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WO2005023231A1 - Proteine de liaison du tractus polypyrimidique favorisant la biogenese d'un granule insulino-secretoire - Google Patents

Proteine de liaison du tractus polypyrimidique favorisant la biogenese d'un granule insulino-secretoire Download PDF

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Publication number
WO2005023231A1
WO2005023231A1 PCT/EP2004/010167 EP2004010167W WO2005023231A1 WO 2005023231 A1 WO2005023231 A1 WO 2005023231A1 EP 2004010167 W EP2004010167 W EP 2004010167W WO 2005023231 A1 WO2005023231 A1 WO 2005023231A1
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Prior art keywords
precursor
ptb
derivative
biologically active
cells
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Michele Solimena
Klaus-Peter Knoch
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Technische Universitaet Dresden
Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Technische Universitaet Dresden
Max Planck Gesellschaft zur Foerderung der Wissenschaften
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/507—Pancreatic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • Polypyrimidine tract binding protein promotes insulin secretory granule biogenesis
  • the present invention relates to a method for stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of promoting the presence of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells or neurons.
  • PTB polypyrimidine tract binding protein
  • the method alternatively or further comprises promoting the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells. It is also preferred that said promotion comprises the promotion of the nucleocytoplasmic transport of PTB.
  • the invention in another aspect, relates to a method of screening for an agent capable of stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds promote the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell.
  • PTB polypyrimidine tract binding protein
  • Comprised by the invention are further methods of screening for an agent useful as a cure for diabetes, sleeping disorders or depression as well as various medical uses of an agent capable of the promotion/reduction of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof.
  • PTB polypyrimidine tract binding protein
  • the reduction or downregulation of PTB or said biologically active fragment or derivative thereof is considered.
  • SGs secretory granules
  • Chemical stimuli induce the fusion of SGs with the plasma membrane and the release of their content into the extracellular space.
  • peptide hormones are co-translationally translocated into the lumen of the rough endoplasmic reticulum, then they are carried to the Golgi complex and finally they are sorted into nascent SGs.
  • peptide hormones can undergo multiple post-translational modifications, including proteolytic cleavage and glycosylation.
  • the generation of SGs is a slow process, which requires more than 30 minutes.
  • sustained stimulation leads to a progressive depletion of SGs, cells must quickly activate transcriptional and post- transcriptional mechanisms to renew their pool of these organelles.
  • ⁇ -cells of pancreatic islets are the endocrine cells that produce insulin, the most important hormone for the control of glucose homeostasis in vertebrates.
  • Glucose stimulates both the Ca 2+ -dependent exocytosis of insulin SGs as well as the biosynthesis of insulin and other SG components [1 ,2], including chromogranin A [1], and the prohormone convertases 1/3 (PC1/3) [3,4] and 2 (PC2) [4].
  • Glucose enhances both the transcription [5-8] and the translation [9] of the insulin gene. Increased translation, in particular, accounts entirely for up-regulating insulin biosynthesis during the first 2 hours following stimulation [10,11].
  • Such increase results from the stimulation of initiation, elongation and signal-recognition-particle- mediated translocation of the nascent preproinsulin polypeptide into the lumen of the endoplasmic reticulum [2,11] as well as from a reduced degradation of insulin mRNA [8].
  • glucose stimulation increases the stability of insulin mRNA by inducing the binding of polypyrimidine-tract binding protein (PTB) to its 3'-UTR [12,13].
  • PTB polypyrimidine-tract binding protein
  • PTB also known as heterogeneous nuclear ribonucleoprotein I
  • PTB is a pre-mRNA splicing repressor [14,15], which has also been implicated in cap-independent translation [16,17], cytoplasmic RNA transport [18], poly (A) site cleavage [19], and mRNA stability [12].
  • the PTB gene encodes a protein of 59 kD with four RNA recognition motif domains [20].
  • PKA 3',5'-cAMP-dependant protein kinase K
  • the present invention relates to a method for stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of promoting the presence of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells (i.e. said endocrine cells or neurons, in this and all pertinent following embodiments).
  • PTB polypyrimidine tract binding protein
  • the term "stimulating production of secretory granules (SGs) in peptide hormone- secreting endocrine cells or neurons” refers to the induction or enhancement of a process in peptide hormone-secreting endocrine cells or neurons that leads to increase of production of such granules of at least 20%, preferably at least 30%, more preferred at least 50%, even more preferred at least 75% and most preferred at least 100% as compared to a physiological, normal and optionally non-pathogenic status of corresponding endocrine cells or neurons.
  • the increase may be at least 200% such as at least 500%.
  • the extent of the production of secretory granules can be monitored by methods well known in the art including microscopy (more specifically confocal microscopy or electron microscopy), measurement of the expression of one or more components of the secretory granules at the protein or the mRNA level, and determining the amount of insulin, e.g. using Western blotting, a radio-immuno assay (RIA) or an ELISA assay.
  • RIA radio-immuno assay
  • ELISA assay ELISA assay
  • peptide hormone-secreting endocrine cells or neurons refers to any mammalian and preferably human cell that naturally produces and secretes peptide hormones or neuropeptides. Also included are precursors of such cells as well as cells which in their normal context in the body do not secrete such peptides but which have been manipulated to secrete such peptides, for example by genetic engineering. In principle, one option in this regard envisages reprogramming a somatic cell to transdifferentiate into a peptide-secreting endocrine cell or a neuron. It is preferred in accordance with the present invention that peptide hormone-secreting endocrine cells or neurons are human cells that naturally produce and secrete peptide hormones or neuropeptides.
  • promoting the presence in general means that a certain level of PTB or a biologically active fragment or derivative thereof is maintained or produced in the cytoplasm of the recited cells. Accordingly, the invention envisages that, in one alternative, an existing level of PTB or a biologically active fragment or derivative thereof in the cytoplasm is maintained or essentially maintained, such as maintained to at least 70%, preferably to at least 80%, more preferred to at least 90% and most preferred to at least 95% such as 100%. Important options to maintain an existing level of PTB or a biologically active fragment or derivative thereof in the cytoplasm are described in connection with preferred embodiments of the method of the invention further below.
  • PTB or a biologically active fragment or derivative thereof is actively produced inside the cell and/or shifted from the nucleus into the cytoplasm.
  • the level of PTB or a biologically active fragment or derivative thereof may be enhanced in a cell or in the cytoplasm. For example, if the cell naturally does not produce PTB, production may be stimulated by introducing a vector capable of expressing PTB into said cell.
  • the levels of PTB may also be enhanced in a cell that naturally produces PTB by way of expression from an introduced vector.
  • Important options how the level of PTB may be enhanced by production of PTB or a biologically active fragmen or derivative thereof are, again, discussed in connection with preferred embodiment further below.
  • the term "enhancing" in connection with the production of PTB relates both to the enhancement of levels of PTB already existing in the cytoplasm and to the de novo production of PTB (or a biologically active fragment or derivative thereof) in a cell.
  • the level of PTB required in a cell that is necessary for stimulating production of secretory granules may vary from cell type to cell type.
  • a biologically active fragment of PTB refers to a portion of PTB that maintains the function of stimulation SG production.
  • the fragment comprises (i.e. preferably extending not more than 10 amino acids beyond) or consists of PTB RNA binding domains 3 and 4, encompassing amino acid 361-563.
  • the term "derivative" of PTB refers to a molecule that is modified in its primary amino acid sequence as compared to the naturally occurring PTB molecule or a fragment thereof as indicated above such as a mutein but retains the function of SG stimulation.
  • accession number 26294 in Unigene database at http://www.ncbi.nlm.nih.gov/entrez, and references cited therein, including: Gil.A., Sharp.P.A., Jamison, S.F. and Garcia- Blanco.M.A. (1991) Characterization of cDNAs encoding the polypyrimidine tract- binding protein. Genes Dev. 5, 1224-1236.
  • amino acids in certain positions of an amino acid sequence may be exchanged by different amino acids expected not to (essentially) change the higher order structure of the corresponding peptide or polypeptide.
  • Such an exchange includes the exchange of alanine and valine, for example.
  • the skilled artisan would primarily consider to exchange amino acids of the same polarity.
  • the active center of a peptide or polypeptide will not be affected by such an exchange.
  • the establishment or availability of the three-dimensional structure will assist the choice of options for exchanging certain amino acids without the concomitant expectation of a change in structure and/or function. Tests based on the teachings of this invention can be used to check for a change in the function of the peptide or polypeptide.
