EP1558639A2 - Genes dont l'expression augmente en reaction a une stimulation par l'hormone liberatrice de la corticotropine - Google Patents

Genes dont l'expression augmente en reaction a une stimulation par l'hormone liberatrice de la corticotropine

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Publication number
EP1558639A2
EP1558639A2 EP03809730A EP03809730A EP1558639A2 EP 1558639 A2 EP1558639 A2 EP 1558639A2 EP 03809730 A EP03809730 A EP 03809730A EP 03809730 A EP03809730 A EP 03809730A EP 1558639 A2 EP1558639 A2 EP 1558639A2
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EP
European Patent Office
Prior art keywords
seq
crh
protein
cell
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP03809730A
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German (de)
English (en)
Inventor
Pieter Johan Peeters
Hinrich Wilhelm Helmut GÖHLMANN
Sigrid Maria Alice Swagemakers
Frederik Lucien Pieter Fierens
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Janssen Pharmaceutica NV
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Janssen Pharmaceutica NV
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Priority to EP03809730A priority Critical patent/EP1558639A2/fr
Priority claimed from PCT/EP2003/011793 external-priority patent/WO2004039835A2/fr
Publication of EP1558639A2 publication Critical patent/EP1558639A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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

  • Corticotropin releasing hormone a 41 -amino acid polypeptide plays a central •role in the regulation of the hypothalamic-pituitary-adrenal axis, mediating the endocrine responses to various stressors.
  • Hypothalamic neurons release CRH into the hypophyseal portal system in response to stress, stimulating the secretion and biosynthesis of pituitary adrenocorticotropin (ACTH) leading to increased adrenal glucocorticoid production (1).
  • ACTH pituitary adrenocorticotropin
  • the expression levels are typically assessed using an oligonucleotide probe that binds to the aforementioned polynucleotides, preferably using array technology methods.
  • Figure 3 A heat map depicting genes that are changed upon CRH treatment. Values were calculated by dividing the intensity of each sample by the intensity of the DMSO sample at the corresponding time point. These calculated ratio are converted into a color ramp using on a log 2 scale, hi this way the different timing of induction of expression becomes apparent. Genes showing a 2-fold change after 30 minutes of treatment with CRH were called “early responders”, “intermediate responders” show a change after 1 to 2 hours of treatment and “late responders” show a response after 2 hours or more.
  • Figure 4 Overview of a selection of genes induced by CRH grouped by pathway or function as discussed in the text. Values were calculated by dividing the intensity of each sample by the intensity of the DMSO sample at the corresponding time point. These calculated ratio are converted into a color ramp using on a log 2 scale and depicted in a heat map.
  • Figure 5 Induction of Rgs2 by CRH in AtT-20 cells. Induction is calculated in comparison to levels observed in AtT-20 cells before any treatment. On top array data obtained for Rgs2 are shown. Below, levels of Rgs2 mRNA are shown as measured by quantitative RT-PCR on the same samples as used for array experiments and as measured on a repeated experiment.
  • the term "compound” or “agent” means a biological or chemical compound such as a simple or complex organic molecule, a peptide, a protein or an oligonucleotide.
  • a “test compound” as used herein refers to a “compound” or “agent” used in a method according to the invention to assess whether said compounds modulates CRH signalling activity.
  • CRH signaling refers to the cellular changes in gene transcription after activation of the corticotropin releasing hormone receptor by CRH in said cell. It induces a CRH specific gene expression profile. Changes at the transcriptional level can be assessed either at the protein level or at the gene, RNA level.
  • CH response activity refers in general to the change of a detectable cellular parameter as a result of the exposure of said cell to CRH. Detectable cellular parameters include amongst others, changes in membrane potential, changes in enzyme activity of an enzyme that modulates CRH signalling a said cell, changes in expression levels of a protein according to the invention or changes in the amount of second messengers such as cGMP, cAMP, Ca 2+ or IP 3 .
  • the term “analog” or “functional analog” refers to a modified form of protein according to the invention in which at least one amino acid substitution has been made such that said analog retains substantially the same biological activity as the unmodified protein in vivo and/or in vitro.
  • polypeptides are well known in the art and include, for example covalent modification of reactive organic site contained within the polypeptide with a secondary chemical moiety.
  • cross-linking reagents are useful to react to amino, carboxyl, or aldehyde residues to introduce, for example an affinity tag such as biotin, a fluorescent dye, or to conjugate the polypeptide to a solid phase surface (for example to create an affinity resin)
  • “Partially complementary” refers to the aforementioned relationship in which one of the two single-stranded nucleic acid molecules is shorter in length than the other such that a portion of one of the molecules remains single-stranded.
  • the term “conservative substitution” or “conservative amino acid substitution” refers to a replacement of one or more amino acid residue(s) in a parent protein without affecting the biological activity of the parent molecule based on the art recognized substitutability of certain amino acids (See e.g. M. Dayhoff, In Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3, pgs 345-352, 1978).
