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WO2007045906A1 - Protéines précurseurs de peptide dérivé de rf-amide et peptides de type rf-amide - Google Patents

Protéines précurseurs de peptide dérivé de rf-amide et peptides de type rf-amide Download PDF

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Publication number
WO2007045906A1
WO2007045906A1 PCT/GB2006/003926 GB2006003926W WO2007045906A1 WO 2007045906 A1 WO2007045906 A1 WO 2007045906A1 GB 2006003926 W GB2006003926 W GB 2006003926W WO 2007045906 A1 WO2007045906 A1 WO 2007045906A1
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Prior art keywords
polypeptide
seq
nucleic acid
disease
acid molecule
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Inventor
Kinsey Maundrell
Mark Douglas Davies
Richard Joseph Fagan
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Ares Trading SA
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Ares Trading SA
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • This invention relates to a novel protein, termed INSP207, herein identified as a RFamide- related peptide precursor protein and to the use of this protein and nucleic acid sequence from the encoding gene in the diagnosis, prevention and treatment of disease.
  • This invention also relates to the RFamide-related peptides generated by cleavage of the INSP207 RFamide-related peptide precursor protein.
  • bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
  • Various institutions and commercial organisations are examining sequence data as they become available and significant discoveries are being made on an on-going basis. However, there remains a continuing need to identify and characterise further genes and the polypeptides that they encode, as targets for research and for drug discovery.
  • the RFamides represent a diverse family of bioactive peptides distinguishable by an RFamide (arginine-phenyalanine-amide) structure at their C-termini.
  • the first member of this diverse family to be identified was the cardioexcitatory neuropeptide from the bivalve mollusc MacrocalHsta nimbosa (Price DA and Greenberg MJ, Structure of a molluscan cardioexcitatory neuropeptide. Science. 1977 Aug 12;197(4304):670-l).
  • RFamides have been described throughout the animal kingdom (Li et al., FMRFamide- related neuropeptide gene family in Caenorhabditis elegans.
  • RFamide-related peptides are processed from a larger secreted precursor protein. Each RFamide-related peptide terminates with the sequence
  • Arg-X-Gly where X may be Phe, Leu or Ser.
  • the terminal glycine residue acts as an amide donor resulting in an amidated mature peptide product.
  • Either di- or monobasic proteolytic cleavage sites flank the RFamide-related peptides and allow excision from the precursor protein.
  • the secreted precursor protein comprises a secretory signal peptide sequence upstream of the RFamide-related peptide sequences.
  • RFamide-related peptides described to date act as high affinity ligands for G-protein coupled receptors. These include the prolactin releasing peptide (PrRP) (Hinuma et al, A prolactin-releasing peptide in the brain. Nature. 1998 May 21;393(6682):272-6), NPFF and NPAF morphine-modulating peptides (Yang et al, Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci U S A.
  • KiSS-I Metastasis suppressor gene KiSS-I encodes peptide ligand of a G-protein-coupled receptor. Nature. 2001 May 31;411(6837):613-7).
  • KiSS-I Metastasis suppressor gene KiSS-I encodes peptide ligand of a G-protein-coupled receptor. Nature. 2001 May 31;411(6837):613-7.
  • These peptides have been implicated in modulating diverse biological systems such as pain, cellular metastasis, cardioexitation, neuromodulation and the control of hormone release as well as effects on feeding behaviour (Dockray, Exp. Physiol. 2004, 89(3):229-35.
  • the invention is based on the discovery that the INSP207 protein is a RFamide-related peptide (RFRP) precursor protein. Cleavage of this novel RFamide-related peptide precursor protein produces novel RFamide-related peptides.
  • RFRP RFamide-related peptide
  • (i) comprises the amino acid sequence as recited in SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and/or SEQ ID NO: 10;
  • (ii) is a fragment thereof which is a RFamide-related peptide precursor protein or an RFamide-related peptide, or has an antigenic determinant in common with the • polypeptides of (i); or
  • the RFamide-related peptide according to part ii) above produced by cleavage of the RFamide-related peptide precursor protein of the first embodiment of the invention:
  • (i) comprises the amino acid sequence as recited in SEQ ID NO:12, SEQ ID NO: 15 or SEQ ID NO: 18; or (ii) is a functional equivalent of (i).
  • a polypeptide which consists of the amino acid sequence as recited in SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO:18.
  • the polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "INSP207 polypeptide sequence exon 1".
  • the polypeptide having the sequence recited in SEQ ID NO:4 is referred to hereafter as "INSP207 polypeptide sequence exon 2".
  • the polypeptide having the sequence recited in SEQ ID NO:6 is referred to hereafter as "INSP207 polypeptide sequence”.
  • Proteolytic cleavage of the INSP207 RF amide-related peptide precursor protein generates three RFamide-related peptides, herein termed "INSP207 neuropeptide 1", “INSP207 neuropeptide 2" and "INSP207 neuropeptide 3".
  • the polypeptide having the sequence recited in SEQ ID NO: 12 is referred to hereafter as "INSP207 neuropeptide 1".
  • the polypeptide having the sequence recited in SEQ ID NO: 15 is referred to hereafter as "INSP207 neuropeptide 2".
  • the polypeptide having the sequence recited in SEQ ID NO: 18 is referred to hereafter as "INSP207 neuropeptide 3".
  • the polypeptides having the sequences recited in SEQ ID NO:13, SEQ ID NO:16 and SEQ ID NO:19 are referred to herein as "amidated INSP207 peptide products”.
  • INSP207 polypeptides as used herein includes polypeptides comprising the INSP207 polypeptide sequence exon 1, the INSP207 polypeptide sequence exon 2, the INSP207 polypeptide sequence, the INSP207 neuropeptide 1, the INSP207 neuropeptide 2 and the INSP207 neuropeptide 3.
  • INSP207 polypeptides as used herein also includes the amidated INSP207 peptide products.
  • the RFamide-related precursor protein of the present invention (INSP207) is predicted to be a secreted protein possessing a secretory leader sequence at its N-terminus (see Examples and Figures).
  • the INSP207 polypeptide sequence exon 1 without this postulated signal sequence is recited in SEQ DD NO: 8.
  • the full length polypeptide sequence without this postulated signal sequence is recited in SEQ ID NO: 10.
  • the polypeptide sequence recited in SEQ ID NO: 8 is referred to hereafter as the INSP207 mature polypeptide exon 1.