  • a derivative of PTB may be produced by peptidomimetics; see for further teaching in this regard, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
  • the present invention also relates to a method for reducing production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of reducing the presence and/or activity of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells.
  • PTB polypyrimidine tract binding protein
  • reducing production of secretory granules (SGs) in peptide hormone- secreting endocrine cells or neurons refers to the reduction or stop of a process in peptide hormone-secreting endocrine cells or neurons that leads to reduction of production of such granules of at least 10%, such as at least 20%, preferably at least 30%, more preferred at least 50%, even more preferred at least 75% and most preferred at least 95% as compared to a physiological, normal and optionally non- pathogenic status of corresponding endocrine cells or neurons.
  • PTB activity can now be treated by blocking PTB activity or by reducing PTB levels in the cytoplasm of appropriate cells.
  • diseases include hyperprolactinemia or acromegalia as well as depression or sleeping disorders.
  • the first two diseases result from tumors of endocrine cells that oversecrete certain hormones, such as prolactin or growth hormone in the case of the two diseases mentioned above.
  • it is intended to pursue the selective down-regulation of PTB in these cells to reduce hormone production. This measure will provide an assistant or alternative means for surgery, which is presently the therapy of choice. Reducing the level or activity of PTB or a biologically active fragment or derivative thereof may be accomplished by a variety of means all of which are to be considered as preferred embodiments in accordance with the invention.
  • These means preferably include inhibition of transcription, for example by using antisense constructs, RNAi, intercellular blocking by antibodies, inactivation of the PTB gene, to name some options.
  • reducing includes a reduction of at least 10%, preferably at least 30%, more preferred at least 50%, even more preferred at least 80% and particularly preferred at least 90% of the normal level. Most preferred is that the reduction of intracellular level is achieved to at least 95% such as at least 98%.
  • Reduction of the levels includes down-regulation of the gene by appropriate means as well as complete blockage of expression in further alternatives.
  • Prevention of the nucleoplasmic transport of PTB is another means of reducing the cytoplasmic levels of PTB. Further included are enhancement of degradation or prevention of phophorylation of PTB.
  • the present invention thus also relates to a method for reducing or blocking production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of reducing the presence and/or the activity of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells.
  • PTB polypyrimidine tract binding protein
  • the PTB or a fragment or derivative thereof or an agent promoting/reducing the presence/activity will, for these purposes, preferably be formulated into a pharmaceutical composition.
  • the pharmaceutical composition may, in general, be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s).
  • the pharmaceutical composition may be particularly useful for the treatment of any disease that is causally interrelated with lack of production, diminished production or retarded production of secretory granules (or, in other recited embodiments, with overproduction).
  • the pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients and/or diluents.
  • suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration.
  • said administration is carried out by injection and/or delivery, e.g., to a site in the pancreas or into a brain artery or directly into brain tissue.
  • the compositions may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the brain.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute.
  • compositions of the invention may be administered locally or systemically.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition may comprise further agents depending on the intended use of the pharmaceutical composition.
  • a nucleic acid molecule and preferably a DNA molecule encoding said PTB or fragment or derivative thereof or (poly) peptides used in the enhancement or reduction of presence or activity of PTB may formulated into a pharmaceutical composition.
  • the nucleic acid molecule is eventually to be introduced into the desired cells.
  • Appropriate formulations include those wherein 10 6 to 10 12 copies of the DNA molecule, advantageously comprised in an appropriate vector are administered per dose.
  • the vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells.
  • the nucleic acid molecules may be joined to a vector containing selectable markers for propagation in a host.
  • a plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon-based clusters, such as fullerens.
  • a virus it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells; see, for general information in this regard, Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (2001).
  • the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof.
  • Expression of said polynucleotide comprises transcription of the polynucleotide, preferably into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells (a less preferred embodiment) comprise, e.g., the lac, trp or tac promoter in E.
  • coli and examples for regulatory elements permitting expression in eukaryotic host cells (the more preferred embodiment) are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV- enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Preferred promoters are the natural promoter of PTB as well as promoters allowing the tissue specific expression of PTB such as promoters active in pancreatic ⁇ -cells or neuronal cells.
  • regulatory elements may also comprise transcription termination signals, such as the SV40-poIy-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM ⁇ , pRc/CMV, pcDNAI , pcDNA3 (Invitrogen), pSPORTI (GIBCO BRL).
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector into targeted cell population.
  • viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus.
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (2001).
  • the polynucleotides or vectors can be reconstituted into liposomes for delivery to target cells.
  • isolated fractions thereof refers to fractions of eukaryotic or prokaryotic cells or tissues which are capable of transcribing or transcribing and translating RNA from the vector.
  • Said fractions comprise proteins which are required for transcription of RNA or transcription of RNA and translation of said RNA into a polypeptide.
  • Said isolated fractions may be, e.g., nuclear and cytoplasmic fractions of eukaryotic cells such as of reticulocytes. Kits for transcribing and translating RNA which encompass the said isolated fractions of cells or tissues are commercially available, e.g., as TNT reticulolysate (Promega).
  • the vector may be a gene transfer or gene targeting vector.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in- vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911- 919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res.
  • said vectors and/or gene delivery systems are also described in gene therapy approaches in neurological tissue/cells (see, inter alia Bl ⁇ mer, J. Virology 71 (1997) 6641-6649) or in the hypothalamus (see, inter alia, Geddes, Front Neuroendocrinol. 20 (1999), 296-316 or Geddes, Nat. Med. 3 (1997), 1402-1404).
  • Further suitable gene therapy constructs for use in neurological cells/tissues are known in the art, for example in Meier (1999), J. Neuropathol. Exp. Neural. 58, 1099-1110.
  • the above described modes for introducing the vector into a desired target tissue or cell also find application if the introduction is for the purposes of gene therapy.
  • the nucleic acid molecules and vectors may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell.
  • viral vectors e.g. adenoviral, retroviral
  • electroporation e.g. ballistic
  • ballistic e.g. gene gun
  • a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention.
  • the introduction and gene therapeutic approach should, preferably, lead to the expression of a functional PTB or fragment or derivative thereof.
  • the method of the invention may be put into practice in vivo, ex vivo or in vitro (this holds also true for further embodiments of the invention discussed further below).
  • In vivo applications have been discussed above. Ex vivo applications refer, inter alia, to embodiments wherein cells or tissues are treated outside the body in order to stimulate the production of SGs in accordance with the invention and wherein said cells or tissues, upon successful stimulation or successful modification allowing the stimulation in vivo (for example, by way of an inducible promoter) subsequently are re-introduced into the body;
  • In vitro applications include those that allow the further investigation of the mechanism by which PTB stimulates SG production. These investigations will allow the identification of further molecules interacting with PTB and resulting in the production of SGs. Such further molecules also include members of a protein cascade that only indirectly interact with PTB.
  • the method further comprises promoting/reducing the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells.
  • the invention also envisages to alternatively or additionally promote the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells. Promoting the activity of PTB may be achieved by a variety of means wherein preferred means are addressed herein below and include the meaning of promoting PTB from an inactive or less active into an active or more active form. Additional means include the mutagenesis of the protein (e.g. on the DNA level by site-directed mutagenesis) followed by a test for an enhanced activity (which may be based on tests performed in the appended examples) thus enhancing the intrinsic activity of PTB. It is of note that the term PTB when used alone, in accordance with the invention would optionally also have the meaning of a fragment or derivative of PTB as defined elsewhere.
  • the invention contemplates solely the enhancement of the activity by any of the means discussed herein below or above.
  • reduction of PTB levels if reduction of PTB levels is desired, then it may be sufficient not to reduce the levels of PTB but to solely reduce the activity of the molecule.
  • a reduction of overproduction of SGs if a reduction of overproduction of SGs is envisaged, then either the presence or the activity of PTB or said fragment or derivative thereof or both may be reduced.
  • promotion/reduction comprises the promotion/reduction of the nucleocytoplasmic transport of PTB or of a biologically active fragment or derivative thereof.
  • promotion i.e., for example, enhancement of or instigation of nucleocytoplasmic transport is a particularly suitable means for stimulating production of SGs.
  • the nucleocytoplasmic transport of PTB or of said biological active fragment or derivative thereof will be reduced or abolished.
  • the promotion/reduction of the nucleocytoplasmic transport may be achieved by different means.
