WO2003073063A2 - Modulation de la signalisation du recepteur de l'insuline par ciblage de helic1 - Google Patents

Modulation de la signalisation du recepteur de l'insuline par ciblage de helic1 Download PDF

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WO2003073063A2
WO2003073063A2 PCT/US2003/003400 US0303400W WO03073063A2 WO 2003073063 A2 WO2003073063 A2 WO 2003073063A2 US 0303400 W US0303400 W US 0303400W WO 03073063 A2 WO03073063 A2 WO 03073063A2
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helicl
assay
agent
assay system
activity
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WO2003073063A3 (fr
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Lisa C. Kadyk
Eric C. Kong
Carol L. O'brien
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Exelixis, Inc.
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins

Definitions

  • Insulin is the central hormone governing metabolism in vertebrates (reviewed in
  • insulin is secreted by the beta cells of the pancreas in response to elevated blood glucose levels, which normally occur following a meal.
  • the immediate effect of insulin secretion is to induce the uptake of glucose by muscle, adipose tissue, and the liver.
  • a longer-term effect of insulin is to increase the activity of enzymes that synthesize glycogen in the liver and triglycerides in adipose tissue.
  • Insulin can exert other actions beyond these "classic" metabolic activities, including increasing potassium transport in muscle, promoting cellular differentiation of adipocytes, increasing renal retention of sodium, and promoting production of androgens by the ovary. Defects in the secretion and/or response to insulin are responsible for the disease diabetes mellitus, which is of enormous economic significance. Within the United States, diabetes mellitus is the fourth most common reason for physician visits by patients; it is the leading cause of end-stage renal disease, non-traumatic limb amputations, and blindness in individuals of working age (Warram et al., 1995, In Joslin's Diabetes Mellitus, Kahn and Weir, eds., Philadelphia, Lea & Febiger, pp.
  • 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 of genes, pathways, and cellular processes, have direct relevance to more complex vertebrate organisms. Identification of novel functions of genes involved in particular pathways in such model organisms can directly contribute to the understanding of the correlative pathways in mammals and of methods of modulating them (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.
  • Drosophila and C. elegans are not susceptible to human pathologies, various experimental models can mimic the pathological states. A correlation between the pathology model and the modified expression of a Drosophila or C. elegans gene can identify the association of the human ortholog with the human disease.
  • a genetic screen is performed in an invertebrate model organism displaying a mutant (generally visible or selectable) phenotype due to mis-expression - generally reduced, enhanced or ectopic expression - of a known gene (the "genetic entry point"). Additional genes are mutated in a random or targeted manner. When an additional gene mutation changes the original mutant phenotype, this gene is identified as a "modifier" that directly or indirectly interacts with the genetic entry point and its associated pathway. If the genetic entry point is an ortholog of a human gene associated with a human pathology, such as lipid metabolic disorders, the screen can identify modifier genes that are candidate targets for novel therapeutics.
  • T ⁇ R insulin receptor
  • KIAA0111 has been annotated as having DEAD/DEAH box helicase homology. Helicases are involved in unwinding nucleic acids. DEAD/DEAH box RNA helicases have been implicated in many cell processes, including transcription, splicing, ribosome biogenesis, nucleocytoplasmic transport, translation, and RNA decay (de la Cruz, et al., 1999, Trends Biochem Sci 24: 192-198). A S. cerevisiae DEAD-box helicase, Fallp, is involved in 40S ribosomal subunit biogenesis. It is located in the nucleolus, and mutants have decreased numbers of 40S subunits.
  • HELICl helicasel
  • HELICl -modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress HELICl gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
  • HELICl modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with an HELICl polypeptide or nucleic acid.
  • candidate HELICl modulating agents are tested with an assay system comprising a HELICl polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate INR modulating agents.
  • the assay system may be cell-based or cell-free.
  • HELICl -modulating agents include HELICl related proteins (e.g. dominant negative mutants, and biotherapeutics); HELICl -specific antibodies; HELICl -specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with HELICl or compete with HELICl binding partner (e.g. by binding to an HELICl binding partner).