  • “Fragment thereof refers to a fragment, piece, or sub-region of a nucleic acid or protein molecule whose sequence is disclosed herein, such that said fragment comprises 5 or more amino acids, or 10 or more nucleotides that are contiguous in the parent protein or nucleic acid molecule.
  • “Functional fragment” as used herein refers to an isolated sub-region, or fragment of a protein disclosed herein, or sequence of amino acids that, for example, comprises a functionally distinct region such as an active site for a receptor.
  • primer is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC). Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • an array of oligonucleotides probes comprising a nucleotide sequence encoding a protein that modulates CRH signalling or fragments thereof can be constructed to conduct efficient screening of gene expression.
  • 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, Vol 274, pp 610-613 (1996)).
  • an array of oligonucleotides probes comprising a nucleotide sequence encoding a protein that modulates CRH signalling or fragments thereof can be constructed to conduct efficient screening of gene expression.
  • 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, Vol 274, pp 610-613 (1996)).
  • changes in gene transcription are determined at mRNA level.
  • 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 via PCR, RT-PCR; RNase protection; Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well- known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assay techniques that can be used to determine the presence of protein derivatives or variants comprise amongst others mass spectrometry.
  • the method to assay the amount of protein that modulates CRH signaling is using an antibody which binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ 3D NO.2, SEQ 3D A, SEQ 3D NO.6, SEQ 3D NO.8, SEQ 3D NO.10, SEQ 3D NO.12, SEQ 3D NO.14, SEQ 3D NO.16, SEQ 3D NO.18, SEQ 3D NO.20, SEQ 3D NO.22, SEQ 3D NO.24, SEQ 3D NO.26, SEQ 3D NO.28, SEQ 3D NO.30, SEQ 3D NO.32, SEQ 3D NO.34, SEQ 3D NO.36, SEQ 3D NO.38, SEQ 3D NO.40, SEQ 3D NO.42, SEQ 3D NO.44, SEQ 3D NO.46 and SEQ 3D NO.48.
  • a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ 3
  • the antibodies according to the invention are used in conventional immunological techniques.
  • Suitable immunological techniques are well known to those skilled in the art and include for example, ELISA, Western Blot analysis, competitive or sandwich immunoassays and the like, as is otherwise well known they all depend on the formation of an antigen-antibody immune complex wherein for the purpose of the assay, the antibody can be detectable labeled with, e.g. radio, enzyme or fluorescent labels or it can be immobilized on insoluble carriers.
  • radioisotopes In measuring methods using labelling substances, radioisotopes, enzymes, fluorescent substances, luminous substances, etc. are used as labelling agents.
  • the radioisotopes include 125 I, 131 I, 3 H and 14 C.
  • Enzymes are usually made detectable by conjugation of an appropriate substrate that, in turn catalyzes a detectable reaction. Examples thereof include, for example, beta-galactosidase, beta- glucosidase, alkaline phosphatase, peroxidase and malate deydrogenase, preferably horseradish peroxidase.
  • the luminous substances include, for example, luminol, luminol derivatives, luciferin, aequorin and luciferase.
  • the avidin-biotin systems can also be used for labelling the antibodies and immunogens of the present invention.
  • the present invention provides for a method of identifying and obtaining compounds that alter the CRH signalling response activity in a cell, comprising: a) contacting a cell which expresses at least one protein comprising an amino acid sequence selected from the group consisting of SEQ 3D NO.2, SEQ 3D A, SEQ 3D NO.6, SEQ 3D NO.8, SEQ 3D NO.10, SEQ 3D NO.12, SEQ 3D NO.14, SEQ 3D
  • the activity of the cell may be assessed using electrophysiological methods. Therefore, proteins modulating CRH signalling in a cell can be characterised using whole cell and single channel electrophysiology.
  • the host cells in the aforementioned screening method express a protein selected from the group consisting of SEQ 3D No. 26, SEQ ID No.28, SEQ 3D No. 30, SEQ ID No. 32 and SEQ 3D NO.34.
  • the host cells are Xenopus oocytes and the electrophysiological measurement consists of measuring the membrane current using the voltage clamp technique at distinct membrane potentials. Changes in enzyme activity of an enzyme that modulates CRH signalling a said cell, can generally be measured or assayed in terms of the catalytic effect the enzyme produces, that is the conversion of its substrate into reaction product.
  • the kinase may also be provided by expression of a plasmid, which encodes the kinase protein.
  • Standard molecular biology techniques may be used to construct operable plasmids encoding the kinase protein and to express the plasmid in cells (Sambrook, et al., 1989, In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • the phosphate source does not have bound thereon a detectable label; instead, phosphorylated kinase substrate may be distinguished from non- phosphorylated kinase substrate, for instance by recognition of one form of the substrate, but not the other, by an antibody.
  • the phosphatase in the assay of the invention may be provided as a protein or it may be provided in the assay mixture as an mRNA encoding said phosphatase.
