WO2003066813A2 - MINRs AS MODIFIERS OF INSULIN RECEPTOR SIGNALING AND METHODS OF USE - Google Patents

MINRs AS MODIFIERS OF INSULIN RECEPTOR SIGNALING AND METHODS OF USE Download PDF

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WO2003066813A2
WO2003066813A2 PCT/US2003/003389 US0303389W WO03066813A2 WO 2003066813 A2 WO2003066813 A2 WO 2003066813A2 US 0303389 W US0303389 W US 0303389W WO 03066813 A2 WO03066813 A2 WO 03066813A2
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mtnr
tnr
assay
agent
signaling
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PCT/US2003/003389
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French (fr)
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WO2003066813A3 (en
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Arthur Brace
Agnes V. Eliares
Kimberly Carr Ferguson
Cynthia Seidel-Dugen
Felipa A. Mapa
Donald Ruhrmund
Jianfeng Wu
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Exelixis, Inc.
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Priority to US10/504,120 priority Critical patent/US20060088829A1/en
Priority to CA002473499A priority patent/CA2473499A1/en
Priority to EP03710857A priority patent/EP1472515A4/en
Priority to AU2003215007A priority patent/AU2003215007A1/en
Priority to JP2003566164A priority patent/JP2005532787A/en
Publication of WO2003066813A2 publication Critical patent/WO2003066813A2/en
Publication of WO2003066813A3 publication Critical patent/WO2003066813A3/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Definitions

  • the FYVE-domain protein Rabenosyn-5 has been shown to be an effector of Rab5 and Rab4, physically connecting early endosomes and receptor recycling to the cell surface (De Renzis et al., 2002, Nat. Cell Biol. 4:124-133).
  • Insulin-responsive tissues express several Rab isoforms, including Rab3b, Rab4, Rab5, and Rab8. Of these isoforms, only Rab4 has been shown to play a role in mediating insulin actions within the cell, including insulin-stimulated GLUT4 translocation to the cell membrane (Knight et al., 2000, Endocrinology 141:208-218).
  • membrane association of Rab5 is altered in skeletal muscle isolated from insulin resistant and Type 2 diabetic patients (Bao et al, 1998, Horm. Metab. Res. 30:656-662).
  • CAF-1 catabolite repressor protein (CCR4)-associative factor 1
  • CCR4-associative factor 1 also known as a CCR4-NOT transcription complex subunit 7
  • CCR4-associative factor 1 is a component of a complex of proteins that interact with the RNA polymerase II holoenzyme to regulate gene expression (Albeit 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).
  • candidate TNR pathway modulating agents are further tested using a second assay system that detects changes in activity associated with TNR signaling.
  • the second assay system may use cultured cells or non-human animals.
  • the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the TNR pathway.
  • MTNR polypeptide refers to a full-length MTNR protein or a fragment or derivative thereof that is “functionally active,” meaning that the MTNR protein derivative or fragment exhibits one or more functional activities associated with a full- length, wild-type MTNR 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 MTNR fragment or derivative displays one or more biological activities associated with MTNR proteins such as enzymatic activity, signaling activity, ability to bind natural cellular substrates, etc.
  • the nucleotide sequence encoding a MTNR 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 MTNR 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.
  • 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).
  • 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.
  • 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 TNR pathway, as animal models of disease and disorders implicating defective TNR 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 MTNR function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered MTNR expression that receive candidate therapeutic agent.
  • animal models having defective TNR function can be used in the methods of the present invention.
  • a TNR knockout mouse can be used to assess, in vivo, the activity of a candidate TNR modulating agent identified in one of the in vitro assays described below.
  • the candidate TNR modulating agent when administered to a model system with cells defective in TNR function, produces a detectable phenotypic change in the model system indicating that the TNR function is restored.
  • MTNR-modulating agent is any agent that modulates MTNR function, for example, an agent that interacts with MTNR to inhibit or enhance MTNR activity or otherwise affect normal MTNR function.
  • MTNR function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity.
  • the MTNR - modulating agent specifically modulates the function of the MTNR.
  • specific modulating agent specifically modulates the function of the MTNR.
  • specific modulating agent are used herein to refer to modulating agents that directly bind to the MTNR polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the MTNR.
  • 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 TNR 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.
  • MTNR-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the TNR pathway and related disorders, as well as in validation assays for other MTNR-modulating agents.
  • MTNR- interacting proteins affect normal MTNR function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity.
  • MTNR-interacting proteins are useful in detecting and providing information about the function of MTNR proteins, as is relevant to TNR related disorders, such as diabetes (e.g., for diagnostic means).
  • a MTNR-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with an MTNR, such as a member of the MTNR pathway that modulates MTNR expression, localization, and/or activity.
  • MTNR-modulators include dominant negative forms of MTNR-interacting proteins and of MTNR proteins themselves.
  • Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous MTNR-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 MTNR-interacting protein may be an exogenous protein, such as an MTNR-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). MTNR antibodies are further discussed below.
  • a MTNR-interacting protein specifically binds an MTNR protein.
  • a MTNR-modulating agent binds an MTNR substrate, binding partner, or cofactor.
  • the protein modulator is an MTNR specific antibody agonist or antagonist.
  • the antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify MTNR modulators.
  • the antibodies can also be used in dissecting the portions of the MTNR pathway responsible for various cellular responses and in the general processing and maturation of the MTNR.
  • Antibodies that specifically bind MTNR polypeptides can be generated using known methods.
  • the antibody is specific to a mammalian ortholog of MTNR polypeptide, and more preferably, to human MTNR.
  • 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 MTNR which are particularly antigenic can be selected, for example, by routine screening of MTNR 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; Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequence of a MTNR.
  • Monoclonal antibodies with affinities of 10 8 M “1 preferably 10 9 M “1 to 10 10 M “1 , or stronger can be made by standard procedures as described (Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577).
  • Antibodies may be generated against crude cell extracts of MTNR or substantially purified fragments thereof. If MTNR fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an MTNR protein.
  • MINR-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.
  • Chimeric antibodies specific to MTNR polypeptides can be made that contain different portions from different animal species.
  • 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., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604- 608; Takeda et al., Nature (1985) 31:452-454).
  • MINR-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.
  • 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).
  • MTNR-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA)* which generally inhibit MTNR activity.
  • Preferred antisense oligomers interfere with the function of MTNR nucleic acids, such as DNA replication, transcription, MTNR RNA translocation, translation of protein from the MTNR RNA, RNA splicing, and any catalytic activity in which the MTNR RNA participates.
  • the antisense oligomer is an oligonucleotide that is sufficiently complementary to a MTNR mRNA to bind to and prevent translation from the MTNR mRNA, preferably by binding to the 5' untranslated region.
  • MTNR-specific antisense oligonucleotides preferably range from at least 6 to about 200 nucleotides.
  • 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.
  • 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. 15, 485-490 (2001); Hammond, S.
  • 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 MINR-specific antisense oligomer is used in an assay to further elucidate the function of MTNR in TNR signaling.
  • Zebrafish is a particularly useful model for the study of TNR 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 TNR signaling.
  • a MINR-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 MTNR nucleic acid or protein.