  • the promotion of the presence, activity and preferably nucleocytoplasmic transport comprises the activation of phosphorylation of PTB (or, if reduction is envisaged, activation of phosphorylation should be avoided or stalled.)
  • protein kinase A is a prime example of a phosphorylating agent and will thus separately be addressed below
  • the invention envisages the phophorylation by different kinases, alternatively or in addition to the phosphorylation by protein kinase A.
  • the means to effect phosphorylation by these different enzymes can be based on the same principles that are described in detail for protein kinase A below.
  • Activation of phosphorylation includes the de novo synthesis of any of these enzymes, the activation of inactive molecules, the prevention of degradation, the enhancement of stability and the enhancement of the intrinsic activity, e.g. by providing muteins of such enzymes.
  • PTB phosphorylation can also be enhanced by inhibiting the activity of a dephosphorylating agent, such as a phosphatase. Reduction of PTB presence or activity in the cytosol or its nucleocytoplasmic translocation could e.g. be induced by a) inhibiting the kinase activity; b) activating PTB dephosphorylation. This could be accomplished using similar strategies as for its phosphorylation, i.e through a specific dephosphorylating agent such as a phosphatase.
  • PKA phosphorylates serine residue 16 of PTB. It was further demonstrated in accordance with the invention that cAMP-dependent phosphorylation of PTB is ERK1/2 independent.
  • said promotion of the nucleocytoplasmic transport of PTB or of said biologically active fragment or derivative thereof is effected by promoting the presence or activity of protein kinase A (PKA) in said cell.
  • PKA protein kinase A
  • Protein kinase A has been shown in the art to activate PTB.
  • the term "promotion of the presence” has the same meaning in accordance with this invention as explained above and elsewhere in connection with the promotion of the presence of PTB, with the exception, of course, that here the promotion of the presence of PKA is addressed.
  • promoting the presence of PKA includes the prevention or reduction of PKA degradation, or enhancing the stability of PKA.
  • PKA does not only include naturally occurring PKA but, in addition, molecules that retain the kinase function and substrate specificity of naturally occurring PKA. Such molecules include fragments or derivatives such as muteins of naturally occurring PKA.
  • promotion of the presence of protein kinase A in said cell is effected by overexpression or activation of protein kinase A.
  • Promotion of the activity includes enhancement of the intrinsic activity of the enzyme. Again, if reduction is desired instead of promotion, the presence or activity of protein kinase A should be diminished or abolished.
  • cAMP influences phosphorylation of PTB in INS-1 cells and induces ICA512 expression said neuroendocrine cell line. If reduction of the presence of PTB is desired, then cAMP levels should be decreased or the cell should be contacted with a drug that decreases or abolishes the expression of PKA in said cell.
  • cAMP also refers to derivatives of cAMP having the same or essentially the same biological function and that are preferably well known in the art.
  • cAMP levels in the cell may be increased by conventional means. Drugs that induce the overexpression of PTB or lead to in increase of cAMP levels include cAMP analogs that are commercially available. cAMP levels can also be increased by activators of adenylate cyclase, which produces cAMP, or by ligands of the G- protein coupled receptors, which, in turn, stimulate adenylate cyclase activity. Another way to increase cAMP levels is to inhibit the activity of phosphodiesterase, which hydrolizes cAMP into AMP.
  • PKA activity may be favored by reducing agents, such as DTT or ⁇ -mercaptoethanol, while oxidative agents inhibit PKA activity by promoting its glutathionylation (see: Humphries KM, Juliano C, Taylor SS. 2002. Regulation of cAMP-dependent protein kinase activity by glutathionylation. J Biol Chem. 277:43505-11 ; and Kopperud R, Krakstad C, Selheim F, Doskeland SO. 2003 cAMP effector mechanisms. Novel twists for an 'old' signaling system. FEBS Lett. 3;546:121-6). All these possibilities are included within the scope of the present invention.
  • the reverse means as outlined above may be employed.
  • Another preferred embodiment of the present invention relates to a method wherein the promotion comprises the transient or stable expression of PTB or of said biologically active fragment or derivative thereof from an exogenously introduced vector.
  • This preferred embodiment of the invention may be put into practice according to conventional techniques of molecular biology and requires, optionally after cloning of the PTB gene and insertion into a vector, the introduction into the cell (see above) and the expression of PTB or of said biologically active fragment or derivative thereof from an expression vector.
  • Expression vectors useful in this embodiment of the invention and options for the introduction of such vectors into suitable cells have been described herein above.
  • the promotion comprises enhancing the stability of PTB or of said biologically active fragment or derivative thereof or reducing degradation of PTB or of said biologically active fragment or derivative thereof in said cell. If reduction is required, then the corresponding embodiment comprises decreasing the stability of PTB or of said biologically active fragment or derivative thereof or stimulating degradation or said biologically active fragment or derivative thereof in said cell.
  • the enhancement of the stability of the protein may be achieved by agents that inhibit proteasome activity. Reduction of the degradation comprises methods wherein PTB degrading proteins are inhibited such as by RNAi or antisense oligonucleotides [see, for example, 25].
  • the enhancement and reduction respectively, amount to at least 25% of the amount of PTB present in the cytoplasm, preferably at least 50%, more preferred at least 75% and most preferred at least 90%.
  • an increase of more than 100% is advantageously envisaged.
  • a further preferred embodiment of the invention relates to a method wherein the promotion comprises introducing PTB or a biologically active fragment or derivative thereof into the cell.
  • the protein or peptidomimetic is directly introduced into the desired cell or tissue.
  • Available processes for such an introduction include microinjection techniques or protein transduction procedures. See, for example, Lindsay MA. Peptide-mediated cell delivery: application in protein target validation. 2002. Curr Opin Pharmacol. 2:587-94.
  • the promotion of the presence or activity of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells comprises the inhibition of dephosphorylation of PTB (or in the case of reduction the stimulation of dephosphorylation of PTB) or said fragment or derivative (as long as said derivative is a (poly)peptide, i.e. a peptide (up to 30 amino acids) or a polypeptide, the term "polypeptide" having the same meaning therein as the term "protein).
  • PTB polypyrimidine tract binding protein
  • said PTB is the 59 kD isoform having four RNA recognition motif domains of PTB. This is the largest isoform arising from several alternatively spliced variants [44,45]. Particularly preferred is a method wherein said endocrine cells to be employed in accordance with the invention are pancreatic ⁇ -cells.
  • said promotion is effected by glucose or glucagon-like peptide-1 (GLP-1).
  • GLP-1 Glucagon-like peptide-1
  • GLP-1 is produced by intestinal L-cells, which secrete it in response to food intake.
  • the hormone affects the physiology of multiple organs, including pancreatic islets. Specifically, it rapidly potentiates insulin secretion and its biosynthesis. It also promotes islet cell growth and islet neogenesis.
  • said method requires that the secretory peptide contained in said secretory granules is insulin, amylin or a peptide hormone or neuropeptide derived from one of the following precursors: ADM precursor, Agouti switch protein precursor, Agouti-related protein precursor, Apelin precursor, Atrial natriuretic factors, Beta-neoendorphin-dynorphin precursor, Brain natriuretic peptide precursor, Calcitonin gene-related peptide I precursor, Calcitonin gene-related peptide, II precursor, Calcitonin precursor, Cholecystokinin precursor, Chromogranin A precursor, Cocaine- and amphetamine- regulated transcript protein precursor, Corticoliberin precursor, Corticotropin- lipotropin precursor, Cortistatin precursor, FMRFamide-related peptides precursor, FMRFamide-related peptides precursor, Follistatin precursor, Follitropin beta chain precursor, Galanin
  • Glicentin-related polypeptide (residues 21-50): RSLQDTEEKS RSFSASQADP LSDPDQMNED b) Glucagon (residues 53-81): HSQGTFTS DYSKYLDSRR AQDFVQWLMN T c) Glucagon-like peptide 1 (residues 98-127): HAE GTFTSDVSSY LEGQAAKEFI AWLVKGR d) Glucagon-like peptide 2 (residues 146-178): HADGS FSDEMNTILD NLAARDFINW LIQTKITD
  • Corticoliberin (residues 154-194 ): SEEPPIS LDLTFHLLRE VLEMARAEQL AQQAHSNRKL MEII
  • NPP (residues 27-102): WCLE SSQCQDLTTE SNLLECIRAC KPDLSAETPM FPGNGDEQPL TENPRKYVMG HFRWDRFGRR NSSSSGSSGA GQ b) Melanotropin gamma (residues 77-87): YVMG HFRWDRF c) melanotropin alpha (residues 138-150): SYS MEHFRWGKPV d) Corticotropin (residues 138-176): SYS MEHFRWGKPV GKKRRPVKVY PNGAEDESAE AFPLEF e) Lipotropin beta (residues 179-267): EL TGQRLREGDG PDGPADDGAG AQADLEHSLL VAAEKKDEGP YRMEHFRWGS PPKDKRY
  • Orexin A (residues 34-66): QPLPDCC RQKTCSCRLY ELLHGAGNHA AGILTL b) Orexin B (residues 70-97): R SGPPGLQGRL QRLLQASGNH AAGILTM
  • Ghrelin (residues 24-51 ): GSSFLSP EHQRVQQRKE SKKPPAKLQP R
  • a comprehensive list of accession numbers associated with the above hormones, neuropeptides and precursors thereof is provided by Table 3. Further information about the hormones/peptides/precursors can be obtained from these sources.