  • the polypeptide sequence recited in SEQ ID NO: 10 is referred to hereafter as the INSP207 mature polypeptide.
  • This first aspect of the invention also includes fusion proteins that incorporate polypeptide fragments and variants of these polypeptide fragments as defined above, provided that said fusion proteins comprise a RFamide-related precursor protein or RFamide-related peptide of the present invention.
  • the "RFamide-related peptide precursor protein or an RFamide-related peptide” may be a molecule containing a RFamide-like domain detected with an e-value lower than 0.1, 0.01, 0.001, 0.0008, 0.0001, 0.00001, 0.000001 or 0.0000001.
  • RFamide-related peptide denotes that the peptide in question possesses a sequence of amino acids associated with RFamide peptides, for example, a R-F or R-L amino acid pair at its C-terminus.
  • an RFamide-related peptide of the invention is amidated at its C-terminus. This results in the loss of the C terminal glycine.
  • the sequences of these amidated peptide products are presented in SEQ ID NO:13, SEQ ID NO: 16 and SEQ ID NO: 19.
  • Activity of the polypeptides of the invention can be evaluated by measuring their ability to bind G-protein-coupled receptors.
  • the activity of an INSP207 polypeptide of the present invention, alone or as part of a fusion protein, a fragment thereof and/or an antagonist thereof can be confirmed in at least one of the following assays: in an assay measuring prolactin (PRL) secretion (Hinuma et al. Nat Cell Biol 2000; 2:703-8), or - in an assay measuring pain modulation (Liu et al. J Biol Chem 2001; 276:36961-
  • PRL prolactin
  • the polypeptides of the first aspect of the invention may further comprise a histidine tag.
  • the histidine tag is found at the C-terminal of the polypeptide.
  • the histidine tag comprises 1-10 histidine residues ⁇ e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues). More preferably the histidine tag comprises 6 histidine residues.
  • An "antigenic determinant" of the present invention may be a part of a polypeptide of the present invention, which binds to an antibody-combining site or to a T-cell receptor (TCR).
  • TCR T-cell receptor
  • an "antigenic determinant" may be a site on the surface of a polypeptide of the present invention to which a single antibody molecule binds.
  • an antigen has several or many different antigenic determinants and reacts with antibodies of many different specificities.
  • the antibody is immunospecific to a polypeptide of the invention.
  • the antibody is immunospecific to a polypeptide of the invention, which is not part of a fusion protein.
  • the antibody is immunospecific to INSP207 or a fragment thereof.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the "antigenic determinant" refers to a particular chemical group on a polypeptide of the present invention that is antigenic, i.e. that elicit a specific immune response.
  • the invention provides a purified nucleic acid molecule which encodes a RFamide-related peptide precursor protein or RFamide-related peptide of the first aspect of the invention.
  • purified nucleic acid molecule preferably refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "purified nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence.
  • the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.
  • genomic DNA are specifically excluded from the scope of the invention.
  • genomic DNA larger than 10 kbp (kilo base pairs), 50 kbp, 100 kbp, 150 kbp, 200 kbp, 250 kbp or 300 kbp are specifically excluded from the scope of the invention.
  • the "purified nucleic acid molecule" consists of cDNA only.
  • the purified nucleic acid molecule comprises the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSP207 polypeptide sequence exon 1), SEQ ID NO:3 (encoding the INSP207 polypeptide sequence exon 2), SEQ ID NO:5 (encoding the INSP207 polypeptide sequence), SEQ ID NO:7 (encoding the INSP207 mature polypeptide sequence exon 1), SEQ ID NO:9 (encoding the INSP207 mature polypeptide sequence), SEQ ID NO: 11 (encoding the INSP207 neuropeptide 1), SEQ ID NO:14 (encoding the INSP207 neuropeptide 2), SEQ ID NO: 17 (encoding the INSP207 neuropeptide 3), or is a redundant equivalent or fragment of any one of these sequences.
  • the invention further provides that the purified nucleic acid molecule consists of the nucleic acid sequence as recited in SEQ ID NO:1 (encoding the INSP207 polypeptide sequence exon 1), SEQ ID NO:3 (encoding the INSP207 polypeptide sequence exon 2),
  • SEQ ID NO:5 (encoding the INSP207 polypeptide sequence)
  • SEQ ID NO:7 (encoding the
  • INSP207 mature polypeptide sequence exon 1) SEQ ID NO:9 (encoding the INSP207 mature polypeptide sequence), SEQ ID NO: 11 (encoding the INSP207 neuropeptide 1), SEQ ID NO:14 (encoding the INSP207 neuropeptide 2), SEQ ID NO:17 (encoding the
  • INSP207 neuropeptide 3 is a redundant equivalent or fragment of any one of these sequences.
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the invention provides a ligand which binds specifically to, and which preferably inhibits the activity of, a RF amide-related precursor protein or RF amide-related peptide of the first aspect of the invention.
  • the sixth aspect of the invention also provides a ligand which binds specifically to, and which preferably inhibits the activity of, a complex of the fifteenth aspect of the invention.
  • the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Such compounds may be identified using the assays and screening methods disclosed herein.
  • a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
  • the identification of the function of the INSP207 polypeptide allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease.
  • Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention.
  • Another aspect of this invention resides in the use of an INSP207 gene or polypeptide as a target for the screening of candidate drag modulators, particularly candidate drugs active against RFamide-related peptide precursor protein or RFamide-related peptide related disorders.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of RFamide-related peptide precursor protein or RFamide-related peptide related disorders, comprising determining the ability of a compound to bind to an INSP207 gene or polypeptide, or a fragment thereof.
  • a further aspect of this invention resides in methods of screening of compounds for therapy of RFamide-related peptide precursor protein or RFamide-related peptide related disorders, comprising testing for modulation of the activity of an INSP207 gene or polypeptide, or a fragment thereof.
  • the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis of diseases in which RFamide- related peptide precursor proteins or RFamide-related peptides are implicated.
  • Such diseases include cell proliferative disorders including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours, myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis 1 sarcoma, pain, opoid tolerance and other pain-related disorders, cardiovascular diseases, psychiatric disorders such as, depression, schizophrenia, anxiety, mania, neurological disorders such as Alzheimer's disease, neurodegenerative diseases, stroke, pain, migraine and epilepsy, neuromuscular disorders, disorders of the regulation of behaviour involving feeding, sleep, arousal, pain and stress, metabolic disorders, disorders relating to the limbic system including obesity, anorexia, bulimia, sleep disorders, sexual dysfunction and drug addiction, endocrine disorders such as estrogen deficiency, infertility, premature labour, birth complication, dysmenorrhea, premen
  • the disease is one in which RF amide-related peptide precursor proteins or RFamide-related peptides are implicated, such as a neurological disorder.