  • secondary assays further assess the activity of a MTNR-modulating agent identified by a primary assay and may confirm that the modulating agent affects MTNR in a manner relevant to TNR signaling.
  • MINR-modulators will be directly tested in a "secondary assay,” without having been identified or confirmed in a "primary assay.”
  • the assay system comprises contacting a suitable assay system comprising a MTNR 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 MTNR activity, and hence TNR signaling.
  • a statistically significant difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates MTNR activity, and hence the TNR signaling.
  • the MTNR 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.
  • Binding Assays A variety of 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).
  • 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).
  • Protein phosophatases catalyze the removal of a gamma phosphate from a serine, threonine or tyrosine residue in a protein substrate. Since phosphatases act in opposition to kinases, appropriate assays monitor the removal of a phosphate from a protein substrate. In one example, 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).
  • Other appropriate assays for may monitor lipid phosphatase activity and may use labeled, such as fluorescently labeled or radio-labeled substrates to detect removal of a phosphate from a phosphatidylinositol substrate.
  • an assay uses "FlashPlate" technology (U.S. Patent No.
  • 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).
  • Alternative assays may detect GAP function in endosome trafficking by monitoring movement of a cargo molecule, which may be labeled (Sonnichsen et al., 2000, J Cell Biol 149:901-14).
  • Kinase assays detect kinase activity, the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine or threonine residue in a protein substrate. Radioassays, which monitor the transfer from [gamma- 32 P or - 33 P]ATP, may be used to assay kinase activity.
  • ATP adenosine triphosphate
  • 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 (KTRA) 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 l;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.
  • Release factor activity assays may detect in vitro release factor activity (see, e.g., Seit-Nebi et al. 2001, Nucleic Acids Res 29:3982-7; Frolova et al. 1994, Nature 372:701-3; Caskey et al. 1974, Methods Enzymol 30:293-303).
  • Cell-based screening assays usually require systems for recombinant expression of MTNR 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 MTNR 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 MTNR polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein.
  • the MTNR polypeptide can be full length or a fragment thereof that retains functional MINR activity.
  • the MTNR polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag.
  • the MTNR polypeptide is preferably human MTNR, or is an ortholog or derivative thereof as described above.
  • the screening assay detects candidate agent-based modulation of MTNR interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has MTNR -specific binding activity, and can be used to assess normal MTNR gene function.
  • a binding target such as an endogenous or exogenous protein or other substrate that has MTNR -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 MTNR mRNA expression.
  • appropriate primary assays are binding assays that test the antibody's affinity to and specificity for the MTNR 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 MINR-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.
  • primary assays may test the ability of the nucleic acid modulator to inhibit MTNR gene expression, preferably mRNA expression.
  • expression analysis comprises comparing MTNR expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express MTNR) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art.
  • RNA expression may be confirmed 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 ah, Biotechniques (1999) 26:112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm DH and Guiseppi-Elie, ACurr Opin Biotechnol 2001, 12:41-47).
  • 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 ah, Biotechniques (1999) 26:112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm DH and Guiseppi-Elie, ACur
  • Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the MTNR 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).
  • Secondary assays may be used to further assess the activity of a MTNR- modulating agent identified by any of the above methods to confirm that the modulating agent affects MTNR in a manner relevant to TNR signaling.
  • MTNR- 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 MTNR.
  • Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express MTNR) in the presence and absence of the candidate modulator.
  • such assays test whether treatment of cells or animals with a candidate MTNR-modulating agent results in changes in TNR signaling, in comparison to untreated (or mock- or placebo-treated) cells or animals. Changes in TNR signaling may be detected as modifications to TNR pathway components, or changes in their expression or activity.
  • Assays may also detect an output of normal or defective TNR 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 TNR or interacting pathways, or pathways associated with TNR signaling or an output of TNR signaling.
  • Cell-based assays may use a variety of insulin-sensitive mammalian cells and may detect endogenous TNR signaling or may rely on recombinant expression of TNR and/or other TNR 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-TI-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 TNR.
  • muscle cells such as L6 myotubes
  • CHO cells engineered to over- express TNR.
  • 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 MTNR - modulating agent alters TNR 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 MTNR function increases insulin sensitivity.
  • TNR 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 ⁇ , glycogen synthetase, lipoprotein lipase, etc; PEPCK is specifically down-regulated in response to TNR 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 luciferase, GFP or other fluorescent proteins, beta-galactosidase, etc.) under control of regulatory sequences (e.g., enhancer/promoter regions) of an insulin responsive gene.
  • a reporter gene such as luciferase, GFP or other fluorescent proteins, beta-galactosidase, etc.
  • regulatory sequences e.g., enhancer/promoter regions
  • TNR signaling may also be detected by measuring the activity of components of the TNR-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 KI et al., 1998, J Biol Chem 273:17491- 7).
  • Other suitable assays measure cellular uptake of glucose (typically labeled glucose) in response to insulin stimulation. Adipocytes are preferred for these assays.
  • Assays also measure translocation of glucose transporter (GLUT) 4, which is a primary mediator of insulin-induced glucose uptake, primarily in muscle and adipocytes, and which specifically translocates to the cell surface following insulin stimulation.
  • GLUT 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 MTNR 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 TNR 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 TNR signaling pathway, such as TNR or the insulin receptor substrate (IRS) develop symptoms of diabetes, and show hepatic lipid accumulation (fatty liver) and, frequently, increased plasma lipid levels (Nishina et al., 1994, Metabolism 43:549-553; Michael et al., 2000, Mol Cell 6:87-97; Bruning JC et al., 1998, Mol Cell 2:559-569).
  • 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.
  • 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 inco ⁇ oration 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). nsulin 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 [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.).
  • TNR signaling provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders associated with TNR signaling and for the identification of subjects having a predisposition to such diseases and disorders. Any method for assessing MTNR expression in a sample, as previously described, may be used.
  • Such methods may, for example, utilize reagents such as the MTNR oligonucleotides and antibodies directed against MTNR, as described above for: (1) the detection of the presence of MTNR gene mutations, or the detection of either over- or under-expression of MTNR mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of MTNR gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in a biological pathway mediated by MTNR.
  • reagents such as the MTNR oligonucleotides and antibodies directed against MTNR, as described above for: (1) the detection of the presence of MTNR gene mutations, or the detection of either over- or under-expression of MTNR mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of MTNR gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in
  • the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in MTNR expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for MTNR 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 modifiers of the TNR 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).
  • LDH lactate dehydrogenase
  • lactate dehydrogenase expression was used as a surrogate for TNR pathway activity.
  • the screen identified "modifier" genes, whose knock-down by RNAi produced a changes in LDH expression. Genes whose disruption by RNAi produced an increase in LDH expression were identified as candidate negative effectors of TNR pathway activity, while those whose disruption decreased LDH expression were candidate positive effectors.
  • Potential modifiers were retested in triplicate in a confirmation experiment using RT-PCR analysis of LDH, as well as a sodium/phosphate cotransporter ("CG 4726," Gl 10727399; amino acid sequence in Gl 7296119), whose expression was also found to decrease following RNAi-based disruption of TNR.
