WO2002006313A2 - Regulation of human glutamate receptor delta-1 subunit - Google Patents

Regulation of human glutamate receptor delta-1 subunit Download PDF

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Publication number
WO2002006313A2
WO2002006313A2 PCT/EP2001/008102 EP0108102W WO0206313A2 WO 2002006313 A2 WO2002006313 A2 WO 2002006313A2 EP 0108102 W EP0108102 W EP 0108102W WO 0206313 A2 WO0206313 A2 WO 0206313A2
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subunit
glutamate receptor
polypeptide
receptor delta
seq
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PCT/EP2001/008102
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French (fr)
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WO2002006313A3 (en
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Sophia Kossida
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Bayer Aktiengesellschaft
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Publication of WO2002006313A3 publication Critical patent/WO2002006313A3/en

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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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the regulation of human glutamate-like receptors to provide therapeutic effects.
  • glutamate receptor classification schemes are based on pharmacological criteria which serve to define five receptor subtypes or classes: those activated by N-methyl-D-aspartic acid (NMD A), kainic acid (KA), ⁇ -amino-3-hydroxy-5-methyl- isoxazole-4-propionic acid (AMP A, formally called the quisqualic acid or QUIS receptor), 2-amino-4-phosphonobutyric acid (AP4 or APB), and 1-amino-cyclo- pentyl-l,3-dicarboxylic acid (ACPD).
  • NMD A N-methyl-D-aspartic acid
  • KA kainic acid
  • AMP A ⁇ -amino-3-hydroxy-5-methyl- isoxazole-4-propionic acid
  • AP4 or APB 2-amino-4-phosphonobutyric acid
  • ACPD 1-amino-cyclo- pentyl-l,3-dicarboxylic acid
  • ACPD receptor subtype which has the properties of a metabotropic receptor.
  • This class of glutamate receptors alters synaptic physiology via GTP-binding proteins and the second messengers diacylglycerol and inositol 1,4,5-triphosphate (Gundersen et al., Proc. R. Soc. London Ser. B 221, 127, 1984; Sladeczek et al, Nature 317, 111, 1985; Nicoletti et al, J. Neurosci. 6, 1905, 1986; Sugiyama et al, Nature 325, 531, 1987).
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a glutamate receptor delta- 1 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence shown in SEQ ID NO:7; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and the amino acid sequence shown in SEQ ID NO: 8.
  • a test compound which binds to the glutamate receptor delta- 1 subunit polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the activity of the glutamate receptor delta- 1 subunit.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a glutamate receptor delta- 1 subunit polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9; the nucleotide sequence shown in SEQ ID NO: 9; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; the nucleotide sequence shown in SEQ ID NO: 10; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 11 ; the nucleotide sequence shown in SEQ ID NO:l 1 nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 12; and the nucleotide sequence shown in SEQ ID NO: 12.
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the glutamate receptor delta- 1 subunit through interacting with the glutamate receptor delta- 1 subunit mRNA.
  • a test compound is contacted with a glutamate receptor delta- 1 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid 5 sequence shown in SEQ ID NO: 7; the amino acid sequence shown in SEQ ID NO:7; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and the amino acid sequence shown in SEQ ID NO: 8. re
  • a glutamate receptor delta- 1 subunit activity of the polypeptide is detected.
  • a test compound which increases glutamate receptor delta- 1 subunit activity of the polypeptide relative to glutamate receptor delta- 1 subunit activity in the absence of the test compound is thereby identified as a potential agent for increasing 15 extracellular matrix degradation.
  • a test compound which decreases glutamate receptor delta- 1 subunit activity of the polypeptide relative to glutamate receptor delta- 1 subunit activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • a test compound is contacted with a glutamate receptor delta- 1 subunit product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide 5 sequence shown in SEQ ID NO: 9; the nucleotide sequence shown in SEQ ID NO: 9; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; the nucleotide sequence shown in SEQ ID NO: 10; 0 nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 11 ; and the nucleotide sequence shown in SEQ ID NO:l 1 ; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 12; and the nucleotide sequences
  • a test compound which binds to the glutamate receptor delta- 1 subunit product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a glutamate receptor delta- 1 subunit polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9; the nucleotide sequence shown in SEQ ID NO: 9; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; the nucleotide sequence shown in SEQ ID NO: 10; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 11; the nucleotide sequence shown in SEQ ID NO:l 1; nucleotide sequences
  • Glutamate receptor delta- 1 subunit activity in the cell is thereby decreased.
  • the invention thus provides reagents and methods for regulating a human glutamate receptor delta- 1 subunit.
  • Such reagents and methods can be used ter alia, to treat or prevent epilepsy, schizophrenia and other mood disorders, neurodegenerative diseases such as Huntington's disease and Alzheimer's disease, ischemia, pain, benign prostate hyperplasia and urinary incontinence.
  • Fig. 1 shows the amino acid sequence of the rattus norvegicus Sprague-
  • Dawley glutamate receptor delta- 1 subunit identified with the Accesseion No. U08255 (SEQ ED NO: 1).
  • Fig. 2 shows the DNA-sequence of a glutamate receptor delta- 1 subunit polypeptide (SEQ ID NO:2).
  • Fig. 3 shows the DNA-sequence of a glutamate receptor delta- 1 subunit polypeptide (SEQ ID NO:3).
  • Fig. 4 shows the DNA-sequence of a glutamate receptor delta- 1 subunit polypeptide (SEQ ID NO:4).
  • Fig. 5 shows the amino acid sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:5).
  • Fig. 6 shows the amino acid sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:6).
  • Fig. 7 shows the amino acid sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:7).
  • Fig. 8 shows the amino acid sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:8).
  • Fig. 9 shows the DNA-sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:9).
  • Fig. 10 shows the DNA-sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO: 10).
  • Fig. 11 shows the DNA-sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO: 11).
  • Fig. 12 shows the exons which encompass the first approximately 190 amino acids of glutamate receptor delta-1 subunit polypeptide.
  • Fig. 13 shows the protein sequences of glutamate receptor delta- 1 subunit polypeptide.
  • Fig. 14 shows the BLASTP alignment of glutamate receptor delta- 1 subunit polypeptide with the rat protein identified with GenBank Accession No. U08255.
  • Fig. 15 shows the DNA-sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO: 12).
  • the invention relates to an isolated polynucleotide encoding a glutamate" receptor delta- 1 subunit polypeptide and being selected from the group consisting of: a) a polynucleotide encoding a glutamate receptor delta- 1 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence shown in SEQ ID NO: 7; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and the amino acid sequence shown in SEQ ID NO: 8.
  • glutamate receptor delta-1 subunit Previously identified glutamate receptor delta-1 subunit presently has the status of an orphan receptor. Villmann et al, Eur. J. Neurosci. 11, 11765-78, 1999. In mice, this receptor is expressed in inner hair cells and in type I and type II vestibular hair cells, suggestion a functional role in neurotransmission in hair cells. Saffieddine & Wenthold, J Neurosci. 17, 7523-31, 1999. It is believed that the human glutamate receptor delta-1 subunit can be used to develop treatments for various diseases, to develop diagnostic assays for these diseases, and to provide new tools for basic research especially in the fields of medicine and biology.
  • the present invention can be used to develop new drugs to treat or prevent epilepsy, schizophrenia and other mood disorders, neurodegenerative diseases such as Huntington's disease and Alzheimer's disease, ischemia, pain, benign prostate hyperplasia and urinary incontinence.
  • neurodegenerative diseases such as Huntington's disease and Alzheimer's disease
  • ischemia ischemia
  • pain benign prostate hyperplasia
  • urinary incontinence Polypeptides
  • Glutamate receptor delta-1 subunit variants which are biologically active, i.e., retain a ligand-binding function and/or a neurotransmission function, also are glutamate receptor delta-1 subunit polypeptides.
  • glutamate receptor delta-1 subunit variants Preferably, naturally or non-naturally occurring glutamate receptor delta-1 subunit variants have amino acid sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to an amino acid sequence shown in SEQ ID NOS:5, 6, 7, or 8.
  • Percent identity between a putative glutamate receptor delta-1 subunit variant and an amino acid sequence of SEQ ID NOS:5, 6, 7, or 8 is determined with the Needleman/Wunsch algorithm (Needleman and Wunsch, J.Mol. Biol. 48; 443-453, 1970) using a Blosum62 matrix with a gap creation penalty of 8 and a gap extension penalty of 2 (S. Henikoff and J.G. Henikoff, Proc. Natl. Acad. Sci. USA 89:10915- 10919, 1992).
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids.
  • Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a glutamate receptor delta-1 subunit polypeptide can be found using computer programs well known in the art, such as
  • Fusion proteins are useful for generating antibodies against glutamate " receptor delta-1 subunit amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of a glutamate receptor delta-1 subunit polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a glutamate receptor delta-1 subunit fusion protein comprises two protein segments fused together by means of a peptide bond.
  • the first protein segment comprises at least 5, 6, 8, 10, 25, or 50 or more contiguous amino acids of a glutamate receptor delta-1 subunit polypeptide.
  • Contiguous amino acids for use in a fusion protein can be selected from the amino acid sequence shown in SEQ ID NOS:5, 6, 7, or 8 or from a biologically active variant of those sequences, such as those described above.
  • the first protein segment also can comprise full-length glutamate receptor delta-1 subunit.
  • the second protein segment can be a full-length protein or a protein fragment or polypeptide.
  • Proteins commonly used in fusion protein construction include ⁇ - galactosidase, ⁇ -glucuronidase, green fluorescent protein (GFP), auto fluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV- G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4
  • a fusion protein also can be engineered to contain a cleavage site located between the glutamate receptor delta-1 subunit polypeptide-encoding sequence and the heterologous protein sequence, so that the glutamate receptor delta-1 subunit polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two protein segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises a coding sequence from NOS:2, 3, 4, 9, 10, and 11 in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz
  • Species homologs of human glutamate receptor delta-1 subunit can be obtained using glutamate receptor delta-1 subunit polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of glutamate receptor delta- 1 subunit, and expressing the cDNAs as is known in the art.
  • glutamate receptor delta-1 subunit polynucleotides described below
  • a glutamate receptor delta-1 subunit polynucleotide can be single- or double- stranded and comprises a coding sequence or the complement of a coding sequence for a glutamate receptor delta-1 subunit polypeptide. Coding sequences for a glutamate receptor delta-1 subunit polypeptide are shown in SEQ ID NOS:2, 3, 4, 9, 10, 11 and 12.
