidate the function of particular genes (see, for example, U.S. Pat. No. 6,165,790). Nucleic acid modulators are also used, for example, to distinguish between functions of various members of a biological pathway. For example, antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and man and have been demonstrated in numerous clinical trials to be safe and effective (Milligan JF, et al, Current Concepts in Antisense Drug Design, J Med Chem. (1993) 36:1923-1937; Tonkinson JL et al., Antisense
  • an MSREBP-specific nucleic acid modulator is used in an assay to further elucidate the role of the MSREBP in the SREBP pathway, and/or its relationship to other members of the pathway.
  • an MSREBP-specific antisense oligomer is used as a therapeutic agent for treatment of SREBP-related disease states.
  • an "assay system” encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event.
  • primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the MSREBP nucleic acid or protein.
  • secondary assays further assess the activity of a MSREBP modulating agent identified by a primary assay and may confirm that the modulating agent affects MSREBP in a manner relevant to the SREBP pathway. In some cases, MSREBP modulators will be directly tested in a secondary assay.
  • the screening method comprises contacting a suitable assay system comprising an MSREBP polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e.g. binding activity), which is based on the particular molecular event the screening method detects.
  • a reference activity e.g. binding activity
  • a statistically significant difference between the agent- biased activity and the reference activity indicates that the candidate agent modulates MSREBP activity, and hence the SREBP pathway.
  • the MSREBP polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above.
  • the type of modulator tested generally determines the type of primary assay.
  • screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam GS et al, Curr Opin Chem Biol (1997) 1:384-91 and accompanying references).
  • cell-based refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction.
  • cell free encompasses assays using substantially purified protein (either endogenous or recombinantly produced), partially purified or crude cellular extracts.
  • Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (e.g., receptor-ligand binding), transcriptional activity (e.g., using a reporter gene), enzymatic activity (e.g., via a property of the substrate), activity of second messengers, immunogenicty and changes in cellular morphology or other cellular characteristics.
  • Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, colorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.
  • Cell-based screening assays usually require systems for recombinant expression of MSREBP and any auxiliary proteins demanded by the particular assay. Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility.
  • Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes.
  • the binding specificity of the interacting protein to the MSREBP protein may be assayed by various known methods such as substrate processing (e.g. ability of the candidate MSREBP-specific binding agents to function as negative effectors in MSREBP-expressing cells), binding
  • R 1 equilibrium constants usually at least about 10 M " , preferably at least about 10 M " , more preferably at least about 10 9 M “1 ), and immunogenicity (e.g. ability to elicit MSREBP specific antibody in a heterologous host such as a mouse, rat, goat or rabbit).
  • immunogenicity e.g. ability to elicit MSREBP specific antibody in a heterologous host such as a mouse, rat, goat or rabbit.
  • binding may be assayed by, respectively, substrate and ligand processing.
  • the screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of a MSREBP polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein.
  • the MSREBP polypeptide can be full length or a fragment thereof that retains functional MSREBP activity.
  • the MSREBP polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag.
  • the MSREBP polypeptide is preferably human MSREBP, or is an ortholog or derivative thereof as described above.
  • the screening assay detects candidate agent-based modulation of MSREBP interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has MSREBP -specific binding activity, and can be used to assess normal MSREBP gene function.
  • a binding target such as an endogenous or exogenous protein or other substrate that has MSREBP -specific binding activity
  • Suitable assay formats that may be adapted to screen for MSREBP modulators are known in the art.
  • Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes PB, Curr Opin Chem Biol (1998) 2:597-603; Sundberg SA, Curr Opin Biotechnol 2000, 11:47-53).
  • screening assays uses fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer. These systems offer means to monitor protein-protein or DNA-protein interactions in which the intensity of the signal emitted from dye-labeled molecules depends upon their interactions with partner molecules (e.g., Selvin PR, Nat Struct Biol (2000) 7:730-4; Fernandes PB, supra; Hertzberg RP and Pope AJ, Curr Opin Chem Biol (2000) 4:445-451).
  • fluorescence technologies including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer.