  • a small molecule modulator is identified using an enzymatic assay.
  • the screening assay system is selected from a binding assay, a hepatic lipid accumulation assay, a plasma lipid accumulation assay, an adipose lipid accumulation assay, a plasma glucose level assay, a plasma insulin level assay, and insulin sensitivity assay.
  • candidate INR pathway modulating agents are further tested using a second assay system that detects changes in activity associated with INR signaling.
  • the second assay system may use cultured cells or non-human animals, hi specific embodiments, the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the INR pathway.
  • the invention further provides methods for modulating the HELICl function and/or the INR pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a HELICl 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 INR pathway.
  • HELICl The association of HELICl with INR signaling was identified using a C. elegans model for defective insulin receptor function.
  • Y65B4A.6 Genebank Identifier [GI] 17510385
  • HELICl the C. elegans ortholog of human KIAA0111
  • HELICl genes i.e., nucleic acids and polypeptides
  • therapy involves increasing signaling through INR in order to treat pathologies related to diabetes and/or metabolic syndrome.
  • the invention provides in vitro and in vivo methods of assessing HELICl function, and methods of modulating (generally inhibiting or agonizing) HELICl activity, which are useful for further elucidating USER signaling and for developing diagnostic and therapeutic modalities for pathologies associated with INR signaling.
  • pathologies associated with INR signaling encompass pathologies where INR signaling contributes to maintaining the healthy state, as well as pathologies whose course may be altered by modulation of the INR signaling.
  • Human HELICl sequences used in the invention are provided in Genbank as Genbank identifier (GI) numbers GI 7661919 (SEQ ID NO:l) for nucleic acid and GI 7661920 (SEQ ID NO:2) for protein sequences.
  • GI Genbank identifier
  • the term "HELICl polypeptide” refers to a full-length HELICl protein or a fragment or derivative thereof that is "functionally active,” meaning that the HELICl protein derivative or fragment exhibits one or more functional activities associated with a full-length, wild-type HELICl protein.
  • a fragment or derivative may have antigenicity such that it can be used in immunoassays, for immunization, for generation of inhibitory antibodies, etc, as discussed further below.
  • a functionally active HELICl fragment or derivative displays one or more biological activities associated with HELICl proteins such as enzymatic activity, signaling activity, ability to bind natural cellular substrates, etc.
  • Preferred HELICl polypeptides display enzymatic (helicase) activity.
  • a functionally active HELICl polypeptide is a HELICl derivative capable of rescuing defective endogenous HELICl activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species.
  • the fragments preferably comprise a HELICl domain, such as a C- or N-terminal or catalytic domain, among others, and preferably comprise at least 10, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous amino acids of a HELICl protein.
  • a preferred HELICl fragment comprises a catalytic domain.
  • Functional domains can be identified using the PFAM program (Bateman A et al, 1999 Nucleic Acids Res 27:260-262; website at pfam.wustl.edu).
  • HELICl nucleic acid refers to a DNA or RNA molecule that encodes a HELICl polypeptide.
  • the HELICl polypeptide or nucleic acid or fragment thereof is from a human, but it can be an ortholog or derivative thereof with at least 70%, preferably with at least 80%, preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with a human HELICl.
  • 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).
  • Programs for multiple sequence alignment such as CLUSTAL (Thompson JD et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees.
  • 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 e.g., using software by ProCeryon, Biosciences, Salzburg, Austria
  • protein folding may also identify potential orthologs.
  • a gene duplication event follows speciation, a single gene in one species, such as C. elegans, may correspond to multiple genes (paralogs) in another, such as human.
  • the term "orthologs" encompasses paralogs.
  • percent (%) sequence identity with respect to a specified subject sequence, or a specified portion thereof, 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. Biol. (1997) 215:403-410; http.7/blast. wustl.edu/blast/README.html) with search parameters set to default values.
  • the 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.
  • a conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected.