  • nucleotide sequences encoding human glutamate receptor delta-1 subunit polypeptides, as well as homologous nucleotide sequences which are at least about
  • polynucleotide sequences shown in SEQ ID NOS:2, 3, 4, 9, 10, 11 and 12 also are glutamate receptor delta-1 subunit polynucleotides.
  • Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • cDNA Complementary DNA molecules, species homologs, and variants of glutamate receptor delta-1 subunit polynucleotides which encode biologically active glutamate receptor delta-1 subunit polypeptides also are glutamate receptor delta-1 subunit polynucleotides.
  • Variants and homologs of the glutamate receptor delta-1 subunit polynucleotides described above also are glutamate receptor delta-1 subunit polynucleotides.
  • homologous glutamate receptor delta-1 subunit polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known glutamate receptor delta-1 subunit polynucleotides under stringent conditions, as is known in the art.
  • homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
  • Species homologs of the glutamate receptor delta-1 subunit polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of glutamate receptor delta-1 subunit polynucleotides can be identified, for example, by screening human cDNA expression libraries. II is well known that the T m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973).
  • Variants of human glutamate receptor delta-1 subunit polynucleotides or glutamate receptor delta-1 subunit polynucleotides of other species can therefore be identified by hybridizing a putative homologous glutamate receptor delta-1 subunit polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NOS:2, 3, 4, 9, 10, 11 or 12 or the complements thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising glutamate receptor delta-1 subunit polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to glutamate receptor delta-1 subunit polynucleotides or their complements following stringent hybridization and/or wash conditions also are glutamate receptor delta-1 subunit polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • T m of a hybrid between a glutamate receptor delta-1 subunit polynucleotide having a nucleotide sequence shown in SEQ ID NOS:2, 3, 4, 9, 10, 11 and 12 or the complements thereof and a polynucleotide sequence which is at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
  • Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C.
  • Highly stringent wash conditions include, for example, 0.2X SSC at 65 oC.
  • a naturally occurring glutamate receptor delta-1 subunit polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated glutamate receptor delta-1 subunit polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise glutamate receptor delta-1 subunit nucleotide sequences.
  • Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • Glutamate receptor delta-1 subunit cDNA molecules can be made with standard molecular biology techniques, using glutamate receptor delta-1 subunit mRNA as a template. Glutamate receptor delta-1 subunit cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of glutamate receptor delta-1 subunit polynucleotides using either human genomic DNA or cDNA as a template.
  • glutamate receptor delta-1 subunit polynucleotides can be synthesized.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a glutamate receptor delta-1 subunit polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:5, 6, 7, or 8 or a biologically active variant.
  • the partial sequences of SEQ ID NOS:2, 3, 4, 9, 10, and 11 or their complements can be used to identify the corresponding full length gene from which they were derived.
  • the partial sequences can be nick-translated or end-labeled with 32 P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al, eds., Elsevier Press, N.Y.,
  • a lambda library prepared from human tissue can be screened directly with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al, 1989, pg. 1.20).
  • pBluescript Stratagene Cloning Systems, La Jolla, Calif. 92037
  • Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector.
  • Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis.
  • the complete sequence of the clones can be determined, for example after exonuclease III digestion
  • PCR-based methods can be used to extend the nucleic acid sequences encoding the disclosed portions of human glutamate receptor delta-1 subunit to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991).
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • Glutamate receptor delta-1 subunit polypeptides can be obtained, for example, by purification from human neuronal cells, by expression of glutamate receptor delta-1 subunit polynucleotides, or by direct chemical synthesis.
  • Glutamate receptor delta-1 subunit polypeptides can be purified, for example, from human neuronal cells or cell lines or from cells which have been transfected with a glutamate receptor delta-1 subunit polynucleotide. Brain, particularly amygdala, corpus callosum, and hippocampus provide useful sources of human glutamate receptor delta-1 subunit polypeptides. A purified glutamate receptor delta-1 subunit polypeptide is separated from other compounds which normally associate with the glutamate receptor delta-1 subunit polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art.
  • a glutamate receptor delta-1 subunit polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding glutamate receptor delta-1 subunit polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y, 1989.
  • TMV TMV
  • bacterial expression vectors e.g., Ti or pBR322 plasmids
  • animal cell systems e.g., TMV, TMV, TMV, TMV, TMV, or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions ⁇ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a glutamate receptor delta-1 subunit polypeptide, vectors based on S V40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected depending upon the use intended for a glutamate receptor delta-1 subunit polypeptide. For example, when a large quantity of a glutamate receptor delta-1 subunit polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • a sequence encoding a glutamate receptor delta-1 subunit polypeptide can be ligated in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pIN vectors Van Heeke &
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH
  • Plant and Insect Expression Systems the expression of sequences encoding glutamate receptor delta-1 subunit polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al,
  • An insect system also can be used to express a glutamate receptor delta-1 subunit polypeptide.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in
  • Sequences encoding glutamate receptor delta-1 subunit polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of glutamate receptor delta-1 subunit polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect & frugiperda cells or Trichoplusia larvae in which glutamate receptor delta-1 subunit polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994). Mammalian Expression Systems
  • a number of viral-based expression systems can be used to express glutamate receptor delta-1 subunit polypeptides in mammalian host cells.
  • sequences encoding glutamate receptor delta-1 subunit polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a glutamate receptor delta-1 subunit polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding glutamate receptor delta-1 subunit polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a glutamate receptor delta-1 subunit polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl. Cell Differ. 20, 125-162, 1994).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed glutamate receptor delta-1 subunit polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding, and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express glutamate receptor delta-1 subunit polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced glutamate receptor delta-1 subunit sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.
  • herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 213-31, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tk ⁇ or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci.
  • npt confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB, allows cells to utilize indole in place of tryptophan; hisD, allows cells to utilize histinol in place of histidine (Hartman &
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).
  • marker gene expression suggests that a glutamate receptor delta-1 subunit polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a glutamate receptor delta-1 subunit polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode the glutamate receptor delta-1 subunit polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a glutamate receptor delta-1 subunit polypeptide under the control of a single promoter.
  • Expression of the marker gene in response to induction or selection usually indicates expression of a glutamate receptor delta-1 subunit polynucleotide.
  • host cells which contain a glutamate receptor delta-1 subunit polynucleotide and which express a glutamate receptor delta-1 subunit polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a glutamate receptor delta-1 subunit polypeptide can be detected by DNA-DNA or
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding the glutamate receptor delta-1 subunit polypeptide to detect transformants which contain a glutamate receptor delta-1 subunit polynucleotide.
  • a variety of protocols for detecting and measuring the expression of a glutamate receptor delta-1 subunit polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a glutamate receptor delta-1 subunit polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn.,
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding glutamate receptor delta-1 subunit polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a glutamate receptor delta-1 subunit polypeptide can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a glutamate receptor delta-1 subunit polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode glutamate receptor delta-1 subunit polypeptides can be designed to contain signal sequences which direct secretion of glutamate receptor delta-1 subunit polypeptides through a prokaryotic or eukaryotic cell membrane.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension affinity purification system
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the glutamate receptor delta-1 subunit polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a glutamate receptor delta-1 subunit polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp. Purif.
  • enterokinase cleavage site provides a means for purifying the glutamate receptor delta-1 subunit polypeptide from the fusion protein.
  • Vectors which contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.
  • Sequences encoding a glutamate receptor delta-1 subunit polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-113, 1980; Horn et al. Nucl. Acids
  • a glutamate receptor delta-1 subunit polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of glutamate receptor delta-1 subunit polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983).
  • the composition of a synthetic glutamate receptor delta-1 subunit polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the glutamate receptor delta-1 subunit polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein. Production of Altered Polypeptides
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter glutamate receptor delta-1 subunit polypeptide- encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • An antibody which specifically binds to an epitope of a glutamate receptor delta-1 subunit polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.
  • an antibody which specifically binds to a glutamate receptor delta-1 subunit polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to glutamate receptor delta-1 subunit polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a glutamate receptor delta-1 subunit polypeptide from solution.
  • BCG Bacilli Calmette-Gueri ⁇
  • Corynebacterium parvum axe especially useful.
  • Monoclonal antibodies which specifically bind to a glutamate receptor delta-1 subunit polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984).
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985).
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues.
  • rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described in GB2188638B.
  • Antibodies which specifically bind to a glutamate receptor delta-1 subunit polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant D ⁇ A methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; ⁇ icholls et al, 1993, J. Immunol. Meth. 165, 81-91).
  • Antibodies which specifically bind to glutamate receptor delta-1 subunit polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a glutamate receptor delta-1 subunit polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense Oligonucleotides can be affinity purified by passage over a column to which a glutamate receptor delta-1 subunit polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of glutamate receptor delta-1 subunit gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al,
  • Modifications of glutamate receptor delta-1 subunit gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of a glutamate receptor delta-1 subunit gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a glutamate receptor delta-1 subunit polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent glutamate receptor delta-1 subunit nucleotides, can provide sufficient targeting specificity for glutamate receptor delta-1 subunit mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular glutamate receptor delta-1 subunit polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a glutamate receptor delta-1 subunit polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515,
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a glutamate receptor delta-1 subunit polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the glutamate receptor delta-1 subunit polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et «/., EP 321,201).
  • Specific ribozyme cleavage sites within a glutamate receptor delta-1 subunit RNA target can be identified by scanning the glutamate receptor delta-1 subunit target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate glutamate receptor delta-1 subunit RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • the nucleotide sequence shown in SEQ ID NO:l and its complement provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease glutamate receptor delta-1 subunit expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells. Screening Methods
  • the invention provides methods for identifying modulators, i.e., candidate or test compounds which bind to glutamate receptor delta-1 subunit polypeptides or polynucleotides and/or have a stimulatory or inhibitory effect on, for example, expression or binding activity of the glutamate receptor delta-1 subunit polypeptide or polynucleotide, so as to regulate a biological function.
  • Regulation of human glutamate receptor delta-1 subunit is useful, for example, for preventing or treating epilepsy, schizophrenia and other mood disorders, neurodegenerative diseases such as Huntington's disease and Alzheimer's disease, ischemia, pain, benign prostate hyperplasia and urinary incontinence.