  • transcriptional activity is detected using quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems).
  • a transcriptional reporter e.g., luciferase, GFP, beta-galactosidase, etc.
  • a responsive gene regulatory sequence e.g., Berg M et al, 2000, J Biomol Screen, 5:71-76. Proteins that are part of a transcriptional complex may also be assayed for binding activity (i.e., to other members of the complex).
  • assays are available to detect the activity of proteins that have specific binding activity.
  • Exemplary assays use fluorescence polarization, fluorescence polarization, and laser scanning techniques to measure binding of fluorescently labeled proteins, peptides, or other molecules (Lynch BA et al., 1999, Anal Biochem 275:62-73; Li HY, 2001, J Cell Biochem 80:293-303; Zuck P et al., Proc Natl Acad Sci USA 1999, 96: 11122-11127).
  • binding activity is detected using the scintillation proximity assay (SPA), which uses a biotinylated peptide probe captured on a streptavidin coated SPA bead and a radio-labeled partner molecule.
  • SPA scintillation proximity assay
  • the assay specifically detects the radio-labeled protein bound to the peptide probe via scintillant immobilized within the SPA bead (Sonatore LM et al., 1996, Anal Biochem 240:289-297).
  • GAP assay GAP proteins stimulate GTP hydrolysis to GDP.
  • Exemplary assays may monitor GAP activity, for instance, via a GTP hydrolysis assay, using labeled GTP (e.g., Jones S et al, Molec Biol Cell (1998) 9:2819-2837).
  • Phosphatase assay Preferred phosphatase assays detect phosphatase activity, the removal of a gamma phosphate from a serine or threonine residue of a protein substrate.
  • the dephosphorylation of a fluorescently labeled peptide substrate allows trypsin cleavage of the substrate, which in turn renders the cleaved substrate significantly more fluorescent (Nishikata M et al., Biochem J (1999) 343:35-391).
  • fluorescence polarization monitors direct binding of the phosphatase with the target; increasing concentrations of phosphatase increases the rate of dephosphorylation, leading to a change in polarization (Parker GJ et al., (2000) J Biomol Screen 5:77-88).
  • Protein kinases catalyze the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue in a protein substrate.
  • Radioassays which monitor the transfer from [gamma- 32 P or - 33 P]ATP, are frequently used to assay kinase activity.
  • Separation of the phospho-labeled product from the remaining radio-labeled ATP can be accomplished by various methods including SDS- polyacrylamide gel electrophoresis, filtration using glass fiber filters or other matrices which bind peptides or proteins, and adsorption/binding of peptide or protein substrates to solid-phase matrices allowing removal of remaining radiolabeled ATP by washing.
  • a scintillation assay monitors the transfer of the gamma phosphate from [gamma - 33 P] ATP to a biotinylated peptide substrate.
  • the substrate is captured on a streptavidin coated bead that transmits the signal (Beveridge M et al, J Biomol Screen (2000) 5:205- 212).
  • This assay uses the scintillation proximity assay (SPA), in which only radio-ligand bound to receptors tethered to the surface of an SPA bead are detected by the scintillant immobilized within it, allowing binding to be measured without separation of bound from free ligand.
  • SPA scintillation proximity assay
  • Other assays for protein kinase activity may use antibodies that specifically recognize phosphorylated substrates.
  • the kinase receptor activation (KIRA) assay measures receptor tyrosine kinase activity by ligand stimulating the intact receptor in cultured cells, then capturing solubilized receptor with specific antibodies and quantifying phosphorylation via phosphotyrosine ELISA (Sadick MD, Dev Biol Stand (1999) 97: 121-133).
  • TRF time-resolved fluorometry
  • the amount of phosphorylation is then detected using time- resolved, dissociation-enhanced fluorescence (Braunwalder AF, et al., Anal Biochem 1996 Jul 1;238(2): 159-64).
  • Generic assays may be established for protein kinases that rely upon the phosphorylation of substrates such as myelein basic protein, casein, histone, or synthetic peptides such as polyGlutamate/Tyrosine and radiolabeled ATP.