  • 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 glut-imine 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; Smith and Waterman, 1981, J. of MolecBiol., 147:195- 197; Nicholas et al., 1998, "A tutorial on Searching Sequence Databases and Sequence Scoring Methods” (website at 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 SEQ ID NO: 1.
  • 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. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al, Molecular Cloning, Cold Spring Harbor (1989)).
  • a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of any one of SEQ ID NO: 1 under stringent 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/wl yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for 1 h 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% PNP, 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 jug/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.
  • HELICl nucleic acids and polypeptides useful for identifying and testing agents that modulate HELICl function and for other applications related to the involvement of HELICl in INR signaling.
  • HELICl nucleic acids 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.
  • HELICl polypeptides A wide variety of methods are available for obtaining HELICl polypeptides.
  • the intended use for the polypeptide will dictate the particulars of expression, production, and purification methods.
  • production of polypeptides for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of polypeptides for antibody generation may require structural integrity of particular epitopes.
  • Expression of polypeptides to be purified for screening or antibody production may require the addition of specific tags (t.e., generation of fusion proteins).
  • Overexpression of a HELICl polypeptide for cell- based assays used to assess HELICl function, such as involvement in tubulogenesis may require expression in eukaryotic cell lines capable of these cellular activities.
  • the nucleotide sequence encoding a HELICl polypeptide can be inserted into any appropriate vector for expression of the inserted protein-coding sequence.
  • the necessary transcriptional and translational signals can derive from the native HELICl 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.
  • a host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.
  • the HELICl polypeptide may be optionally expressed as a fusion or chimeric product, joined via a peptide bond to a heterologous protein sequence.
  • the heterologous sequence encodes a transcriptional reporter gene (e.g., GFP or other fluorescent proteins, luciferase, beta-galactosidase, etc.).
  • a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame 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-
  • HELICl polypeptide can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis).
  • native HELICl 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 HELICl or other genes associated with INR signaling.
  • 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).
  • the methods of this invention may use non-human animals that have been genetically modified to alter expression of HELICl and/or other genes known to be involved in INR signaling.
  • Preferred genetically modified animals are mammals, particularly mice or rats.
  • Preferred non-mammalian species include Zebrafish, C. elegans, and Drosophila.
  • the altered HELICl or other gene expression results in a detectable phenotype, such as modified levels of INR signaling, modified levels of plasma glucose or insulin, or modified lipid profile as compared to control animals having normal expression of the altered gene.
  • the genetically modified animals can be used to further elucidate INR signaling, in animal models of pathologies associated with INR signaling, and for in vivo testing of candidate therapeutic agents, as described below.
  • Preferred genetically modified animals are transgenic, at least a portion of their cells harboring non-native nucleic acid that is present either as a stable genomic insertion or as an extra-chromosomal element, which is typically mosaic.
  • Preferred transgenic animals have germ-line insertions that are stably transmitted to all cells of progeny animals.
  • Non-native nucleic acid is introduced into host animals by any expedient method.
  • Methods of making transgenic animals are well-known in the art (for 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 HELICl gene that results in a decrease of HELICl function, preferably such that HELICl 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 HELICl gene is used to construct a homologous recombination vector suitable for altering an endogenous HELICl gene in the mouse genome.
  • homologous recombination vector suitable for altering an endogenous HELICl 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; Simms 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 HELICl gene, e.g., by introduction of additional copies of HELICl, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the HELICl 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 INR pathway, as animal models of disease and disorders implicating defective INR 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 HELICl function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered HELICl expression that receive candidate therapeutic agent.
  • I-n addition to the above-described genetically modified animals having altered HELICl function, animal models having defective USER function (and otherwise normal HELICl function), can be used in the methods of the present invention.
  • a INR knockout mouse can be used to assess, in vivo, the activity of a candidate INR modulating agent identified in one of the in vitro assays described below.
  • the candidate INR modulating agent when administered to a model system with cells defective in INR function, produces a detectable phenotypic change in the model system indicating that the INR function is restored.