  • the phosphorylated substrate is typically labeled with a detectable phosphate residue. Labels which are useful include, but are not limited to, radioactive labels, such as ⁇ ' 2' ?, 31 S , and non-radioactive labels, such as biotin and the like.
  • the substrate preferably consists of a peptide substrate, phosphorylated at a tyrosine or serine residue, typically labeled with ⁇ ' 2' ?.
  • separation of non-dephosphorylated phosphopeptide from free radioactive phosphorus released by dephosphorylation of phosphopeptide is effected by centrifugation, following termination of the dephosphorylation reaction by the addition of substances including nonradioactive phosphates and charcoal.
  • Radioactivity in the supernate is determined by means well known to those of ordinary skill in the art. Based upon the amount of radioactivity added to the assay mixture initially via the phosphopeptide and the amount of radioactivity detected at the end of the assay as radioactivity released by dephosphorylation, the phosphatase enzymatic activity of the sample assayed may be calculated.
  • Levels of second messengers can be determined using art known techniques either in whole cells or cellular extracts comprising one of the aforementioned proteins.
  • a further method to identify a compound capable to alter CRH signalling in a cell is based on the use of a gene, such as a reporter gene, operably linked to a gene promoter or regulatory sequence element thereof characterized in that said gene promoter or regulatory sequence element comprises a transcription factor binding site, wherein said transcription factor is capable of modulating CRH signalling in a cell.
  • a gene such as a reporter gene
  • the transcription factor capable of modulating CRH signalling in a cell is being selected from SEQ 3D NO.2, SEQ 3D NO .4, SEQ 3D NO.6 and SEQ 3D NO.8.
  • the present invention provides a recombinant DNA molecule comprising the gene promoter region as defined above.
  • Reporter genes such as, for example, a firefly luciferase, D-galactosidase, alkaline phosphatase, the bacterial chloramphenicol acetyl transferase or green fluorescent protein reporter gene, can be used to determine transcriptional activity in screening assays according to the invention (see, for example, Goeddel (ed.), Methods Enzymol., Vol. 185, San Diego -.Academic Press, Inc. (1990); see also Sambrook, supra).
  • the reporter gene is the firefly luciferase gene.
  • the invention also provides a vector comprising the recombinant DNA molecule as defined above, as well as a host cell stably transformed with such a vector, or generally with the recombinant DNA molecule according to the invention.
  • vector refers to any carrier of exogenous DNA that is useful for transferring the DNA into a host cell for replication and/or appropriate expression of the exogenous DNA by the host cell.
  • the invention provides a method for identification of a compound modulating CRH signalling activity, said method comprising the steps: (i) contacting a candidate agent with a gene promoter region as defined above; and (ii) determining whether said candidate agent modulates expression of the detectable product, such modulation being indicative for an agent capable of modulating CRH signalling activity.
  • the detectable product refers either to the gene encoded protein or to the product of a reporter gene such as luciferase, ⁇ -galactosidase or green fluorescent protein.
  • the present invention encompasses an isolated and purified nucleic acid molecule comprising a member selected from a group consisting of:
  • nucleic acid molecule encoding a protein that modulates CRH signalling comprising a nucleotide sequence of which is degenerated as a result of the genetic code to a nucleotide sequence of a polynucleotide of any of (a) to (d).
  • polynucleotides include a polynucleotide consisting essentially of a polynucleotide sequence selected from SEQ 3D No.45, SEQ 3D No.47 or SEQ 3D No.49. Accordingly, in a further aspect, the present invention provides for an isolated polynucleotide, comprising: a) a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 90% identity, yet more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity to an amino acid sequence selected from the group consisting of SEQ 3D No. 46 and SEQ 3 No.48; b) a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, more preferably at least 90% identity,
  • Identity or similarity are relationships between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al, (1984) Nucleic Acids Research 12(1), 387), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al, (1990) J. Molec. Biol. 215, 403).
  • the nucleic acid sequence encoding a protein capable of modulating CRH activity, or fragment thereof can be isolated from a tissue in which said gene is expressed, such as but not limited to, brain, heart, kidney, pancreas, liver and skin. Said sequence can also be isolated from mammals other than human and mouse. Other cells and cell lines may also be suitable for use to isolate mammalian purine permease cDNA. Selection of suitable cells may be done by screening for CRH modulating activity in cell extracts or in whole cell assays, as described herein. Cells that possess CRH modulating activity in any one of these assays may be suitable for the isolation of purine permease DNA or mRNA.
  • any of a variety of procedures known in the art may be used to moleculary clone DNA encoding a protein according to the invention.
  • mRNA is isolated, and first strand cDNA synthesis is carried out.
  • a second round of DNA synthesis can be carried out for the production of the second strand.
  • an isolated cDNA can be obtained.
  • the double-stranded cDNA can be cloned into any suitable vector, for example, a plasmid, thereby forming a cDNA libary.