  • ELISA enzyme-linked immunosorbant assay
  • screening assays described for small molecule modulators may also be used to test antibody modulators.
  • primary assays may test the ability of the nucleic acid modulator to inhibit or enhance MSREBP gene expression, preferably mRNA expression.
  • expression analysis comprises comparing MSREBP expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express MSREBP) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art.
  • MSREBP mRNA expression is reduced in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al., Biotechniques (1999) 26: 112-125;
  • nucleic acid modulator e.g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al., Biotechniques (1999) 26: 112-125;
  • Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the MSREBP protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra). In some cases, screening assays described for small molecule modulators, particularly in assay systems that involve MSREBP mRNA expression, may also be used to test nucleic acid modulators. Secondary Assays
  • MSREBP-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a modulating agent on a particular genetic or biochemical pathway or to test the specificity of the modulating agent's interaction with MSREBP.
  • 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. In general, such assays test whether treatment of cells or animals with a candidate MSREBP-modulating agent results in changes in the SREBP pathway in comparison to untreated (or mock- or placebo-treated) cells or animals. Certain assays use "sensitized genetic backgrounds", which, as used herein, describe cells or animals engineered for altered expression of genes in the SREBP or interacting pathways.
  • Cell based assays may use a variety of mammalian cell types capable of SREBP signaling, including HEK-293 cells, CHO cells, primary hepatocytes, or hepatocytic cell lines such as McA-RH7777 (DeBose-Boyd et al., 2001, PNAS 98:1477-1482) or HEPG2 (Kotzka et al., 2000J. Lipid Res. 41:99-108). Cell based assays may detect endogenous SREBP pathway activity or may rely on recombinant expression of SREBP pathway components.
  • Cell based assays typically use culture condition that permit SREBP signaling, such as low cholesterol or low PUFA conditions, or glucose- or insulin- stimulation.
  • Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.
  • 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.
  • transgenic reporter construct comprising sequences encoding a reporter gene (such as luciferase, GFP or other fluorescent proteins, beta-galactosidase, etc.) under control of regulatory sequences (e.g., enhancer/promoter regions) of an SREBP transcriptional target gene.
  • a reporter gene such as luciferase, GFP or other fluorescent proteins, beta-galactosidase, etc.
  • regulatory sequences e.g., enhancer/promoter regions
  • 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
  • SREBP cleavage is monitored as secretion of AP, which is detected using a standard alkaline phosphatase assay (Sakai J., et al., 1998, Mol. Cell 2:505-514).
  • a fusion construct is generated in which the transcriptional activator domain of SREBP is replaced with another transcriptional activator domain, such as yeast GAL4.
  • the substituted domain which is preferably from a different species, specifically activates transcription of a reporter gene under the control of responsive regulatory sequences, such as UAS if GAL4 is used.
  • assays measure candidate modulators' effects on the functional output of SREBP signaling, such as lipid accumulation and lipid metabolism.
  • lipid accumulation is measured by staining fixed cells with Oil Red O (Foretz et al., 1999, PNAS 96:12737-12742).
  • lipid synthesis is monitored by measuring C14 acetate incorporation into either cholesterol or fatty acids (Pai, J-T et al., 1998, J. Biol. Chem. 273:26138-26148).
  • a variety of non-human animal models of lipid metabolic disorders may be used to test candidate ERFl modulators.
  • Such models typically use genetically modified animals that have been engineered to mis-express (e.g., over-express or lack expression in) genes involved in lipid metabolism, adipogenesis, and/or the SREBP pathway.
  • particular feeding conditions, and/or administration or certain biologically active compounds may contribute to or create animal models of lipid and/or metabolic disorders.
  • Assays generally required systemic delivery of the candidate modulators, such as by oral administration, injection (intravenous, subcutaneous, intraperitoneous), bolus administration, etc.
  • assays use mouse models of diabetes and/or insulin resistance.