  • the invention provides methods to identify agents that interact with and/or modulate the function of HELICl and/or INR signaling. Such agents are useful in a variety of diagnostic and therapeutic applications associated with INR signaling, as well as in further analysis of the HELICl protein and its contribution to INR signaling. Accordingly, the invention also provides methods for modulating INR signaling comprising the step of specifically modulating HELICl activity by administering a HELICl-interacting or -modulating agent.
  • a "HELICl -modulating agent” is any agent that modulates HELICl function, for example, an agent that interacts with HELICl to inhibit or enhance HELICl activity or otherwise affect normal HELICl function.
  • the HELICl function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity.
  • the HELICl - modulating agent specifically modulates the function of the HELICl.
  • the phrases "specific modulating agent”, “specifically modulates”, etc., are used herein to refer to modulating agents that directly bind to the HELICl polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the HELICl. These phrases also encompasses modulating agents that alter the interaction of the HELICl with a binding partner, substrate, or cofactor (e.g.
  • the HELICl- modulating agent is a modulator of the USER pathway (e.g. it restores and/or upregulates INR function) and thus is also a INR-modulating agent.
  • Preferred HELICl -modulating agents include small molecule chemical agents,
  • HELICl-interacting proteins including antibodies and other biotherapeutics, and nucleic acid modulators, including antisense oligomers and RNA.
  • 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 Molecule Modulators 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 HELICl 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 HELICl-modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151:1947-1948).
  • Small molecule modulators identified from screening assays, as described below, 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 INR signaling.
  • 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.
  • HELICl-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the INR pathway and related disorders, as well as in validation assays for other HELICl-modulating agents, hi a preferred embodiment, HELICl-interacting proteins affect normal HELICl function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, HELICl-interacting proteins are useful in detecting and providing information about the function of HELICl proteins, as is relevant to INR related disorders, such as diabetes (e.g., for diagnostic means).
  • a HELICl-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with an HELICl, such as a member of the HELICl pathway that modulates HELICl expression, localization, and/or activity.
  • HELICl -modulators include dominant negative forms of HELICl-interacting proteins and of HELICl proteins themselves.
  • Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous HELICl-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 HELICl-interacting protein may be an exogenous protein, such as an HELIC1- 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).
  • HELICl antibodies are further discussed below.
  • a HELICl-interacting protein specifically binds an HELICl protein.
  • a HELICl-modulating agent binds an HELICl substrate, binding partner, or cof actor.
  • the protein modulator is an HELICl specific antibody agonist or antagonist.
  • the antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify HELICl modulators.
  • the antibodies can also be used in dissecting the portions of the HELICl pathway responsible for various cellular responses and in the general processing and maturation of the HELICl .
  • Antibodies that specifically bind HELICl polypeptides can be generated using known methods.
  • the antibody is specific to a mammalian ortholog of HELICl polypeptide, and more preferably, to human HELICl.
  • 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 HELICl which are particularly antigenic can be selected, for example, by routine screening of HELICl 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;
  • HELICl fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an HELICl protein.
  • HELICl -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.
  • HELICl -specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELIS A) using immobilized corresponding HELICl polypeptides.
  • an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELIS A) using immobilized corresponding HELICl polypeptides.
  • Other assays such as radioimmunoassays or fluorescent assays might also be used.
  • Chimeric antibodies specific to HELICl 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).
  • HELICl -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., it 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 about 10 mg/ml. Immunotherapeutic methods are further described in the literature (US Pat. No. 5,859,206; WO0073469).
  • HELICl-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit HELICl activity.
  • Preferred antisense oligomers interfere with the function of HELICl nucleic acids, such as DNA replication, transcription, HELICl RNA translocation, translation of protein from the HELICl RNA, RNA splicing, and any catalytic activity in which the HELICl RNA participates.
  • the antisense oligomer is an oligonucleotide that is sufficiently complementary to a HELICl mRNA to bind to and prevent translation from the HELICl mRNA, preferably by binding to the 5' untranslated region.