  • the invention provides assays for screening test compounds which bind to or modulate the binding activity of a glutamate receptor delta-1 subunit polypeptide or a glutamate receptor delta-1 subunit polynucleotide.
  • a test compound preferably binds to a glutamate receptor delta-1 subunit polypeptide or polynucleotide.
  • a test compound decreases a glutamate receptor delta-1 subunit ligand binding activity of a glutamate receptor delta-1 subunit polypeptide or expression of a glutamate receptor delta-1 subunit polynucleotide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinanfiy, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • High Throughput Screening Test compounds can be screened for the ability to bind to glutamate receptor delta-1 subunit polypeptides or polynucleotides or to affect glutamate receptor delta-1 subunit ligand binding activity or glutamate receptor delta-1 subunit gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995).
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel.
  • beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule which binds to and occupies the active site of a glutamate receptor delta-1 subunit polypeptide, thereby making the active site inaccessible to substrate such that normal biological binding activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the glutamate receptor delta-1 subunit polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound which is bound to the glutamate receptor delta-1 subunit polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • binding of a test compound to a glutamate receptor delta-1 subunit polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a glutamate receptor delta-1 subunit polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS).
  • Determining the ability of a test compound to bind to a glutamate receptor delta-1 subunit polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995).
  • BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • a glutamate receptor delta-1 subunit polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs. For example, in one construct a polynucleotide encoding a glutamate receptor delta-1 subunit polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein (“prey" or "sample”) can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • a DNA sequence that encodes an unidentified protein "prey" or "sample”
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the glutamate receptor delta-1 subunit polypeptide.
  • a reporter gene e.g., LacZ
  • glutamate receptor delta-1 subunit polypeptide or polynucleotide
  • test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • a glutamate receptor delta-1 subunit polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated glutamate receptor delta-1 subunit polypeptides, polynucleotides, or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce
  • antibodies which specifically bind to a glutamate receptor delta-1 subunit polypeptide, polynucleotides, or a test compound, but which do not interfere with a desired binding site, such as the active site of the glutamate receptor delta-1 subunit polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to a glutamate receptor delta-1 subunit polypeptide or test compound, enzyme-linked assays which rely on detecting a glutamate receptor delta-1 subunit activity of the glutamate receptor delta-1 subunit polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Any cell which comprises a glutamate receptor delta-1 subunit polynucleotide or polypeptide can be used in a cell-based assay system.
  • a glutamate receptor delta-1 subunit polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, including cell lines such as the HCN-1A, HCN-2, CATH.a, Neuro-2a, and
  • PC 12 (Clontech), can be used, can be used.
  • An intact cell is contacted with a test compound. Binding of the test compound to a glutamate receptor delta-1 subunit polypeptide or polynucleotide is determined as described above, after lysing the cell to release the glutamate receptor delta-1 subunit polypeptide-or polynucleotide-test compound complex.
  • Test compounds can be tested for the ability to increase or decrease an activity of a human glutamate receptor delta-1 subunit polypeptide, such as ligand gated ion channel activity. Such activity is measured as is known in the art, for example, before and after contacting either a purified glutamate receptor delta-1 subunit polypeptide, a cell extract, or an intact cell with a test compound.
  • a test compound which decreases glutamate receptor delta-1 subunit binding to a ligand, such as kainate, by at least about 10, preferably about 50, more preferably about 75, -90, or 100% is identified as a potential antagonist of the receptor;
  • a test compound which increases glutamate receptor delta-1 subunit binding to the ligand by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agonist of the receptor.
  • test compounds which increase or decrease glutamate receptor delta-1 subunit gene expression are identified.
  • a glutamate receptor delta-1 subunit polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the glutamate receptor delta-1 subunit polynucleotide is determined.
  • the level of expression of glutamate receptor delta-1 subunit mRNA or polypeptide in the presence of the test compound is compared to the level of expression of glutamate receptor delta-1 subunit mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison.
  • polypeptide products of a glutamate receptor delta-1 subunit polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a glutamate receptor delta-1 subunit polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses a glutamate receptor delta-1 subunit polynucleotide can be used in a cell-based assay system.
  • the glutamate receptor delta-1 subunit polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • Either a primary culture or an established cell line, including cell lines such as the HCN-1A, HCN-2, CATH.a, Neuro-2a, and PC 12 (Clontech), can be used.
  • compositions of the invention can comprise, for example, a glutamate receptor delta-1 subunit polypeptide, Glutamate receptor delta-1 subunit polynucleotide, antibodies which specifically bind to glutamate receptor delta-1 subunit activity, or mimetics, agonists, antagonists, or inhibitors of glutamate receptor delta-1 subunit activity.
  • the compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • a filler or binders such as lactose or starches
  • lubricants such as talc or magnesium stearate
  • stabilizers optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • Glutamate is involved in epileptogenesis. Overstimulation of glutamate receptors can lead to seizures and excitotoxin injury throughout the CNS and in particular the hippocampus. Antagonists of glutamate receptor delta-1 subunit can act to prevent or treat epilepsy.
  • Glutamate plays a role in ischemic brain damage and compounds that decrease the accumulation of glutamate or block its postsynaptic effects can ameliorate ischemic injury and other forms of acute neuronal degenerative diseases like hypoxia/hypoglycemia, traumatic brain injury, and stroke.
  • Antagonists of glutamate receptor delta-1 subunit can act to prevent or treat brain damage.
  • Excitotoxicity the overstimulation of glutamate receptors resulting in the death of neurons, participates in the pathogenesis of chronic neurodegenerative disorders including Huntington's disease, Alzheimer's disease, and Parkinson's disease.
  • Antagonists of glutamate receptor delta-1 subunit can act to prevent or treat excitotoxicity and therefore can prevent or treat chronic neurodegenerative disorders.
  • the excitatory amino acids glutamate and aspartate have been implicated in the transmission of acute and chronic pain. Blocking the activity of glutamate receptor delta-1 subunit can prevent or treat pain, and in particular chronic pain disorders. Pain which can be treated includes that which arises from pancreatitis, interstitial cystitis from various origin, including infection, dysfunctional bladder epithelium, neurogenic disturbances, bladder mastocytosis, allergic/immune/auto-immune causes, endocrine, food intolerance, painful bladder disease causing suprapubic, urethral, or pelvic pain, including interstitial cystitis, endometriosis, bacterial cystitis, outlet obstruction, dysmenorrhea, IBS (Irritable Bowel Syndrome), and Crohn's Disease, as well as pain syndromes of the pelvic cavity, e.g.
  • Neuropathologic pain includes that arising in connection with multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke status, vascular lesion in the brain and spinal cord, including infarct, hemorrhage, and vascular malformation.
  • post mastectomy pain reflex sympathetic dystrophy (RSD), trigeminal neuralgia, radioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy), vasculitic neuropathy (e.g., secondary to connective tissue disease), paraneoplastic polyneuropathy associated e.g.
  • Headache including migraine with aura, migraine without aura, episodic and chronic tension-type headache, cluster headache, and chronic paroxysmal hemicrania, as well as hyperirritability of the tracheobronchial tree, can be treated by regulating human glutamate receptor delta-1 subunit activity.
  • dementias including Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia including Pick's disease, Parkinsonism, progressive nuclear palsy, corticobasal degeneration, motoneuron disease dementia, including ALS, Huntington's disease, multiple sclerosis, small-vessel cerebrovascular disease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff s psychosis can be treated.
  • This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a polypeptide-binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • a liposome is between about 100 and 500 ran, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a tumor cell, such as a tumor cell ligand exposed on the outer surface of the liposome. Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151).
  • polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al Trends in Biotechnol 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci.
  • a polynucleotide encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated
  • DNA transfer transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases glutamate receptor delta-1 subunit binding activity relative to glutamate receptor delta-1 subunit binding activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED5o.
  • compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED5 0 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts of any particular reagent can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for polypeptides or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • the polynucleotide of SEQ ID NO: 12 is inserted into the expression vector pCEV4 and the expression vector pCEV4-glutamate receptor delta-1 subunit polypeptide obtained is transfected into human embryonic kidney 293 cells.
  • Binding reaction mixtures are incubated for one hour at 30 °C.
  • the reaction is stopped by filtration through GF/B filters treated with 0.5% polyethyleneimine, using a cell harvester. Radioactivity is measured by scintillation counting, and data are analyzed by a computerized non-linear regression program.
  • Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 100 nM of unlabeled peptide. Protein concentration is measured by the Bradford method using Bio-Rad Reagent, with bovine serum albumin as a standard. It is shown that the polypeptide of SEQ ID NO: 1
  • the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of a recombinant human glutamate receptor delta-1 subunit in yeast.
  • the encoding DNA sequence is derived from the nucleotide sequence shown in SEQ ID NO:9, 10, 11 or 12.
  • the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag, and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added.
  • the modified DNA sequence is ligated into pPICZB.
  • This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter.
  • the resulting pPICZ/md-His6 vector is used to transform the yeast.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks, and the recombinantly produced protein isolated from the culture by affinity chromatography
  • Glutamate receptor delta-1 subunit polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Glutamate receptor delta-1 subunit polypeptides comprise an amino acid sequence shown in SEQ ID NO:5, 6, 7, or 8.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a glutamate receptor delta-1 subunit polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound was not incubated is identified as a compound which binds to a glutamate receptor delta-1 subunit polypeptide.
  • a human embryonic 293 kidney cell line is transfected with a glutamate receptor delta-1 subunit polynucleotide.
  • Test compounds comprising potential glutamate receptor delta-1 subunit antagonists or agonists are prepared. Forty-eight hours after transfection, agonist-activated and antagonist-deactivated currents in the presence of various ligands, such as kainate, are measured using standard patch clamp techniques in the whole-cell configuration. See Sommers et al. EMBO J. 11, 1651-1656, 1992;
  • Keinanen et al Science. 249, 556-560 (1990); Sommer et al, Science. 249, 1580-1585; Verdoorn et al, Science. 252, 1715-1718, 1991.
  • Currents in the presence of a test agonist or antagonist and in the absence of a test compound are collected, recorded and analyzed.
  • Test compounds that evoke currents in the presence of the ligand are identified as agonists of glutamate receptor delta-1 subunit and compounds that depress currents in the presence of the ligand are identified as antagonists of glutamate receptor delta-1 subunit.