  • a list of diseases associated with the hormones/peptides/precursors is provided by Table 4. It is to be understood in accordance with the present invention, that the invention comprises the treatment of any of the diseases associated with any of these hormones/peptides/precursors by manipulating PTB presence/activity as described for the treatment of type-1 and type-2 diabetes, sleeping disorders or depression elsewhere in this specification with the exception that different cells are targeted, if appropriate.
  • the skilled artisan is well aware which type of cell is to be targeted for each disease; see also the above cited textbooks for further guidance.
  • the present invention also relates to a method of treating or preventing type-1 or type-2 diabetes comprising stimulating production of insulin-containing secretory granules in pancreatic ⁇ -cells wherein said stimulation comprises the step of promoting the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in the cytoplasm of said ⁇ -cells.
  • PTB polypyrimidine tract binding protein
  • the promotion of the presence is mandatory whereas the promotion of the activity is optional.
  • the invention contemplates solely the enhancement of the activity by any of the means discussed herein above. The same holds true for any of the further embodiments referred to in this specification wherein the feature "promoting the presence or activity" occurs, also in connection with other compounds such as protein kinase A.
  • the present invention relates to the use of an agent of the promotion of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in ⁇ -cells for the preparation of a pharmaceutical composition for treating or preventing type-1 or type-2 diabetes.
  • PTB polypyrimidine tract binding protein
  • the invention further relates to the use of an agent of the reduction of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in hypothalamic neurons for the preparation of a pharmaceutical composition for treating or preventing sleep disorders or depression.
  • PTB polypyrimidine tract binding protein
  • cortocoliberin also known as corticotropin-releasing hormone (CRH)
  • CRH corticotropin-releasing hormone
  • CRH corticotropin-releasing hormone
  • ACTH and ⁇ -endorphin corticotropin-releasing hormones
  • Excess release of CRH has been implicated in sleep disorders and depression. Reducing the presence/activity of PTB in the cytoplasm of hypothalamic neurons is therefore expected to be beneficial for the treatment of these common disorders, by downregulating CHR expression and release from SGs.
  • transgenic mouse in which the presence /activity of PTB or part thereof in the cytoplasm of hypothalamic neurons is enhanced is expected to establish an animal model for sleep disorder and depression.
  • These animal models could be useful for the screening of drugs for the therapy of sleep disorders and depression in humans.
  • said agent is GLP-1 (for diabetes) or CRH (for sleeping disorders or depression).
  • agents are disclosed throughout this specification. If reduction is desired, then, also siRNA technology, antibodies, ribozymes, antisense molecules, derivatives or fragments of antibodies such as Fab fragments, F(ab')2, scFvs, protease, etc. and other means described herein may be used. Similarly, if promotion of the presence or activity is desired, further means have been described throughout this specification.
  • the promotion in the case of diabetes treatment, also for the following preferred embodiments
  • reduction for sleeping disorders or depression, also for the following preferred embodiments
  • the promotion/reduction comprises the activation/reduction or prevention of phosphorylation of PTB or said fragment or derivative thereof (in the case of sleeping disorders and depression, dephosphorylation is also considered).
  • said promotion/reduction of the nucleocytoplasmic transport of PTB or of said biologically active fragment or derivative thereof is effected by promoting/reducing the presence or activity of protein kinase A in said cell.
  • the promotion/reduction of the presence or activity of protein kinase A in said cell is effected by the oyerexpression or activation of protein kinase A/reduction of expression of protein kinase A.
  • said overexpression of protein kinase A is effected by overexpressing PKA from a vector, by increasing cAMP levels in the cell or by contacting the cell with a drug that induces the overexpression PKA or the increase of cAMP levels in said cell.
  • a drug that induces the overexpression PKA or the increase of cAMP levels in said cell In the case of reducing PTB levels, preferably cAMP levels will be decreased.
  • Still another preferred embodiment of the latter method of the invention or the use of the invention requires that the promotion comprises the transient or stable expression of PTB or of said biologically active fragment or derivative thereof from an exogenously introduced vector.
  • An additional preferred embodiment of the method of the invention or the use of the invention necessitates that the promotion/reduction comprises enhancing/reducing the stability of PTB or of said biologically active fragment or derivative thereof in said cell or reducing/enhancing degradation of PTB or of said biologically active fragment or derivative thereof in said cell.
  • the promotion comprises introducing PTB or a biologically active fragment or derivative thereof into the cell.
  • a further preferred embodiment of the invention requires a method or the use wherein the promotion/reduction comprises the inhibition/activation of dephosphorylation of PTB.
  • the present invention relates to a method of screening for an agent capable of stimulating production of secretory granules in peptide hormone- secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds promote the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell.
  • Contacting of the cell in step (a) will be done, for example, under conditions that allow the uptake of the one or more compounds into the cell.
  • conditions are provided where the compounds are allowed to interact with the surface receptors of the plasma membrane.
  • the conditions may be the same.
  • Appropriate conditions include physiological conditions such as incubation in physiological saline. If more than one compound is contacted with the cell and the number of compounds tests positive in step (b), it is advantageous to repeat the experiment by incubating the cell with only one compound at the time. In so far, a direct relation between the compound tested and its capability to promote the presence of PTB can be established.
  • the assessment in step (b) can be established by a variety of means.
  • the presence of PTB in the cytoplasm can be observed by microscopic means or biochemical subcellular fractionation. If microscopic assessment is envisaged, it is advantageous to label PTB or said biologically active fragment or derivative thereof.
  • Means of labeling proteins and cells are well known in the art and include labeling by tags such as myc-tag, His-tag or Flag-tag. Labels further include phosphorescent or fluorescent labels. Activity can be measured, e.g by the increase of SGs in the cells.
  • An additional means envisaged by the present invention is to test the binding activity of PTB to mRNA encoding secretory granule components, such as ICA512, and stabilization or translation of mRNA encoding secretory granule components, such as ICA512, upon PTB binding.
  • Said method of screening can be accomplished in a high-throughput manner.
  • Robotic equipment for that purpose is known in the art and available from a number of suppliers.
  • Cells are grown in wells of plates containing arrays of 96, 384, 1536 or more wells. Transfer of the well-plates from incubators, addition of test compounds, optional washing steps as well determining the read-out is performed in an automated fashion without requiring user interference using hundreds of thousands to millions of compounds in days to weeks.
  • the present invention relates to a method of screening for an agent capable of reducing production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds reduce the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell.
  • PTB polypyrimidine tract binding protein
  • the invention pertains to a method for screening for an agent useful as a cure for sleeping disorders or depression comprising the steps of , (a) contacting an animal carrying a transgene encoding polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof under the control of a promoter that is active in hypothalamic neurons with one or more compounds; and (b) assessing whether said one or more compounds reduce the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said neurons.
  • PTB polypyrimidine tract binding protein
  • Monitoring of presence or activity, respectively, of PTB or of said biologically active fragment or derivative thereof may be effected by determining the level of protein expression of PTB or of said biologically active fragment or derivative thereof or by determining the phosphorylation status thereof, respectively, in a sample taken from the transgenic animal by using, for example, methods disclosed herein and exemplified in the Examples.
  • the agent to be screened can be contained in libraries of small molecules, such as organic or inorganic small molecules which may be commercially available.