  • RF amide-related peptide precursor proteins or RFamide-related peptides are implicated, such as a neurological disorder.
  • These molecules may also be used in the manufacture of a medicament for the treatment of such diseases.
  • moieties of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention may also be used in the manufacture of a medicament for the treatment of such diseases.
  • the moieties of the present invention may have particular utility in the therapy or diagnosis of disorders/diseases (the two terms are used interchangeably herein) such as neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours, myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma, pain, opoid tolerance and other pain-related disorders, cardiovascular diseases, psychiatric disorders such as, depression, schizophrenia, anxiety, mania, neurological disorders such as Alzheimer's disease,
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
  • a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
  • a ligand such as an antibody
  • PCR polymerase chain reaction
  • the disease diagnosed by a method of the ninth aspect of the invention is a disease in which RFamide-related peptide precursor proteins or RFamide-related peptides are implicated, as described above.
  • the invention provides for the use of a polypeptide of the first aspect of the invention as a RFamide-related peptide precursor protein or as a RFamide-related peptide. Suitable uses of the polypeptides of the invention as a RFamide-related peptide precursor protein include use as a vehicle for the efficient production of the RFamide- related peptides of the present invention.
  • Suitable uses of the polypeptides of the invention as a RFamide-related peptide include use as a modulator of physiological processes such as pain sensation, hormone secretion, cardioexcitation or cellular metastasis and also as a investigative reagent for example in methods for identifying and characterising (de- orphanising) 7TM G-protein-coupled receptors (see below).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically- acceptable carrier.
  • the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, including, but not limited to neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours, myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma, pain, opoid tolerance and other pain-related disorders, cardiovascular diseases, psychiatric disorders such as, depression, schizophrenia, anxiety, mania, neurological disorders such as Alzheimer's
  • a disease including, but not
  • the disease is a disease in which RFamide-related peptide precursor proteins or RFamide-related peptides are implicated, as described above.
  • the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
  • the polypeptide, nucleic acid molecule, vector, host cell, ligand or compound administered to the patient should be an agonist.
  • the polypeptide, nucleic acid molecule, vector, host cell, ligand or compound administered to the patient should be an antagonist. Examples of such antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.
  • the disease is a disease in which RFamide-related peptide precursor proteins or RFamide-related peptides are implicated, as described above.
  • the INSP207 polypeptides are RFamide-related peptide precursor proteins or RFamide- related peptides and thus have roles in many disease states. Antagonists of the INSP207 polypeptides are of particular interest as they provide a way of modulating these disease states.
  • “functional equivalent” refers to a protein or nucleic acid molecule that possesses functional or structural characteristics that are substantially similar to a polypeptide or nucleic acid molecule of the present invention.
  • a functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional equivalent” is intended to include the fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a molecule.
  • the "functional equivalent” may be a protein or nucleic acid molecule that exhibits any one or more of the functional activities of the polypeptides of the present invention.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays substantially similar activity compared with INSP207 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the "functional equivalent” may be a protein or nucleic acid molecule that displays identical or higher activity compared with INSP207 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the “functional equivalent” may be a protein or nucleic acid molecule that displays 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% or more activity compared with INSP207 or fragments thereof in a suitable assay for the measurement of biological activity or function.
  • the "functional equivalent” may be a protein or polypeptide capable of exhibiting a substantially similar in vivo or in vitro activity as the polypeptides of the invention.
  • the "functional equivalent” may be a protein or polypeptide capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the polypeptides of the invention would.
  • a "functional equivalent” would be able, in an immunoassay, to diminish the binding of an antibody to the corresponding peptide (i.e., the peptide the amino acid sequence of which was modified to achieve the "functional equivalent") of the polypeptide of the invention, or to the polypeptide of the invention itself, where the antibody was raised against the corresponding peptide of the polypeptide of the invention.
  • An equimolar concentration of the functional equivalent will diminish the aforesaid binding of the corresponding peptide by at least about 5%, preferably between about 5% and 10%, more preferably between about 10% and 25%, even more preferably between about 25% and 50%, and most preferably between about 40% and 50%.
  • functional equivalents can be fully functional or can lack function in one or more activities.
  • variations can affect the function, for example, of the activities of the polypeptide that reflect its possession of a RFamide-like domain.
  • the Applicant considers that the RFamide-related polypeptides of the first aspect of the invention bind G-protein-coupled receptors.
  • the polypeptides of the invention are therefore predicted to be ligands for an orphan G-protein-coupled receptor.
  • the invention provides a complex of a polypeptide according to the first aspect of the invention with a G-protein-coupled receptor or receptors.
  • the invention therefore also provides a method of identifying and characterising a 7TM G- protein-coupled receptor comprising: a) contacting an orphan G-protein-coupled receptor with a polypeptide of the first aspect of the invention; and b) detecting binding.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptides of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
  • the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
  • the polypeptide of the first aspect of the invention may form part of a fusion protein.
  • a fusion protein may contain one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production.
  • the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • a polypeptide of the invention that may comprise a sequence having at least 85% of homology with INSP207, is a fusion protein.
  • fusion proteins can be obtained by cloning a polynucleotide encoding a polypeptide comprising a sequence having at least 85% of homology with INSP207 in frame to the coding sequences for a heterologous protein sequence.
  • heterologous when used herein, is intended to designate any polypeptide other than a human INSP207 polypeptide.
  • heterologous sequences that can be comprised in the fusion proteins either at the N- or C-terminus, include: extracellular domains of membrane-bound protein, immunoglobulin constant regions (Fc regions), multimerization domains, domains of extracellular proteins, signal sequences, export sequences, and sequences allowing purification by affinity chromatography.
  • heterologous sequences are commercially available in expression plasmids since these sequences are commonly included in fusion proteins in order to provide additional properties without significantly impairing the specific biological activity of the protein fused to them (Terpe K 5 2003, Appl Microbiol Biotechnol, 60:523-33).
  • additional properties are a longer lasting half-life in body fluids, the extracellular localization, or an easier purification procedure as allowed by the a stretch of Histidines forming the so-called "histidine tag" (Gentz et al.