  • oligonucleotides comprising at least 11 contiguous nucleotides selected from SEQ ID NO:9, 10, 11 or 12 is performed on a Pharmacia Gene Assembler series synthesizer using the phosphoramidite procedure (Uhlmann et al, Chem. Rev. 90, 534-83, 1990). Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate-buffered saline (PBS) at the desired concentration. Purity of these oligonucleotides is tested by capillary gel electro- phoreses and ion exchange HPLC. Endotoxin levels in the oligonucleotide preparation are determined using the Limulus Amebocyte Assay (Bang, Biol. Bull. (Woods Hole, Mass.) 105, 361-362, 1953).
  • the antisense oligonucleotides are administered to a patient with epilepsy.
  • the severity of the patient's epilepsy is decreased.

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Abstract

Reagents and methods for regulating human glutamate receptor delta-1 subunit are provided. Such reagents and methods can be used inter alia, to treat or prevent epilepsy, schizophrenia and other mood disorders, neurodegenerative diseases such as Huntington's disease and Alzheimer's disease, ischemia, pain, benign prostate hyperplasia and urinary incontinence.

Description

_ ι _
REGULATION OF HUMAN GLUTAMATE RECEPTOR DELTA-1 SUBUNIT
TECHNICAL FIELD OF THE INVENTION
The invention relates to the regulation of human glutamate-like receptors to provide therapeutic effects.
BACKGROUND OF THE INVENTION
L-glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (CNS). L-glutamate opens cation channels that mediate fast excitatory synaptic responses and establish and maintain synaptic plasticity underlying learning and memory. These cation channels also mediate cell death resulting from excessive glutamate release in the CNS due to acute injury or environmental excitotoxins. Thus, glutamate receptors are involved in the developmental plasticity processes and long term potentiation. Further, the continuous activation of glutamate receptors can contribute to the pathogenesis of diseases such as ischemia, pain, epilepsy, schizophrenia, Huntington's disease, Parkinson's disease and Alzheimer's disease.
See U.S. Patent 5,945,509.
Currently, glutamate receptor classification schemes are based on pharmacological criteria which serve to define five receptor subtypes or classes: those activated by N-methyl-D-aspartic acid (NMD A), kainic acid (KA), α-amino-3-hydroxy-5-methyl- isoxazole-4-propionic acid (AMP A, formally called the quisqualic acid or QUIS receptor), 2-amino-4-phosphonobutyric acid (AP4 or APB), and 1-amino-cyclo- pentyl-l,3-dicarboxylic acid (ACPD). The effects of glutamate are mediated primarily through interactions with cation-selective, ionotropic receptors (Foster and Fagg, Brain Res. 7, 103-64, 1984; Strange, Biochem. J. 249, 309-18, 1988). An exception is the ACPD receptor subtype which has the properties of a metabotropic receptor. This class of glutamate receptors alters synaptic physiology via GTP-binding proteins and the second messengers diacylglycerol and inositol 1,4,5-triphosphate (Gundersen et al., Proc. R. Soc. London Ser. B 221, 127, 1984; Sladeczek et al, Nature 317, 111, 1985; Nicoletti et al, J. Neurosci. 6, 1905, 1986; Sugiyama et al, Nature 325, 531, 1987).
The electrophysiological and pharmacological properties of the glutamate receptors have been extensively studied and are now well established. See, for example, Foster and Fagg, 1984; Cotman et al, Trends Neurosci. 10, 263, 1987; Mayer and Westbrook, Prog. Neurobiol. 28, 197, 1987; Watkins and Olvermann, Trends
Neurosci. 10, 265, 1987; and Blair et al, Science 242, 577, 1988.
Because of the role of glutamate in various pathologic disorders, there is a need in the art to identify members of the glutamate receptor family which can be regulated to provide therapeutic effects.
SUMMARY OF THE INVENTION
It is an object of the invention to provide reagents and methods of regulating a human glutamate receptor delta- 1 subunit. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention is a glutamate receptor delta- 1 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence shown in SEQ ID NO:7; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and the amino acid sequence shown in SEQ ID NO: 8.
Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a glutamate receptor delta- 1 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence shown in SEQ ID NO:7; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and the amino acid sequence shown in SEQ ID NO: 8.
Binding between the test compound and the glutamate receptor delta- 1 subunit polypeptide is detected. A test compound which binds to the glutamate receptor delta- 1 subunit polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the activity of the glutamate receptor delta- 1 subunit. Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a polynucleotide encoding a glutamate receptor delta- 1 subunit polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9; the nucleotide sequence shown in SEQ ID NO: 9; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; the nucleotide sequence shown in SEQ ID NO: 10; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 11 ; the nucleotide sequence shown in SEQ ID NO:l 1 nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 12; and the nucleotide sequence shown in SEQ ID NO: 12.
Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the glutamate receptor delta- 1 subunit through interacting with the glutamate receptor delta- 1 subunit mRNA.
Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a glutamate receptor delta- 1 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid 5 sequence shown in SEQ ID NO: 7; the amino acid sequence shown in SEQ ID NO:7; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and the amino acid sequence shown in SEQ ID NO: 8. re
A glutamate receptor delta- 1 subunit activity of the polypeptide is detected. A test compound which increases glutamate receptor delta- 1 subunit activity of the polypeptide relative to glutamate receptor delta- 1 subunit activity in the absence of the test compound is thereby identified as a potential agent for increasing 15 extracellular matrix degradation. A test compound which decreases glutamate receptor delta- 1 subunit activity of the polypeptide relative to glutamate receptor delta- 1 subunit activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
0 Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a glutamate receptor delta- 1 subunit product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide 5 sequence shown in SEQ ID NO: 9; the nucleotide sequence shown in SEQ ID NO: 9; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; the nucleotide sequence shown in SEQ ID NO: 10; 0 nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 11 ; and the nucleotide sequence shown in SEQ ID NO:l 1 ; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 12; and the nucleotide sequence shown in SEQ ID NO: 12.
Binding of the test compound to the glutamate receptor delta- 1 subunit product is detected. A test compound which binds to the glutamate receptor delta- 1 subunit product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a glutamate receptor delta- 1 subunit polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9; the nucleotide sequence shown in SEQ ID NO: 9; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; the nucleotide sequence shown in SEQ ID NO: 10; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 11; the nucleotide sequence shown in SEQ ID NO:l 1; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 12; and the nucleotide sequence shown in SEQ ID NO: 12.
Glutamate receptor delta- 1 subunit activity in the cell is thereby decreased.
The invention thus provides reagents and methods for regulating a human glutamate receptor delta- 1 subunit. Such reagents and methods can be used ter alia, to treat or prevent epilepsy, schizophrenia and other mood disorders, neurodegenerative diseases such as Huntington's disease and Alzheimer's disease, ischemia, pain, benign prostate hyperplasia and urinary incontinence.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the amino acid sequence of the rattus norvegicus Sprague-
Dawley glutamate receptor delta- 1 subunit identified with the Accesseion No. U08255 (SEQ ED NO: 1). Fig. 2 shows the DNA-sequence of a glutamate receptor delta- 1 subunit polypeptide (SEQ ID NO:2).
Fig. 3 shows the DNA-sequence of a glutamate receptor delta- 1 subunit polypeptide (SEQ ID NO:3). Fig. 4 shows the DNA-sequence of a glutamate receptor delta- 1 subunit polypeptide (SEQ ID NO:4). Fig. 5 shows the amino acid sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:5). Fig. 6 shows the amino acid sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:6). Fig. 7 shows the amino acid sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:7).
Fig. 8 shows the amino acid sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:8). Fig. 9 shows the DNA-sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO:9). Fig. 10 shows the DNA-sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO: 10). Fig. 11 shows the DNA-sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO: 11). Fig. 12 shows the exons which encompass the first approximately 190 amino acids of glutamate receptor delta-1 subunit polypeptide. Fig. 13 shows the protein sequences of glutamate receptor delta- 1 subunit polypeptide. Fig. 14 shows the BLASTP alignment of glutamate receptor delta- 1 subunit polypeptide with the rat protein identified with GenBank Accession No. U08255.
Fig. 15 shows the DNA-sequence of a glutamte receptor delta 1 subunit polypeptide (SEQ ID NO: 12).
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to an isolated polynucleotide encoding a glutamate" receptor delta- 1 subunit polypeptide and being selected from the group consisting of: a) a polynucleotide encoding a glutamate receptor delta- 1 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence shown in SEQ ID NO: 7; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and the amino acid sequence shown in SEQ ID NO: 8. b) a polynucleotide comprising the sequence of SEQ ID NOS: 9, 10, 1 1 or 12; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).
Furthermore, it has been discovered by the present applicant that a novel glutamate receptor delta- 1 subunit, particularly a human glutamate receptor delta- 1 subunit, is a discovery of the present invention. Human glutamate receptor delta-1 subunit is 98% identical over 791 amino acids to the Rattus norvegicus Sprague-Dawley glutamate receptor delta-1 subunit mRNA identified with GenBank Accession No. "U08255. Coding sequences for human glutamate receptor delta-1 subunit are provided in SEQ ID NOS:2, 3, 4, 9, 10, 11 and 12.
Previously identified glutamate receptor delta-1 subunit presently has the status of an orphan receptor. Villmann et al, Eur. J. Neurosci. 11, 11765-78, 1999. In mice, this receptor is expressed in inner hair cells and in type I and type II vestibular hair cells, suggestion a functional role in neurotransmission in hair cells. Saffieddine & Wenthold, J Neurosci. 17, 7523-31, 1999. It is believed that the human glutamate receptor delta-1 subunit can be used to develop treatments for various diseases, to develop diagnostic assays for these diseases, and to provide new tools for basic research especially in the fields of medicine and biology. Specifically, the present invention can be used to develop new drugs to treat or prevent epilepsy, schizophrenia and other mood disorders, neurodegenerative diseases such as Huntington's disease and Alzheimer's disease, ischemia, pain, benign prostate hyperplasia and urinary incontinence. Polypeptides
Human glutamate receptor delta-1 subunit polypeptides according to the invention comprise at least the amino acid sequences shown in SEQ ID NOS:5, 6, 7, or 8, a portion of one of those sequences, or a biologically active variant, as described below. Thus, a glutamate receptor delta-1 subunit polypeptide can be a portion of a receptor molecule, a full-length glutamate receptor delta-1 subunit molecule, or a fusion protein comprising all or a portion of a glutamate receptor delta-1 subunit molecule.