  • libraries comprising antibodies or functional fragments or derivatives thereof i.e. fragments or derivatives maintaining the binding specificity of the original antibody
  • libraries of aptamers or peptides might be employed. The skilled artisan is of course free to use any other starting point of desired compounds for use in the screen assays described throughout the specification.
  • the animal to be employed is a non-human animal.
  • rodent hypothalamic neurons carrying the transgene can be isolated and grown in culture. Their response to drugs in term of PTB presence in the cytoplasm, expression of CRH and secretory granules may be monitored by microscopy or biochemically.
  • the invention pertains to a method for screening for an agent useful as a cure for type-1 or type-2 diabetes comprising the steps of (a) contacting an anirrial carrying a transgene encoding polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof under the control of a promoter that is active in pancreatic ⁇ - cells with one or more compounds; and (b) assessing whether said one or more compounds increase the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cells.
  • PTB polypyrimidine tract binding protein
  • said promoter is an inducible promoter.
  • inducible promoter is well known in the art and denotes a promoter the activity of which can be influenced by external parameters such as upon the addition of a drug.
  • vasopressin promoter is the vasopressin promoter.
  • this vasopressin gene promoter may be used (see: Murphy, D. & Wells, S. 2003. In Vivo Gene Transfer Studies on the Regulation and Function of the Vasopressin and Oxytocin Genes. Journal of Neuroendocrinology 15, 109-125).
  • CRH and vasopressin are produced by the same hypothalamic neurons (see: Whitnall MH, Mezey E, Gainer H. 1985. Co- localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles. Nature 317 :248-50).
  • said derivative of PTB is a fusion protein of PTB or a biologically active fragment thereof and a detectable marker.
  • fusion proteins The making of fusion proteins is well known in the art and requires the availability of the coding sequence of PTB and the fusion partner.
  • a nucleic acid molecule encoding a fusion protein may be prepared.
  • PTB may be fused, for example, to a marker such as a tag.
  • Appropriate tags have been discussed above, and include FLAG, Myc, HIS and others. Markers that can be used in accordance with the invention also include radioactive markers or phosphorescent markers.
  • detectable marker is a fluorescent compound.
  • Appropriate fluorescent compounds include Green Fluorescent Protein (GFP) and derivatives thereof.
  • step (b) comprises the assessment of nucleocytoplasmic transport of PTB.
  • said endocrine cells are pancreatic ⁇ -cells.
  • a particularly preferred embodiment is a method wherein the peptide is insulin.
  • said method further comprises the step of
  • This embodiment of the invention advantageously allows the quantitative assessment of the tested compound with respect to its capability to induce/reduce SG formation.
  • said one or more compounds are members of a library of compounds.
  • Useful libraries include those libraries of small molecules, either organic or inorganic or of peptides. Suitable libraries are commercially available, for example from
  • the method of the invention thus also allows for testing whether the compound may affect not only the presence of PTB in the cell but also whether it can (de)activate
  • step (c) comprises the step of
  • step (d) testing the efficacy of a compound assessed as being capable of promoting or reducing the presence or activity of PTB in step (b) in the stimulating of the production of secretory granules in peptide hormone-secreting endocrine cells or neurons in an animal model.
  • This additional step will allow significant insights in the in vivo applicability of the compound that tested positive in an in vitro assay. Parameters like toxicity, half-life etc. that have to be tested in pre-clinical trials may be assessed in this step. Suitable
  • non-human animals foremost include laboratory mice and rats such as available from Charles River Laboratories etc.
  • the invention envisages that the method comprises the further step of improvement or of refining the pharmacological properties of the identified promoting or reducing agent (in the following also referred to as “compound” or “drug”) , by the method as described herein above, said method comprising the optionally the steps of said methods and:
  • Steps (1) and (2) can be carried out according to conventional protocols.
  • a protocol for site directed mutagenesis is described in Ling MM, Robinson BH. (1997) Anal. Biochem. 254: 157-178.
  • the use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships is reviewed in Szklarz and Halpert (1997) Life Sci.
  • Chimeric proteins are generated by ligation of the corresponding DNA fragments via a unique restriction site using the conventional cloning techniques described in Sambrook (1989), loc. cit..
  • a fusion of two DNA fragments that results in a chimeric DNA fragment encoding a chimeric protein can also be generated using the gateway- system (Life technologies), a system that is based on DNA fusion by recombination.
  • gateway- system Life technologies
  • a prominent example of molecular modeling is the structure-based design of compounds binding to HIV reverse transcriptase that is reviewed in Mao, Sudbeck, Venkatachalam and Uckun (2000). Biochem. Pharmacol. 60: 1251-1265.
  • identification of the binding site of said drug by site-directed mutagenesis and chimerical protein studies can be achieved by modifications in the PTB primary sequence that affect the drug affinity; this usually allows to precisely map the binding pocket for the drug.
  • step (2) the following protocols may be envisaged: Once the effector site for drugs has been mapped, the precise residues interacting with different parts of the drug can be identified by combination of the information obtained from mutagenesis studies (step (1)) and computer simulations of the structure of the binding site provided that the precise three-dimensional structure of the drug is known (if not, it can be predicted by computational simulation). If said drug is itself a peptide, it can be also mutated to determine which residues interact with other residues in the (poly)peptide of interest.
  • the drug can be modified to improve its binding affinity or its potency and specificity. If, for instance, there are electrostatic interactions between a particular residue of the PTB of interest and some region of the drug molecule, the overall charge in that region can be modified to increase that particular interaction.
  • Identification of binding sites may be assisted by computer programs. Thus, appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the (poly)peptide by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.
  • activators of the expression of PTB can be used for the design of peptidomimetic activators (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).
  • said pharmacological properties of the identified inhibitor or antagonist is further improved or refined by peptidomimetics.
  • the method of the invention can further include modifying a compound identified, improved of refined by the method as described herein above as a lead compound to achieve (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g.
  • the invention moreover contemplates in another preferred embodiment a method wherein the compound identified and optionally further modified as indicated above is formulated into a pharmaceutical composition.
  • FIG. 1 Glucose-stimulated induction of pro-ICA512 in insulinoma and rat islet cells.
  • a-d Western blots for pro-ICA512 and g-tubulin on Triton X-100 extracts of INS-1 cells (a, b) or purified pancreatic islets (c, d) kept either in resting buffer or stimulated with 25mM glucose for the indicated times, with (b) or without 5 ⁇ g/ml actinomycin D (AmD) (a, c, d). Cells were incubated at 37 °C (a, b, c) or at 19 °C (d).
  • Figure 2 Stabilization of rat ICA512 mRNA by PTB.
  • a Quantitative RT-PCR for ICA512 and insulin with 1mg total RNA from resting or glucose-stimulated INS-1 cells. Amounts were normalized against b-actin.
  • b c, Autoradiographies (b) and quantification by phosphoimaging (c) of 32 P-labeled ICA512 mRNA 3'-UTR incubated for the indicated times with cytosolic extracts from INS-1 cells or islets which were either kept in resting buffer or glucose-stimulated for 60 or 120 min.
  • Electrophoretic mobility shift assays of biotinylated RNA-oligonucleotides including either the wild-type (wt1 , wt2) or mutated (ml , m2) first or second consensus sites for PTB binding in the 3'- UTR of ICA512 mRNA.
  • EMSA Electrophoretic mobility shift assays
  • oligonucleotides Prior to EMSA, oligonucleotides were incubated with cytosolic extracts from resting (-) or glucose- stimulated (+) INS-1 cells, e, Binding of wt1 or wt2 oligonucleotides to PTB as measured by ELISA.
  • PTB was captured with a specific antibody from cytosolic extracts of resting or 120 min glucose-stimulated INS-1 cells
  • f Immunoblots for PTB and D-tubulin in INS-1 cell cytosolic extracts: lane 1 , 120 min resting cells; lane 2: 120 min stimulated cells; lane 3: 120 min stimulated cells + PTB-immunodepletion; lane 4: 120 min stimulated cells + mock-immunodepletion.
  • g Binding of 32 P-labeled full-length ICA512 mRNA 3'-UTR to the cytosolic extracts 1-4 shown in (f).
  • h Decay assays as in (c) upon incubation of ICA512 mRNA 3'-UTR with cytosolic extracts 1-4 shown in (f).
  • FIG. 3 Stability of PC1/3, PC2 and CPE mRNAs. Quantification by phosphoimaging of 32 P-labeled PC1/3, PC2 and CPE mRNA 3'-UTRs incubated for the indicated times with cytosolic extracts from INS-1 cells which were either kept in resting buffer or glucose-stimulated for 60 or 120 min.