  • 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 lists the modifiers and their orthologs.
  • MTNR 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 MTNR activity.
  • 33 P-labeled MTNR 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 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 MTNR 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).
  • 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 (MultiScreen, Millipore). The filters are subsequently washed with phosphate-buffered saline, dilute phosphoric acid (0.5%) or other suitable medium to remove excess radiolabeled ATP. Scintillation cocktail is added to the filter plate and the incorporated radioactivity is quantitated by scintillation counting (Wallac/Perkin Elmer). Activity is defined by the amount of radioactivity detected following subtraction of the negative control reaction value (EDTA quench).

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Abstract

Human MINR genes are identified as modulators of INR signaling and thus are therapeutic targets for disorders associated with defective INR signaling. Methods for identifying modulators of MINR, comprising screening for agents that modulate the activity of MINR are provided.

Description

MINRs AS MODIFIERS OF INSULIN RECEPTOR SIGNALING AND METHODS OF USE
REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional patent applications 60/354,824 filed 2/6/2002, 60/358,217 filed 2/20/2002, 60/358,189 filed 2/20/2002, 60/358,126 filed 2/20/2002, 60/358,995 filed 2/21/2002, 60/358,756 filed 2/21/2002, 60/358,765 filed 2/21/2002, 60/359,531 filed 2/25/2002, 60/360,222 filed 2/26/2002, 60/360,224 filed 2/26/2002, 60/360,167 filed 2/26/2002, and 60/360,166 filed 2/26/2002. The contents of the prior applications are hereby incorporated in their entirety.
BACKGROUND OF THE INVENTION
Insulin is the central hormone governing metabolism in vertebrates (reviewed in Steiner et al., 1989, In Endocrinology, DeGroot, eds. Philadelphia, Saunders: 1263-1289). In humans, 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. 201-215; Kahn et al., 1996, Annu. Rev. Med. 47:509-531; Kahn, 1998, Cell 92:593-596). Beyond its role in diabetes mellitus, the phenomenon of insulin resistance has been linked to other pathogenic disorders including obesity, ovarian hyperandrogenism, and hypertension. Within the pharmaceutical industry, there is interest in understanding the molecular mechanisms that connect lipid defects and insulin resistance. Hyperlipidemia and elevation of free fatty acid levels correlate with "Metabolic Syndrome," defined as the linkage between several diseases, including obesity and insulin resistance, which often occur in the same patients and which are major risk factors for development of Type 2 diabetes and cardiovascular disease. Current research suggests that the control of lipid levels, in addition to glucose levels, may be required to treat Type 2 Diabetes, heart disease, and other manifestations of Metabolic Syndrome (Santomauro AT et al., Diabetes (1999) 48:1836-1841).
The ability to manipulate and screen the genomes of model organisms such as 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. 2000 Nov 2;408(6808):37-8; Ivanov IP, et al., EMBO J 2000 Apr 17; 19(8): 1907-17; Vajo Z et al., Mamm Genome 1999 Oct; 10(10): 1000-4; Miklos GL and Rubin GM, Cell 1996, 86:521- 529; Mechler BM et al., 1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74; Watson KL., et al., 1994 J Cell Sci. 18: 19-33; Miklos GL, and Rubin GM. 1996 Cell 86:521-529; Wassarman DA, et al., 1995 Curr Opin Gen Dev 5: 44-50; and Booth DR. 1999 Cancer Metastasis Rev. 18: 261-284). While 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.
In one example, 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.
Genetic screens may utilize RNA interference (RNAi) techniques, whereby introduction of exogenous double stranded (ds) RNA disrupts the activity of genes containing homologous sequences and induce specific loss-of -function phenotypes (Fire et al., 1998, Nature391:806-811). 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). •
The insulin receptor (INR) signaling pathway has been extensively studied in C. elegans. Signaling through daf-2, the C. elegans INR ortholog, mediates various events, including reproductive growth and normal adult life span (see, e.g., US PAT NO 6,225,120; Tissenbaum HA and Ruvkun G, 1998, Genetics 148:703-17; Ogg S and Ruvkun G, 1998, Mol Cell 2:887-93; Lin K et al, 2001, Nat Genet 28:139-45).
NOT2 and 5. Cerevisiae ortholog CDC36 are 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. 2001, J. Biol. Chem. 276: 7541-7548). The Regena (Rga) gene of Drosophila is an ortholog of NOT2, and 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).
Myotubularins (MYT) belong to a conserved family of proteins from several organisms, including human, Drosophila, and C. elegans (Laporte et al. 1998, Hum. Molec. Genet. 7:1703-1712; Laporte et al., 2001 Trends in Genetics 17:221-228). The human family consists of at least 10 genes, and Drosophila and C. elegans each have 6 myotubularin related genes. Myotubularins have active site residues that are consistent with both protein and lipid phosphatase activity, and have been shown to have these activities biochemically (Laporte et al. 1998, 2001). In addition, it has been suggested based on experimental evidence in yeast that myotubularin might down regulate PI-3- kinase activity. In yeast, myotubularin has a strong preference for PtdIns3P as a substrate (Taylor et al. 2000, Proc. Natl. Acad. Sci. USA 97:8910-8915). Conserved residues in the catalytic domain are consistent with its activity as a monophosphoinositide phosphatase, and mutation of these residues abolishes lipid phosphatase activity in vitro (Taylor et al., 2000; Laporte et al., 2001). In addition, a mutant form of human myotubularin, when introduced into yeast, co-immunoprecipitated with the yeast PI-3 kinase, suggesting that myotubularin might directly affect PI-3 kinase activity (Blondeau et al. 2000, Hum. Mol. Genet. 9: 2223-2229). The Drosophila myotubularin gene of Gl 17737395 falls into the human MTM1/MTMR2 subgroup and it is the only Drosophila gene in this subgroup. MTM1 mutations are associated with the disease X-linked myotubular myopathy (Laporte et al. 1996, Nat. Genet. 13:175-182), which results in the disorganization of muscle fibers. The mutations that have been found in MTM1 in patients are missense mutations that, for the most part, affect residues that are conserved between the human and the Drosophila protein. Mutations in MTMR2 result in Charcot- Marie-Tooth disease, which affects the myelination of motor and sensory neurons (Bolino et al. 2000, Nat. Genet. 25:17-19).
DNMT1 is an enzyme that maintains mammalian DNA methylation and is also a component of a repressive transcriptional complex. DNMT associated protein (DMAP1) was identified in a yeast two-hybrid screen for proteins that interact with DNMT1. DMAPl has intrinsic transcriptional repressive activity and also binds to the tumor suppressor gene TSG101. TSG101 has been shown to act as a transcriptional co- repressor involved in the silencing of nuclear hormone induced genes, and also may function in late endosomal trafficking (Roundtree et al., 2000, Nature Genetics 25:269- 277).