  • Mice carrying knockouts of genes in the leptin pathway such as ob (leptin) or db (leptin receptor), or the insulin signaling pathway, such as the insulin receptor (InR) or insulin receptor substrate (IRS)
  • the insulin receptor InR
  • insulin receptor substrate insulin receptor substrate
  • Certain susceptible wild type mice such as C57BIJ6, exhibit similar symptoms when fed a high fat diet (Linton and Fazio, 2001, Current Opinion in Lipidology 12:489-495). Accordingly, appropriate assays using these models test whether administration of a candidate modulator alters, preferably decreases lipid accumulation in the liver. Lipid levels in plasma and adipose tissue may also be tested.
  • SREBP pathway activity may be tested by examining changes in the transcription of SREBP target genes in the liver.
  • target genes are associated with fatty acid metabolism and include acetyl CoA carboxylase, fatty acid synthase, ATP citrate lyase, glycerol -3-phosphate acyltransferase, glucose-6-phosphate dehydrogenase, malic enzyme, and stearoyl-CoA desaturase-l,etc. (Shimomura I et al, 1999, supra).
  • HMG-CoA synthase HMG-CoA reductase
  • squalene synthase lipoprotein lipase
  • LDLR low-density lipoprotein receptor
  • mice that overexpress a constitutively active form of SREBP under control of the PEPCK promoter develop display fatty liver.
  • LDLR low-density lipoprotein receptor
  • plasma lipids increase as well (Horton JD et al, 1999, J Clin Invest 103:10677-1076). Assays using these mice may measure both hepatic and plasma lipid levels.
  • assays use mouse models of lipoprotein biology and cardiovascular disease.
  • mouse knockouts of apolipoprotein E (apoE) display elevated plasma cholesterol and spontaneous arterial lesions (Zhang SH, 1992, Science 258:468-471).
  • Transgenic mice over-expressing cholesterol ester transfer protein (CETP) also display increased plasma lipid levels (specifically, very-low-density lipoprotein [VLDL] and low-density lipoprotein [LDL] cholesterol levels) and plaque formation in arteries (Marotti KR et al., 1993, Nature 364:73-75).
  • VLDL very-low-density lipoprotein
  • LDL low-density lipoprotein
  • Assays using these models may test whether administration of candidate modulators alters plasma lipid levels, such as by decreasing levels of the pro-atherogenic LDL and VLDL, increasing HDL, or by decreasing overall lipid (including trigyceride) levels. Additionally histological analysis of arterial morphology and lesion formation (i.e., lesion number and size) may indicate whether a candidate modulator can reduce progression and/or severity of atherosclerosis.
  • mice models for atherosclerosis including knockouts of Apo-Al, PPARgamma, and scavenger receptor (SR)-Bl in LDLR- or ApoE-null background (reviewed in, e.g., Glass CK and Witztum JL, 2001, Cell 104:503-516).
  • SR scavenger receptor
  • 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.).
  • the invention also provides methods for modulating the SREBP pathway in a cell, preferably a cell pre-determined to have defective or impaired SREBP function (e.g. due to overexpression, underexpression, or misexpression of SREBP, or due to gene mutations), comprising the step of administering an agent to the cell that specifically modulates MSREBP activity.
  • the modulating agent produces a detectable phenotypic change in the cell indicating that the SREBP function is restored.
  • the phrase "function is restored", and equivalents, as used herein, means that the desired phenotype is achieved, or is brought closer to normal compared to untreated cells. For example, with restored SREBP function, adipogenesis or lipid metabolism may normalize, or be brought closer to normal relative to untreated cells.
  • the invention also provides methods for treating disorders or disease associated with impaired SREBP function by administering a therapeutically effective amount of an MSREBP -modulating agent that modulates the SREBP pathway.
  • the invention further provides methods for modulating MSREBP function in a cell, preferably a cell pre-determined to have defective or impaired MSREBP function, by administering an MSREBP -modulating agent. Additionally, the invention provides a method for treating disorders or disease associated with impaired MSREBP function by administering a therapeutically effective amount of an MSREBP -modulating agent.
  • Various expression analysis methods can be used to diagnose whether MSREBP expression occurs in a particular sample, including Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR, and microarray analysis, (e.g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al, Biotechniques (1999) 26: 112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001, 12:41-47).