  • a suitable vector such as a plasmid or phage for propagation in prokaryotic or eukaryotic cells.
  • Suitable cloning vectors are well known and are widely available. It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating the nucleic acid sequences according to the invention.
  • Other types of libraries include, but are not limited to, cDNA libraries derived from other cells, from organisms other than human and mouse, and genomic DNA libraries that include YAC (yeast artificial chromosome) and cosmid libraries. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in T.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is short at the 5' end of the cDNA. This is a consequence of reverse transcriptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a DNA copy of the mRNA template during the 1 st strand cDNA synthesis.
  • the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the polynucleotide sequences according to the invention and will be capable of hybridizing to DNA encoding the desired protein, even in the presence of DNA oligonucleotides with mismatches. DNA isolated by these methods can be used to screen DNA libraries from a variety of cell types, from invertebrate and vertebrate sources, and to isolate homologous genes.
  • polypeptides In a further embodiment this invention relates to a polypeptide in a substantially pure form which modulate CRH signalling wherein said polypeptide is encoded by an isolated and purified nucleic acid molecule according to the invention.
  • the poiypeptide has the amino acid sequence selected from the group consisting of SEQ 3D NO 46, SEQ 3D NO 48 and functional analogs thereof
  • the protein according to the invention includes all possible amino acid variants encoded by the nucleic acid according to the invention including a polypeptide encoded by said molecule and having conservative amino acid changes.
  • Purified biologically active protein that modulates CRH signallings may have several different physical forms.
  • the polypeptides according to the invention may exist as full-length nascent or unprocessed polypeptides, or as partially processed polypeptides or combinations of processed polypeptides.
  • the full-length nascent polypeptide may be post-translationally modified, amongst other, by specific proteolytic cleavage events that result in the formation of fragments of the full-length nascent polypeptide.
  • a fragment, or physical association of fragments may have the full biological activity associated with proteins according to the invention; however, the degree of CRH modulating activity may vary between individual fragments.
  • fragments characterized by structural or functional attributes of the polypeptide are also preferred in this aspect of the invention.
  • polynucleotides according to the invention may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce a protein that modulates CRH signalling.
  • Techniques for such manipulations are fully described in Maniatis, T, et al., supra, and are well known in the art.
  • the isolated and purified nucleic acid molecules, according to the invention, encoding a protein which modulates CRH signalling may be cloned into an expression vector for expression in a recombinant host cell.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, amphibian cells such as Xenopus oocytes, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines.
  • this invention relates to a recombinant host cell containing a recombinantly cloned nucleic acid molecule encoding a protein that modulates CRH signalling or functional analog thereof.
  • the recombinant host cell according to the invention contains a nucleic acid molecule which is either genomic DNA or has a nucleotide sequence selected from a group consisting of: (SEQ 3D NO:45); (SEQ 3D NO 47); (SEQ 3D NO:49); and functional analogs thereof.
  • the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, lipofection, and electroporation.
  • the expression vector- containing cells are clonally propagated and analyzed to determine whether they produce a protein that modulates CRH signalling.
  • Identification of permeases expressing host cell clones may be done by several means, including but not limited to immunological reactivity with antibodies directed against the polypeptides according to the invention, and the presence of host cell-associated mammalian purine permease activity.
  • a mammalian cell that already comprises in its genome a nucleic acid molecule encoding a protein that modulates CRH signalling as described above, but does not express the same or not in an appropriate manner due to, e.g., a weak promoter, and introduce into the mammalian cell a regulatory sequence such as a strong promoter in close proximity to the endogenous nucleic acid molecule encoding said purine permease polypeptide so as to induce expression of the same.
  • a recombinant host cell containing a polynucleotide encoding a protein which modulates CRH signalling under the control of a heterologous transcription and/or regulatory sequence or protein, would be another embodiment of this invention.
  • the present invention is aiming at providing de novo and/or increased expression of polypeptides that modulate CRH signalling.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis, Basic Methods In Molecular Biology (1986). It is specifically contemplated that polypeptides, which modulate CRH signalling may in fact be expressed by a host cell lacking a recombinant vector.
  • expression of poiynucleotides according to the invention may also be performed using in vitro produced synthetic mRNA.
  • Synthetic mRNA or mRNA isolated from cells capable of modulating CRH signaling can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being generally preferred.
  • transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press.
  • the DNA of the embryonal membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe; see supra.
  • the reduction is achieved by an anti-sense, sense, ribozyme, co-suppression and/or dominant mutant effect.
  • Antisense and “antisense nucleotides” means DNA or RNA constructs which block the expression of the naturally occurring gene product.
  • polynucleotide according to the invention opens up the possibility to produce transgenic non-human animals with a reduced level of the protein as described above and, thus, with a defect in phosphate metabolism.
  • nucleic acid molecule encoding the antisense-RNA is preferably of homologous origin with respect to the animal species used for transformation.
  • nucleic acid molecules which display a high degree of homology to endogenously occurring nucleic acid molecules encoding a protein that modulates CRH signalling are also possible.
