WO2001004142A2 - Nouveaux composes - Google Patents

Nouveaux composes Download PDF

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Publication number
WO2001004142A2
WO2001004142A2 PCT/EP2000/006253 EP0006253W WO0104142A2 WO 2001004142 A2 WO2001004142 A2 WO 2001004142A2 EP 0006253 W EP0006253 W EP 0006253W WO 0104142 A2 WO0104142 A2 WO 0104142A2
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polypeptide
sequence
polynucleotide
seq
isolated
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PCT/EP2000/006253
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WO2001004142A3 (fr
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Hugh Jonathan Herdon
Joanne Rachel Evans
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Smithkline Beecham P.L.C.
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Publication of WO2001004142A2 publication Critical patent/WO2001004142A2/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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

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 drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics", that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superceding earlier approaches based on “positional cloning”. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
  • the present invention relates to GC42, in particular GC42 polypeptides and GC42 polynucleotides, recombinant materials and methods for their production.
  • Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, schizophrenia, Alzheimer's disease, multi-infarct & other dementias, attention deficit hyperactivity disorders, age-related cognitive decline & other cognitive disorders, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, stroke, 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 GC42 imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate GC42 activity or levels.
  • the present invention relates to GC42 polypeptides.
  • polypeptides include:
  • polypeptide sequence of SEQ ID NO:2 (e) the polypeptide sequence of SEQ ID NO:2; and (f) an isolated polypeptide having or comprising a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2; (g) fragments and variants of such polypeptides in (a) to (f); with the proviso that the isolated polypeptide does not have the amino acid sequence of SEQ ID NO:4.
  • 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 psychiatric or neurological diseases. Reliable neurotransmission in the nervous system depends on rapid termination of transmitter action following postsynaptic activation. In some cases this is achieved by metabolism of the neurotransmitter, as in the case of acetylcholine and neuropeptides. In many cases, however, including catecholamines, serotonin and some amino acids (e.g.
  • the neurotransmitter is efficiently removed into the presynaptic terminal or surrounding glial cells by neurotransmitter transporters, membrane-bound polypeptides located in the plasma membrane. Modulation of neurotransmitter transport enables synaptic transmission to be increased or decreased by altering the levels of neurotransmitter in the synaptic cleft, and blockade of transport is an established approach to the treatment of psychiatric and neurological illness.
  • Drugs that act by this mechanism include the tricyclic antidepressants, which act on monoamine transporters in general, and the selective serotonin uptake inhibitors (SSRIs) (Lesch KP and Bengel D, CNS Drugs 4(1995), 302-322).
  • a GABA transport inhibitor tiagabine
  • tiagabine has recently been identified as a potential therapy for epilepsy (Lesch KP and Bengel D, CNS Drugs 4(1995), 302-322). This indicates that the neurotransmitter transporter family has an established, proven history as therapeutic targets.
  • cDNAs encoding a number of Na/Cl-dependent neurotransmitter transporters e.g. for serotonin, catecholamine, amino acid (glycine, GABA) have been described .
  • This class of transporter is very similar, containing twelve potential transmembrane helices and an external loop with 3-4 glycosylation sites between transmembrane segments 3 and 4.
  • the amino acid sequence is about 60-80% identical to other members within a subfamily and about 40% identical to members between two subfamilies (Liu et al., Proc. Natl. Acad. Sci. USA, (1992), 89:6639-6643).
  • Transporters of amino acids such as glycine share about 40-50% homology with all members of the neurotransmitter transporter superfamily.
  • GlyT-1 has about 50% amino acid sequence identity with GlyT-2.
  • GlyT-la amino acid sequence identity with GlyT-2.
  • GlyT-lb amino acid sequence identity with GlyT-2.
  • GlyT-lc variants of GlyT-1 (GlyT-la, GlyT-lb, GlyT-lc), which arise through alternate exon use and differ in their N-terminal regions (Kim et al, Mol. Pharmacol. (1994), 45, 608-617).
  • Glycine is a major transmitter in the nervous system. Glycine can have both inhibitory and excitatory functions, which are mediated by two different types of receptor, each associated with a different class of glycine transporter.
  • the excitatory function of glycine is mediated by "strychnine-insensitive" glycine receptors, which are part of the NMD A receptor complex that mediates some of the actions of glutamate, the major excitatory transmitter in the central nervous system.
  • Glycine acts as a "co-agonist" at the NMDA receptor complex, and agonist occupation of these "strychnine-insensitive" glycine receptors is a pre-requisite for glutamatergic neurotransmission via the NMDA receptor complex.
  • glycine receptor This type of glycine receptor is widely distributed throughout the brain, and is associated with the GlyT-1 transporter. Conversely, the inhibitory action of glycine is mediated by "strychnine-sensitive" glycine receptors. These receptors are found mainly in the spinal cord, brainstem and cerebellum, and are associated with the GlyT-2 transporter (Zafra et al, Mol. Neurobiol. (1997) 14, 117-142).
  • NMDA receptor complex has important functions m memory and learning (e g Rison & Stanton, Neurosci Biobehav Rev (1995) 19, 533-552) There is also good evidence for the role of NMDA receptor hypofunction in schizophrenia (e g Ishimaru & Toru, CNS Drugs (1997) 7, 46-67, Duncan et al, Brain Res Rev (1999) 29, 250-264) Conversely, excessive activity at NMDA receptors is likely to play a part in diseases which may involve excitotoxic cell death, such as stroke and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntmgton's disease and amyotrophic lateral sclerosis
  • GlyT- 1 transporter modulation of transporter function would produce changes in the degree of activation of strychnine-insensitive glycme receptors on the NMDA receptor complex, and thus influence NMDA glutamatergic transmission (Bergeron et al, Proc Natl Acad Sci (1998) 95, 15730-15734) Therefore, modulation of GlyT- 1 transporter function could alleviate symptoms associated with schizophrenia & other psychoses, Alzheimer's disease, multi- mfarct & other dementias, attention deficit hyperactivity disorders, age-related cognitive decline & other cognitive disorders, Parkinson's disease, Huntmgton's disease, amyotrophic lateral sclerosis, stroke or head trauma
  • a therapeutic target for treatment of such diseases could be provided by a polypeptide which has the properties of a glycine transporter, in particular of the GlyT-1
  • GC42 activity a polypeptide of the present invention exhibits at least one biological activity of GC42
  • 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 amino acids from the amino acid sequence of SEQ ID NO 2, or an isolated polypeptide comp ⁇ sing an amino acid sequence having at least 30, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO 2