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 TSCl or TSC2 genes (Consortium T.E.C.T.S., 1993, Cell 75:1305-1315; van Slegtenhorst et al. 1997, Science 277:805-808). TSCl encodes hamartin, TSC2 encodes tuberin, and there is evidence that the human proteins interact in vitro (Plank et al 1998, Cancer Res. 58: 4766-4770; van Slegtenhorst 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). The Rap/ran-GAP domain is also found in the GTPase activating protein (GAP) responsible for the activation of nuclear Ras-related regulatory proteins Rapl, Rsrl and Ran in vitro , which affects cell cycle progression. Gigas (GIG) is the Drosophila ortholog of TCS2. 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). In addition, cells in a GIG mutant differentiate normally, but are 2-3 times the normal size. Overexpression of the Drosophila TSCl and TSC2 (GIG) genes leads 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 TSCl 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 TSCl 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.
RAB 5 is a member of the Ras superfamily of GTPases, which have been implicated in vesicle trafficking (Somsel Rodman and Wandinger-Ness, 2000, J. Cell Sci. 113: 183-192). The endocytic pathway is important for uptake of nutrients, regulation of cell surface receptors, the recycling of proteins used in the secretory pathway. RAB5 is associated with the clathrin-coated vesicles and early endosomes and functions to regulate endocytic internalization and early endosome fusion (Woodman, 2000, Traffic 9:695-701). The FYVE-domain protein Rabenosyn-5 has been shown to be an effector of Rab5 and Rab4, physically connecting early endosomes and receptor recycling to the cell surface (De Renzis et al., 2002, Nat. Cell Biol. 4:124-133). Insulin-responsive tissues express several Rab isoforms, including Rab3b, Rab4, Rab5, and Rab8. Of these isoforms, only Rab4 has been shown to play a role in mediating insulin actions within the cell, including insulin-stimulated GLUT4 translocation to the cell membrane (Knight et al., 2000, Endocrinology 141:208-218). There is some evidence that membrane association of Rab5 is altered in skeletal muscle isolated from insulin resistant and Type 2 diabetic patients (Bao et al, 1998, Horm. Metab. Res. 30:656-662).
Drosophila SNAP is an ortholog of human alpha-Soluble NSF gene (alpha-SNAP or "aSNAP). In Drosophila, SNAP is known to be a part of the conserved SNARE complex necessary for secretory vesicle fusion with the plasma membrane (Ordway et al., 1994, PNAS USA 91:5715-5719). There are no loss-of-f unction mutations reported in Drosophila, but mutations in NSF, the primary protein SNAP is responsible for recruiting, are defective in motor behavior and display paralysis (Littleton et al. 1998, Neuron 21: 401-413). In vertebrates, it has been demonstrated that SNAPs play a role in the association of the SNARE complex in trans during vesicle docking (Xu et al. 1999, EMBO J. 18: 3293-3304). SNAPs are responsible for recruiting and stimulating NSF, the ATPase responsible for disassembly and recycling of the SNARE complex (Sudlow et al. 1996, FEBS Lett 393: 185-188; Barnard et al 1997, J. Cell Biology 139: 875-883; Cheatham 2000, Trends in Endocrinol. Metab. 11:356-361). Together, SNAP and NSF are responsible for increasing the rate of exocytosis dramatically. It has been shown that although beta-SNAP in vertebrates is similar to alpha-SNAP, alpha-SNAP increases exocytosis more than beta-SNAP (Xu et al. 2002, J. Neurosci 22:53-61). Mutational analysis of alpha-SNAP shows a requirement for Leucine 294. alpha-SNAP (L294A) acted as a dominant mutant by associating with the SNARE complex and NSF normally but blocking the ATPase dependent stimulation of exocytosis by exogenous alpha-SNAP (Barnard et al 1997, supra).
CAF-1 (catabolite repressor protein (CCR4)-associative factor 1), also known as a CCR4-NOT transcription complex subunit 7, is a component of a complex of proteins that interact with the RNA polymerase II holoenzyme to regulate gene expression (Albeit et al., 2000, Nucleic Acids Res. 28:809-817). The complex also contains CCR4 and NOT proteins, among others. In addition to the global regulation of RNA polymerase II transcription, 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. cerevisiae, and may function in early steps of mRNA turnover by initiating the shortening of the poly(A) tail (Tucker et al., Cell 104:377-386).
VAMPs are members of the SNARE protein family, which are critical proteins in membrane fusion for both regulated and constitutive vesicle trafficking. VAP33 (VAMP-associated proteins of 33 kDa) proteins bind VAMPs and SNAREs (Weir et al. 2001, Biochem Biophys Res Commun 286:616-21). Mammalian VAP33 (VAP-A) is widely expressed in multiple tissues and appears to be associated with the ER and microtubules, as well as trafficking vesicles (Weir et al. 1998, Biochem. J. 333:247-251). There are three known human isoforms of VAP33. VAP-A and -B are encoded by distinct genes and are approximately 60% identical; VAP-C is a splice variant of VAP-B, which lacks the C-terminal transmembrane domain (Nishimura et al. 1999, Biochem. Biophys. Res. Commun. 254:21-26). VAP33 has been shown to play a pivotal role in insulin-stimulated GLUT4 translocation to the cell surface in L6 myoblasts and 3T3-L1 adipocytes (Foster et al. 2000, Traffic 6:512-521). There is also evidence that the yeast homolog SCS2 is required for inositol metabolism (Kagiwada et al. 1998, J. Bacteriol. 180:1700-1708).
PP2 (also called 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). Finally there is evidence that 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 an ortholog of 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. 109:3001-3012), and 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).
CSNKl, 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). CSNKl 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). CSNKl 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 an ortholog of human CSNKl, 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. 2002, Neuron 33:193-203).
ERF1 (eucaryotic release factor 1) 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).
All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incoφorated herein in their entireties.
SUMMARY OF THE INVENTION
We have discovered genes that modify the INR pathway in Drosophila cells, and identified their human orthologs, hereinafter referred to as Modifiers of insulin receptor signaling (MINR). The invention provides methods for utilizing these INR modifier genes and polypeptides to identify MTNR-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired INR function and/or MINR function. Preferred MTNR-modulating agents specifically bind to MTNR polypeptides and restore INR function. Other preferred MTNR-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress MTNR gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
MTNR modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with an MTNR polypeptide or nucleic acid. In one embodiment, candidate MTNR modulating agents are tested with an assay system comprising a MTNR 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. MTNR-modulating agents include MTNR related proteins (e.g. dominant negative mutants, and biotherapeutics); MTNR -specific antibodies; MTNR -specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with MTNR or compete with MTNR binding partner (e.g. by binding to an MTNR binding partner). In one specific embodiment, a small molecule modulator is identified using a binding assay. In specific embodiments, the screening assay system is selected from 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.
In another embodiment, candidate TNR pathway modulating agents are further tested using a second assay system that detects changes in activity associated with TNR signaling. The second assay system may use cultured cells or non-human animals. In specific embodiments, the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the TNR pathway.
The invention further provides methods for modulating the MTNR function and/or the TNR pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a MTNR 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 TNR pathway.
DETAILED DESCRIPTION OF THE INVENTION
We used a cellular RNAi screen to identify modifiers of the TNR pathway and signaling activity. Modulators of the TNR pathway were identified, followed by identification of their orthologs. Accordingly, modifiers of insulin receptor signaling (MTNR) genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of disorders related to TNR signaling. In one example, therapy involves increasing signaling through TNR in order to treat pathologies related to diabetes and/or metabolic syndrome.
