WO2001019854A2 - Nouveaux composes - Google Patents

Nouveaux composes Download PDF

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Publication number
WO2001019854A2
WO2001019854A2 PCT/GB2000/003541 GB0003541W WO0119854A2 WO 2001019854 A2 WO2001019854 A2 WO 2001019854A2 GB 0003541 W GB0003541 W GB 0003541W WO 0119854 A2 WO0119854 A2 WO 0119854A2
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Prior art keywords
polypeptide
sequence
polynucleotide
seq
isolated
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PCT/GB2000/003541
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English (en)
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WO2001019854A3 (fr
Inventor
Jeffrey Hill
David Malcolm Duckworth
Maurice Farmer
Menelas Pangalos
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Smithkline Beecham P.L.C.
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Priority claimed from GBGB9921833.1A external-priority patent/GB9921833D0/en
Priority claimed from GB0006545A external-priority patent/GB0006545D0/en
Application filed by Smithkline Beecham P.L.C. filed Critical Smithkline Beecham P.L.C.
Priority to EP00962668A priority Critical patent/EP1144440A3/fr
Publication of WO2001019854A2 publication Critical patent/WO2001019854A2/fr
Publication of WO2001019854A3 publication Critical patent/WO2001019854A3/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
  • the present invention relates to NTT7, in particular NTT7 polypeptides and NTT7 polynucleotides, recombinant materials and methods for their production.
  • NTT7 polypeptides and NTT7 polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, psychiatric disorders, anxiety, depression, schizophrenia, phobias, panic disorder, obsessive compulsive disorder, Parkinson's disease, CNS disorders, neurological disorders, stroke, pain, neuropathic pain, sleep disorders, and diseases in which neurotransmitters are implicated, hereinafter referred to as "diseases of the invention”.
  • the invention relates to methods for identifying agonists and antagonists (e.g., inhibitors) using the materials provided by the invention, and treating conditions associated with NTT7 imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate NTT7 activity or levels.
  • the present invention relates to NTT7 polypeptides.
  • Such polypeptides include:
  • Polypeptides of the present invention are believed to be members of the neurotransmitter transporter family of polypeptides. They are therefore of interest because of the established, proven history of neurotransmitter transporters as therapeutic targets for the treatment of psychiat ⁇ c diseases. Neurotransmitter transporters are cruicial in the control of neurotransmitter levels in the synapse, thus playing a pivotal role in the control and regulation of neuronal excitability and fi ⁇ ng. Modulation of these proteins with specific pharmacological agents can therefore be used as a means of regulating communication between nerve terminals in diseases where specific neurotransrrutters have been implicated.
  • NTT7 The biological properties of the NTT7 are hereinafter referred to as "biological activity of NTT7" or "NTT7 activity"
  • a polypeptide of the present invention exhibits at least one biological activity of NTT7
  • Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including all alle c forms and splice variants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non- conservative, or any combination thereof. Particularly preferred variants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acids are inserted, substituted, or deleted, in any combination.
  • Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO. 2, or an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ED NO. 2
  • Preferred fragments are biologically active fragments that mediate the biological activity of NTT7, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or lmmunogenic in an animal, especially in a human.
  • polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • the polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histid e residues, or an additional sequence for stability during recombinant production.
  • Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurring sources, from genetically engineered host cells comprising expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesizers, or a combination of such methods. Means for prepa ⁇ ng such polypeptides are well understood in the art
  • the present invention relates to NTT7 polynucleotides.
  • Such polynucleotides include
  • Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comprising an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ED NO. 1, or an isolated polynucleotide comp ⁇ sing an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ED NO: 1.
  • Preferred va ⁇ ants of polynucleotides of the present invention include splice va ⁇ ants, allelic va ⁇ ants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • Polynucleotides of the present invention also include polynucleotides encoding polypeptide va ⁇ ants that comp ⁇ se the amino acid sequence of SEQ ED NO.2 and m which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination.