  • Figure 4 Depletion of SGs in INS-1 cells upon RNA interference for PTB.
  • a, b Western blotting for PTB, various SG components (a) or housekeeping proteins (b) in INS-1 cells treated (T) or untreated (U) by RNAi for PTB.
  • c Insulin in the medium and in the total protein extracts of INS-1 cells treated or untreated by RNAi for PTB, as measured by RIA.
  • d Immunofluorescence for PTB (red), insulin (green) and ICA512 (red) in INS-1 cells treated or untreated by RNAi for PTB. Nuclei were counterstained in blue with DAPI.
  • e Electron micrographs of INS-1 cells untreated (U) and treated (T) by RNAi for PTB. In untreated INS-1 cells secretory granules (arrows) are mostly aligned along the plasma membrane.
  • Figure 5 Nucleocytoplasmic translocation of PTB upon glucose stimulation of b-cells.
  • a-c Low (a, c) and high power (b) immunofluorescence images for PTB (red) in INS- 1 cells (a, b) and islets (c) incubated either in resting buffer or glucose stimulated for 120 min. Nuclei were counterstained in blue with DAPI.
  • d, e Western blotting for PTB and g-tubulin on both nuclear and cytosolic fractions (d) or cytosolic fractions only (e) from islets that were incubated in resting buffer or glucose-stimulated for 120 min. Islets were cultured in vitro for 1 day (d) or 3, 6 and 9 days (e).
  • FIG. 6 Glucose-regulated luciferase expression in INS-1 cells upon inclusion of PTB binding sites.
  • a Schematic drawing of firefly luciferase cDNA constructs in pGL3-Basic.
  • pGL3 PC2 3'-UTR and pGL3 PC2 5'-UTR included the corresponding UTRs from rat PC2.
  • Two additional pGL3 constructs were generated with mutations in the PTB binding sites of PC2 (pGL3 PC2 3'-UTR mut and pGL3 PC2 5'-UTR mut).
  • b RLU in resting or 120 min stimulated INS-1 cells transfected with the pGL3 constucts shown in (a).
  • RLU in resting or stimulated INS-1 cells transfected with pGL3-Basic was equal to 100 %.
  • Electrophoretic mobility shift assay of the biotinylated RNA- oligonucleotide wt1 following incubation with nuclear extracts from INS-1 cells kept at rest or glucose stimulated for 30, 60 or 120 min. There was no electrophoretic mobility shift of the oligo wt1 , indicating that nuclear PTB, even from stimulated cells, was not competent for binding the corresponding consensus motif in ICA512 mRNA 3'-UTR.
  • FIG. 8a Western blotting for cytosolic and nuclear PTB, chromogranin A and ⁇ - tubulin in .
  • INS-1 cells untreated (-) or treated with siRNA oligos 1+2, 3 or 4 for PTB. Left and right panels are from different experiments.
  • the additional siRNA oligos 3 and 4 for rat PTB were selected by Cenix Biosciences with a proprietary algorithm and chemically synthesized by Ambion.
  • siRNA oligo 3 sense oligo, 5'- GGUGAUAACAGGAGCACAGdTdT; antisense oligo, 5'-
  • siRNA oligo 4 sense oligo, 5'-
  • b Quantitation of PTB, SG and control proteins upon transfection of INS-1 cells with siRNA oligos 3 and 4 for PTB.
  • Cells were transfected with either 1 ⁇ g siRNA oligo 3 or 4. All proteins were quantified by western blotting and normalized against ⁇ -tubulin, except insulin, which was measured by RIA. The values are from two independent experiments, c, Quantitation of SG proteins and luciferase activity upon treatment of INS-1 cells with the indicated siRNA oligos.
  • Control scrambled 21-mer siRNA oligos were synthesized with the Silencer siRNA Construction Kit (Ambion) using the following cDNA primers: sense primer, 5'-AATGCTCGACATGACAGACGGCCTGTCTC; antisense primer, 5'- AACCGTCTGTCATGTCGAGCACCTGTCTC.
  • Firefly luciferase (F-Luc) was knockdown by RNAi with the following chemically synthesized siRNA oligo (kind gift from Cenix Bioscience): sense oligo, 5'-CUUACGCUGAGUACUUCGA-dTdT; antisense oligo, 5'-UCGAAGUACUCAGCGUAAG-dTdT.
  • INS-1 cells Two days before transfection of siRNA oligo for firefly luciferase, INS-1 cells were co-transfected by electroporation (Amaxa) with pGL3-Basic and phRL vectors for co-expression of firefly and renilla luciferase, respectively. Insulin content was measured by RIA, while the other proteins were quantified by immunoblotting and normalized against ⁇ - tubulin.
  • results shown are from six (siRNA oligos 1+2 for PTB or control scrambled siRNA oligos) or four (siRNA oligo for firefly luciferase) independent experiments, d, Western blotting for the ER markers calnexin and PDI in INS-1 cells incubated with resting (R) or stimulating (S) buffer for 120 min. Unlike most SG proteins, calnexin and PDI are not up-regulated by glucose stimulation, despite the presence of a consensus motif for PTB binding in their mRNA 3'-UTR. Equal loading of proteins was verified by western blotting for ⁇ -tubulin. e, Immunofluorescence for PTB (red), insulin (green) and ICA512 (red) in INS-1 cells untransfected or transfected with siRNA oligos 3 or 4. Nuclei were counterstained in blue with DAPI.
  • Figure 9 RT-PCR for PTB on 1 ⁇ g total RNA from INS-1 cells, rat islets and Jurkat cells.
  • RNA templates were obtained from cells kept at rest or glucose-stimulated for the indicated times.
  • specific primers flanking the entire open reading frame of rat PTB were used: forward, 5'-ATGGACGGCATCGTCCCAG; reverse, 5'-
  • FIG. 10 cAMP-dependent induction of ICA512 and other secretory granule markers in INS-1 cells.
  • A mRNA expression of insulin, ICA512, PC1 and CPE.
  • B to D protein expression of ICA512.
  • PTB translocation induces the expression of insulin secretory granule marker.
  • Glucose-stimulated activation of PTB promotes the stability of ICA512 mRNA and upregulates ICA512 expression.
  • the findings made in accordance with the invention originated from the study of ICA512/IA-2, a receptor tyrosine phosphatase-like protein associated with insulin SGs, and neurosecretory granules in general [21]. Cleavage by a furin-like convertase of pro-ICA512 leads to the generation of a transmembrane fragment (ICA512 TMF) of 65 kD, which is enriched in SGs [21 ,22]. Glucose stimulation of rat insulinoma INS-1 cells induces the biosynthesis of pro-ICA512 [23]. This induction was already apparent in cells stimulated for 30 minutes and persisted upon stimulation for 120 minutes.
  • ICA512 mRNA was increased 1.46, 1.33 and 2.31 folds in INS-1 cells stimulated with 25 mM glucose for 30, 60 and 120 minutes, respectively. This increase exceeded that of insulin mRNA, which after 120 minutes stimulation was enhanced 1.43 fold (Fig. 2a).
  • glucose stimulation promoted the stability of ICA512 mRNA.
  • the labeled 3'-UTR of rat ICA512 mRNA was incubated with cytosolic extracts from INS-1 cells that have been either incubated in resting buffer (no glucose) or with 25 mM glucose for 30, 60 or 120 minutes.
  • ICA512 mRNA 3'-UTR contains two evolutionary conserved consensus binding sites for PTB (fig. 2d). We asked then whether glucose stimulation stabilizes ICA512 mRNA by promoting the binding of PTB to its 3'-UTR.
  • the cytosolic extracts of cells stimulated for 120 minutes displayed a binding activity that caused a similar retardation of both oligonucleotides in electrophoretic mobility shift assays (fig. 2d, arrows).
  • ICA512 mRNA as measured by quantitative real-time PCR is induced by 1mM IBMX, which inhibits phosphodiesterase activity and therefore leads to an increase of intracellular cAMP levels.
  • IBMX induction was also observed for another SG protein (prohormone convertase 1/3, PC1/3), but not in case of carboxypeptidase E, another SG marker.
  • IBMX induction of ICA512 and PC1/3 mRNAs was prevented by treatment with H89, which inhibits cAMP-dependent PKA. Furthermore, it has been found that the glucose- and cAMP-induced expression of ICA512 are additive. Specifically, Panel B in Fig.