The invention provides in vitro and in vivo methods of assessing MTNR function, and methods of modulating (generally inhibiting or agonizing) MTNR activity, which are useful for further elucidating TNR signaling and for developing diagnostic and therapeutic modalities for pathologies associated with TNR signaling. As used herein, pathologies associated with INR signaling encompass pathologies where TNR signaling contributes to maintaining the healthy state, as well as pathologies whose course may be altered by modulation of the TNR signaling.
MINR nucleic acids and polypeptides
Sequences related to MTNR 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 (Example 1).
The term "MTNR polypeptide" refers to a full-length MTNR protein or a fragment or derivative thereof that is "functionally active," meaning that the MTNR protein derivative or fragment exhibits one or more functional activities associated with a full- length, wild-type MTNR protein. As one example, 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. Preferably, a functionally active MTNR fragment or derivative displays one or more biological activities associated with MTNR proteins such as enzymatic activity, signaling activity, ability to bind natural cellular substrates, etc. In one embodiment, a functionally active MTNR polypeptide is a MTNR derivative capable of rescuing defective endogenous MTNR activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species. If MTNR fragments are used in assays to identify modulating agents, the fragments preferably comprise a MTNR 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 MTNR protein. A preferred MTNR 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).
The term "MTNR nucleic acid" refers to a DNA or RNA molecule that encodes a MTNR polypeptide. Preferably, the MTNR 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 MTNR. 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. In a phylogenetic tree representing multiple homologous sequences from diverse species (e.g., retrieved through BLAST analysis), 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) may also identify potential orthologs. In evolution, when 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. As used herein, the term "orthologs" encompasses paralogs. As used herein, "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://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 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.
Alternatively, 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 Molec.Biol., 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. 3:353-358, National Biomedical Research Foundation, Washington, D.C, USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). Smith-Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two). From the data generated the "Match" value reflects "sequence identity."
Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of a MTNR. 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)). In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of a MTNR 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 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 μg/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). In other embodiments, 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 μg/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%o Ficoll, 0.2% BSA, 100 μg/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. Alternatively, 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 μg/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.
Isolation, Production, Expression, and Mis-expression of MINR Nucleic Acids and Polypeptides
MTNR nucleic acids and polypeptides, useful for identifying and testing agents that modulate MTNR function and for other applications related to the involvement of MTNR in TNR signaling. MTNR 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.
A wide variety of methods are available for obtaining MTNR polypeptides. In general, the intended use for the polypeptide will dictate the particulars of expression, production, and purification methods. For instance, 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 (i.e., generation of fusion proteins). Overexpression of a MTNR polypeptide for cell- based assays used to assess MTNR function, such as involvement in tubulogenesis, may require expression in eukaryotic cell lines capable of these cellular activities. Techniques for the expression, production, and purification of proteins are well known in the art; any suitable means therefor may be used (e.g., Higgins SJ and Hames BD (eds.) Protein Expression: A Practical Approach, Oxford University Press Inc., New York 1999; Stanbury PF et al., Principles of Fermentation Technology, 2nd edition, Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan JE et al, Current Protocols in Protein Science (eds.), 1999, John Wiley & Sons, New York; U.S. Pat. No. 6,165,992).
The nucleotide sequence encoding a MTNR polypeptide can be inserted into any appropriate vector for expression of the inserted protein-coding sequence. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native MTNR 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 MTNR polypeptide may be optionally expressed as a fusion or chimeric product, joined via a peptide bond to a heterologous protein sequence. In one application 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) 310:105-111).
An MTNR polypeptide can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis). Alternatively, native MTNR 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 MTNR or other genes associated with TNR signaling. As used herein, 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).
Genetically modified animals
The methods of this invention may use non-human animals that have been genetically modified to alter expression of MTNR and/or other genes known to be involved in TNR signaling. Preferred genetically modified animals are mammals, particularly mice or rats. Preferred non-mammalian species include Zebrafish, C. elegans, and Drosophila. Preferably, the altered MTNR or other gene expression results in a detectable phenotype, such as modified levels of TNR 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 TNR signaling, in animal models of pathologies associated with TNR 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. No., 4,945,050, by Sandford et al.; for transgenic Drosophila see Rubin and Spradling, Science (1982) 218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see Berghammer AJ. et al., A Universal Marker for Transgenic Insects (1999) Nature 402:370-371; for transgenic Zebrafish see Lin S., Transgenic Zebrafish, Methods Mol Biol. (2000); 136:375-3830); for microinjection procedures for fish, amphibian eggs and birds see Houdebine and Chourrout, Experientia (1991) 47:897-905; for transgenic rats see Hammer et al., Cell (1990) 63: 1099-1112; and for culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection see, e.g., Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed., TRL Press (1987)). 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).
In one embodiment, the transgenic animal is a "knock-out" animal having a heterozygous or homozygous alteration in the sequence of an endogenous MTNR gene that results in a decrease of MTNR function, preferably such that MTNR 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.- For example, a mouse MTNR gene is used to construct a homologous recombination vector suitable for altering an endogenous MTNR 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). In a preferred embodiment, 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).
In another embodiment, 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 MTNR gene, e.g., by introduction of additional copies of MTNR, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the MTNR gene. Such regulatory sequences 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. One example of such 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. Another example of 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). In a preferred embodiment, 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 TNR pathway, as animal models of disease and disorders implicating defective TNR 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 MTNR function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered MTNR expression that receive candidate therapeutic agent.
In addition to the above-described genetically modified animals having altered MTNR function, animal models having defective TNR function (and otherwise normal MTNR function), can be used in the methods of the present invention. For example, a TNR knockout mouse can be used to assess, in vivo, the activity of a candidate TNR modulating agent identified in one of the in vitro assays described below. Preferably, the candidate TNR modulating agent when administered to a model system with cells defective in TNR function, produces a detectable phenotypic change in the model system indicating that the TNR function is restored.
MINR Modulating agents
The invention provides methods to identify agents that interact with and/or modulate the function of MTNR and/or TNR signaling. Such agents are useful in a variety of diagnostic and therapeutic applications associated with TNR signaling, as well as in further analysis of the MTNR protein and its contribution to TNR signaling. Accordingly, the invention also provides methods for modulating TNR signaling comprising the step of specifically modulating MTNR activity by administering a MTNR-interacting or - modulating agent.
As used herein, a "MTNR-modulating agent" is any agent that modulates MTNR function, for example, an agent that interacts with MTNR to inhibit or enhance MTNR activity or otherwise affect normal MTNR function. MTNR function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a preferred embodiment, the MTNR - modulating agent specifically modulates the function of the MTNR. The phrases "specific modulating agent", "specifically modulates", etc., are used herein to refer to modulating agents that directly bind to the MTNR polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the MTNR. These phrases also encompasses modulating agents that alter the interaction of the MTNR with a binding partner, substrate, or cofactor (e.g. by binding to a binding partner of an MTNR, or to a protein/binding partner complex, and altering MTNR function). In a further preferred embodiment, the MTNR- modulating agent is a modulator of the TNR pathway (e.g. it restores and/or upregulates TNR function) and thus is also a TNR-modulating agent.