  • the present invention provides polynucleotides that are RNA transc ⁇ pts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that
  • (a) comprises an RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO.2
  • (b) is the RNA transcript of the DNA sequence encoding the polypeptide of SEQ ED
  • (c) comprises an RNA transc ⁇ pt of the DNA sequence of SEQ ID NO.1 ;
  • (d) is the RNA transcnpt of the DNA sequence of SEQ ED NO- 1 ,
  • the polynucleotide sequence of SEQ ED NO: 1 shows homology with Bos taurus bv7-3 orphan transporter (Genbank Entry AB020854).
  • the polynucleotide sequence of SEQ ID NO: 1 is a cDNA sequence that encodes the polypeptide of SEQ ED NO.2.
  • the polynucleotide sequence encoding the polypeptide of SEQ ED NO.2 may be identical to the polypeptide encoding sequence of SEQ ID NO.1 or it may be a sequence other than SEQ ED NO: 1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ED NO:2.
  • the polypeptide of the SEQ ED NO:2 is related to other proteins of the neurotransmitter transporter family, having homology and or structural simila ⁇ ty with Bos taurus bv7-3 orphan transporter (Genbank Entry AB020854)
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one NTT7 activity.
  • Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library de ⁇ ved from mRNA in cells of human melanocyte and cerebellum, (see for instance, Sambrook et al.. Molecular Cloning. A Laboratory Manual, 2nd Ed., Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N Y. (1989))
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbra ⁇ es or can be synthesized using well known and commercially available techniques.
  • the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence that facilitates pu ⁇ fication of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and desc ⁇ bed in Gentz et al, Proc Natl Acad Sci USA (1989) 86.821-824, or is an HA tag
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transc ⁇ bed, non-translated sequences, splicing and polyadenylation signals, nbosome binding sites and sequences that stabilize mRNA.
  • Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ED NO 1, may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers for a nucleic acid amplification reaction (for instance, PCR).
  • probes and p ⁇ mers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence simila ⁇ ty to SEQ ID NO 1 , typically at least 95% identity
  • Preferred probes and p ⁇ mers will generally comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides.
  • Particularly preferred probes will have between 30 and 50 nucleotides
  • Particularly preferred p ⁇ mers will have between 20 and 25 nucleotides.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process comp ⁇ sing the steps of screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in O.lx SSC at about 65°C.
  • the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof, preferably of at least 15 nucleotides.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus. 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 first strand cDNA synthesis.
  • PCR Nucleic acid amplification
  • PCR Nucleic acid amplification
  • the PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adapter specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence).
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Polynucleotides may be introduced into host cells by methods described in many standard laboratory manuals, such
  • Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micro-injection, canonic pid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as Streptococci, Staphvlococci, E coli, Streptomvces and Bacillus subt ⁇ is cells, fungal cells, such as yeast cells and Asperg ⁇ lus cells, insect cells such as Drosophda S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS. HeLa, C127.
  • bacte ⁇ al cells such as Streptococci, Staphvlococci, E coli, Streptomvces and Bacillus subt ⁇ is cells
  • fungal cells such as yeast cells and Asperg ⁇ lus cells
  • insect cells such as Drosophda S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS. HeLa, C127.
  • a great va ⁇ ety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses.
  • papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression
  • anv system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used
  • the approp ⁇ ate polynucleotide sequence may be inserted into an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al , (ibid)
  • Approp ⁇ ate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum,
  • a polypeptide of the present invention is to be expressed for use m screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested p ⁇ or to use in the screening assay If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and pu ⁇ fy the polypeptide If produced intracellularly, the cells must first be lysed before the polypeptide is recovered
  • Polypeptides of the present invention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng lntracellular synthesis, isolation and/or pu ⁇ fication
  • Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene Detection of a mutated form of the gene characte ⁇ zed by the polynucleotide of SEQ ID NO 1 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene Individuals carrying mutations in the gene may be detected at the DNA level by a va ⁇ ety of techniques well known m the art Nucleic 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 it may be amplified enzymatically by using PCR, preferably RT-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 NTT7 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
  • DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, for instance, Myers et al., Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401).
  • An array of oligonucleotides probes comprising NTT7 polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Such arrays are preferably high density arrays or grids.
  • 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, 274, 610-613 (1996) and other references cited therein. Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention.
  • Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • 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 radio-immunoassays, competitive-binding assays. Western Blot analysis and ELISA assays.