  • HELICl -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, a chimeric mixture of DNA and RNA, 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 phosphorothioate morpholino oligomer (PMO).
  • PMOs are assembled from four different morpholino subunits, each of which containing 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 inter-subunit linkages. Methods of producing and using PMOs and other antisense oligonucleotides are well known in the art (e.g.
  • 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. Methods relating to the use of RNAi to silence genes in C.
  • Nucleic acid modulators are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to specifically inhibit gene expression, are often used to elucidate the function of particular genes (see, e.g., US 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 humans and have been demonstrated in numerous clinical trials to be safe and effective (Milligan JF et al, 1993, J Med Chem 36: 1923-1937; Tonkinson JL et al, 1996, Cancer Invest 14:54-65).
  • a HELICl -specific antisense oligomer is used in an assay to further elucidate the function of HELICl in INR signaling.
  • Zebrafish is a particularly useful model for the study of INR signaling using antisense oligomers.
  • PMOs are used to selectively inactive one or more genes in vivo in the Zebrafish embryo. By injecting PMOs into Zebrafish at the 1-16 cell stage candidate targets emerging from the Drosophila screens are validated in this vertebrate model system.
  • PMOs are used to screen the Zebrafish genome for identification of other therapeutic modulators of INR signaling.
  • a HELIC1- specific antisense oligomer is used as a therapeutic agent for treatment of metabolic pathologies.
  • an "assay system” encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event or events.
  • primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the HELICl nucleic acid or protein.
  • secondary assays further assess the activity of a HELICl-modulating agent identified by a primary assay and may confirm that the modulating agent affects HELICl in a manner relevant to INR signaling.
  • the assay system comprises contacting a suitable assay system comprising a HELICl polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity, which is based on the particular molecular event the assay system detects.
  • the method further comprises detecting the same type of activity in the presence of a candidate agent ("the agent-biased activity of the system"). A difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates HELICl activity, and hence INR signaling.
  • HELICl 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).
  • the term "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 cellular extracts, 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.
  • 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, Curr Opin Chem Biol (1998) 2:597-603; 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.
  • Suitable assay formats that may be adapted to screen for HELICl modulators are known in the art. Appropriate assays monitor enzymatic (helicase) activity.
  • an assay for RNA helicase activity uses the scintillation proximity (SPA) assay to detect the displacement of a radio-labeled oligonucleotide from single stranded RNA (Kyono K et al, Anal Biochem (1998) 257: 120-126).
  • SPA scintillation proximity
  • 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, 1998, supra; Sundberg SA, Curr Opin Biotechnol 2000, 11:47-53).
  • Cell-based screening assays usually require systems for recombinant expression of HELICl and any auxiliary proteins demanded by the particular assay.
  • Cell-free assays often use recombinantly produced purified or substantially purified proteins.
  • 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 HELICl protein may be assayed by various known methods, including binding equilibrium constants (usually at least about 10 7 M "1 , preferably at least about 10 8 M "1 , more preferably at least about 10 9 M "1 ), and immunogenic properties.
  • 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 HELICl polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein.
  • the HELICl polypeptide can be full length or a fragment thereof that retains functional HELICl activity.
  • the HELICl polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag.
  • the HELICl polypeptide is preferably human HELICl, or is an ortholog or derivative thereof as described above.
  • the screening assay detects candidate agent-based modulation of HELICl interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has HELICl -specific binding activity, and can be used to assess normal HELICl gene function.
  • a binding target such as an endogenous or exogenous protein or other substrate that has HELICl -specific binding activity
  • Certain screening assays may also be used to test antibody and nucleic acid modulators; for nucleic acid modulators, appropriate assay systems involve HELICl mRNA expression.
  • appropriate primary assays are binding assays that test the antibody's affinity to and specificity for the HELICl 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 methods for detecting HELICl -specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.
  • primary assays may test the ability of the nucleic acid modulator to inhibit HELICl gene expression, preferably mRNA expression.