  • the homology is preferably higher than 80%, particularly higher than 90% and still more preferably higher than 95%.
  • the reduction of the synthesis of a protein according to the invention in the transgenic mammalian cells can result in an alteration in, e.g., adenine reabsorption. In transgenic animals comprising such cells this can lead to various physiological, developmental and/or morphological changes.
  • This invention further relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the polynucleotide of SEQ 3D NO: 1, SEQ 3D NO 3, SEQ 3D NO.5, SEQ 3D NO.7, SEQ 3D NO.9, SEQ 3D NO.ll, SEQ 3D NO.13, SEQ 3D NO.15, SEQ 3D NO.17, SEQ 3D NO.19, SEQ 3D NO.21, SEQ 3D NO.23, SEQ 3D NO.25, SEQ 3D NO.27, SEQ 3D NO.
  • Nuc3eic acids 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 or other amplification techniques prior to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to labeled mammalian purine permease nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in capilary electrophoresis columns or gels, with or without denaturing agents, or by direct DNA sequencing (e.g., Myers et al, Science (1985)230:1242).
  • Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well- known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assay techniques that can be used to determine the presence of protein derivatives or variants comprise amongst others mass spectrometry.
  • the present invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject related to a disorder of prolonged CRH exposure comprising: (a) determining the presence or amount of expression of the polypeptide or a derivative thereof according to the invention in a biological sample; and (b) diagnosing a pathological condition or susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide or of a derivative thereof.
  • the present invention provides a method of diagnosing a CRH induced gene expression profile in an individual, said method comprising; a) obtaining a biological sample of said individual; and b) determine the amount of at least one protein that modulates corticotropin releasing hormone (CRH) signaling in said biological sample; whereby the protein that modulates corticotropin releasing hormone (CRH) signaling is being selected from the group consisting of SEQ 3D NO.2, SEQ 3D 4, SEQ 3D NO.6, SEQ 3D NO.8, SEQ 3D NO.10, SEQ 3D NO.12, SEQ 3D NO.14, SEQ 3D NO.16, SEQ 3D NO.18, SEQ 3D NO.20, SEQ 3D NO.22, SEQ 3D NO.24, SEQ 3D NO.26, SEQ 3D NO.28, SEQ 3D NO.30, SEQ 3D NO.32, SEQ 3D NO.34, SEQ 3D NO.36, SEQ 3D NO.38, SEQ 3D
  • the proteins having the amino acid sequences SEQ 3D NO.2, SEQ 3D NO.4, SEQ 3D NO.6, SEQ 3D NO.8, SEQ 3D NO.10, SQ 3D NO.12, SEQ 3D NO.14, SEQ 3D NO.16, SEQ 3D NO.18, SEQ 3D NO.20, SEQ 3D NO.22, SEQ 3D NO.24, SEQ 3D NO.26, SEQ 3D NO.28, SEQ 3D NO.30, SEQ 3D NO.32, SEQ 3D NO.34, SEQ 3D NO.36, SEQ 3D NO.38, SEQ 3D NO.40, SEQ 3D NO.42, SEQ ID NO.44 and SEQ 3D NO.48.
  • the amount of said proteins is determined either at the protein level, preferably using antibodies that bind thereto, or at the gene transcription level, preferably using probes that bind to a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ 3D NO.2, SEQ 3D 4, SEQ 3D NO.6, SEQ 3D NO.8, SEQ 3D NO.10, SEQ 3D NO.12, SEQ 3D NO.14, SEQ 3D NO.16, SEQ 3D NO.18, SEQ 3D NO.20, SEQ 3D NO.22, SEQ 3D NO.24, SEQ 3D NO.26, SEQ 3D NO.28, SEQ 3D NO.30, SEQ 3D NO.32, SEQ 3D NO.34, SEQ 3D NO.36, SEQ 3D NO.38, SEQ 3D NO.40, SEQ 3D NO.42, SEQ 3D NO.44, SEQ 3D NO.46 and SEQ 3D NO.48.
  • the CRH induced gene expression profile is determined by assessing the level of gene transcription of a gene comprising a nucleic acid sequence selected from the group consisting of SEQ 3D NO: 1, SEQ 3D NO 3, SEQ 3D NO.5, SEQ 3D NO.7, SEQ 3D NO.9, SEQ 3D NO.ll, SEQ 3D NO.13, SEQ 3D NO.15, SEQ 3D NO.17, SEQ 3D NO.19, SEQ 3D NO.21, SEQ 3D NO.23, SEQ 3D NO.25, SEQ 3D NO.27, SEQ 3D NO.
  • a probe which binds, preferably selectively binds to a polynucleotide selected from the group consisting of SEQ 3D NO: 1, SEQ 3D NO 3, SEQ ID NO.5, SEQ 3D NO.7, SEQ 3D NO.9, SEQ 3D NO.ll, SEQ 3D NO.13, SEQ 3D NO.15, SEQ 3D NO.17, SEQ 3D NO.19, SEQ 3D NO.21, SEQ 3D NO.23, SEQ 3D NO.25, SEQ 3D NO.27, SEQ 3D NO.