  • the present invention relates to a diagnostic kit comprising: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ED NO: 1 , or a fragment or an RNA transcript thereof;
  • polypeptide of the present invention preferably the polypeptide of SEQ ED NO:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ED NO:2.
  • kits may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.
  • the polynucleotide sequences of the present invention are valuable for chromosome localisation studies.
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene
  • the polynucleotide sequences of the present invention are also valuable tools for tissue expression studies Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them
  • the techniques used are well known in the art and include in situ hyd ⁇ dization techniques to clones arrayed on a g ⁇ d, such as cDNA microarray hyb ⁇ dization (Schena er /, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR
  • TAQMAN Trade mark
  • the polypeptides of the present invention are expressed in brain (amygdala, caudate nucleus, cerebellum, corpus callosum. frontal cortex, occipital cortex, hippocampus, hypothalamus, nucleus accumbens, putamen, substantia nigra, thalamus, whole brain), foetal brain and pituitary gland with lower levels in spinal cord and placenta
  • brain as mygdala, caudate nucleus, cerebellum, corpus callosum. frontal cortex, occipital cortex, hippocampus, hypothalamus, nucleus accumbens, putamen, substantia nigra, thalamus, whole brain
  • a further aspect of the present invention relates to antibodies
  • the polypeptides of the invention or their fragments, or cells expressing them, can be used as immunogens to produce antibodies that are immunospecific for polypeptides of the present invention
  • Antibodies generated against polypeptides of the present invention may be obtained by administe ⁇ ng the polypeptides or epitope-bea ⁇ ng fragments, or cells to an animal, preferably a non- human animal, using routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hyb ⁇ doma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hyb ⁇ doma technique (Cole et al , Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R Liss. Inc . 1985)
  • the present invention relates to a method for inducing an lmmunological response in a mammal that comp ⁇ ses inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not
  • An immunological response in a mammal may also be induced by a method comp ⁇ ses delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention
  • One way of administe ⁇ ng the vector is by accelerating it into the desired cells as a coating on particles or otherwise
  • Such nucleic acid vector may comp ⁇ se DNA, RNA
  • Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention hereinbefore mentioned. It is therefore useful to identify compounds that stimulate or inhibit the function or level of the polypeptide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide. Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the invention as hereinbefore mentioned.
  • Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical hbra ⁇ es, collections of chemical compounds, and natural product mixtures
  • Such agonists or antagonists so-identified may be natural or modified substrates, gands, receptors, enzymes, etc., as the case may be, of the polypeptide, a structural or functional mimetic thereof (see Coligan et al., Current Protocols in Immunology 1(2). Chapter 5 (1991)) or a small molecule.
  • Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1 ,000 daltons. and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bea ⁇ ng the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve measu ⁇ ng or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g. agonist or antagonist)
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Further, the screening methods may simply comp ⁇ se the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measu ⁇ ng a NTT7 activity in the mixture, and compa ⁇ ng the NTT7 activity of the mixture to a control mixture which contains no candidate compound
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method desc ⁇ bed by Schullek et al, Anal Biochem., 246, 20-29, (1997)
  • Fusion proteins such as those made from Fc portion and NTT7 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al , J Mol Recognition, 8.52-58 (1995), and K Johanson et al., J Biol Chem, 270(16) 9459-9471 ( 1995))
  • the polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells.
  • an ELISA assay may be constructed for measu ⁇ ng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • a polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or pu ⁇ fication, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any Standard methods for conducting such assays are well understood in the art
  • antagonists of polypeptides of the present invention include antibodies or, m some cases, o gonucleotides or proteins that are closely related to the gands, substrates, receptors. enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the hgands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • Screening methods may also involve the use of transgenic technology and NTT7 gene.
  • the art of constructing transgenic animals is well established.
  • the NTT7 gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts.
  • transgenic animals are so-called “knock-in” animals in which an animal gene is replaced by the human equivalent within the genome of that animal Knock-in transgenic animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target
  • Other useful transgenic animals are so-called “knock-out” animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled.
  • the gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal.