  • expression analysis comprises comparing HELICl expression in like populations of cells (e.g. , two pools of cells that endogenously or recombinantly express HELICl) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art.
  • HELICl mRNA expression is reduced in cells treated with the nucleic acid modulator (e.g. , Current
  • Secondary assays may be used to further assess the activity of a HELICl- modulating agent identified by any of the above methods to confirm that the modulating agent affects HELICl in a manner relevant to INR signaling.
  • HELICl - 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 modulator on a particular genetic or biochemical pathway or to test the specificity of the modulator's interaction with HELICl.
  • Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express HELICl) in the presence and absence of the candidate modulator, hi general, such assays test whether treatment of cells or animals with a candidate HELICl-modulating agent results in changes in INR signaling, in comparison to untreated (or mock- or placebo-treated) cells or animals. Changes in INR signaling may be detected as modifications to INR pathway components, or changes in their expression or activity.
  • Assays may also detect an output of normal or defective INR signaling, used herein to encompass immediate outputs, such as glucose uptake, or longer-term effects, such as changes in glycogen and triglycerides metabolism, adipocyte differentiation, or development of diabetes or other INR-related pathologies.
  • Certain assays use sensitized genetic backgrounds, used herein to describe cells or animals engineered for altered expression of genes in the INR or interacting pathways, or pathways associated with INR signaling or an output of INR signaling.
  • Cell-based assays may use a variety of insulin-sensitive mammalian cells and may detect endogenous INR signaling or may rely on recombinant expression of USER and/or other INR pathway components.
  • Exemplary insulin-sensitive cells include adipocytes, hepatocytes, and pancreatic beta cells.
  • Suitable adipocytes include 3T3 LI cells, which are most commonly used for insulin sensitivity assays, as well as primary cells from mice or human biopsy.
  • Suitable hepatocytes include the rat hepatoma H4-U-E cell line.
  • Suitable beta cells include rat INS-1 cells with optimized glucose-sensitive insulin secretion (such as clone 823-13, Hohmeier et al, 2000, Diabetes 49:424).
  • Suitable cells include muscle cells, such as L6 myotubes, and CHO cells engineered to over- express INR.
  • muscle cells such as L6 myotubes
  • CHO cells engineered to over- express INR.
  • factors such as glucosamine, free fatty acids or TNF alpha, which induce an insulin resistant state.
  • Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.
  • Cell based assays generally test whether treatment of insulin responsive cells with the HELICl - modulating agent alters INR signaling in response to insulin stimulation ("insulin sensitivity"); such assays are well-known in the art (see, e.g., Sweeney et al., 1999, J Biol Chem 274: 10071). In a preferred embodiment, assays are performed to determine whether inhibition of HELICl function increases insulin sensitivity.
  • USER signaling is assessed by measuring expression of insulin- responsive genes.
  • Hepatocytes are preferred for these assays.
  • Many insulin responsive genes are known (e.g., p85 PI3 kinase, hexokinase TJ, glycogen synthetase, lipoprotein lipase, etc; PEPCK is specifically down-regulated in response to INR signaling).
  • Any available means for expression analysis may be used.
  • mRNA expression is detected.
  • Taqman analysis is used to directly measure mRNA expression.
  • transgenic reporter construct comprising sequences encoding a reporter gene (such as lucif erase, GFP or other fluorescent proteins, beta-galactosidase, etc.) under control of regulatory sequences (e.g., enhancer/promoter regions) of an insulin responsive gene.
  • regulatory sequences e.g., enhancer/promoter regions
  • INR signaling may also be detected by measuring the activity of components of the INR-signaling pathway, which are well-known in the art (see, e.g., Kahn and Weir, Eds., Joslin's Diabetes Mellitus, Williams & Wilkins, Baltimore, MD, 1994).
  • Suitable assays may detect phosphorylation of pathway members, including IRS, PI3K, Akt, GSK3 etc., for instance, using an antibody that specifically recognizes a phosphorylated protein.