  • an array of oligonucleotides probes comprising a nucleotide sequence according to the invention or fragments thereof can be constructed to conduct efficient screening of the level of gene transcription in the sample of an individual.
  • the present invention relates to a diagnostic ldt which comprises:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ 3D NO: 45, SEQ 3D NO: 47, SEQ 3D NO: 49 or a fragment thereof;
  • b a nucleotide sequence complementary to that of (a);
  • polypeptide of the present invention preferably the polypeptide of SEQ 3D NO:46, SEQ 3D NO 48 or a fragment thereof;
  • kits may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or suspectability to a disease, particularly CRH metabolism related disorders such as CRH induced stress or depression.
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • the nucleotide sequences of the present invention are also valuable for tissue localisation. Such techniques allow the determination of expression patterns of the polypeptides according to the invention in tissues by detection of the mRNAs that encode them. These techniques include in situ hybridziation techniques and nucleotide amplification techniques, for example PCR. Such techniques are well known in the art. Results from these studies provide an indication of the normal functions of the polypeptides in the organism.
  • polypeptides of the invention or their fragments or analogs thereof, or cells expressing them, can also be used as immunogens to produce antibodies immunospecific for polypeptides of the present invention.
  • 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.
  • this invention provides a monospecific antibody immunologically reactive with a mammalian purine permease.
  • said antibody is immunologically reactive with a polypeptide having an amino acid sequence selected from a group consisting of: (SEQ ID NO 46); (SEQ ID NO:48); and functional analogs thereof or said antibody blocks activity of a protein that modulates CRH signalling.
  • single chain antibodies such as those described in U.S. Patent No.4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention.
  • transgenic mice, or other organisms, including other mammals may be used to express humanized antibodies.
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
  • Antibodies against polypeptides of the present invention may also be employed to treat the CRH metabolism related disorders.
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgD, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG I, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with for instance blood clotting factor Xa.
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy.
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914. Tlierapeutic Utility
  • a further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention.
  • the vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, 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 formulations may be presented in unit- dose or multi-dose containers, for example, 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 vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • expression of the gene encoding proteins which modulate CRH signai3ing can be inhibited using expression blocking techniques.
  • antisense sequences either internally generated or externally administered (see, for example, O'Connor, J.Neurochem (1991) 56:560 ;Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • oligonucleotides which form triple helices can be supplied (see, for example, Lee et al, Nucleic Acids Res (1979)6:3073; Cooney et al.., Science (1988)241:456; Dervan et al, Science (1991)251:1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • Synthetic antisense or triplex oligonucleotides may comprise modified bases or modified backbones. Examples of the latter include methylphosphonate, phosphorothioate or peptide nucleic acid backbones.
  • RNA with partial or fully double-stranded character is introduced into the cell or into the extracellular environment.
  • Inhibition is specific in that a nucleotide sequence from a portion of the target gene is chosen to produce inhibitory RNA.
  • the RNA may comprise one or more strands of polymerized ribonucleotide; it may include modifications to either the phosphate-sugar backbone or the nucleoside.
  • the double- stranded structure may be formed by a single self -complementary RNA strand or two complementary strands.
  • Inhibition is sequence-specific in that the nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
  • RNA containing a nucieotide sequence identical to a portion of the target sequence is preferred. Examples of RNA inhibition technology can be found in International Patent Application WO 99/32619.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996)6(4), 527-33.) Synthetic ribozymes can be designed to specifically cleave the aforementiond mRNAs at selected positions thereby preventing translation of said 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 to provide protection from ribonuclease degradation, for example, 2'-O-methyl RNA, and may contain modified bases.
  • a therapeutically effective amount of a compound which activates a polypeptide of the present invention i.e., an agonist as described above
  • a pharmaceutically acceptable carrier i.e., a pharmaceutically acceptable carrier
  • gene therapy may be employed to effect the endogenous production of mammalian purine permease by the relevant cells in the subject.
  • a polynucleotide of the invention may be engineered for expression in a replication-defective retroviral vector, as discussed above.
  • the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present invention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • composition will be adapted to the route of administration, for instance by a systemic or an oral route.
  • Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of patches, salves, pastes, gels, and the like.
  • the dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
  • Microarray hybridization - cRNA was prepared as follows. Reverse transcription was performed on lO ⁇ g of total RNA for lh at 42°C using a T7-oligo(dT) 24 -primer and Superscript ⁇ RT (Invitrogen Life Technologies). Second strand cDNA synthesis was done for 2 h at 16°C using Escherichia coli DNA Polymerase I, DNA ligase and RNAseH (Invitrogen Life Technologies). After phenol-chloroform extraction using phase-lock gel (Eppendorf) in vitro transcription was performed for 6 h at 37°C using the Bioarray high-yield RNA transcript labeling kit with Biotin labeled ribonucleotides (Enzo Diagnostics).