  • the heterologous sequence can be eliminated by a proteolytic cleavage, for example by inserting a proteolytic cleavage site between the protein and the heterologous sequence, and exposing the purified fusion protein to the appropriate protease.
  • the INSP207 polypeptide may be purified by means of a hexa-histidine peptide fused at the C-terminus of INSP207.
  • the fusion protein comprises an immunoglobulin region
  • the fusion may be direct, or via a short linker peptide which can be as short as 1 to 3 amino acid residues in length or longer, for example, 13 amino acid residues in length.
  • Said linker may be a tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13 -amino acid linker sequence comprising GIu- Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ ID NO: 20) introduced between the sequence of the substances of the invention and the immunoglobulin sequence.
  • the resulting fusion protein has improved properties, such as an extended residence time in body fluids ⁇ i.e. an increased half-life), increased specific activity, increased expression level, or the purification of the fusion protein is facilitated.
  • the protein is fused to the constant region of an Ig molecule.
  • it is fused to heavy chain regions, like the CH2 and CH3 domains of human IgGl, for example.
  • Other isoforms of Ig molecules are also suitable for the generation of fusion proteins according to the present invention, such as isoforms IgG2 or IgG4, or other Ig classes, like IgM or IgA, for example. Fusion proteins may be monomeric or multimeric, hetero- or homomultimeric.
  • the functional derivative comprises at least one moiety attached to one or more functional groups, which occur as one or more side chains on the amino acid residues.
  • the moiety is a polyethylene (PEG) moiety. PEGylation may be carried out by known methods, such as the ones described in WO99/55377, for example.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.
  • modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
  • the modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
  • the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
  • polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include, where the polypeptide is a naturally occurring polypeptide, isolated naturally-occurring polypeptides (for example purified from cell culture) and also recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
  • isolated does not denote the method by which the polypeptide is obtained or the level of purity of the preparation. Thus, such isolated species may be produced recombinantly, isolated directly from the cell or tissue of interest or produced synthetically based on the determined sequences.
  • the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the INSP207 polypeptides.
  • Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity” indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity” indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
  • Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the INSP207 polypeptides.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Such substitutions are among Ala, VaI, Leu and He; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
  • any substitution should be preferably a "conservative” or “safe” substitution, which is commonly defined a substitution introducing an amino acids having sufficiently similar chemical properties (e.g. a basic, positively charged amino acid should be replaced by another basic, positively charged amino acid), in order to preserve the structure and the biological function of the molecule.
  • the literature provide many models on which the selection of conservative amino acids substitutions can be performed on the basis of statistical and physico-chemical studies on the sequence and/or the structure of proteins (Rogov SI and Nekrasov AN, 2001).
  • non-conservative mutations can be also introduced in the polypeptides of the invention with different purposes. Mutations reducing the affinity of the RFamide-related protein may increase its ability to be reused and recycled, potentially increasing its therapeutic potency (Robinson CR, 2002). Immunogenic epitopes eventually present in the polypeptides of the invention can be exploited for developing vaccines (Stevanovic S,
  • amino acids derivatives included in peptide mimetics are those defined in Table 2.
  • a non-exhaustive list of amino acid derivatives also include aminoisobutyric acid (Aib), hydroxyproline (Hyp), 1,2,3,4-tetrahydro- isoquinoline-3-COOH, indoline-2carboxylic acid, 4-difluoro-proline, L- thiazolidine-4- carboxylic acid, L-homoproline, 3,4-dehydro-proline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine, and phenylglycine.
  • amino acid derivative is intended an amino acid or amino acid-like chemical entity other than one of the 20 genetically encoded naturally occurring amino acids.
  • the amino acid derivative may contain substituted or non-substituted, linear, branched, or cyclic alkyl moieties, and may include one or more heteroatoms.
  • the amino acid derivatives can be made de novo or obtained from commercial sources (Calbiochem- Novabiochem AG 5 Switzerland; Bachem, USA).
  • polypeptides of the first aspect of the invention typically have a degree of sequence identity with the INSP207 polypeptides, or with active fragments thereof, of greater than 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98% or 99%, respectively.
  • the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
  • the Inpharmatica Genome Threader technology that forms one aspect of the search tools used to generate the BiopendiumTM search database may be used (see PCT application WO 01/69507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP207 polypeptides, are predicted to be RFamide-related peptide precursor proteins, by virtue of sharing significant structural homology with the INSP207 polypeptides.
  • significant structural homology is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above.
  • polypeptides of the first aspect of the invention also include fragments of the INSP207 polypeptides and fragments of the functional equivalents of the INSP207 polypeptides, provided that those fragments are RFamide-related peptide precursor proteins or RF amide- related peptides or have an antigenic determinant in common with the INSP207 polypeptides.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the INSP207 polypeptides or one of their functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.
  • Nucleic acid fragments according to the invention are preferably 10-400 nucleotides in length, preferably 20-300 nucleotides, preferably 50-200 nucleotides, preferably 75-100 nucleotides in length.
  • Polypeptide fragments according to the invention are preferably 5-130 amino acids in length, preferably 10-100, preferably 25-75, preferably 40-50 amino acids in length.
  • Fragments of the full length INSP207 polypeptides may consist of combinations of 1 or more neighbouring exon sequences in the INSP207 polypeptide sequences, respectively. For example, such combinations include exons 1 and 2. Such fragments are included in the present invention.
  • fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • the fragment of the invention When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region.
  • certain preferred embodiments relate to a fragment having a pre- and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment.
  • several fragments may be comprised within a single larger polypeptide.
  • polypeptides of the present invention or their immunogenic fragments can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are irnmunospecific for the polypeptides.
  • ligands such as polyclonal or monoclonal antibodies
  • Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography.
  • the antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention.
  • substantially greater affinity we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for known RFamide-related peptide precursor proteins or RFamide-related peptides.
  • the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 3 -fold, 10 4 - fold, 10 5 -fold or 10 6 -fold greater for a polypeptide of the invention than for known RFamide-related peptide precursor proteins or RFamide-related peptides.
  • a polypeptide of the invention may be immunised with a polypeptide of the first aspect of the invention.
  • the polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically.
  • the polypeptide can be conjugated to a carrier protein. Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin.
  • the coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography.
  • Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
  • Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc.
  • Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed.
  • genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
  • Chimeric antibodies, in which non-human variable regions are joined or fused to human constant regions see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
  • the antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239, 1534 (1988); Kabat et al, J. Immunol., 147, 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci. USA, 88, 34181 (1991); and Hodgson et al, Bio/Technology, 9, 421 (1991)).
  • humanisation see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239, 1534 (1988); Kabat et al, J. Immunol., 147, 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
  • the antibody may be a "bispecific" antibody, that is, an antibody having two different antigen binding domains, each domain being directed against a different epitope.
  • Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • nucleic acid molecules of the second and third aspects of the invention are those which encode a polypeptide sequence as recited in SEQ ED NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO:15 and SEQ ID NO:18 and functionally equivalent polypeptides. These nucleic acid molecules may be used in the methods and applications described herein.
  • the nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences. The nucleic acid molecules may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • nucleic acid molecule also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drag Des. 8:53-63).
  • a nucleic acid molecule which encodes a polypeptide of this invention may be identical to the coding sequence of one or more of the nucleic acid molecules disclosed herein.
  • These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:15 or SEQ ID NO:18.
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:15 or SEQ ID NO:18.
  • Examples of preferred such nucleic acid molecules include the nucleic acid sequences presented in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 17.
  • nucleic acid molecules may include, but are not limited to, the coding sequence for the INSP207 polypeptides alone; the coding sequence for the INSP207 polypeptides and additional coding sequences, such as those encoding an additional or substituted leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the INSP207 polypeptides, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability.
  • the nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
  • nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention.
  • a nucleic acid molecule may be a naturally-occurring variant such as a naturally-occurring allelic variant, or the molecule may be a variant that is not known to occur naturally.
  • non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions.
  • the substitutions, deletions or insertions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
  • the nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide).
  • DNA shuffling by random fragmentation and PCR re-assembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences.
  • Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules
  • antisense molecules such as oligonucleotides
  • oligonucleotides can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56,
  • hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. [supra]).
  • the inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al. [supra]).
  • a substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-511).
  • Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (15OmM NaCl, 15mM trisodium citrate), 5OmM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the INSP207 polypeptides and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to such coding sequences, or is a nucleic acid molecule that is complementary thereto.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98%, 99% or more identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the INSP207 polypeptides.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the INSP207 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
  • the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the INSP207 polypeptides is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992).
  • Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 17), are particularly useful probes.
  • Such probes may be labelled with an analytically- detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • probes the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al, PNAS USA 85, 8998-9002, 1988).
  • RACE Rapid Amplification of cDNA Ends
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size- selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • the nucleic acid molecules of the present invention may be used for chromosome localisation.
  • a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation. Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • RNA interference RNA interference
  • Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
  • Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies.
  • the vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
  • any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et al., (supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • HACs Human artificial chromosomes
  • Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems.
  • Cell-free translation systems can also be employed to produce the polypeptides of the invention.
  • nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, ⁇ supra). Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998).
  • expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
  • the encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment.
  • a control sequence such as a signal peptide or leader sequence
  • These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Leader sequences can be removed by the bacterial host in post-translational processing. hi addition to control sequences, it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
  • Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3 1 untranslated regions. These interact with host cellular proteins to carry out transcription and translation.
  • Such regulatory sequences may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJoIIa, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • stable expression is preferred.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), Cl 27, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • ATCC American Type Culture Collection
  • baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).
  • Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
  • plant cell culture and whole plant genetic expression systems known in the art. Examples of suitable plant cellular genetic expression systems include those described in US 5,693,506; US 5,659,122; and US 5,608,143.
  • yeast cells for example, S. cerevisiae
  • Aspergillus cells examples include yeast cells (for example, S. cerevisiae) and Aspergillus cells.
  • antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. MoI. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA- DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
  • sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
  • Suitable reporter molecules or labels include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
  • the polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well-known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
  • Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
  • purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et at (1992), Prot. Exp. Purif.
  • the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffmity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • the polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques.
  • Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention.
  • Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et at, Current Protocols in Immunology 1(2): Chapter 5 (1991).
  • Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • the INSP207 polypeptides of the present invention may modulate a variety of physiological and pathological processes, including processes such as the sensation of pain, secretion of hormones, cellular metastasis (including cancer cell metastasis), control of pro-lactin release, sleep-cycle and feeding-cycle control, cardioexitation and neuromodulation.
  • the biological activity of the INSP207 polypeptides can be examined in systems that allow the study of such modulatory activities, using a variety of suitable assays.
  • suitable assays for observing the effect of the INSP207 polypeptides of the present invention on cellular metastasis, one can employ one or more of the methods described in Ohtaki et al, Nature. 2001 May 31 ;411 (6837):613-7 or the publications referred to therein.
  • the INSP207 polypeptides of the present invention may also be used for the identification and characterisation of receptors, particularly 7TM G-protein-linked receptors, which interact with the INSP207 polypeptides of the present invention.
  • Suitable methods of identification and characterisation include, but are not limited to, those described in Hinuma et al, Nat Cell Biol. 2000 Oct;2(10):703-8, Jiang et al, J. Biol. Chem. 2003 Jul;278(30):27652-7 and the International patent application published as WO 01/17958 or the publications referred to therein.
  • the INSP207 polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the INSP207 polypeptides include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose-dependent manner.
  • the degree of dose-dependent activity need not be identical to that of the INSP207 polypeptides, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the INSP207 polypeptides.
  • simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor.
  • competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
  • Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
  • Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays.
  • Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signalling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • the polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
  • a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
  • the efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance (supplied by Biacore AB, Uppsala, Sweden) and spectroscopy.
  • Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
  • this invention relates to the use of a INSP207 polypeptide or fragment thereof, whereby the fragment is preferably a INSP207 gene-specific fragment, for isolating or generating an agonist or stimulator of the INSP207 polypeptide for the treatment of an immune related disorder, wherein said agonist or stimulator is selected from the group consisting of:
  • a specific antibody or fragment thereof including: a) a chimeric, b) a humanized or c) a fully human antibody, as well as; 2. a bispecific or multispecific antibody,
  • an antibody-mimetic such as a) an anticalin or b) a f ⁇ bronectin-based binding molecule (e.g. trinectin or adnectin).
  • Anticalins are also known in the art (Vogt et al., 2004). Fibronectin-based binding molecules are described in US6818418 and WO2004029224. Furthermore, the test compound may be of various origin, nature and composition, such as any small molecule, nucleic acid, lipid, peptide, polypeptide including an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody, a single chain (e.g. scFv) or single domain antibody or an antibody-mimetic such as an anticalin or fibronectin-based binding molecule (e.g. trinectin or adnectin), etc., in isolated form or in mixture or combinations.