  • Assays may also detect a change in the specific signaling activity of pathway components (e.g., kinase activity of PI3K, GSK3, Akt, etc.).
  • Kinase assays, as well as methods for detecting phosphorylated protein substrates are well known in the art (see, e.g., Ueki K et al, 2000, Mol Cell Biol;20: 8035-46).
  • assays measure glycogen synthesis in response to insulin stimulation, preferably using hepatocytes.
  • Glycogen synthesis may be assayed by various means, including measurement of glycogen content, and determination of glycogen synthase activity using labeled, such as radio-labeled, glucose (see, e.g., Aiston S and Agius L, 1999, Diabetes 48:15-20; Rother Kl et al., 1998, J Biol Chem 273:17491-7).
  • glucose transporter GLUT 4
  • GLUT4 glucose transporter
  • Such assays may detect endogenous GLUT4 translocation using GLUT4-specific antibodies or may detect exogenously introduced, epitope-tagged GLUT4 using an antibody specific to the particular epitope (see, e.g., Sweeney, 1999, supra; Quon MJ et al., 1994, Proc Natl Acad Sci U S A 91:5587-91).
  • ELISA see, e.g., Bergsten and Hellman, 1993, Diabetes 42:670-4
  • RIA radioimmunoassay
  • a variety of non-human animal models of metabolic disorders may be used to test candidate HELICl 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 USER signaling 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 USER signaling pathway, such as USER or the insulin receptor substrate (IRS)
  • Leptin the leptin pathway
  • USER signaling pathway such as USER or the insulin receptor substrate (IRS)
  • UFS insulin receptor substrate
  • Certain susceptible wild type mice such as C57BL/6, 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.
  • Methods for assaying lipid content typically by FPLC or colorimetric assays (Shimano H et al, 1996, J Clin Invest 98:1575-1584; Hasty et al., 2001, J Biol Chem 276:37402-37408), and lipid synthesis, such as by scintillation measurement of incorporation of radio-labeled substrates (Horton JD et al, 1999, J Clin Invest 103:1067-1076), are well known in the art.
  • Other useful assays test blood glucose levels, insulin levels, and insulin sensitivity (e.g., Michael MD, 2000, Molecular Cell 6: 87). Insulin sensitivity is routinely tested by a glucose tolerance test or an insulin tolerance test.
  • 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 [NLDL] and low-density lipoprotein [LDL] cholesterol levels) and plaque formation in arteries (Marotti KR et al., 1993, Nature 364:73-75).
  • 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 NLDL, 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.).
  • HELICl is implicated in USER signaling provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders associated with USER signaling and for the identification of subjects having a predisposition to such diseases and disorders. Any method for assessing HELICl expression in a sample, as previously described, may be used.
  • Such methods may, for example, utilize reagents such as the HELICl oligonucleotides and antibodies directed against HELICl, as described above for: (1) the detection of the presence of HELICl gene mutations, or the detection of either over- or under-expression of HELICl mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of HELICl gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in a biological pathway mediated by HELIC 1.
  • reagents such as the HELICl oligonucleotides and antibodies directed against HELICl
  • the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in HELICl expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for HELICl 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-based screen was used to identify modifiers (suppressors) of the larval arrest (dauer-formation) phenotype of loss-of -function mutations in daf-2, the insulin- receptor in C. elegans (Kimura KD, et al,. 1997, Science 277:942).
  • the screen used two worm strains, each containing a missense mutation in the ligand-binding domain of the worm insulin receptor. When late larval or adult animals are raised to a restrictive temperature, their progeny arrest as dauer larvae (an alternate developmental fate that normally occurs only in adverse conditions).
  • RNAi treatment of these strains involved RNAi treatment of these strains with dsRNA derived from cDNA or exon-rich genomic fragments of worm genes in order to cause reduction-of-function of these genes.
  • Potential suppressors were identified as those genes that, when knocked down by RNAi treatment, allowed growth of the insulin-receptor mutant strains rather than larval arrest.
  • 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.