Biologically Active Variants
Glutamate receptor delta-1 subunit variants which are biologically active, i.e., retain a ligand-binding function and/or a neurotransmission function, also are glutamate receptor delta-1 subunit polypeptides. Preferably, naturally or non-naturally occurring glutamate receptor delta-1 subunit variants have amino acid sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to an amino acid sequence shown in SEQ ID NOS:5, 6, 7, or 8. Percent identity between a putative glutamate receptor delta-1 subunit variant and an amino acid sequence of SEQ ID NOS:5, 6, 7, or 8 is determined with the Needleman/Wunsch algorithm (Needleman and Wunsch, J.Mol. Biol. 48; 443-453, 1970) using a Blosum62 matrix with a gap creation penalty of 8 and a gap extension penalty of 2 (S. Henikoff and J.G. Henikoff, Proc. Natl. Acad. Sci. USA 89:10915- 10919, 1992).
Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a glutamate receptor delta-1 subunit polypeptide can be found using computer programs well known in the art, such as
DNASTAR software. Whether an amino acid change results in a biologically active glutamate receptor delta-1 subunit polypeptide can readily be determined by assaying for ligand-gated ion channel function. See U.S. Patent 5,945,509.
Fusion Proteins
Fusion proteins are useful for generating antibodies against glutamate " receptor delta-1 subunit amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of a glutamate receptor delta-1 subunit polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
A glutamate receptor delta-1 subunit fusion protein comprises two protein segments fused together by means of a peptide bond. The first protein segment comprises at least 5, 6, 8, 10, 25, or 50 or more contiguous amino acids of a glutamate receptor delta-1 subunit polypeptide. Contiguous amino acids for use in a fusion protein can be selected from the amino acid sequence shown in SEQ ID NOS:5, 6, 7, or 8 or from a biologically active variant of those sequences, such as those described above. The first protein segment also can comprise full-length glutamate receptor delta-1 subunit.
The second protein segment can be a full-length protein or a protein fragment or polypeptide. Proteins commonly used in fusion protein construction include β- galactosidase, β-glucuronidase, green fluorescent protein (GFP), auto fluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV- G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4
DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the glutamate receptor delta-1 subunit polypeptide-encoding sequence and the heterologous protein sequence, so that the glutamate receptor delta-1 subunit polypeptide can be cleaved and purified away from the heterologous moiety.
A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two protein segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises a coding sequence from NOS:2, 3, 4, 9, 10, and 11 in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz
Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
Identification of Species Homologs Species homologs of human glutamate receptor delta-1 subunit can be obtained using glutamate receptor delta-1 subunit polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of glutamate receptor delta- 1 subunit, and expressing the cDNAs as is known in the art. Polynucleotides
A glutamate receptor delta-1 subunit polynucleotide can be single- or double- stranded and comprises a coding sequence or the complement of a coding sequence for a glutamate receptor delta-1 subunit polypeptide. Coding sequences for a glutamate receptor delta-1 subunit polypeptide are shown in SEQ ID NOS:2, 3, 4, 9, 10, 11 and 12.
Degenerate nucleotide sequences encoding human glutamate receptor delta-1 subunit polypeptides, as well as homologous nucleotide sequences which are at least about
50, 55, 60, 65, 70, preferably about 75, 90, 96, or 98% identical to the nucleotide sequences shown in SEQ ID NOS:2, 3, 4, 9, 10, 11 and 12 also are glutamate receptor delta-1 subunit polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of glutamate receptor delta-1 subunit polynucleotides which encode biologically active glutamate receptor delta-1 subunit polypeptides also are glutamate receptor delta-1 subunit polynucleotides.
Identification of Polynucleotide Variants and Homologs
Variants and homologs of the glutamate receptor delta-1 subunit polynucleotides described above also are glutamate receptor delta-1 subunit polynucleotides. Typically, homologous glutamate receptor delta-1 subunit polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known glutamate receptor delta-1 subunit polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions~2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each— homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
Species homologs of the glutamate receptor delta-1 subunit polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of glutamate receptor delta-1 subunit polynucleotides can be identified, for example, by screening human cDNA expression libraries. II is well known that the Tm of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973). Variants of human glutamate receptor delta-1 subunit polynucleotides or glutamate receptor delta-1 subunit polynucleotides of other species can therefore be identified by hybridizing a putative homologous glutamate receptor delta-1 subunit polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NOS:2, 3, 4, 9, 10, 11 or 12 or the complements thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising glutamate receptor delta-1 subunit polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
Nucleotide sequences which hybridize to glutamate receptor delta-1 subunit polynucleotides or their complements following stringent hybridization and/or wash conditions also are glutamate receptor delta-1 subunit polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated Tm of the hybrid under study. The Tm of a hybrid between a glutamate receptor delta-1 subunit polynucleotide having a nucleotide sequence shown in SEQ ID NOS:2, 3, 4, 9, 10, 11 and 12 or the complements thereof and a polynucleotide sequence which is at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
Tm = 81.5 °C - 16.6(log10[Na+]) + 0.41(%G + C) - 0.63(%formamide) - 600/1), where / = the length of the hybrid in basepairs.
Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C. Highly stringent wash conditions include, for example, 0.2X SSC at 65 oC.
Preparation of Polynucleotides
A naturally occurring glutamate receptor delta-1 subunit polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated glutamate receptor delta-1 subunit polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise glutamate receptor delta-1 subunit nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
Glutamate receptor delta-1 subunit cDNA molecules can be made with standard molecular biology techniques, using glutamate receptor delta-1 subunit mRNA as a template. Glutamate receptor delta-1 subunit cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of glutamate receptor delta-1 subunit polynucleotides using either human genomic DNA or cDNA as a template.
Alternatively, synthetic chemistry techniques can be used to synthesize glutamate receptor delta-1 subunit polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a glutamate receptor delta-1 subunit polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:5, 6, 7, or 8 or a biologically active variant.
Obtaining Full-Length Polynucleotides
The partial sequences of SEQ ID NOS:2, 3, 4, 9, 10, and 11 or their complements can be used to identify the corresponding full length gene from which they were derived. The partial sequences can be nick-translated or end-labeled with 32P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al, eds., Elsevier Press, N.Y.,
1986). For example, a lambda library prepared from human tissue can be screened directly with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al, 1989, pg. 1.20).
Both methods are well known in the art. Briefly, filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured, and the DNA is fixed to the filters. The filters are hybridized with the labeled probe using hybridization conditions described by Davis et al, 1986. The partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification. The resulting autoradio- grams are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque. The colonies or plaques are selected and expanded, and the DNA is isolated from the colonies for further analysis and sequencing. Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector. Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis.
Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined, for example after exonuclease III digestion
(McCombie et al, Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.
Various PCR-based methods can be used to extend the nucleic acid sequences encoding the disclosed portions of human glutamate receptor delta-1 subunit to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer
Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 ° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
Another method which can be used to retrieve unknown sequences is that of Parker et al, Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
Obtaining Polypeptides
Glutamate receptor delta-1 subunit polypeptides can be obtained, for example, by purification from human neuronal cells, by expression of glutamate receptor delta-1 subunit polynucleotides, or by direct chemical synthesis.
Protein Purification
Glutamate receptor delta-1 subunit polypeptides can be purified, for example, from human neuronal cells or cell lines or from cells which have been transfected with a glutamate receptor delta-1 subunit polynucleotide. Brain, particularly amygdala, corpus callosum, and hippocampus provide useful sources of human glutamate receptor delta-1 subunit polypeptides. A purified glutamate receptor delta-1 subunit polypeptide is separated from other compounds which normally associate with the glutamate receptor delta-1 subunit polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. Purification of recombinant GluR5, a similar receptor, is taught in Bettler et al, Neuron. 5, 583-595 (1990). A preparation of purified glutamate receptor delta-1 subunit polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. Expression of Polynucleotides
To express a glutamate receptor delta-1 subunit polypeptide, a glutamate receptor delta-1 subunit polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding glutamate receptor delta-1 subunit polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y, 1989.
A variety of expression vector/host systems can be utilized to contain and express sequences encoding a glutamate receptor delta-1 subunit polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculo virus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,
TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
The control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions ~ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a glutamate receptor delta-1 subunit polypeptide, vectors based on S V40 or EBV can be used with an appropriate selectable marker.
Bacterial and Yeast Expression Systems
In bacterial systems, a number of expression vectors can be selected depending upon the use intended for a glutamate receptor delta-1 subunit polypeptide. For example, when a large quantity of a glutamate receptor delta-1 subunit polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding a glutamate receptor delta-1 subunit polypeptide can be ligated in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke &
Schuster, J Biol. Chem. 264, 5503-5509, 1989 or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used. For reviews, see Ausubel et al. (1989) and Grant et al, Methods Enzymol 153, 516-544, 1987.
Plant and Insect Expression Systems If plant expression vectors are used, the expression of sequences encoding glutamate receptor delta-1 subunit polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al,
EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).
An insect system also can be used to express a glutamate receptor delta-1 subunit polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in
Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding glutamate receptor delta-1 subunit polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of glutamate receptor delta-1 subunit polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect & frugiperda cells or Trichoplusia larvae in which glutamate receptor delta-1 subunit polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994). Mammalian Expression Systems
A number of viral-based expression systems can be used to express glutamate receptor delta-1 subunit polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding glutamate receptor delta-1 subunit polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a glutamate receptor delta-1 subunit polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.
Sci. 81, 3655-3659, 1984). If desired, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells.
Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
Specific initiation signals also can be used to achieve more efficient translation of sequences encoding glutamate receptor delta-1 subunit polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a glutamate receptor delta-1 subunit polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl. Cell Differ. 20, 125-162, 1994).
Host Cells A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed glutamate receptor delta-1 subunit polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding, and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express glutamate receptor delta-1 subunit polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced glutamate receptor delta-1 subunit sequences.
Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.
Any number of selection systems can be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 213-31, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes which can be employed in tk~ or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci. 77, 3567-70, 1980) npt confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB, allows cells to utilize indole in place of tryptophan; hisD, allows cells to utilize histinol in place of histidine (Hartman &
Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers- such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).