  • cRNA samples were purified on Qiagen Rneasy columns followed by fragmentation for 35 min at 95°C. cRNA yields were between 50 and lOO ⁇ g. Samples were processed on GeneChips (Affymetrix, Santa Clara, CA). In order to check the quality of each sample, 5 ⁇ g of labeled cRNA was run on Test2-arrays. Actual experiments were performed on Murine Genome U74Av2 arrays, containing probe sets interrogating approximately 12,000 full-length mouse genes and EST clusters from the UniGene database (Build 74). Hybridization was performed using 15 ⁇ g cRNA for 16 h at 45 °C under continuous rotation.
  • Arrays were stained in Affymetrix Fluidics stations using Streptavidin/Phycoerythrin (SAPE) followed by staining with anti-streptavidin antibody and a second SAPE staining. Subsequently arrays were scanned with a HP- Laserscanner and data were analyzed with the Microarray Suite Software (Affymetrix). No scaling or normalization was performed at this stage. Quality of the experiment was assessed based on the percentages present calls across all samples which was on average 47.06 ⁇ 2.45%. The cytoplasmic ⁇ -actin and GAPDH 573' ratios were 1.10+0.08 and 0.93 ⁇ 0.05 respectively.
  • this alignment sets the average intensity of one array to the average measured across all arrays, compensating for array to array variations in hybridization, washing and staining, ultimately allowing a reasonable comparison between arrays.
  • Weighted spectral mapping is an unsupervised multivariate analysis method which includes double-centering of the data combined with a specialized visualization representing the two highest principle components. Even though double-centering removes the "size" component of the array data, this information is reintroduced in the visualization via the area of the symbols representing the size of the respective samples and genes. This method allows the reduction of a large microarray dataset and provides means to visually inspect and thereby identify clusters of genes and/or subjects in the data (7).
  • c-fos mRNA levels were determined by RTq on RNA from the different treatments and time points before array experiments were carried out.
  • exposure to CRH elicited a transient surge in c-fos transcription, with levels already going down after 0.5 to 1 h (see figure 1)(8;9).
  • This response was almost completely suppressed in the presence of R121919.
  • 0.1% DMSO induced c-fos expression, however levels were between 5 to 10 times lower compared to CRH induced expression.
  • transcription factors e.g. hairy/enhancer-of- split related 1 (Heyl), nuclear factor regulated by interleukin 3 (N3PIL3), cAMP responsive element modulator (CREM) and prostate specific ets transcription factor (Pse)
  • receptor and channel regulators e.g. Ras-related GTP-binding protein (GEM) and receptor (calcitonin) activity modifying protein 3 (RAMP3)
  • secreted peptides e.g. adrenomedullin, calcitonin, cholecystokinin
  • proteins involved in intracellular signaling e.g.
  • G-protein signaling 2 Rgs2
  • Pde4b cAMP specific phosphodiesterase 4B
  • Pde4b inositol 1,4,5-triphosphate receptor 1
  • P 3 R1 the regulatory subunit phosphatidylinositol 3-kinase
  • Other interesting regulated genes comprise Period homolog Perl, fibroblast growth factor receptor 2 (Fgfr2), serum glucocorticoid regulated kinase and serum-inducible kinase (figure 4).
  • Fgfr2 fibroblast growth factor receptor 2
  • serum glucocorticoid regulated kinase serum glucocorticoid regulated kinase
  • FIG. 4 all responders identified according to above mentioned criteria were up regulated after exposure to CRH. This induction was transient and nearly all of the induced genes return to baseline after 4 to 8 hr.
  • CREM-ICER c-Fos. Furthermore the kinetic profile of the induction of these genes correspond with the observed maximal transcription rate by CREB after 0.5 hr of cAMP formation.
  • the induction of CREM-ICER constitutes a negative feedback mechanism in attenuating transcriptional response to cAMP.
  • CREM-ICER in response to acute stress in the intermediate lobe of the pituitary gland.
  • Mice deficient for CREM-ICER show a chronic increase of beta- endorphin levels suggesting that CREM-ICER induction may be involved in the modulation of gene expression in response to stress (10).
  • Rgs2 reduces odor ant-elicited cAMP production, not by acting on G ⁇ but by directly inhibiting the activity of adenylyl cyclase type HI.
  • Rgs2 was originally identified as an immediate early response gene in activated T lymphocytes, studies in Rgs2 deficient mice indicate that it also plays a role in the modulation of stress related behavior as these mice show increased anxiety and aggression (11).
  • the induction of cAMP specific phosphodiesterase 4B (Pde4b) can be categorized under negative feedback, directly attenuating the cAMP signal.
  • Another important second messenger generated upon stimulation with CRH is Ca 2+ .