  • I-n yeast the best hit from BLAST was a helicase called Fallp, which has been implicated in biogenesis of the 40S ribosomal subunit (Kressler, et al. 1997, supra). None of the genes of the top BLAST hits in humans, mice, or Drosophila has been experimentally characterize ⁇ i each of these species , the next most closely related protein is translation initiation factor eIF4A.
  • Human eIF4A genes are presented in Genbank entries GI 14735107, GI 4503529, and GI 4503531 ; mouse eIF4A is presented in GI 2507330; Drosophila eIF4A is presented in GI 7297048.
  • RNA helicases involved in some other process that negatively regulates insulin signaling or one of the outputs of insulin signaling.
  • these helicases may have a role in transcription or translation of one or a few proteins that are negative regulators of these pathways.
  • Such specific effects of helicase activity on a subclass of RNAs are not unprecedented.
  • several RNA helicases in C. elegans have been found to have specific effects on the switch from spermatogenesis to oogenesis during development (Puoti and Kimble, 2000, Proc. Natl. Acad. Sci. 97:3276-81).
  • Fluorescently-labeled HELICl peptide/substrate are added to each well of a 96- well microtiter plate, along with a test compound of choice in a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Changes in fluorescence polarization, determined by using a Fluorolite FPM-2 Fluorescence Polarization Microtiter System (Dynatech Laboratories, Inc), relative to control values indicates the test compound is a candidate modifier of HELICl activity.
  • 33 P-labeled HELICl peptide is added in an assay buffer (100 mM KC1, 20 mM HEPES pH 7.6, 1 mM MgCl 2 , 1% glycerol, 0.5% NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors) along with a compound of interest to the wells of a Neutralite-avidin coated assay plate, and incubated at 25°C for 1 hour. Biotinylated substrate is then added to each well, and incubated for 1 hour. Reactions are stopped by washing with PBS, and counted in a scintillation counter.
  • assay buffer 100 mM KC1, 20 mM HEPES pH 7.6, 1 mM MgCl 2 , 1% glycerol, 0.5% NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors
  • proteins bound to the beads are directly solubilized by boiling in SDS sample buffer, fractionated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinyhdene 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).

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Abstract

Selon l'invention, des gènes HELIC1 humains sont identifiés en tant que modulateurs de la signalisation INR et sont ainsi des cibles thérapeutiques pour des troubles associés aux problèmes de signalisation INR. L'invention a pour objet des procédés pour identifier des modulateurs de HELIC1, comprenant le criblage d'agents qui modulent l'activité de HELIC1.
PCT/US2003/003400 2002-02-25 2003-02-05 Modulation de la signalisation du recepteur de l'insuline par ciblage de helic1 WO2003073063A2 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
US5856111A (en) * 1994-02-28 1999-01-05 Max-Planck-Gessellschaft Zur Forderung Der Wissenschaften E.V. Methods for identifying modulators of insulin receptor phosphorylation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5856111A (en) * 1994-02-28 1999-01-05 Max-Planck-Gessellschaft Zur Forderung Der Wissenschaften E.V. Methods for identifying modulators of insulin receptor phosphorylation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ESPANEL X. ET AL.: 'The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities' PROTEIN SCIENCE vol. 11, 2002, pages 2326 - 2334, XP002974254 *
SALITURO G.M. ET AL.: 'Discovery of a small molecule insulin receptor activator' RECENT PROG. HORM. RES. vol. 56, 2001, pages 107 - 126, XP002974253 *
SELL S.M. ET AL.: 'Insulin-inducible changes in the relative ratio of PTP1B splice variants' MOL. GENET. METAB. vol. 66, no. 3, March 1999, pages 189 - 192, XP002974251 *
SKOREY K.I. ET AL.: 'Development of a robust scintillation proximity assay for protein tyrosine phosphatase 1B using the catalytically inactive (C215S) mutant' ANAL. BIOCHEM. vol. 291, no. 2, April 2001, pages 269 - 278, XP002974252 *

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