  • an antibody such as a chimeric, humanized or fully human antibody or an antibody fragment, peptide- or non- peptide mimetic derived therefrom as well as a bispecific or multispecific antibody
  • the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
  • the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
  • the various moieties of the invention i.e. the polypeptides of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, a compound of the seventh aspect of the invention
  • the various moieties of the invention may be useful in the therapy or diagnosis of diseases.
  • one or more of the following assays may be carried out.
  • test compound refers to the test compound as being a protein/polypeptide
  • test compound a person skilled in the art will readily be able to adapt the following assays so that the other moieties of the invention may also be used as the "test compound”.
  • compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
  • a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X.
  • X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.
  • compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drags or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by
  • compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule. If the activity of the polypeptide of the invention is in excess in a particular disease state, several approaches are available.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • such antagonists are antibodies. Most preferably, such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question may be administered.
  • the polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and nitrons) of the gene encoding the polypeptide.
  • triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • triplex DNA Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY).
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et ah, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and undine which are not as easily recognised by endogenous endonucleases.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition.
  • a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
  • Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
  • Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient.
  • in vivo gene therapy does not require isolation and purification of a patient's cells.
  • the therapeutic gene is typically "packaged" for administration to a patient.
  • Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479.
  • a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector.
  • This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
  • Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
  • vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the vaccine formulations of the invention may be presented in unit-dose or multi-dose containers.
  • sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • jet injection see, for example, www.powderject.com
  • jet injection may also be useful in the formulation of vaccine compositions.
  • nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol, 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included.
  • Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
  • Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et al, Genomics, 5, 874-879 (1989)).
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • point mutations and other sequence variations, such as polymorphisms can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (see Cotton et al, Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401).
  • mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N. Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane.
  • Fluorescence in situ hybridization is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al, Science, 250, 559-562 (1990), and Trask et al, Trends, Genet., 7, 149-154 (1991)).
  • an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996), VoI 274, pp 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93: 10614-10619).
  • Oligonucleotide pairs may range from two to over one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al).
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
  • diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
  • Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules known in the art may be used, several of which are described above.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit of the present invention may comprise: (a) a nucleic acid molecule of the present invention; (b) a polypeptide of the present invention; or (c) a ligand of the present invention.
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease in which RFamide-related peptide precursor proteins or RFamide-related peptides are implicated.
  • diseases include cell proliferative disorders including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours, myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis 1 sarcoma, pain, opoid tolerance and other pain-related disorders, cardiovascular diseases, psychiatric disorders such as, depression, schizophrenia, anxiety, mania, neurological disorders such as Alzheimer's disease, neurodegenerative diseases, stroke, pain, migraine and epilepsy, neuromuscular disorders, disorders of the regulation of behaviour involving feeding, sleep, arousal, pain and stress, metabolic disorders, disorders relating to the limbic system including obesity, anorexia, bulimia,
  • Figure 1 INSP207 (SEQ ID NO:6) showing the RFG motif (boxed) and the mono-/dibasic sites (underlined). The predicted cleavage site for the signal peptide is indicated by an arrow.
  • Figure 2 Multiple sequence alignment of the INSP207 neuropeptides 1-3 (SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18) with other known RFamide sequences.
  • the sequences which are aligned with neuropeptides 1-3 of INSP207 are derived from following precursor proteins: HUM_NPSF (015130); HUM_NPFF (015130);
  • MUS_NPSF Q9WVA8; RAT_NPSF (Q9WVA9); RATJSIPFF (Q9WVA9); MUS_NPFF
  • HUM_RFRP1 Q9HCQ7
  • BOV_RFRP1 Q9GM96
  • HUM_RFRP2 Q9HCQ7
  • BOV_RFRP2 (Q9GM96); HUM_RFRP3 (Q9HCQ7); HUM_P518RF (NP_937823);
  • HUM_KISS1 (Q15726); MUS_KISS1 (Q6Y4S4); BOV_PRP (P81264); RAT_PRP
  • the C terminal glycine residue is amidated (-NH2) on the mature active forms of the neuropeptide sequences.
  • the INSP207 amino acid sequence comprises a RFG motif in addition to the predicted signal peptide.
  • INSP207 also comprises predicted dibasic and monobasic protease cleavage sites ( Figure 1). The combination of these motifs within the INSP207 amino acid sequence provides very strong evidence in support of the prediction that the INSP207 amino acid sequence represents a RFamide-related peptide precursor protein.
  • INSP207 neuropeptide 1, INSP207 neuropeptide 2 and INSP207 neuropeptide 3 peptides are RFamide-related peptides generated by post-translational cleavage of the INSP207 amino acid sequence between Lysine 62 at the C-terminus and the basic sites in the positions 31, 54, or 57, respectively, at the N-terminus.
  • INSP207-derived neuropeptides can be aligned with some Rfamide neuropeptides known in the literature ( Figure 2).
  • the human prolactin releasing peptide stimulates prolactin release and regulates the expression of prolactin through its receptor hGR3 / GPRlO (Hinuma S et al, Nature 393 (6682), 212-216 (1998)).
  • the Carassius auratus RF amide C-RFa has an excitatory effect on visceral muscle tissues offish, newt, quail, and rat (Fujimoto M et al, Biochem Biophys Res Commun 1998 Jan 14;242(2):436-40).
  • Human cDNA templates for example, from a variety of normal human tissue total RNA samples (which can be purchased from Clontech, Stratagene, Ambion, Biochain Institute and prepared in-house) using an enzyme such as Superscript II RNase H- Reverse Transcriptase (Invitrogen).
  • Human cDNA libraries in bacteriophage lambda ( ⁇ ) vectors
  • ⁇ bacteriophage lambda
  • Pairs of specific PCR primers can be designed for amplifying the complete coding sequence of the virtual cDNA using software such as the Primer Designer Software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA). PCR primers were optimized to have a Tm close to 55 ⁇ 1O 0 C and a GC content of 40-60%. Primers should be selected which have high selectivity for the target sequence (INSP207) with little or no non-specific priming. PCR can then be used to amplify the gene sequence of interest.
  • the presence of the transcripts for INSP207 may be investigated by PCR of cDNA from different human tissues.