  • Tissues having a disease or disorder implicating defective SREBP signaling that express an MSREBP are identified as amenable to treatment with an MSREBP modulating agent.
  • the SREBP defective tissue overexpresses an MSREBP relative to normal tissue.
  • a Northern blot analysis of mRNA from tumor and normal cell lines, or from tumor and matching normal tissue samples from the same patient, using full or partial MSREBP cDNA sequences as probes can determine whether particular tumors express or overexpress MSREBP.
  • the TaqMan® is used for quantitative RT-PCR analysis of MSREBP expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).
  • reagents such as the MSREBP oligonucleotides, and antibodies directed against an MSREBP, as described above for: (1) the detection of the presence of MSREBP gene mutations, or the detection of either over- or under-expression of MSREBP mRNA relative to the non-disorder state; (2) the detection of either an over- or an under- abundance of MSREBP gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the pathway mediated by MSREBP.
  • the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in MSREBP expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for MSREBP expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of the disease or disorder.
  • the probe may be either DNA or protein, including an antibody.
  • RNAi screen was used to identify the association of MSREBPs with the SREBP pathway. Briefly, the screen involved treating cells from the Dmel line, a derivative of the Drosophila S2 cell line that thrives in serum-free media, with dsRNA corresponding to predicted Drosophila genes, in order to effect disruption of these genes (Adams et al., 2000, Science 287:2185-95). Duplicate wells of cells in a multi-well plate were treated with dsRNA corresponding to individual Drosophila genes (methods were essentially as described in Clemens et al., 2000, supra).
  • modifier genes are candidate positive effectors of SREBP pathway activity. Potential modifiers were retested in triplicate. The dsRNA used for the confirmation experiment was produced from a PCR product generated using different primers to the candidate modifier gene than were used to produce the original result. Table 1 is a list of the modifiers and their human orthologs.
  • proteins bound to the beads are solubilized by boiling in SDS sample buffer, fractionated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane and blotted with the indicated antibodies.
  • the reactive bands are visualized with horseradish peroxidase coupled to the appropriate secondary antibodies and the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech).
  • a purified or partially purified MSREBP is diluted in a suitable reaction buffer, e.g., 50 mM Hepes, pH 7.5, containing magnesium chloride or manganese chloride (1-20 mM) and a peptide or polypeptide substrate, such as myelin basic protein or casein (1-10 ⁇ g/ml).
  • a suitable reaction buffer e.g., 50 mM Hepes, pH 7.5, containing magnesium chloride or manganese chloride (1-20 mM) and a peptide or polypeptide substrate, such as myelin basic protein or casein (1-10 ⁇ g/ml).
  • the final concentration of the kinase is 1-20 nM.
  • the enzyme reaction is conducted in microtiter plates to facilitate optimization of reaction conditions by increasing assay throughput. A 96-well microtiter plate is employed using a final volume 30-100 ⁇ l.
  • the reaction is initiated by the addition of 33 P-gamma-ATP (0.5 ⁇ Ci/ml) and incubated for 0.5 to 3 hours at room temperature. Negative controls are provided by the addition of EDTA, which chelates the divalent cation (Mg2 + or Mn 2+ ) required for enzymatic activity. Following the incubation, the enzyme reaction is quenched using EDTA. Samples of the reaction are transferred to a 96-well glass fiber filter plate

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Abstract

L'invention concerne des gènes humains modificateurs des protéines SREBP (MSREBP), identifiés comme modulateurs de la voie des protéines SREBP. Ces gènes constituent des cibles thérapeutiques dans les troubles liés à une fonction défectueuse des protéines SREBP. L'invention concerne également des procédés d'identification de modulateurs de protéines SREBP. Ces procédés consistent à effectuer un criblage pour trouver des agents qui modulent l'activité des gènes MSREBP.
PCT/US2003/003384 2002-02-06 2003-02-05 Genes msrebp utilises en tant que modificateurs de la voie des proteines srebp, et procedes d'utilisation associes WO2003066811A2 (fr)

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