  • Transgenic animal technology also offers a whole animal expression-cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention
  • Screening kits for use in the above described methods form a further aspect of the present invention
  • Such screening kits comprise. (a) a polypeptide of the present invention
  • polypeptide is preferably that of SEQ ED NO 2
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library
  • Isolated means altered “by the hand of man” from its natural state, J e , if it occurs in nature, it has been changed or removed from its o ⁇ ginal environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated even if it is still present in said organism, which organism may be living or non-living
  • Polynucleotide generally refers to any poly ⁇ bonucleotide (RNA) or polydeox ⁇ bonucleotide (DNA), which may be unmodified or modified RNA or DNA
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hyb ⁇ d molecules comp ⁇ sing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions
  • polynucleotide refers to t ⁇ ple-stranded regions comprising RNA or DNA or both RNA and DNA
  • the term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, t ⁇ tylated bases and unusual bases such as inosine A va ⁇ ety of modifications may be made to DNA and RNA, thus, "polynucleotide”
  • Polypeptide refers to any polypeptide comp ⁇ sing two or more am o acids joined to each other by peptide bonds or modified peptide bonds, I e , peptide isosteres
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or o gomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids
  • Polypeptides include am o acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may
  • polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation.
  • “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide.
  • “Fragment” of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ED NO: 1.
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof.
  • a typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val. lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr.
  • a variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ribosylation and the like. Embodiments include methylation of the N-terminal amino acid, phosphorylations of serines and threonines and modification of C-terminal glycines. "Allele ' refers to one of two or more alternative forms of a gene occur ⁇ ng at a given locus in the genome
  • Polymorphism ' refers to a va ⁇ ation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population
  • Single Nucleotide Polymorphism refers to the occu ⁇ ence of nucleotide variability at a single nucleotide position in the genome, within a population
  • An SNP may occur within a gene or within intergenic regions of the genome SNPs can be assayed using Allele Specific Amplification (ASA)
  • ASA Allele Specific Amplification
  • a common p ⁇ mer is used in reverse complement to the polymorphism being assayed This common p ⁇ mer can be between 50 and 1500 bps from the polymorphic base
  • the other two (or more) p ⁇ mers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism
  • Two (or more) PCR reactions are then conducted on sample DNA each using the common primer
  • RNA molecules produced from RNA molecules initially transc ⁇ bed from the same genomic DNA sequence but which have undergone alternative RNA splicing
  • Alternative RNA splicing occurs when a primary RNA transc ⁇ pt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mRNA molecule each of that may encode different amino acid sequences
  • the term splice variant also refers to the proteins encoded by the above cDNA molecules "Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compa ⁇ ng the sequences In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared "% Identity" - For sequences where there is not an exact co ⁇ espondence, a "% identity"
  • Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences
  • similar ⁇ ty means a compa ⁇ son between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other This likelihood has an associated "score” from which the "% similanty” of the two sequences can then be determined.
  • programs available in the Wisconsin Sequence Analysis Package, version 9 1 (Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wisconsin, USA), for example the programs BESTFTT and GAP, may be used to determine the % identity between two poly
  • BESTFTT uses the "local homology" algorithm of Smith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482-489, 1981) and finds the best single region of similarity between two sequences.
  • BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
  • GAP aligns two sequences, finding a "maximum simila ⁇ ty", according to the algo ⁇ thm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970).
  • GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length.
  • the parameters "Gap Weight” and “Length Weight” used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
  • % identities and similarities are determined when the two sequences being compared are optimally aligned.
  • NCBI National Center for Biotechnology Information
  • NCBI National Center for Biotechnology Information
  • FASTA FASTA
  • BLOSUM62 amino acid substitution mat ⁇ x Henikoff S and Henikoff J
  • the program BESTFTT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore desc ⁇ bed
  • Identity Index is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence
  • a candidate polynucleotide sequence having, for example, an Identity Index of 0.95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and trans version, or insertion.