  • the INSP207 transcripts may be present at very low levels in the samples tested. Therefore, extreme care is needed in the design of experiments to establish the presence of a transcript in various human tissues as a small amount of genomic contamination in the RNA preparation will provide a false positive result.
  • all RNA should be treated with DNAse prior to use for reverse transcription.
  • a control reaction may be set up in which reverse transcription was not undertaken (a -ve RT control). For example, 1 ⁇ g of total RNA from each tissue may be used to generate cDNA using Multiscript reverse transcriptase (ABI) and random hexamer primers.
  • ABSI Multiscript reverse transcriptase
  • a control reaction is set up in which all the constituents are added except the reverse transcriptase (-ve RT control).
  • PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls.
  • INSP207-specific primers may readily be designed on the basis of the sequence information provided herein. The presence of a product of the correct molecular weight in the reverse transcribed sample together with the absence of a product in the minus RT control may be taken as evidence for the presence of a transcript in that tissue. Any suitable cDNA libraries may be used to screen for the INSP207 transcripts, not only those generated as described above.
  • tissue distribution pattern of the INSP207 polypeptides will provide further useful information in relation to the function of those polypeptides.
  • overexpression or knock-down of the expression of the polypeptides in cell lines may be used to determine the effect on transcriptional activation of the host cell genome.
  • Dimerisation partners, co-activators and co-repressors of the INSP207 polypeptide may be identified by immunoprecipitation combined with Western blotting and immunoprecipitation combined with mass spectroscopy. Work is also currently underway to test the INSP207-derived neuropeptides and find relevant activities and receptors in mammalians as described in the literature on this subject (Dockray GJ, Exp Physiol.
  • the neuropeptides can be produced by any method known in the art (Bray BL, Nat Rev Drug Discov. 2003, 2(7):587-93; Casi G and Hilvert D, Curr Opin Struct Biol. 2003, 13(5):589-94; European patent application Nos. 1347053 and 1391518).
  • Example 3 RT-PCR from human multi-tissue mRNA 3.1.1 Preparation of a human multi-tissue cDNA template A preparation of human RNA was prepared by mixing approximately lO ⁇ g total RNA from each of the following sources:
  • RNA was fractionated by chromatography on a pre-packed oligo-dT column (Stratagene) according to the protocol supplied by the manufacturer. Approximately 400 ⁇ g total RNA yielded 12.6 ⁇ g polyA+ mRNA which was aliquotted and frozen at -8O 0 C.
  • the gene specific cDNA primer for INSP207, AS397 was pooled with gene specific cDNA primers for 9 other predictions, each at a final concentration of IpM.
  • the pooled cDNA primer set was diluted 10 fold into 50 ⁇ l of a mixutre containing 1 x RT buffer, 500 ⁇ M each dNTPs, lOU/ ⁇ l RNAguard (Pharmacia) and l ⁇ g denatured ⁇ olyA + RNA prepared as described above.
  • cDNA synthesis was initiated by addition of 1OU Omniscript reverse transcriptase (Qiagen) and allowed to proceed for Ih at 37°C. At the end of the reaction, 5 ⁇ l of the cDNA mix was used for PCR amplification as described below.
  • Top strand (AS398) and bottom strand (AS399) PCR primers were designed to span the entire predicted coding sequence of INSP207. BamHI restrictions sites were added at the 5' end of each primer since no internal sites for this enzyme were predicted.
  • a reaction mixture was set up containing 1 x PCR buffer, 0.2mM each dNTP, 0.5 ⁇ M each PCR primer, 5 ⁇ l cDNA above, and the PCR reaction was initiated by addition of 5U PfuTurbo (Stratagene). Cycling conditions were: 95°C 3min (1 cycle); 95 0 C 30 sec, 50 0 C 30 sec, 75°C 70 sec (35 cycles); 75°C 10 min (1 cycle).
  • the precipitated DNA was recovered by centrifugation, washed in 70% ethanol, dried under vacuum and ligated in a final volume of lO ⁇ l using the Rapid Ligation Kit (Roche Diagnostics) according to the protocol supplied by the manufacturers.
  • the ligation mixture was then used to transform E. coli strain JMlOl as follows: 50 ⁇ l aliquots of competent JMlOl cells were thawed on ice and l ⁇ l or 5 ⁇ l of the ligation mixture reaction was added. The cells was incubated for 40 min on ice and then heat shocked by incubation at 42°C for 2min. 1ml of warm (room temperature) L-Broth (LB) was added and samples were incubated for a further 1 h at 37°C.
  • the transformation mixture was then plated on LB plates containing ampicillin (lOO ⁇ g/ml) PTG (0.1 ⁇ M) and X-gal (50 ⁇ g/ml) and incubated overnight at 37 0 C. Single white colonies were chosen for plasmid isolation.
  • Miniprep plasmid DNA was prepared from 5ml cultures using a Biorobot 8000 robotic system (Qiagen) according to the manufacturer's instructions. Plasmid DNA was eluted in 80 ⁇ l of sterile water. The DNA concentration was measured using an Eppendorf BO photometer or Spectramax 190 photometer (Molecular Devices).
  • Miniprep#28 was taken as a representative clone for mutagenesis.
  • mutagenesis primers were designed to delete the sequence corresponding to the unspliced intronic sequence from the cloned miniprep 28 described above.
  • the top strand mutagenesis primer, AS426 was used with the 3' primer AS399; in a second PCR reaction, the bottom strand mutagenesis primer, AS427 was used with the 5' primer AS398 (see Table 3).
  • PCR products of approximately 0.23kb and 0.20kb were obtained from reactions 1 and 2 respectively, and purified using the Wizard Cleanup system as described above.
  • PCR In a third PCR reaction, 2 ⁇ l purified DNA from each of reactions 1 and 2 were combined and amplified in the presence of AS398 and AS399. All PCR reactions were performed in a reaction mixture containing 1 x PCR buffer, 0.2mM each dNTP, 0.5 ⁇ M each PCR primer, and DNA templates as described above. PCR was initiated by addition of 5U PfuTurbo (Stratagene) and cycling conditions were: 95 0 C 3min (1 cycle); 95 0 C 30 sec, 50 0 C 30 sec, 75°C 60 sec (25 cycles); 75 0 C 10 min (1 cycle).
  • the amplified products from the final PCR reaction were fractionated by electrophoresis in 0.8% agarose gels and a band of the predicted size of 0.43kb was excised and subcloned into the BamHI site of Bluescript BSK " as described above (section 3.2). Sequence analysis confirmed the mature full length cDNA coding sequence predicted for INSP207.