  • Preferred fragments are biologically active fragments that mediate the biological activity of GC42, including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • 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 histidine 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 composing expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods Means for prepa ⁇ ng such polypeptides are well understood in the art
  • the present invention relates to GC42 polynucleotides
  • Such polynucleotides include
  • Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comp ⁇ sing an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID 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 ID NO 1
  • Preferred vanants of polynucleotides of the present invention include splice vanants, allehc vanants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs)
  • Polynucleotides of the present invention also include polynucleotides encoding polypeptide vanants that compnse the ammo acid sequence of SEQ ID NO 2 and 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 acid residues are substituted, deleted or added, in any combination
  • the present invention provides polynucleotides that are RNA transcripts of the
  • RNA polynucleotide that (a) comprises an RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO: 1
  • (b) is the RNA transc ⁇ pt of the DNA sequence encoding the polypeptide of SEQ ID NO 2,
  • (c) comprises an RNA transcript of the DNA sequence of SEQ ID NO: 1;
  • (d) is the RNA transcript of the DNA sequence of SEQ ID NO: 1; and RNA polynucleotides that are complementary thereto.
  • polynucleotide sequence of SEQ ID NO:l shows homology with Glycine Transporter Type lc
  • the polynucleotide sequence of SEQ ID NO:l is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2.
  • the polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ID NO: 1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polypeptide of the SEQ ID NO:2 is related to other proteins of the Neurotransmitter Transporter family, having homology and/or structural similarity with Glycine Transporter Type lc (GB accession P48067 (the glycine transporter type lc of this accession is named sodium and chloride-dependent glycine transporter); Kim et al.Mol. Pharm. 45:608-17. 1994; shown as SEQ ID NO:4).
  • the polypeptide of the invention shown in SEQ ID NO:2 has 2 amino acid differences from the polypeptide of SEQ ID NO:4 (Kim et al.Mol. Pharm. 45:608-17.
  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one GC42 activity
  • Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library denved from mRNA in cells of human brain and hippocampus (see for instance, Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Spnng Harbor Laboratory Press, Cold Spnng Harbor, N Y (1989))
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries 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 readmg 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 punfication of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc ) and descnbed 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 trans
  • Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO 1 may be used as hybndization probes for cDNA and genomic DNA or as pnmers for a nucleic acid amplification reaction (for instance, PCR) Such probes and pnmers 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 similarity to SEQ ID NO: 1, typically at least 95% identity.
  • Preferred probes and primers will generally comprise 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 primers will have between 20 and 25 nucleotides.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process comprising 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
  • 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 (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. Ix 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 ID 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
  • a combination of gene specific and adaptor specific oligonucleotide primers is then canied out to amplify the "missing" 5' end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers.
  • the PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor 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 analysed 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. For recombinant production, 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 as Davis et al. , Basic Methods in Molecular Biology (1986) and Sambrook et al.(ibid). Prefened methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • bacterial cells such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as Streptococci, Staphylococci, E.
  • expression systems can be used, for instance, chromosomal, episomal and virus- derived systems, e.g. , vectors derived from bacterial plasmids, from bacteriophage, 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 derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
  • the appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those set forth in Sambrook et al., (ibid).
  • Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • a polypeptide of the present invention is to be expressed for use in 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 prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify 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 purified 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 purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification.
  • 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 characterised 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 variety of techniques well known in 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 GC42 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 GC42 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 radioimmunoassays, 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 ID NO: 1, or a fragment or an RNA transcript thereof; (b) a nucleotide sequence complementary to that of (a);
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or
  • 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 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 rnRNAs that encode them.
  • the techniques used are well known in the art and include in situ hydridisation techniques to clones arrayed on a grid, such as cDNA microarray hybridisation (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR.
  • a preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism.
  • 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.
  • 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.
  • Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G.
  • 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 diseases of the invention, amongst others.
  • Polypeptides and polynucleotides of the present invention may also be used as vaccines.
  • the present invention relates to a method for inducing an immunological response in a mammal that comprises 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 comprises 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
  • a method comprises 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 admimstenng the vector is by accelerating it into the desired cells as a coating on particles or otherwise
  • Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybnd
  • a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition)
  • the formulation may further comprise a suitable earner Since a polypeptid