Preferred MTNR-modulating agents include small molecule chemical agents, MTNR-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, 19th 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 MTNR 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 MTNR-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 TNR 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. Additionally, candidate clinical compounds are generated with specific regard to clinical and pharmacological properties. For example, the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
Protein Modulators
Specific MTNR-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the TNR pathway and related disorders, as well as in validation assays for other MTNR-modulating agents. In a preferred embodiment, MTNR- interacting proteins affect normal MTNR function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, MTNR-interacting proteins are useful in detecting and providing information about the function of MTNR proteins, as is relevant to TNR related disorders, such as diabetes (e.g., for diagnostic means).
A MTNR-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with an MTNR, such as a member of the MTNR pathway that modulates MTNR expression, localization, and/or activity. MTNR-modulators include dominant negative forms of MTNR-interacting proteins and of MTNR proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous MTNR-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. 169-203; Fashema SF et al., Gene (2000) 250:1- 14; Drees BL Curr Opin Chem Biol (1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; and U.S. Pat. No. 5,928,868). 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 3rd, Trends Genet (2000) 16:5-8).
An MTNR-interacting protein may be an exogenous protein, such as an MTNR- 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). MTNR antibodies are further discussed below.
In preferred embodiments, a MTNR-interacting protein specifically binds an MTNR protein. In alternative preferred embodiments, a MTNR-modulating agent binds an MTNR substrate, binding partner, or cofactor.
Antibodies
In another embodiment, the protein modulator is an MTNR specific antibody agonist or antagonist. The antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify MTNR modulators. The antibodies can also be used in dissecting the portions of the MTNR pathway responsible for various cellular responses and in the general processing and maturation of the MTNR.
Antibodies that specifically bind MTNR polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ortholog of MTNR polypeptide, and more preferably, to human MTNR. 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 MTNR which are particularly antigenic can be selected, for example, by routine screening of MTNR 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; Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequence of a MTNR. Monoclonal antibodies with affinities of 108 M"1 preferably 109 M"1 to 1010 M"1, or stronger can be made by standard procedures as described (Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577). Antibodies may be generated against crude cell extracts of MTNR or substantially purified fragments thereof. If MTNR fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an MTNR protein. In a particular embodiment, MINR-specific antigens and or immunogens are coupled to carrier proteins that stimulate the immune response. For example, 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. An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.
The presence of MINR-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MTNR polypeptides. Other assays, such as radioimmunoassays or fluorescent assays might also be used.
Chimeric antibodies specific to MTNR 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., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604- 608; Takeda et al., Nature (1985) 31:452-454). 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). 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. Tmmun. 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). MINR-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).
Other suitable techniques for antibody production involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors (Huse et al., Science (1989) 246:1275-1281). As used herein, T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).
The 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). A wide variety of 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. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also, recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567). Antibodies to cytoplasmic polypeptides may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U.S. Pat. No. 6,086,900).
When used therapeutically in a patient, 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. Typically, the amount of antibody administered is in the range of about 0.1 mg/kg -to about 10 mg/kg of patient weight. For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle. Such vehicles 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).
Nucleic Acid Modulators
Other preferred MTNR-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA)* which generally inhibit MTNR activity. Preferred antisense oligomers interfere with the function of MTNR nucleic acids, such as DNA replication, transcription, MTNR RNA translocation, translation of protein from the MTNR RNA, RNA splicing, and any catalytic activity in which the MTNR RNA participates.
In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to a MTNR mRNA to bind to and prevent translation from the MTNR mRNA, preferably by binding to the 5' untranslated region. MTNR-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.
In another embodiment, 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. see WO99/18193; Summerton J, and Weller D, Antisense Nucleic Acid Drug Dev 1997, 7:187-95; Probst JC, Methods 2000, 22:271-281; US PAT NO: 5,325,033; US PAT NO: 5,378,841).
Alternative preferred MTNR nucleic acid modulators are double-stranded RNA species mediating RNA interference (RNAi). 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. 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. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25- 33 (2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619; Elbashir SM, et al., 2001 Nature 411:494-498).
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). Accordingly, in one aspect of the invention, a MINR-specific antisense oligomer is used in an assay to further elucidate the function of MTNR in TNR signaling. Zebrafish is a particularly useful model for the study of TNR signaling using antisense oligomers. For example, 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. In another aspect of the invention, PMOs are used to screen the Zebrafish genome for identification of other therapeutic modulators of TNR signaling. In a further aspect of the invention, a MINR-specific antisense oligomer is used as a therapeutic agent for treatment of metabolic pathologies.
Assay Systems
The invention provides assay systems and screening methods for identifying specific modulators of MTNR activity. As used herein, 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. In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the MTNR nucleic acid or protein. In general, secondary assays further assess the activity of a MTNR-modulating agent identified by a primary assay and may confirm that the modulating agent affects MTNR in a manner relevant to TNR signaling. In some cases, MINR-modulators will be directly tested in a "secondary assay," without having been identified or confirmed in a "primary assay."
In a preferred embodiment, the assay system comprises contacting a suitable assay system comprising a MTNR 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 MTNR activity, and hence TNR signaling. A statistically significant difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates MTNR activity, and hence the TNR signaling. The MTNR polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above
Primary Assays
The type of modulator tested generally determines the type of primary assay. Primary assays for small molecule modulators
For small molecule modulators, 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). As used herein 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. The term "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.
In a preferred embodiment, 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).
Suitable assay formats that may be adapted to screen for MTNR modulators are known in the art.
Binding Assays. A variety of 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). In another example, 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. 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).
Transcriptional activity assays. In one example, transcriptional activity is detected using quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems). In another example, a transcriptional reporter (e.g., luciferase, GFP, beta-galactosidase, etc.) operably linked to a responsive gene regulatory sequence is used (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).
Phosphatase assays. Protein phosophatases catalyze the removal of a gamma phosphate from a serine, threonine or tyrosine residue in a protein substrate. Since phosphatases act in opposition to kinases, appropriate assays monitor the removal of a phosphate from a protein substrate. In one example, 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). In another example, 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). Other appropriate assays for may monitor lipid phosphatase activity and may use labeled, such as fluorescently labeled or radio-labeled substrates to detect removal of a phosphate from a phosphatidylinositol substrate. In one example, an assay uses "FlashPlate" technology (U.S. Patent No. 5,972,595), in which a radio-labeled hydrophobic substrate is immobilized on a solid support in each well of a multi-well plate. Dephosphorylation of the substrate is measured as a decrease in bound radioactivity, which is detected by the close proximity of the scintillant. Other assays for detecting phosphoinositide phosphatase activity are known in the art (see, e.g., US PAT NOs: Patent 6,001,354 and 6,238,903). GAP assays. 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). Alternative assays may detect GAP function in endosome trafficking by monitoring movement of a cargo molecule, which may be labeled (Sonnichsen et al., 2000, J Cell Biol 149:901-14).