  • Glucose-stimulation enhances the stability of mRNAs encoding secretory granule proteins
  • RNA decay assays showed that glucose stimulation stabilizes rat PC1/3 (85% increase) and PC2 (90% increase) mRNA 3'- UTRs even more effectively than ICA512 mRNA 3'-UTR (fig. 3), while it had no effect on the stability of carboxypeptidase E (CPE) mRNA 3'-UTR.
  • CPE carboxypeptidase E
  • CPE is a protease that is partly responsible for the processing of insulin within SGs, but whose expression is not glucose-regulated.
  • the 3'-UTR of CPE mRNA does not contain any consensus for PTB binding and was significantly more stable than the 3'-UTR of ICA12, PC1/3 and PC2 mRNAs.
  • PTB is necessary for secretory granule biogenesis
  • RNA interference RNA interference
  • both rat calnexin and PDI mRNA 3'-UTRs contain a consensus for PTB binding, and yet their expression was not stimulated by glucose (fig. 8d). Consistent with the immunoblot results, light immunomicroscopy on RNAi- treated cells showed a significant reduction of PTB and even more dramatically of insulin and ICA512 (fig. 4d and 8e). These data suggested that PTB may affect the biogenesis of SGs. Quantification by electron microscopy confirmed the virtual absence of SGs in 60% of the cells treated by RNAi for PTB (fig. 4e and table 2).
  • Cells treated by RNAi for PTB could clearly be distinguished in two categories.
  • One group of cells (60%) contained up to 2 granules, with an average number of 0.3 ⁇ 0.1 granules.
  • the other group (40%) contained at least 7 granules / cell profile and had an average number of granules (16.6 ⁇ 1.1) that was not different from the control cells.
  • these numbers correlate well with the transfection efficiency measured by fluorescence microscopy, cells with ⁇ 2 granules are likely to represent cells in which PTB expression was affected by RNAi.
  • Glucose-stimulation induces the nucleocytoplasmic translocation of PTB
  • PTB was identified in accordance with the invention as a factor required for the biogenesis of ⁇ -cell SGs.
  • the 3'-UTR of many SG components and other proteins associated with the neuroendocrine phenotype contains one or more consensus sites for PTB binding and at least in the case of PC2 also in the 5'-UTR.
  • Glucose stimulation of ⁇ -cells promotes the nucleocytoplasmic translocation of PTB as well as the stabilization of mRNAs encoding SG components by the cytoplasmic pool of PTB.
  • mRNA stabilization is a mechanism by which PTB up-regulates the expression of ⁇ -cell SG proteins in response to glucose.
  • PTB activation must depend therefore on other second messengers, such as cAMP, whose increased levels upon glucose metabolism have already been proposed to promote insulin biosynthesis [30,31].
  • cAMP second messengers
  • other post-transcriptional mechanisms should participate in the rapid stimulus- dependent up-regulation of insulin and peptide hormone expression, even before stabilization of their mRNAs by PTB.
  • Such mechanisms may include, for instance, the phosphorylation and dephosphorylation of factors implicated in cap-dependent mRNA translation [32,33].
  • PTB in ⁇ -cells is phosphorylated by PKA, whose activity is cAMP-dependent.
  • PTB includes a consensus site for phosphorylation by PKA on Ser16 within its nuclear export signal.
  • INS-1 cells were labeled with 32 P-orthophosphoric acid.
  • 32 P-labelled PTB was detected by autoradiography (Fig. 11A, top) and immunoblot (Fig. 11 A, middle) following PKA activation with IBMX, which increases cAMP levels.
  • Fig. 11 A bottom, shows the total amount of PTB detected with a pan-PTB antibody.
  • Panel B of Fig. 11 shows that detection of phospho-PTB by Western blot is sensitive to alkaline phosphatase (AP) treatment.
  • AP alkaline phosphatase
  • PKA can activate Erk1/2 through MEK1/2. These kinases, however, are not involved in PTB phosphorylation, because the phospho-PTB bands were still detected following treatment with PD98057 which inhibits MEK1/2 (Fig. 11C).
  • Fig. 12A in resting INS-1 cells, PTB is mostly found in the nucleus, as shown by the. co-localization with DAPI, a fluorescence dye which binds DNA. Stimulation with IBMX leads to the disappearance of PTB immunoreactivity in the nucleus and its increased detection in the cytosol. The nucleocytoplasmic translocation by IBMX can be prevented by co-treatment with H89.
  • Panel B shows that a PTB allele fused with GFP and in which Ser16 was converted into Ala is restricted to the nucleus. Conversely, a PTB mutant in which Ser16 was converted into Asp was mostly enriched in the cytosol.
  • Panel C shows that conversion of Ser16 into Ala correlates with inability of PTB to translocate from the nucleus into the cytosol upon IBMX treatment of INS-1 cells.
  • the first bar in Panel C is indicative of the transfection efficiency in INS-1 cells (around 50%).
  • Panel D shows that in resting INS-1 cells transfected PTB-GFP is restricted to the nucleus.
  • the protein Upon stimulation with IBMX, the protein translocates to the cytosol, similarly to endogenous PTB (as shown in Panel A). This translocation is not inhibited by RNA interference with a scrambled oligo.
  • INS-1 cells were transiently transfected with firefly luciferase constructs, which either carried the original 5'- and 3'-UTRs or also included the corresponding regions of rat PC2 mRNA or rat ICA512 mRNA (Fig. 6a and Fig. 13).
  • the choice of rat PC2 UTRs was convenient because its 3'-UTR and 5'-UTR both contain a single putative PTB binding site.
  • Inclusion of PC2 3'-UTR increased luciferase activity by 8.2 and 28.8 fold in resting and stimulating conditions, respectively (Fig. 6b).
  • firefly luciferase constructs including the 3' UTR PTB consensus binding site of ICA512 or PC2 was enhanced by treatment of INS-1 cells with 1 mM IBMX, as measured by luciferase activity (Fig. 13). Induction by IBMX of transfected luciferase construct including the 3' UTR of either ICA512 or PC2 was inhibited by co-treatment with 10 mM H89.
  • Pancreatic islets were isolated from female Wistar rats by collagenase digestion, purified by density gradient centrifugation, and cultured (400/60mm culture dish) as described previously [38]. INS-1 cells were grown as described [39].
  • cytosolic extracts of INS-1 cells were incubated overnight at 4 °C with 5 ⁇ g monoclonal antibody 1 directed against the C-terminal region of PTB (Zymed Lab) or mouse IgGs (BioRad) prior to the addition of protein G Sepharose (Pharmacia) and centrifugation. Protein concentration in the detergent soluble material was measured using the BCA assay (Pierce).
  • Cell extracts were separated by SDS-PAGE and immunoblotted as described [23] using the following antibodies: mouse monoclonals anti-PTB, anti- ICA512 23 , anti-calnexin (Transduction Laboratories), anti-chromogranin A (Immunon), anti-PDI (Stressgen), anti- ⁇ -tubulin and anti-insulin (Sigma), anti- synaptophysin and anti-synaptobrevin 2 (Synaptic Systems); rabbit polyclonals anti- secretogranin 2 (gift from Dr. W. Huttner), anti-mannosidase II 42 , anti-p58 43 , anti- PC1/3, anti-PC2, and anti-CPE (Chemicon). Chemiluminescence was performed using the Supersignal West Pico Substrate (Pierce) as substrate and detected with a LAS 3000 Bioimaging System (Fuji).
  • rat ICA512, PC1, PC2, and CPE mRNAs were subcloned into pCRII- TOPO with both T7 and SP6 promoters (Invitrogen).
  • [a- 32 P]-UTP labeled RNA was synthesized with the T7-MEGA script kit (Ambion). Decay assays were performed as described [40] with minor modifications by incubating 10 4 cpm labeled in-vitro transcripts with 10 ⁇ g cytosolic extracts from resting or stimulated cells for 0-240 minutes. Reactions were stopped with 6x gel loading buffer and separated on a 6% polyacrylamide-7M urea gel. Dried gels were exposed and quantified with a BAS 180011 phosphoimager (Fuji) using the Image Gauge v3.45 software.