Detecting Expression of Polypeptides
Although the presence of marker gene expression suggests that a glutamate receptor delta-1 subunit polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a glutamate receptor delta-1 subunit polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode the glutamate receptor delta-1 subunit polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a glutamate receptor delta-1 subunit polypeptide under the control of a single promoter.
Expression of the marker gene in response to induction or selection usually indicates expression of a glutamate receptor delta-1 subunit polynucleotide.
Alternatively, host cells which contain a glutamate receptor delta-1 subunit polynucleotide and which express a glutamate receptor delta-1 subunit polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a glutamate receptor delta-1 subunit polypeptide can be detected by DNA-DNA or
DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding the glutamate receptor delta-1 subunit polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding the glutamate receptor delta-1 subunit polypeptide to detect transformants which contain a glutamate receptor delta-1 subunit polynucleotide.
A variety of protocols for detecting and measuring the expression of a glutamate receptor delta-1 subunit polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a glutamate receptor delta-1 subunit polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn.,
1990) and Maddox et /., J Exp. Med. 158, 1211-1216, 1983).
A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding glutamate receptor delta-1 subunit polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a glutamate receptor delta-1 subunit polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Expression and Purification of Polypeptides
Host cells transformed with nucleotide sequences encoding a glutamate receptor delta-1 subunit polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode glutamate receptor delta-1 subunit polypeptides can be designed to contain signal sequences which direct secretion of glutamate receptor delta-1 subunit polypeptides through a prokaryotic or eukaryotic cell membrane.
As discussed above, other constructions can be used to join a sequence encoding a glutamate receptor delta-1 subunit polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension affinity purification system
(Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the glutamate receptor delta-1 subunit polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a glutamate receptor delta-1 subunit polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the glutamate receptor delta-1 subunit polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.
Chemical Synthesis
Sequences encoding a glutamate receptor delta-1 subunit polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-113, 1980; Horn et al. Nucl. Acids
Res. Symp. Ser. 225-232, 1980). Alternatively, a glutamate receptor delta-1 subunit polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of glutamate receptor delta-1 subunit polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic glutamate receptor delta-1 subunit polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the glutamate receptor delta-1 subunit polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein. Production of Altered Polypeptides
As will be understood by those of skill in the art, it may be advantageous to produce glutamate receptor delta-1 subunit polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter glutamate receptor delta-1 subunit polypeptide- encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
Antibodies
Any type of antibody known in the art can be generated to bind specifically to an epitope of a glutamate receptor delta-1 subunit polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')2, and Fv, which are capable of binding an epitope of a glutamate receptor delta-1 subunit polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve noncontiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
An antibody which specifically binds to an epitope of a glutamate receptor delta-1 subunit polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.
Typically, an antibody which specifically binds to a glutamate receptor delta-1 subunit polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to glutamate receptor delta-1 subunit polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a glutamate receptor delta-1 subunit polypeptide from solution.
Glutamate receptor delta-1 subunit polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a glutamate receptor delta-1 subunit polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Gueriή) and Corynebacterium parvum axe especially useful.
Monoclonal antibodies which specifically bind to a glutamate receptor delta-1 subunit polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984).
In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to a glutamate receptor delta-1 subunit polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to glutamate receptor delta-1 subunit polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).
Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.
A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DΝA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Νicholls et al, 1993, J. Immunol. Meth. 165, 81-91).
Antibodies which specifically bind to glutamate receptor delta-1 subunit polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).
Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a glutamate receptor delta-1 subunit polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration. Antisense Oligonucleotides
Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of glutamate receptor delta-1 subunit gene products in the cell.
Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al,
Chem. Rev. 90, 543-583, 1990.
Modifications of glutamate receptor delta-1 subunit gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of a glutamate receptor delta-1 subunit gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex
DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a glutamate receptor delta-1 subunit polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a glutamate receptor delta-1 subunit polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent glutamate receptor delta-1 subunit nucleotides, can provide sufficient targeting specificity for glutamate receptor delta-1 subunit mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular glutamate receptor delta-1 subunit polynucleotide sequence.
Antisense oligonucleotides can be modified without affecting their ability to hybridize to a glutamate receptor delta-1 subunit polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol 10, 152-158, 1992; Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett.
215, 3539-3542, 1987. Ribozymes
Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515,
1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
The coding sequence of a glutamate receptor delta-1 subunit polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the glutamate receptor delta-1 subunit polynucleotide. Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et «/., EP 321,201).
Specific ribozyme cleavage sites within a glutamate receptor delta-1 subunit RNA target can be identified by scanning the glutamate receptor delta-1 subunit target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate glutamate receptor delta-1 subunit RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequence shown in SEQ ID NO:l and its complement provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease glutamate receptor delta-1 subunit expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells. Screening Methods
The invention provides methods for identifying modulators, i.e., candidate or test compounds which bind to glutamate receptor delta-1 subunit polypeptides or polynucleotides and/or have a stimulatory or inhibitory effect on, for example, expression or binding activity of the glutamate receptor delta-1 subunit polypeptide or polynucleotide, so as to regulate a biological function. Regulation of human glutamate receptor delta-1 subunit is useful, for example, for preventing or treating epilepsy, schizophrenia and other mood disorders, neurodegenerative diseases such as Huntington's disease and Alzheimer's disease, ischemia, pain, benign prostate hyperplasia and urinary incontinence.
The invention provides assays for screening test compounds which bind to or modulate the binding activity of a glutamate receptor delta-1 subunit polypeptide or a glutamate receptor delta-1 subunit polynucleotide. A test compound preferably binds to a glutamate receptor delta-1 subunit polypeptide or polynucleotide. For example, a test compound decreases a glutamate receptor delta-1 subunit ligand binding activity of a glutamate receptor delta-1 subunit polypeptide or expression of a glutamate receptor delta-1 subunit polynucleotide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
Test Compounds
Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinanfiy, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl 33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J.
Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin,
Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).
High Throughput Screening Test compounds can be screened for the ability to bind to glutamate receptor delta-1 subunit polypeptides or polynucleotides or to affect glutamate receptor delta-1 subunit ligand binding activity or glutamate receptor delta-1 subunit gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format. Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
Yet another example is described by Salmon et al, Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.
Another high throughput screening method is described in Beutel et al, U.S. Patent
5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly such that the assays can be performed without the test samples running together. Binding Assays
For binding assays, the test compound is preferably a small molecule which binds to and occupies the active site of a glutamate receptor delta-1 subunit polypeptide, thereby making the active site inaccessible to substrate such that normal biological binding activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules. In binding assays, either the test compound or the glutamate receptor delta-1 subunit polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the glutamate receptor delta-1 subunit polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
Alternatively, binding of a test compound to a glutamate receptor delta-1 subunit polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a glutamate receptor delta-1 subunit polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS).
Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a glutamate receptor delta-1 subunit polypeptide. (McConnell et al, Science 257, 1906-1912, 1992).
Determining the ability of a test compound to bind to a glutamate receptor delta-1 subunit polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
In yet another aspect of the invention, a glutamate receptor delta-1 subunit polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300) to identify other proteins which bind to or interact with the glutamate receptor delta- 1 subunit polypeptide and modulate its. activity.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct a polynucleotide encoding a glutamate receptor delta-1 subunit polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the glutamate receptor delta-1 subunit polypeptide.
It may be desirable to immobilize either a glutamate receptor delta-1 subunit polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the glutamate receptor delta-1 subunit polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the glutamate receptor delta-1 subunit polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a glutamate receptor delta-1 subunit polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
In one embodiment, a glutamate receptor delta-1 subunit polypeptide is a fusion protein comprising a domain that allows the glutamate receptor delta-1 subunit polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed glutamate receptor delta-1 subunit polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a glutamate receptor delta-1 subunit polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated glutamate receptor delta-1 subunit polypeptides, polynucleotides, or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce
Chemical). Alternatively, antibodies which specifically bind to a glutamate receptor delta-1 subunit polypeptide, polynucleotides, or a test compound, but which do not interfere with a desired binding site, such as the active site of the glutamate receptor delta-1 subunit polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to a glutamate receptor delta-1 subunit polypeptide or test compound, enzyme-linked assays which rely on detecting a glutamate receptor delta-1 subunit activity of the glutamate receptor delta-1 subunit polypeptide, and SDS gel electrophoresis under non-reducing conditions.
Screening for test compounds which bind to a glutamate receptor delta-1 subunit polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a glutamate receptor delta-1 subunit polynucleotide or polypeptide can be used in a cell-based assay system. a glutamate receptor delta-1 subunit polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, including cell lines such as the HCN-1A, HCN-2, CATH.a, Neuro-2a, and
PC 12 (Clontech), can be used, can be used. An intact cell is contacted with a test compound. Binding of the test compound to a glutamate receptor delta-1 subunit polypeptide or polynucleotide is determined as described above, after lysing the cell to release the glutamate receptor delta-1 subunit polypeptide-or polynucleotide-test compound complex. Glutamate Receptor Delta-1 Subunit Assays
Test compounds can be tested for the ability to increase or decrease an activity of a human glutamate receptor delta-1 subunit polypeptide, such as ligand gated ion channel activity. Such activity is measured as is known in the art, for example, before and after contacting either a purified glutamate receptor delta-1 subunit polypeptide, a cell extract, or an intact cell with a test compound. For example, a test compound which decreases glutamate receptor delta-1 subunit binding to a ligand, such as kainate, by at least about 10, preferably about 50, more preferably about 75, -90, or 100% is identified as a potential antagonist of the receptor; a test compound which increases glutamate receptor delta-1 subunit binding to the ligand by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agonist of the receptor.
Gene Expression
In another embodiment, test compounds which increase or decrease glutamate receptor delta-1 subunit gene expression are identified. A glutamate receptor delta-1 subunit polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the glutamate receptor delta-1 subunit polynucleotide is determined. The level of expression of glutamate receptor delta-1 subunit mRNA or polypeptide in the presence of the test compound is compared to the level of expression of glutamate receptor delta-1 subunit mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of glutamate receptor delta-1 subunit mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of glutamate receptor delta-1 subunit mRNA or polypeptide expression. Alternatively, when expression of glutamate receptor delta-1 subunit mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of glutamate receptor delta-1 subunit mRNA or polypeptide expression. The level of glutamate receptor delta-1 subunit mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptides. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a glutamate receptor delta-1 subunit polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a glutamate receptor delta-1 subunit polypeptide.
Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses a glutamate receptor delta-1 subunit polynucleotide can be used in a cell-based assay system. The glutamate receptor delta-1 subunit polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, including cell lines such as the HCN-1A, HCN-2, CATH.a, Neuro-2a, and PC 12 (Clontech), can be used.
Pharmaceutical Compositions
The invention also provides pharmaceutical compositions which can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a glutamate receptor delta-1 subunit polypeptide, Glutamate receptor delta-1 subunit polynucleotide, antibodies which specifically bind to glutamate receptor delta-1 subunit activity, or mimetics, agonists, antagonists, or inhibitors of glutamate receptor delta-1 subunit activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
Therapeutic Indications and Methods
Glutamate is involved in epileptogenesis. Overstimulation of glutamate receptors can lead to seizures and excitotoxin injury throughout the CNS and in particular the hippocampus. Antagonists of glutamate receptor delta-1 subunit can act to prevent or treat epilepsy.
Glutamate plays a role in ischemic brain damage and compounds that decrease the accumulation of glutamate or block its postsynaptic effects can ameliorate ischemic injury and other forms of acute neuronal degenerative diseases like hypoxia/hypoglycemia, traumatic brain injury, and stroke. Antagonists of glutamate receptor delta-1 subunit can act to prevent or treat brain damage.
Excitotoxicity, the overstimulation of glutamate receptors resulting in the death of neurons, participates in the pathogenesis of chronic neurodegenerative disorders including Huntington's disease, Alzheimer's disease, and Parkinson's disease. Antagonists of glutamate receptor delta-1 subunit can act to prevent or treat excitotoxicity and therefore can prevent or treat chronic neurodegenerative disorders.
Glutamate has been implicated in psychiatric disorders such as schizophrenia and mood disorders. Modification of glutamate receptor delta-1 subunit levels can have an advantageous effect on psychiatric disorders in which glutamate neuro- transmission is abnormally regulated.
The excitatory amino acids glutamate and aspartate have been implicated in the transmission of acute and chronic pain. Blocking the activity of glutamate receptor delta-1 subunit can prevent or treat pain, and in particular chronic pain disorders. Pain which can be treated includes that which arises from pancreatitis, interstitial cystitis from various origin, including infection, dysfunctional bladder epithelium, neurogenic disturbances, bladder mastocytosis, allergic/immune/auto-immune causes, endocrine, food intolerance, painful bladder disease causing suprapubic, urethral, or pelvic pain, including interstitial cystitis, endometriosis, bacterial cystitis, outlet obstruction, dysmenorrhea, IBS (Irritable Bowel Syndrome), and Crohn's Disease, as well as pain syndromes of the pelvic cavity, e.g. vulvodynia, orchialgia, urethral syndrome, prostatodynia, biliary colic, ureteral colic, nephrolithiasis, urinary tract obstruction, incontinence, dysuria, voiding dysfunction of the bladder, and pain due to distension of visceral surfaces, e.g. hepatic or renal capsules, and myocardial infarction.
Neuropathologic pain includes that arising in connection with multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke status, vascular lesion in the brain and spinal cord, including infarct, hemorrhage, and vascular malformation. Similarly, post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgia, radioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy), vasculitic neuropathy (e.g., secondary to connective tissue disease), paraneoplastic polyneuropathy associated e.g. with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia also can be treated. Headache, including migraine with aura, migraine without aura, episodic and chronic tension-type headache, cluster headache, and chronic paroxysmal hemicrania, as well as hyperirritability of the tracheobronchial tree, can be treated by regulating human glutamate receptor delta-1 subunit activity.
Neurodegenerative conditions, such as Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small- vessel cerebrovascular disease are amenable to treatment by regulating human glutamate receptor delta-1 subunit activity. Various dementias, including Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia including Pick's disease, Parkinsonism, progressive nuclear palsy, corticobasal degeneration, motoneuron disease dementia, including ALS, Huntington's disease, multiple sclerosis, small-vessel cerebrovascular disease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff s psychosis can be treated.
This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a polypeptide-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
A reagent which affects glutamate receptor delta-1 subunit binding activity can be administered to a human cell, either in vitro or in vivo, to reduce glutamate receptor delta-1 subunit binding activity. The reagent preferably binds to an expression product of a glutamate receptor delta-1 subunit gene. If the expression product is a polypeptide, for example, the reagent can be an antibody or a small chemical compound. For treatment of human cells ex vivo, a reagent can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung or liver.
A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 106 cells, more preferably about 1.0 μg of DNA per 16 nmol of liposome delivered to about 106 cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about
106 cells. Preferably, a liposome is between about 100 and 500 ran, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a tumor cell, such as a tumor cell ligand exposed on the outer surface of the liposome. Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.
In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al Trends in Biotechnol 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci.
U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).
If the reagent is a single-chain antibody, a polynucleotide encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated
DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.
Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.
If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
Preferably, a reagent reduces expression of a glutamate receptor delta-1 subunit gene or the binding activity of a glutamate receptor delta-1 subunit polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a glutamate receptor delta-1 subunit gene or the binding activity of a glutamate receptor delta-1 subunit polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to glutamate receptor delta-1 subunit-specific mRNA, quantitative RT-PCR, immunologic detection of a glutamate receptor delta-1 subunit polypeptide, or measurement of glutamate receptor delta-1 subunit binding activity.
In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
Determination of a Therapeutically Effective Dose Determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases glutamate receptor delta-1 subunit binding activity relative to glutamate receptor delta-1 subunit binding activity which occurs in the absence of the therapeutically effective dose.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
Therapeutic efficacy and toxicity, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED5o.
Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
Normal dosage amounts of any particular reagent can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for polypeptides or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
The above disclosure generally describes the present invention, and all patents and patent applications cited in this disclosure are expressly incorporated herein. A more complete understanding can be obtained by reference to the following specific examples which are provided for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1
Detection of glutamate receptor delta-1 subunit activity
The polynucleotide of SEQ ID NO: 12 is inserted into the expression vector pCEV4 and the expression vector pCEV4-glutamate receptor delta-1 subunit polypeptide obtained is transfected into human embryonic kidney 293 cells.
These cells are scraped from a culture flask into 5 ml of Tris HC1, 5 mM EDTA, pH 7.5, and lysed by sonication. Cell lysates are centrifuged at 1000 rpm for 5 minutes at 4 °C. The supernatant is centrifuged at 30,000 x g for 20 minutes at 4 °C. The pellet is suspended in binding buffer containing 50 mM Tris HC1, 5 mM MgSO4, 1 mM EDTA, 100 mM NaCl, pH 7.5, supplemented with 0.1 % BSA, 2 μ/ml aprotinin, 0.5 mg/ml leupeptin, and 10 μ/ml phosphoramidon. Optimal membrane suspension dilutions, defined as the protein concentration required to bind less than 10 % of the added radioligand, i.e. glutamate, are added to 96-well polypropylene microtiter plates containing 125I-labeled ligand non-labeled peptides, and binding buffer to a final volume of 250 μ.
In equilibrium saturation binding assays, membrane preparations are incubated in the presence of increasing concentrations (0.1 nM to 4 nM) of 125I-labeled ligand.
Binding reaction mixtures are incubated for one hour at 30 °C. The reaction is stopped by filtration through GF/B filters treated with 0.5% polyethyleneimine, using a cell harvester. Radioactivity is measured by scintillation counting, and data are analyzed by a computerized non-linear regression program.
Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of 100 nM of unlabeled peptide. Protein concentration is measured by the Bradford method using Bio-Rad Reagent, with bovine serum albumin as a standard. It is shown that the polypeptide of SEQ ID
NO: 7 has a glutamate receptor delta-1 subunit activity. EXAMPLE 2
Expression of recombinant human glutamate receptor delta-1 subunit polypeptide
The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of a recombinant human glutamate receptor delta-1 subunit in yeast. The encoding DNA sequence is derived from the nucleotide sequence shown in SEQ ID NO:9, 10, 11 or 12. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag, and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added.
After digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes, the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.
The yeast is cultivated under usual conditions in 5 liter shake flasks, and the recombinantly produced protein isolated from the culture by affinity chromatography
(Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human glutamate receptor delta-1 subunit is obtained. EXAMPLE 3
Identification of a test compound which binds to a glutamate receptor delta-1 subunit polypeptide
Purified glutamate receptor delta-1 subunit polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Glutamate receptor delta-1 subunit polypeptides comprise an amino acid sequence shown in SEQ ID NO:5, 6, 7, or 8. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a glutamate receptor delta-1 subunit polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound was not incubated is identified as a compound which binds to a glutamate receptor delta-1 subunit polypeptide.
EXAMPLE 4
Identification of a test compound which decreases glutamate receptor delta-1 subunit gene expression
A test compound is administered to a culture of neuronal cells and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells incubated for the same time without the test compound provides a negative control.
RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a 32P-labeled glutamate receptor delta-1 subunit-specific probe at 65 ° C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:9, 10, 11 or 12. A test compound which decreases the glutamate receptor delta-1 subunit-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of glutamate receptor delta-1 subunit gene expression.
EXAMPLE 5
Identification of glutamate receptor delta-1 subunit agonists and antagonists
A human embryonic 293 kidney cell line is transfected with a glutamate receptor delta-1 subunit polynucleotide. Test compounds comprising potential glutamate receptor delta-1 subunit antagonists or agonists are prepared. Forty-eight hours after transfection, agonist-activated and antagonist-deactivated currents in the presence of various ligands, such as kainate, are measured using standard patch clamp techniques in the whole-cell configuration. See Sommers et al. EMBO J. 11, 1651-1656, 1992;
Keinanen et al, Science. 249, 556-560 (1990); Sommer et al, Science. 249, 1580-1585; Verdoorn et al, Science. 252, 1715-1718, 1991. Currents in the presence of a test agonist or antagonist and in the absence of a test compound are collected, recorded and analyzed. Test compounds that evoke currents in the presence of the ligand are identified as agonists of glutamate receptor delta-1 subunit and compounds that depress currents in the presence of the ligand are identified as antagonists of glutamate receptor delta-1 subunit.