Kinase assays. Preferred assays detect kinase activity, the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine or threonine residue in a protein substrate. Radioassays, which monitor the transfer from [gamma-32P or -33P]ATP, may be 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. In one example, a scintillation assay monitors the transfer of the gamma phosphate from [gamma -33P] 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. Other assays for protein kinase activity may use antibodies that specifically recognize phosphorylated substrates. For instance, the kinase receptor activation (KTRA) 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). Another example of antibody based assays for protein kinase activity is TRF (time-resolved fluorometry). This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence (Braunwalder AF, et al., Anal Biochem 1996 Jul l;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.
Release factor activity assays. Appropriate assays may detect in vitro release factor activity (see, e.g., Seit-Nebi et al. 2001, Nucleic Acids Res 29:3982-7; Frolova et al. 1994, Nature 372:701-3; Caskey et al. 1974, Methods Enzymol 30:293-303).
Cell-based screening assays usually require systems for recombinant expression of MTNR 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. In certain applications when MTNR-interacting proteins are used in screening assays, the binding specificity of the interacting protein to the MTNR protein may be assayed by various known methods, including binding equilibrium constants (usually at least about 107 M"1, preferably at least about 108 M"1, more preferably at least about 109 M"1), and immunogenic properties. For enzymes and receptors, 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 MTNR polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The MTNR polypeptide can be full length or a fragment thereof that retains functional MINR activity. The MTNR polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The MTNR polypeptide is preferably human MTNR, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of MTNR interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has MTNR -specific binding activity, and can be used to assess normal MTNR gene function.
Certain screening assays may also be used to test antibody and nucleic acid modulators; for nucleic acid modulators, appropriate assay systems involve MTNR mRNA expression. Primary assays for antibody modulators
For antibody modulators, appropriate primary assays are binding assays that test the antibody's affinity to and specificity for the MTNR 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 MINR-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.
Primary assays for nucleic acid modulators
For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit MTNR gene expression, preferably mRNA expression. In general, expression analysis comprises comparing MTNR expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express MTNR) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art. For instance, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), or microarray analysis may be used to confirm that MTNR 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 ah, Biotechniques (1999) 26:112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm DH and Guiseppi-Elie, ACurr Opin Biotechnol 2001, 12:41-47). Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the MTNR 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).
Secondary Assays
Secondary assays may be used to further assess the activity of a MTNR- modulating agent identified by any of the above methods to confirm that the modulating agent affects MTNR in a manner relevant to TNR signaling. As used herein, MTNR- 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 MTNR.
Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express MTNR) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate MTNR-modulating agent results in changes in TNR signaling, in comparison to untreated (or mock- or placebo-treated) cells or animals. Changes in TNR signaling may be detected as modifications to TNR pathway components, or changes in their expression or activity. Assays may also detect an output of normal or defective TNR 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 TNR or interacting pathways, or pathways associated with TNR signaling or an output of TNR signaling.
Cell-based assays
Cell-based assays may use a variety of insulin-sensitive mammalian cells and may detect endogenous TNR signaling or may rely on recombinant expression of TNR and/or other TNR 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-TI-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). Other suitable cells include muscle cells, such as L6 myotubes, and CHO cells engineered to over- express TNR. For certain assay systems it may be useful to treat cells with 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 MTNR - modulating agent alters TNR 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 MTNR function increases insulin sensitivity.
In one example, TNR 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 π, glycogen synthetase, lipoprotein lipase, etc; PEPCK is specifically down-regulated in response to TNR signaling). 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. Alternatively, expression is indirectly monitored from a 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 insulin responsive gene. Methods for making and using reporter constructs are well known.
TNR signaling may also be detected by measuring the activity of components of the TNR-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).
In another example, 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 KI et al., 1998, J Biol Chem 273:17491- 7). Other suitable assays measure cellular uptake of glucose (typically labeled glucose) in response to insulin stimulation. Adipocytes are preferred for these assays. Assays also measure translocation of glucose transporter (GLUT) 4, which is a primary mediator of insulin-induced glucose uptake, primarily in muscle and adipocytes, and which specifically translocates to the cell surface following insulin stimulation. 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).
Other preferred assays detect insulin secretion from beta cells in response to glucose. Such assays typically use ELISA (see, e.g., Bergsten and Hellman, 1993, Diabetes 42:670-4) or radioimmunoassay (RIA; see, e.g., Hohmeier et al., 2000, supra).
Animal Assays
A variety of non-human animal models of metabolic disorders may be used to test candidate MTNR 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 TNR signaling pathway. Additionally, 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.
In one embodiment, 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 TNR signaling pathway, such as TNR or the insulin receptor substrate (IRS), develop symptoms of diabetes, and show hepatic lipid accumulation (fatty liver) and, frequently, increased plasma lipid levels (Nishina et al., 1994, Metabolism 43:549-553; Michael et al., 2000, Mol Cell 6:87-97; Bruning JC et al., 1998, Mol Cell 2:559-569). 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 incoφoration 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). nsulin sensitivity is routinely tested by a glucose tolerance test or an insulin tolerance test.
In another embodiment, assays use mouse models of lipoprotein biology and cardiovascular disease. For instance, 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). 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. Numerous other mouse models for atherosclerosis are available, 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).
In another embodiment, the ability of candidate modulators to alter plasma lipid levels and artherosclerotic progression are tested in mouse models for multiple lipid disorders. For instance, 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.).
Diagnostic Methods
The discovery that MTNR is implicated in TNR signaling provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders associated with TNR signaling and for the identification of subjects having a predisposition to such diseases and disorders. Any method for assessing MTNR expression in a sample, as previously described, may be used. Such methods may, for example, utilize reagents such as the MTNR oligonucleotides and antibodies directed against MTNR, as described above for: (1) the detection of the presence of MTNR gene mutations, or the detection of either over- or under-expression of MTNR mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of MTNR gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in a biological pathway mediated by MTNR.
Thus, in a specific embodiment, the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in MTNR expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for MTNR 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.
EXAMPLES
The following experimental section and examples are offered by way of illustration and not by way of limitation.
I. Drosophila cell RNAi screen
We used a cellular RNAi screen to identify modifiers of the TNR 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). Quantitative RT-PCR using TaqMan® (PE Applied Biosystems) was used to measure expression of the lactate dehydrogenase ("LDH," Gl 1519714; Abu-Shumays and Fristrom, 1997, Dev Genet 20:11-22) gene, which we had previously show to correlate with TNR pathway activity. Specifically, LDH expression was increased when TNR pathway activity was increased by RNAi- based knock-down of negative effectors of TNR signaling (e.g., PTEN, GSK3beta, and AFX), in the presence or absence of insulin. LDH expression was decreased when TNR pathway activity was decreased by RNAi-based knock-down of positive effectors of TNR signaling (e.g., TNR, TRS, AKT). Accordingly, lactate dehydrogenase expression was used as a surrogate for TNR pathway activity. The screen identified "modifier" genes, whose knock-down by RNAi produced a changes in LDH expression. Genes whose disruption by RNAi produced an increase in LDH expression were identified as candidate negative effectors of TNR pathway activity, while those whose disruption decreased LDH expression were candidate positive effectors. Potential modifiers were retested in triplicate in a confirmation experiment using RT-PCR analysis of LDH, as well as a sodium/phosphate cotransporter ("CG 4726," Gl 10727399; amino acid sequence in Gl 7296119), whose expression was also found to decrease following RNAi-based disruption of TNR. 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 lists the modifiers and their orthologs.