  • EMSA electrophoretic mobility shift assays
  • 5'-ACUCUUCAGCCCCUACCCAUCUGCC (1° PTB binding site in rat ICA512, wt1)
  • 5'- ACUCUUCAGCAAAAAGGGAUCUGCC mutated l° PTB binding site, ml
  • 5'-UGUACCUCCCCACUCCCACCAGCCUA II 0 PTB binding site in rat ICA512, wt2
  • 5'-UGUACAUAGGAACUAGGACCAGCCUA mutant IT PTB binding site, m2
  • the binding reaction was carried out using the LightShift Chemoluminescent EMSA Kit (Pierce).
  • RNA oligonucleotides 10mg cytosolic extracts from resting or stimulated INS-1 cells were incubated with 5pmol RNA oligonucleotides in binding buffer including 100mM KCI and 1.5mM MgCI 2 . After 30 min incubation, 5U RNAse T1 were added to each reaction and incubated for 10 min. After additional 10 min incubation with 110mg heparin, the reaction was stopped with 5 ⁇ l loading buffer. Electrophoresis, blotting, UV cross-linking and detection were performed according to manufacturer's instructions.
  • RNA from 6x10 5 INS-1 cells was isolated with TRIZOL (Invitrogen) according to the manufacturer's protocol. 1mg of total RNA was used for the reverse transcriptase reaction with 2mM gene specific antisense primers for b-actin, ICA512 and insulin. The expression of mRNA was analyzed by quantitative real-time PCR using the LightCycler system (Roche Diagnostics) as described [41].
  • pGL3-Basic Promega encoding firefly luciferase and multiple site-directed mutagenesis of PTB binding sites were performed using standard protocols.
  • pGL3 constructs and phRL Promega encoding renilla luciferase were co-transfected into INS-1 cells with Lipofectamine (Invitrogen). Firefly luciferase activity was measured 4 days after transfection and normalized with that of renilla luciferase using the dual luciferase system (Promega) according to manufacturer's instructions.
  • INS-1 cells (6x10 5 /35 mm well) were grown for 2 days before transfection.
  • 21-mer dsRNA oligonucleotides for rat PTB mRNA were synthesized with the Silencer siRNA Construction Kit (Ambion) (for further details see [47]) using the following primers: sense primer 1 , 5'-AAGATACCTAGTGATGTCACTCCTGTCTC; sense primer 2, 5'- AAGGACCGCAAGATGGCACTGCCTGTCTC; antisense primer 1 , 5'- AAAGTGACATCACTAGGTATCCCTGTCTCTC; antisense primer 2, 5'- AACAGTGCCATCTTGCGGTCCCCTGTCTC).
  • INS-1 cells and islets were fixed with 4% paraformaldehyde. After embedding in gelatin, 5 ⁇ m islet sections were prepared. INS-1 cells and islet sections were then permeabilized with 0.2% saponin or 0.3% Triton X-100 and incubated with monoclonal antibodies against PTB, insulin (Sigma) or ICA512 [21] for 1 h. After washing and incubation with goat-anti-mouse Alexa488 or Alexa568 conjugated secondary antibodies (Molecular Probes) nuclei were counterstained with DAPI (Sigma). Images were acquired with a CoolSnap-HQ CCD camera (Roper Scientific) attached to an Olympus BX61 microscope and processed with Metamorph 4.65 (Universal Imaging).
  • INS-1 cells grown on glass coverslips were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard Epon embedding. Surface areas of cell sections were analyzed using a SIS Megaview camera with analysis software installed on a Tecnai12 electron microscope (FEI).
  • FEI Tecnai12 electron microscope
  • the total number of cells per group was from 3 independent experiments. As no differences between these experiments were found by ANOVA, all data were pooled. Statistical analysis was performed using a t-test or, in case variances were not equal, a Welch test. Cells in the RNAi-treated group were compared for granule content and size (p values in brackets) with cells in the untreated group. Each of the two distinct pools of cells in the RNAi-treated group was also independently compared to the untreated group (p values in brackets) and to each other (p values marked with an asterisk).
  • RNA polymerase III RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I. J Cell Biol 129, 1181-1193 (1995).
  • Ghetti, A., Pinol-Roma, S., Michael, W. M., Morandi, C. & Dreyfuss, G. hnRNP I the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs. Nucleic Acids Res 20, 3671-3678 (1992).

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Abstract

La présente invention porte sur un procédé de stimulation de la production de granules sécrétoires dans des cellules ou neurones endocrines sécrétant des hormones peptidiques consistant à favoriser la présence d'une protéine de liaison du tractus polypyrimidique PTB ou un fragment biologiquement actif ou son dérivé dans le cytoplasme desdites cellules ou neurones. Le procédé consiste de préférence à favoriser l'activité de la PTB ou dudit fragment biologiquement actif ou de son dérivé dans le cytoplasme desdites cellules ou neurones. A cet effet, il est préférable de favoriser le transport nucléocytoplasmique de la PTB. Selon une autre variante, l'invention porte sur un procédé de criblage d'un agent apte à stimuler la production de granules sécrétoires dans des cellules ou neurones endocrines sécrétant des hormones peptidiques consistant à (a) mettre en contact une cellule apte à former des granules sécrétoires et à exprimer la protéine de liaison au tractus polypyrimidique PTB ou un fragment biologiquement actif ou son dérivé avec un ou plusieurs composés; et (b) évaluer si un ou plusieurs composés favorisent la présence ou l'activité de ladite protéine PTB ou dudit fragment biologiquement actif ou de son dérivé dans le cytoplasme de ladite cellule. L'invention concerne en outre des procédés de criblage d'un agent que l'on utilise comme traitement pour le diabète, les troubles du sommeil ou la dépression ainsi que pour différents usages médicaux d'un agent apte à promouvoir/réduire la présence ou l'activité de la protéine PTB ou d'un fragment biologiquement actif ou de son dérivé. Selon d'autres modes de réalisation, la réduction ou la régulation par le bas de la PTB ou dudit fragment biologiquement actif ou de son dérivé est prise en considération.
PCT/EP2004/010167 2003-09-10 2004-09-10 Proteine de liaison du tractus polypyrimidique favorisant la biogenese d'un granule insulino-secretoire Ceased WO2005023231A1 (fr)

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CN106324259A (zh) * 2016-09-06 2017-01-11 中国科学院昆明动物研究所 Prokineticin 2作为银屑病生物标志物及其应用
US10828302B2 (en) 2016-03-10 2020-11-10 Janssen Pharmaceutica Nv Methods of treating depression using orexin-2 receptor antagonists
US11059828B2 (en) 2009-10-23 2021-07-13 Janssen Pharmaceutica Nv Disubstituted octahydropyrrolo[3,4-C]pyrroles as orexin receptor modulators
JPWO2023157460A1 (fr) * 2022-02-18 2023-08-24

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US11059828B2 (en) 2009-10-23 2021-07-13 Janssen Pharmaceutica Nv Disubstituted octahydropyrrolo[3,4-C]pyrroles as orexin receptor modulators
USRE48841E1 (en) 2009-10-23 2021-12-07 Janssen Pharmaceutica Nv Disubstituted octahydropyrrolo[3,4-c]pyrroles as orexin receptor modulators
US11667644B2 (en) 2009-10-23 2023-06-06 Janssen Pharmaceutica Nv Disubstituted octahydropyrrolo[3,4-c]pyrroles as orexin receptor modulators
US12378254B2 (en) 2009-10-23 2025-08-05 Janssen Pharmaceutica Nv Disubstituted octahydropyrrolo[3,4-c]pyrroles as orexin receptor modulators
CN103353527A (zh) * 2013-07-01 2013-10-16 浙江大学 检测肿瘤侵袭性的pnc检测试剂盒及其应用
US10828302B2 (en) 2016-03-10 2020-11-10 Janssen Pharmaceutica Nv Methods of treating depression using orexin-2 receptor antagonists
US11241432B2 (en) 2016-03-10 2022-02-08 Janssen Pharmaceutica Nv Methods of treating depression using orexin-2 receptor antagonists
US12201634B2 (en) 2016-03-10 2025-01-21 Janssen Pharmaceutica Nv Methods of treating depression using orexin-2 receptor antagonists
CN106324259A (zh) * 2016-09-06 2017-01-11 中国科学院昆明动物研究所 Prokineticin 2作为银屑病生物标志物及其应用
JPWO2023157460A1 (fr) * 2022-02-18 2023-08-24
WO2023157460A1 (fr) * 2022-02-18 2023-08-24 株式会社ウイルス医科学研究所 Procédé d'acquisition de données sur une réponse auto-immune impliquée dans la dépression, le vieillissement et analogue, et son utilisation

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