  • 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 vanety of sources, for example, cells, cell- free preparations, chemical libranes, collections of chemical compounds, and natural product mixtures Such agonists or antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of the polypeptide, a structural or functional mimetic thereof (see Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)) or a small molecule Such small molecules
  • 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 described by Schullek et al, Anal Biochem., 246, 20-29, (1997)
  • Fusion proteins such as those made from Fc portion and GC42 polypeptide, as hereinbefore descnbed, 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))
  • activity of a neurotransmitter transporter may be determined in cells in culture through use of a radiolabelled substrate. After exposure of the cells to the substrate (added to the culture medium) for a defined length of time, the cells are washed, their contents extracted by treatment with acid or alkali and the cellular accumulation of radiolabel determined by scmtillation spectrometry.
  • maximal activity, substrate concentration required for half maximal activity (Km) and the potency of competing agents e.g. small molecules
  • 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 m 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 cross nking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ l), 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 m the art
  • antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the ligands, 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
  • transgemc technology Screening methods may also involve the use of transgemc technology and GC42 gene
  • the GC42 gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post- lmplantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts
  • Particularly useful transge c animals are so-called "knock-m” animals in which an animal gene is replaced by the human equivalent within the genome of that animal Knock-in transgemc animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target
  • Other useful transgemc 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
  • Screening kits for use in the above described methods form a further aspect of the present invention
  • Such screening kits compnse (a) a polypeptide of the present invention
  • 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, i e , if it occurs in nature, it has been changed or removed from its original 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 matenals 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, hybrid molecules compnsing DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions
  • polynucleotide refers to tnple-stranded regions comprising RNA or DNA or both RNA and DNA
  • 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
  • Polypeptide refers to any polypeptide comprising two or more amino 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, ohgopeptides or oligomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids
  • Polypeptides include amino 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 descnbed 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 be present to the same or varying degrees at several sites in a given polypeptide Also, a given
  • “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 polynucloetide sequence that is shorter than the reference sequence of SEQ ID 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 Generally, alterations are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination A substituted or
  • Polymorphism refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population
  • SNP Single Nucleotide Polymorphism
  • SNP Single Nucleotide Polymorphism
  • ASA Allele Specific Amplification
  • a common pnmer is used in reverse complement to the polymorphism being assayed This common primer can be between 50 and 1500 bps from the polymorphic base
  • the other two (or more) pnmers 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 and one of the Allele Specific Pnmers
  • RNA Vanant refers to cDNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing Alternative RNA splicing occurs when a primary RNA transcnpt undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of that may encode different amino acid sequences
  • 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 comparing the sequences In general, identity refers to an exact nucleotide to nucleotide or ammo 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 conespondence, a “% identity” may be determined
  • the two sequences to be compared are aligned to give a maximum correlation between the sequences This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment
  • a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length
  • “Similanty” is a further, more sophisticated measure of the relationship between two polypeptide sequences
  • “similarity” means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact conespondences 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 conespondence, 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
  • the BLOSUM62 amino acid substitution matrix (Henikoff S and Henikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into amino acid sequences before comparison.
  • the program BESTFIT 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 described.
  • 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 transversion, or insertion. These differences may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides 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 nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described.
  • 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 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one amino 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.
  • n a is the number of nucleotide or amino acid differences
  • x a is the total number of nucleotides or amino acids in SEQ ID NO: 1 or SEQ ID NO:2, respectively
  • I is the Identity Index , • is the symbol for the multiplication operator, and in which any non-integer product of x a and I is rounded down to the nearest integer prior to subtracting it from x a .
  • “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 similarity between the two sequences as hereinbefore defined. Falling within this generic 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 within 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-0 464 533-A discloses fusion proteins comprising 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 in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262].
  • the transporter of the present invention is expressed transiently in human embryonic kidney 293 (HEK293) cells. To maximise expression, typically 5' and 3' untranslated regions (UTRs) are removed from the transporter cDNA prior to transfection. Cells are transiently transfected using lipofectamine. Typically 24-48 hours after transfection, glycine transport activity is assayed as described in Example 2.
  • Suspensions of cells transiently transfected as described in Example 1 are pipetted into polylysine-coated 96 well plates (50,000 cells per well). 4-24 hours later, the wells are washed with standard Krebs/HEPES buffer (KHB) at 23°C. After addition of further KHB to the wells, the plates incubated at 23°C for 10 min. KHB containing appropriate concentrations of a potential inhibitor is then added to the wells, together with 0.5 ⁇ M [ ⁇ H]-glycine. After incubation at 23°C for 2-60 min, the wells are washed with ice-cold KHB. The cells are then solubilised, and the amount of radioactivity present determined using a scintillation counter.
  • KHB standard Krebs/HEPES buffer