Table 1
Figure imgf000039_0001
Figure imgf000040_0001
TI. High-Throughput In Vitro Fluorescence Polarization Assay Fluorescently-labeled MTNR 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 MTNR activity.
UI. High-Throughput In Vitro Binding Assay.
33P-labeled MTNR peptide is added in an assay buffer (100 mM KC1, 20 mM HEPES pH 7.6, 1 mM MgCl2, 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.
TV. Immunoprecipitations and Immunoblotting
For coprecipitation of transfected proteins, 3 x 106 appropriate cells are plated on 10-cm dishes and transfected on the following day with expression constructs. The total amount of DNA is kept constant in each transfection by adding empty vector. After 24 h, cells are collected, washed once with phosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysis buffer containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM - glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl phosphate, 2 mM dithiothreitol, protease inhibitors (complete, Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris is removed by centrifugation twice at 15,000 x g for 15 min. The cell lysate are incubated with 25 μl of M2 beads (Sigma) for 2 h at 4 °C with gentle rocking.
After extensive washing with lysis buffer, proteins bound to the beads are directly 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).
VI. Kinase assay
A purified or partially purified MTNR 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 μg/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 μl. The reaction is initiated by the addition of 33P-gamma-ATP (0.5 μCi/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 Mn2+) 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 (MultiScreen, Millipore). The filters are subsequently washed with phosphate-buffered saline, dilute phosphoric acid (0.5%) or other suitable medium to remove excess radiolabeled ATP. Scintillation cocktail is added to the filter plate and the incorporated radioactivity is quantitated by scintillation counting (Wallac/Perkin Elmer). Activity is defined by the amount of radioactivity detected following subtraction of the negative control reaction value (EDTA quench).

Claims

WHAT IS CLAIMED IS:
1. A method of identifying a candidate TNR signaling modulating agent, said method comprising the steps of:
(a) providing an assay system comprising a MTNR polypeptide or nucleic acid;
(b) contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and
(c) detecting a test agent-biased activity of the assay system, wherein a difference between the test agent-biased activity and the reference activity identifies the test agent as a candidate TNR signaling modulating agent.
2. The method of Claim 1 wherein the assay system includes a screening assay comprising a MTNR polypeptide, and the candidate test agent is a small molecule modulator.
3. The method of Claim 2 wherein the screening assay is a binding assay.
4. The method of Claim 1 wherein the assay system includes a binding assay comprising a MTNR polypeptide and the candidate test agent is an antibody.
5. The method of Claim 1 wherein the assay system includes an expression assay comprising a MTNR nucleic acid and the candidate test agent is a nucleic acid modulator.
6. The method of Claim 5 wherein the nucleic acid modulator is an antisense oligomer.
7. The method of Claim 6 wherein the nucleic acid modulator is a PMO.
8. The method of Claim 1 wherein the assay system comprises cultured cells or a non- human animal expressing MTNR, and wherein the assay system includes an assay that detects an agent-biased change in TNR signaling or an output of TNR signaling.
9. The method of Claim 8 wherein the assay system comprises cultured cells.
10. The method of Claim 9 wherein the assay detects an event selected from the group consisting of expression of insulin-responsive genes, phosphorylation of an TNR signaling pathway component, kinase activity of an TNR signaling pathway component, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion.
11. The method of Claim 8 wherein the assay system comprises a non-human animal.
12. The method of Claim 11 wherein the non-human animal is a mouse providing a model of diabetes and/or insulin resistance.
13. The method of Claim 12 wherein the assay system includes an assay that detects an event selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, adipose lipid accumulation, plasma glucose level, plasma insulin level, and insulin sensitivity.
14. The method of Claim 1, comprising the additional steps of:
(d) providing a second assay system comprising cultured cells or a non-human animal expressing MTNR ,
(e) contacting the second assay system with the test agent of (b) or an agent derived therefrom under conditions whereby, but for the presence of the test agent or agent derived therefrom, the system provides a reference activity; and
(f) detecting an agent-biased activity of the second assay system, wherein a difference between the agent-biased activity and the reference activity of the second assay system confirms the test agent or agent derived therefrom as a candidate TNR signaling modulating agent, and wherein the second assay system includes a second assay that detects an agent-biased change in an activity associated with TNR signaling or an output of TNR signaling.
15. The method of Claim 14 wherein the second assay system comprises cultured cells.
16. The method of Claim 15 wherein the second assay detects an event selected from the group consisting of expression of insulin-responsive genes, phosphorylation of an TNR signaling pathway component, kinase activity of an TNR signaling pathway component, glycogen synthesis, glucose uptake, GLUT4 translocation, and insulin secretion.
17. The method of Claim 14 wherein the second assay system comprises a non-human animal.
18. The method of Claim 17 wherein the non-human animal is a mouse providing a model of diabetes and/or insulin resistance.
19. The method of Claim 18 wherein the second assay system includes an assay that detects an event selected from the group consisting of hepatic lipid accumulation, plasma lipid accumulation, adipose lipid accumulation, plasma glucose level, plasma insulin level, and insulin sensitivity.
20. A method of modulating TNR signaling in a mammalian cell comprising contacting the cell with an agent that specifically binds a MTNR polypeptide or nucleic acid.
21. The method of Claim 20 wherein the agent is administered to a mammalian animal predetermined to have a pathology associated with TNR signaling.
22. The method of Claim 20 wherein the agent is a small molecule modulator, a nucleic acid modulator, or an antibody.
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FOSTER L.J. ET AL.: 'A functional role for VAP-33 in insulin-stimulated GLUT4 traffic' TRAFFIC vol. 1, 2000, pages 512 - 521, XP002225900 *
KNIGHT J.B. ET AL.: 'Expression of a phenylation-deficient Rab4 interferes with propagation of insulin signaling through insulin receptor substrate-1' ENDOCRINOLOGY vol. 141, no. 1, 2000, pages 208 - 218, XP002971999 *
KOWLURU A. ET AL.: 'Activation of acetyl-CoA carboxylase by a glutamate- and magnesium-sensitive protein phosphorylation in the islet beta-cell' DIABETES vol. 50, no. 7, July 2001, pages 1580 - 1587, XP002971998 *
PASQUINELL A.E. ET AL.: 'Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA' NATURE vol. 408, 02 November 2000, pages 86 - 89, XP002204652 *
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WANG X. ET AL.: 'Eukaryotic initiation factor 2B: identification of multiple phosphorylation sites in the epsilon-subunit and their functions in vivo' THE EMBO JOURNAL vol. 20, no. 16, 2001, pages 4349 - 4359, XP002971997 *

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WO2003066813A3 (en) 2004-02-26
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EP1472515A2 (en) 2004-11-03
EP1472515A4 (en) 2006-08-30
AU2003215007A1 (en) 2003-09-02

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