EXAMPLE 6
Treatment of epilepsy with a reagent which specifically binds to a glutamate receptor delta-1 subunit gene product
Synthesis of antisense glutamate receptor delta-1 subunit oligonucleotides comprising at least 11 contiguous nucleotides selected from SEQ ID NO:9, 10, 11 or 12 is performed on a Pharmacia Gene Assembler series synthesizer using the phosphoramidite procedure (Uhlmann et al, Chem. Rev. 90, 534-83, 1990). Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate-buffered saline (PBS) at the desired concentration. Purity of these oligonucleotides is tested by capillary gel electro- phoreses and ion exchange HPLC. Endotoxin levels in the oligonucleotide preparation are determined using the Limulus Amebocyte Assay (Bang, Biol. Bull. (Woods Hole, Mass.) 105, 361-362, 1953).
The antisense oligonucleotides are administered to a patient with epilepsy. The severity of the patient's epilepsy is decreased.

Claims

1. An isolated polynucleotide encoding a glutamate receptor delta-1 subunit polypeptide and being selected from the group consisting of: a) a polynucleotide encoding a glutamate receptor delta-1 subunit polypeptide comprising an amino acid sequence selected form the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; the amino acid sequence shown in SEQ ID NO: 5; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence shown in SEQ ID NO: 6; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence shown in SEQ ID NO: 7; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 8; and the amino acid sequence shown in SEQ ID NO: 8. b) a polynucleotide comprising the sequence of SEQ ID NO: 9, 10, 11 or
12; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b); d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).
2. An expression vector containing any polynucleotide of claim 1.
3. A host cell containing the expression vector of claim 2.
4. A substantially purified glutamate receptor delta-1 subunit polypeptide encoded by a polynucleotide of claim 1.
5. A method for producing a glutamate receptor delta-1 subunit polypeptide, wherein the method comprises the following steps: a) culturing the host cell of claim 3 under conditions suitable for the expression of the glutamate receptor delta-1 subunit polypeptide; and b) recovering the glutamate receptor delta-1 subunit polypeptide from the host cell culture.
6. A method for detection of a polynucleotide encoding a glutamate receptor delta-1 subunit polypeptide in a biological sample comprising the following steps: a) hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting said hybridization complex.
7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.
8. A method for the detection of a polynucleotide of claim 1 or a glutamate receptor delta-1 subunit polypeptide of claim 4 comprising the steps of: contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the glutamate receptor delta-1 subunit polypeptide.
9. A diagnostic kit for conducting the method of any one of claims 6 to 8.
10. A method of screening for agents which decrease the activity of a glutamate receptor delta-1 subunit, comprising the steps of: contacting a test compound with any glutamate receptor delta-1 subunit polypeptide encoded by any polynucleotide of claiml; detecting binding of the test compound to the glutamate receptor delta- 1 subunit polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a glutamate receptor delta-1 subunit.
11. A method of screening for agents which regulate the activity of a glutamate receptor delta-1 subunit, comprising the steps of: contacting a test compound with a glutamate receptor delta-1 subunit polypeptide encoded by any polynucleotide of claim 1; and detecting a glutamate receptor delta-1 subunit activity of the polypeptide, wherein a test compound which increases the glutamate receptor delta-1 subunit activity is identified as a potential therapeutic agent for increasing the activity of the glutamate receptor delta-1 subunit, and wherein a test compound which decreases the glutamate receptor delta-1 subunit activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the glutamate receptor delta-1 subunit.
12. A method of screening for agents which decrease the activity of a glutamate receptor delta-1 subunit, comprising the steps of: contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of glutamate receptor delta-1 subunit.
13. A method of reducing the activity of glutamate receptor delta-1 subunit, comprising the steps of: contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any glutamate receptor delta-1 subunit polypeptide of claim 4, whereby the activity of glutamate receptor delta-1 subunit is reduced.
14. A reagent that modulates the activity of a glutamate receptor delta-1 subunit polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.
15. A pharmaceutical composition, comprising: the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.
16. Use of the pharmaceutical composition of claim 15 for modulating the activity of a glutamate receptor delta-1 subunit in a disease.
17. Use of claim 16 wherein the disease is epilepsy, schizophrenia, neurodegenerative disease, ischemia, pain, benign prostate hyperplasia or urinary incontinence.
18. A cDNA encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8.
19. The cDNA of claim 18 which comprises SEQ ID NOS: 9, 10, 11 or 12.
20. The cDNA of claim 18 which consists of SEQ ID NOS: 9, 10, 11 or 12.
21. An expression vector comprising a polynucleotide which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8.
22. The expression vector of claim 21 wherein the polynucleotide consists of
SEQ ID NOS: 9, 10, 11 or 12.
23. A host cell comprising an expression vector which encodes a polypeptide comprising the amino acid sequence shown in SEQ LD NOS: 5, 6, 7 and 8.
24. The host cell of claim 23 wherein the polynucleotide consists of SEQ ID
NOS: 9, 10, 11 or 12.
25. A purified polypeptide comprising the amino acid sequence shown in SEQ ID
NOS: 5, 6, 7 and 8.
26. The purified polypeptide of claim 25 which consists of the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8.
27. A fusion protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8.
28. A method of producing a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8, comprising the steps of: culturing a host cell comprising an expression vector which encodes the polypeptide under conditions whereby the polypeptide is expressed; and isolating the polypeptide.
29. The method of claim 28 wherein the expression vector comprises SEQ ID NOS: 9, 10, 11 or 12.
30. A method of detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8, comprising the steps of: hybridizing a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NOS: 9, 10, 11 or 12 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and detecting the hybridization complex.
31. The method of claim 30 further comprising the step of amplifying the nucleic acid material before the step of hybridizing.
32. A kit for detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8, comprising: a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NOS: 9, 10, 11 or 12; and instructions for the method of claim 30.
33. A method of detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8, comprising the steps of: contacting a biological sample with a reagent that specifically binds to the polypeptide to form a reagent-polypeptide complex; and detecting the reagent-polypeptide complex.
34. The method of claim 33 wherein the reagent is an antibody.
35. A kit for detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8, comprising: an antibody which specifically binds to the polypeptide; and instructions for the method of claim 33.
36. A method of screening for agents which can modulate the activity of a human glutamate receptor delta-1 subunit, comprising the steps of: contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50%o identical to the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8 and (2) the amino acid sequence shown in SEQ
ID NOS: 5, 6, 7 and 8; and detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential agent for regulating activity of the human glutamate receptor delta-1 subunit.
37. The method of claim 36 wherein the step of contacting is in a cell.
38. The method of claim 36 wherein the cell is in vitro.
39. The method of claim 36 wherein the step of contacting is in a cell-free system.
40. The method of claim 36 wherein the polypeptide comprises a detectable label.
41. The method of claim 36 wherein the test compound comprises a detectable label.
42. The method of claim 36 wherein the test compound displaces a labeled ligand which is bound to the polypeptide.
43. The method of claim 36 wherein the polypeptide is bound to a solid support.
44. The method of claim 36 wherein the test compound is bound to a solid support.
45. A method of screening for agents which modulate an activity of a human glutamate receptor delta-1 subunit, comprising the steps of: contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8 and (2) the amino acid sequence shown in SEQ
ID NOS: 5, 6, 7 and 8; and detecting an activity of the polypeptide, wherein a test compound which increases the activity of the polypeptide is identified as a potential agent for increasing the activity of the human glutamate receptor delta-1 subunit, and wherein a test compound which decreases the activity of the polypeptide is identified as a potential agent for decreasing the activity of the human glutamate receptor delta-1 subunit.
46. The method of claim 45 wherein the step of contacting is in a cell.
47. The method of claim 45 wherein the cell is in vitro.
48. The method of claim 45 wherein the step of contacting is in a cell-free system.
49. A method of screening for agents which modulate an activity of a human glutamate receptor delta-1 subunit, comprising the steps of: contacting a test compound with a product encoded by a polynucleotide which comprises the nucleotide sequence shown in SEQ ID NOS: 9, 10, 11 or 12; and detecting binding of the test compound to the product, wherein a test compound which binds to the product is identified as a potential agent for regulating the activity of the human glutamate receptor delta-1 subunit.
50. The method of claim 49 wherein the product is a polypeptide.
51. The method of claim 49 wherein the product is RNA.
52. A method of reducing activity of a human glutamate receptor delta- 1 subunit, comprising the step of: contacting a cell with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOS: 9, 10, 11 or 12, whereby the activity of a human glutamate receptor delta-1 subunit is reduced.
53. The method of claim 52 wherein the product is a polypeptide.
54. The method of claim 53 wherein the reagent is an antibody.
55. The method of claim 52 wherein the product is RNA.
56. The method of claim 55 wherein the reagent is an antisense oligonucleotide.
57. The method of claim 56 wherein the reagent is a ribozyme.
58. The method of claim 52 wherein the cell is in vitro.
59. The method of claim 52 wherein the cell is in vivo.
60. A pharmaceutical composition, comprising: a reagent which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8; and a pharmaceutically acceptable carrier.
61. The pharmaceutical composition of claim 60 wherein the reagent is an antibody.
62. A pharmaceutical composition, comprising: a reagent which specifically binds to a product of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOS: 9, 10, 11 or 12; and a pharmaceutically acceptable carrier.
63. The pharmaceutical composition of claim 62 wherein the reagent is a ribozyme.
64. The pharmaceutical composition of claim 62 wherein the reagent is an antisense oligonucleotide.
65. The pharmaceutical composition of claim 62 wherein the reagent is an antibody.
66. A pharmaceutical composition, comprising: an expression vector encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NOS: 5, 6, 7 and 8; and a pharmaceutically acceptable carrier.
67. The pharmaceutical composition of claim 66 wherein the expression vector comprises SEQ ID NOS: 9, 10, 11 or 12.
68. A method of treating a glutamate receptor delta-1 subunit dysfunction related disease, wherein the disease is selected from epilepsy, schizophrenia, neurodegenerative disease, ischemia, pain, benign prostate hyperplasia or urinary incontinence, comprising the step of: administering to a patient in need thereof a therapeutically effective dose of a reagent that modulates a function of a human glutamate receptor delta-1 subunit, whereby symptoms of the glutamate receptor delta-1 subunit dysfunction related disease are ameliorated.
69. The method of claim 68 wherein the reagent is identified by the method of claim 36.
70. The method of claim 68 wherein the reagent is identified by the method of claim 45. The method of claim 68 wherein the reagent is identified by the method of claim 49.
PCT/EP2001/008102 2000-07-18 2001-07-13 Regulation of human glutamate receptor delta-1 subunit WO2002006313A2 (en)

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