  • Custom microarrays have been manufactured using Agilent Technologies' (Agilent Technologies Inc, Palo Alto, CA) non-contact in situ synthesis process of printing 60-mer length oligonucleotide probes, base-by-base, from digital sequence files. This is achieved with an inkjet process which delivers extremely small, accurate volumes (picoliters) of the chemicals to be spotted. Standard phosphoramidite chemistry used in the reactions allows for very high coupling efficiencies to be maintained at each step in the synthesis of the full-length oligonucleotide. Precise quantities are reproducibly deposited "on the fly.” This engineering feat is achieved without stopping to make contact with the slide surface and without introducing surface-contact feature anomalies, resulting in consistent spot uniformity and traceability. (Hughes et al. (2001) Nat. Biotech. Apr; 19(4): 342-7. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer).
  • cDNA synthesis and subsequent T7 polymerase amplification of Cyanine 3(5)-CTP labeled cRNA probe was carried out using Agilent's low RNA input fluorescent linear amplification kit from a template of 5 ⁇ g of total RNA according to the kit protocol (version 2 August 2003, Agilent, Palo Alto, CA). cRNA is then fragmented using Agilent's In Situ hybridization kit-plus and hybridized both according to Agilent's protocol (Agilent 60-mer oligo microarray processing protocol version 4.1 April 2004, Agilent, Palo Alto, CA).
  • Remainder of the probes were designed to public domain sequences which are known to be either secreted soluble extracellular proteins or membrane bound proteins with an extracellular domain in contact with the extracellular milieu.

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Abstract

La présente invention concerne une nouvelle protéine, appelée INSP207, identifiée dans la présente invention en tant que protéine précurseur de peptide dérivé de RF-amide, ainsi que l'emploi de cette protéine et de la séquence d'acides nucléiques du gène codant pour cette protéine dans le diagnostic et le traitement prophylactique et thérapeutique d'une maladie. La présente invention concerne en outre les peptides dérivés de RF-amide obtenus par clivage de la protéine précurseur de peptide dérivé de RF-amide INSP207.
PCT/GB2006/003926 2005-10-21 2006-10-23 Protéines précurseurs de peptide dérivé de rf-amide et peptides de type rf-amide Ceased WO2007045906A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948838A (zh) * 2010-08-24 2011-01-19 中山大学 一种鱼类rf神经肽基因、编码的蛋白质及应用
WO2011123652A1 (fr) 2010-04-02 2011-10-06 The Regents Of The University Of Michigan Peptides de type rf-amide et procédés les utilisant

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004026904A1 (fr) * 2002-09-17 2004-04-01 Inpharmatica Limited Proteines precurseurs de peptides associes d'amide rf et peptides d'amide rf

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004026904A1 (fr) * 2002-09-17 2004-04-01 Inpharmatica Limited Proteines precurseurs de peptides associes d'amide rf et peptides d'amide rf

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BRUMWELL GORDON B ET AL: "Immunocytochemically defined populations of neurons progressively increase in size through embryogenesis of Hydra vulgaris", BIOLOGICAL BULLETIN (WOODS HOLE), vol. 203, no. 1, August 2002 (2002-08-01), pages 70 - 79, XP002412273, ISSN: 0006-3185 *
DARMER DOROTHEA ET AL: "Three different prohormones yield a variety of hydra-RFamide (Arg-Phe-NH2) neuropeptides in Hydra magnipapillata", BIOCHEMICAL JOURNAL, vol. 332, no. 2, 1 June 1998 (1998-06-01), pages 403 - 412, XP002412274, ISSN: 0264-6021 *
DATABASE EMBL 14 January 2000 (2000-01-14), WHITEHEAD,S.: "Human DNA sequence from clone RP5-977L11 on chromosome 1p22.3-31.2 Contains the 3' end of a novel gene", XP002412412, retrieved from EMBL accession no. AL136382 *
FUJIMOTO MASAAKI ET AL: "Isolation and characterization of a novel bioactive peptide, carassius RFamide (C-RFa), from the brain of the Japanese crucian carp", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 242, no. 2, 14 January 1998 (1998-01-14), pages 436 - 440, XP002412275, ISSN: 0006-291X *
FUKUSUMI S ET AL: "A new peptidic ligand and its receptor regulating adrenal function in rats", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 278, no. 47, 5 September 2003 (2003-09-05), pages 46387 - 46395, XP002267777, ISSN: 0021-9258 *
GRIMMELIKHUIJZEN C J P ET AL: "ISOLATION OF PYROGLU-LEU-LEU-GLY-GLY-ARG-PHE-AMIDE POL-RFAMIDE A NOVEL NEUROPEPTIDE FROM HYDROMEDUSAE", BRAIN RESEARCH, vol. 475, no. 1, 1988, pages 198 - 204, XP002412272, ISSN: 0006-8993 *
GRIMMELIKHUIJZEN C J P ET AL: "ISOLATION OF PYROGLUTAMYLGLYCYLARGINYLPHENYLALANINAMIDE ANTHO-RFAMIDE A NEUROPEPTIDE FROM SEA ANEMONES", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 83, no. 24, 1986, pages 9817 - 9821, XP002412271, ISSN: 0027-8424 *
HINUMA S ET AL: "A PROLACTIN-RELEASING PEPTIDE IN THE BRAIN", NATURE, NATURE PUBLISHING GROUP, LONDON, GB, vol. 393, 21 May 1998 (1998-05-21), pages 272 - 276, XP002926478, ISSN: 0028-0836 *
JIANG YING ET AL: "Identification and characterization of a novel RF-amide peptide ligand for orphan G-protein-coupled receptor SP9155", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 278, no. 30, 25 July 2003 (2003-07-25), pages 27652 - 27657, XP002267776, ISSN: 0021-9258 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011123652A1 (fr) 2010-04-02 2011-10-06 The Regents Of The University Of Michigan Peptides de type rf-amide et procédés les utilisant
EP2671889A2 (fr) 2010-04-02 2013-12-11 The Regents Of The University Of Michigan Peptides dérivés de rf-amide et procédés associés
US9527889B2 (en) 2010-04-02 2016-12-27 The Regents Of The University Of Michigan RFamide-related peptides and methods thereof
CN101948838A (zh) * 2010-08-24 2011-01-19 中山大学 一种鱼类rf神经肽基因、编码的蛋白质及应用

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