  • CRH CRH triggers a steady-state depolarization stimulated extracellular Ca 2+ entry via voltage-gated Ca 2+ channels and raises intracellular Ca 2+ concentration through release from inositol 1,4,5-triphosphate (InsP3) sensitive Ca 2+ pools(12).
  • InsP3 receptor and the p85 regulatory subunit of phosphatidylinositol 3-kinase are upregulated, possibly accounting for a compensating mechanism for prolonged Ca + signaling.
  • the upregulation of the small G-protein kir/Gem points towards an attenuation of prolonged Ca 2+ signaling.
  • NFTL3/334BP4 by calcineurin/NFAT and CaM kinase signaling, accounting for an increase in NFIL3 mRNa levels upon CRH treatment.
  • B lymphocytes expression of NITL3 is induced by interleukin 3 through both the Raf-mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.
  • NF3L3 inhibits apoptosis in synergy with Bcl-xL dependent pathways.
  • Our data suggest a role for N-FE 3 in prevention of apoptosis in AtT-20 cells.
  • CRH is the most efficacious ACTH secretagogue. Unfortunately the microarrays that were used did not interrogate for POMC levels. However several other prepropeptides mRNAs were found upregulated after CRH administration such as cholecystokinin (CCK) and two calcitonin peptide family members, adrenomedullin (ADM) and calcitonin (CT). Also of interest in this respect is the upregulation of RAMP3. RAMPs control the transport and glycosylation of the calcitonin receptor-like receptor (CRLR). In the case of RAMP3, it has been shown that together with CRLR it generates an ADM receptor.
  • CCK cholecystokinin
  • ADM adrenomedullin
  • CT calcitonin
  • RAMP3 control the transport and glycosylation of the calcitonin receptor-like receptor (CRLR). In the case of RAMP3, it has been shown that together with CRLR it generates an A
  • Upregulation of this gene might play a role in regulating the responsiveness of AtT-20 cells to ADM after CRH exposure or to other extracellular stimuli as it is not known whether RAMP3 might regulate other G-coupled receptors.
  • CCK is secreted by AtT-20 cells, induction of its expression by CRH has not been previously reported (13). Interaction between CCK and CRH has however been intensively studied and demonstrated in panic attacks, depression, anxiety and gastric emptying (14-19). Most of these experiments point towards a role for CRH in mediating the central effects of CCK. Our data indicate that CRH in addition might function as a CCK secretagogue. A very similar situation to that of CCK seems to be case for adrenomedullin as well.

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Abstract

Cette invention, qui a trait, d'une manière générale, à une méthode thérapeutique et diagnostique de la dépression, concerne, plus précisément, des polypeptides ainsi que des polynucléotides codant ces polypeptides, lesquels polypeptides jouent un rôle éminent, s'agissant de médiatiser une réaction endocrine à l'égard de l'hormone libératrice de la corticotropine. Ces polynucléotides et ces polypeptides se révèlent des plus utiles en matière de diagnostic, de traitement et/ou de prévention de la
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WO2000042178A2 (fr) * 1999-01-13 2000-07-20 Dupont Pharmaceuticals Company Blocage des recepteurs de type 2 de la corticoliberine par des antagonistes dans le traitement des troubles psychiatriques et utilisation d'oligonucleotides chimeres antisens dans les etudes in vivo de la fonction genique au niveau du systeme nerveux central

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000042178A2 (fr) * 1999-01-13 2000-07-20 Dupont Pharmaceuticals Company Blocage des recepteurs de type 2 de la corticoliberine par des antagonistes dans le traitement des troubles psychiatriques et utilisation d'oligonucleotides chimeres antisens dans les etudes in vivo de la fonction genique au niveau du systeme nerveux central

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Title
CHRAPKIEWICZ N.B. ET AL: "RAT GENE 33 ANALYSIS OF ITS STRUCTURE MESSENGER RNA AND BASAL PROMOTER ACTIVITY", NUCLEIC ACIDS RESEARCH, vol. 17, no. 16, 1989, OXFORD UNIVERSITY PRESS, SURREY, GB, pages 6651 - 6667, XP001161090 *
DATABASE GENEMEBL [online] 5 April 2001 (2001-04-05), STRAUSBERG ET AL: "Mus musculs RIKEN cDNA 1300002F13, mRNA (cDNA clone MGC:6714 IMAGE:3585640), complete cds.", XP002270084, Database accession no. BC005546 *
LIEBSCH G: "Differential behavioural effects of chronic infusion of CRH1 and CRH2 receptor antisense oligonucleotides into the rat brain", JOURNAL OF PSYCHIATRIC RESEARCH, vol. 33, no. 2, - 1 March 1999 (1999-03-01), ELSEVIER LTD, GB, pages 153 - 163, XP008036948 *
STROHLE A: "The neuroendocrinology of stress and the pathophysiology and therapy of depression and anxiety", NERVENARZT, vol. 74, no. 3, - 1 March 2003 (2003-03-01), pages 279 - 292 *

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