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Abstract

Cette invention concerne des polypeptides et des polynucléotides GC42 ainsi que des procédés d'obtention desdits polypeptides au moyen de techniques recombinantes. L'invention porte également sur l'utilisation de polypeptides et de polynucléotides GC42 dans le cadre d'essais diagnostiques.
PCT/EP2000/006253 1999-07-08 2000-07-04 Nouveaux composes WO2001004142A2 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
AU2004226547B2 (en) * 2003-03-28 2008-10-23 Nissan Chemical Industries,Ltd. T-Type calcium channel blockers

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Publication number Priority date Publication date Assignee Title
US5756348A (en) * 1991-11-12 1998-05-26 Synaptic Pharmaceutical Corporation DNA encoding a glycine transporter and uses thereof
US5824486A (en) * 1996-05-31 1998-10-20 Allelix Neuroscience Inc. Glycine transporter-transfected cells and uses thereof
WO1998046619A1 (fr) * 1997-04-11 1998-10-22 Allelix Neuroscience Inc. Transporteur de la glycine

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US5756348A (en) * 1991-11-12 1998-05-26 Synaptic Pharmaceutical Corporation DNA encoding a glycine transporter and uses thereof
US5824486A (en) * 1996-05-31 1998-10-20 Allelix Neuroscience Inc. Glycine transporter-transfected cells and uses thereof
WO1998046619A1 (fr) * 1997-04-11 1998-10-22 Allelix Neuroscience Inc. Transporteur de la glycine

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GUASTELLA ET AL.: "Cloning, expression, and localization of a rat brain high-affinity glycine transporter" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, vol. 89, 1992, pages 7189-7193, XP002158328 *
KIM K -M ET AL: "CLONING OF THE HUMAN GLYCINE TRANSPORTER TYPE 1: MOLECULAR AND PHARMACOLOGICAL CHARACTERIZATION OF NOVEL ISOFORM VARIANTS AND CHROMOSOMAL LOCALIZATION OF THE GENE IN THE HUMAN AND MOUSE GENOMES" MOLECULAR PHARMACOLOGY,US,BALTIMORE, MD, vol. 45, 1994, pages 608-617, XP002911694 ISSN: 0026-895X cited in the application *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2004226547B2 (en) * 2003-03-28 2008-10-23 Nissan Chemical Industries,Ltd. T-Type calcium channel blockers
KR100870527B1 (ko) * 2003-03-28 2008-11-26 닛산 가가쿠 고교 가부시키 가이샤 Ｔ형 칼슘 채널 저해제

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