  • a candidate polypeptide sequence having, for example, an Identity Index of 0 95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 am o acids of the reference sequence Such differences are selected from the group consisting of at least one am o acid deletion, substitution, including conservative and non- conservative substitution, or insertion These differences may occur at the amino- or carboxy- terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • an average of up to 5 in every 100 of the am o acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed
  • an average of up to 5 in every 100 of the am o acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore desc ⁇ bed
  • n a is the number of nucleotide or amino acid differences
  • x a is the total number of nucleotides or ammo acids in SEQ ED NO 1 or SEQ ED NO 2, respectively
  • “Homolog ' is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence Such relatedness may be quantified by determining the degree of identity and/or similanty between the two sequences as hereinbefore defined Falling within this gene ⁇ c term are the terms "ortholog", and "paralog ' "Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species "Paralog” refers to a polynucleotideor polypeptide that withm the same species which is functionally similar
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof
  • EP-A-0464 533-A discloses fusion proteins comp ⁇ sing various portions of constant region of immunoglobulin molecules together with another human protein or part thereof
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting m, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262]
  • Example 1 Taqman analysis of mRNA tissue distribution
  • NTT7 The expression pattern of NTT7 was investigated using Taqman fluorescent PCR (Perkin Elmer) and human cDNAs prepared in a total of thirty various brain areas and penpheral tissues amygdala, caudate nucleus, cerebellum, co ⁇ us callosum, frontal cortex, occipital cortex, temporal cortex, hippocampus, hypothalamus, nucleus accumbens, putamen, substantia nigra, thalamus, foetal brain, spinal cord, pituitary gland, whole brain, heart, liver, lung, skeletal muscle, kidney, pancreas, spleen, small intestine, placenta, testis, stomach, prostate, uterus
  • NTT7 is seen brain (amygdala, caudate nucleus, cerebellum, co ⁇ us callosum, frontal cortex, occipital cortex, hippocampus, hypothalamus, nucleus accumbens, putamen, substantia nigra, thalamus, whole brain), foetal brain and pituitary gland with lower levels in the spinal cord and placenta Very little or no expression was detected in heart, liver, lung, skeletal muscle, kidney, pancreas, spleen, small intestine, testis, stomach, prostate and uterus
  • Example 2 Cellular localisation A myc-epitope tag was inserted, by PCR, into a putative extracellular loop of the NTT7 protein Immunocytochemical staining of CHO cells transfected with the myc-tagged construct, in the absence of T ⁇ ton X-100, showed that the myc-tagged epitope appeared to be located on the cell surface
  • GIVTPIVDTFKVRKEILTVICC LAFCIGLIFVQRSGNYFVTMFDDYSATLPLLIWI E NIAVCFVYGIDKFMEDLKDMLGFAPSRYYYYMWKYISP MLLSLLIASWNMG SPPGYN

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Abstract

La présente invention concerne des polypeptides et des polynucléotide NTT7 et des procédés permettant de produire les polypeptides précités par des techniques de recombinaison. L'invention se rapporte également à des procédés d'utilisation des polypeptides et polynucléotides NTT7 dans des méthodes diagnostiques.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002029048A2 (fr) * 2000-10-05 2002-04-11 Bayer Aktiengesellschaft Regulation du transporteur du neurotransmetteur humain induit par le sodium

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031539A1 (fr) * 1994-05-16 1995-11-23 Human Genome Sciences, Inc. Vehicule de neurotransmetteur

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995031539A1 (fr) * 1994-05-16 1995-11-23 Human Genome Sciences, Inc. Vehicule de neurotransmetteur

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Title
HILLIER ET AL.: "The WashU-Merck EST Project" EMBL SEQUENCE DATABASE, 27 January 1996 (1996-01-27), XP002161132 HEIDELBERG DE *
SAKATA K.: "bv7-3; orphan transporter short splice variant" EMBL SEQUENCE DATABASE, 29 April 1999 (1999-04-29), XP002161134 HEIDELBERG DE *
UHL ET AL.: "Neurotransmitter transporter family cDNas in a rat midbrain library: 'orphan transporters' suggest sizable structural variations" MOL. BRAIN RES., vol. 16, no. 3-4, 1992, pages 353-359, XP000986881 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002029048A2 (fr) * 2000-10-05 2002-04-11 Bayer Aktiengesellschaft Regulation du transporteur du neurotransmetteur humain induit par le sodium
WO2002029048A3 (fr) * 2000-10-05 2002-11-14 Bayer Ag Regulation du transporteur du neurotransmetteur humain induit par le sodium

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