WO1992015015A1 - Methodes de detection des antagonistes de la galanine - Google Patents

Methodes de detection des antagonistes de la galanine Download PDF

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Publication number
WO1992015015A1
WO1992015015A1 PCT/US1992/001469 US9201469W WO9215015A1 WO 1992015015 A1 WO1992015015 A1 WO 1992015015A1 US 9201469 W US9201469 W US 9201469W WO 9215015 A1 WO9215015 A1 WO 9215015A1
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galanin
sequence
dna
analog
antagonist
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PCT/US1992/001469
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English (en)
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Gary L. Mcknight
Robert A. Smith
Stephan Kowalyk
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Zymogenetic, Inc.
The Boards Of Regents Of The University Of Washington
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Publication of WO1992015015A1 publication Critical patent/WO1992015015A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • the present invention is directed generally toward methods for detecting galanin antagonists, and more specifically, to methods of producing and screening large numbers of potential galanin antagonists through the use of recombinant DNA techniques.
  • Diabetes mellitus a disease in which a major indicator is an elevated blood glucose level, is generally believed to result from low insulin levels and elevated glucagon levels.
  • hyperglycemia in non-insulin dependent diabetes also known as type II diabetes
  • non-obese and obese patients has been shown in the presence of both elevated glucagon and insulin levels.
  • Type II diabetes is a heterogeneous disorder involving pancreatic islet cell dysfunction and insulin resistance. It is characterized by hyperglycemia, a defect in first-phase glucose-induced insulin secretion and impaired glucose utilization. Insulin secretion in response to non-glucose secretagogues is normal or near normal in patients with type II diabetes.
  • Type II diabetes is treated with sulfonyl ureas, which act on ATP-sensitive K + channels, thereby indirectly increasing insulin secretion.
  • sulfonyl ureas which act on ATP-sensitive K + channels, thereby indirectly increasing insulin secretion.
  • These drugs are, however, unsuitable for long term use as patients become increasingly resistant to insulin. It eventually becomes necessary to administer exogenous insulin.
  • Hyperglycemia may also occur in any state of sympathetic neural activation, including stress due to, for example, surgery, trauma, myocardial infarction or burns. Although insulin is sometimes used therapeutically in such cases, its use creates a significant risk of hypoglycemia.
  • the present invention fulfills this need and provides other related advantages.
  • the present invention provides methods for detecting the presence of galanin antagonists.
  • the methods comprise: (a) growing host cells containing a DNA construct capable of directing the expression of a galanin analog, the construct comprising the following operably linked elements: a transcriptional promoter, a DNA sequence encoding a galanin analog, and a transcriptional terminator, under growth conditions suitable for the expression of the galanin analog; (b) isolating the galanin analog encoded by the DNA sequence from the host cells; (c) exposing the isolated galanin analog in the presence of native galanin to a galanin receptor coupled to a response pathway under conditions and for a time sufficient to allow binding of the galanin analog to the receptor and an associated response through the pathway; and (d) detecting a reduction in the inhibition of the response pathway resulting from the binding of the galanin analog to the galanin receptor, relative to the inhibition of the response pathway by native galanin alone and therefrom determining the presence of a galanin antagonist.
  • the DNA construct further comprises a secretory signal sequence operably linked to the DNA sequence encoding a galanin analog.
  • the galanin receptor is membrane-bound in a cell-free extract, such as an insulinoma cell membrane preparation.
  • the galanin receptor is membrane-bound in a whole cell, such as an insulinoma cell.
  • galanin antagonists are produced from a host cell containing a DNA construct capable of directing the expression of a galanin antagonist, the construct comprising the following operably linked elements: a transcriptional promoter, a DNA sequence encoding a galanin antagonist, wherein the sequence encodes one or more amino acid residues that are different than the corresponding amino acid residues in native galanin, and a transcriptional terminator.
  • the present invention provides isolated DNA molecules encoding human galanin and human galanin antagonists.
  • Figure 1 discloses the sequence of the coding strand of a human galanin cDNA, together with the deduced amino acid sequence (Sequence ID Numbers l and 2) .
  • the mature peptide extends from glycine, amino acid number 7, through serine, amino acid number 36. Numbers below the lines refer to amino acids; those on the right margin refer to nucleotides.
  • Figure 2 discloses the representative expression vector pBS114. Abbreviations used include TPI-P, TPI1 promoter; ⁇ , alpha factor signal sequence; TPI-t, TPI1 terminator; and irl, inverted repeat 1 of the 2 micron plasmid.
  • Analog A molecule, other than a native ligand, capable of being bound by the ligand-binding domain of a receptor.
  • the molecule may be chemically synthesized, produced through recombinant DNA methodology or may occur in nature.
  • galanin analogs are galanin-like polypeptides that contain one or more amino acid residues that are different than the corresponding amino acid residues in native galanin and that are capable of binding to a galanin receptor. These differences may comprise deletions, additions and/or substitutions of amino acids relative to native galanin.
  • a response pathway is a biochemical pathway activated in response to external stimuli that is generally, but not always, directly coupled to a membrane-bound receptor.
  • Response pathways generally regulate cellular functions such as extracellular matrix secretion, hormone secretion, chemotaxis, differentiation, and cell division in responsive cells.
  • One such response pathway is the adenylate cyclase response pathway, which is coupled to the membrane-bound galanin receptor. The adenylate cyclase response pathway is inhibited upon binding of galanin to its cellular receptor, thereby reducing intracellular concentrations of cyclic AMP (cAMP) .
  • cAMP cyclic AMP
  • Antagonist A molecule capable of binding to a receptor, but "that does not affect or exhibits a reduced effect on a response pathway within a cell as compared to the native ligand.
  • Galanin antagonists are generally identified by their ability to bind to the galanin receptor and their inability or reduced ability to affect a cellular response pathway. For example, putative galanin antagonists are combined with native galanin and the inhibition of forskolin-stimulated cAMP production or insulin secretion is measured. Galanin antagonists are identified as those molecules that reduce the inhibition of cAMP production or insulin secretion relative to native galanin alone.
  • DNA Construct A DNA molecule, or a clone of such a molecule, either single- or double-stranded that has been modified through human intervention to contain segments of DNA combined and juxtaposed in a manner that as a whole would not otherwise exist in nature.
  • DNA constructs may contain the information necessary to direct the expression of DNA sequences encoding polypeptides of interest. Such DNA constructs, known as expression vectors, will generally include transcriptional promoters, enhancers and transcriptional terminators. DNA constructs containing the information necessary to direct the secretion of a polypeptide will also contain at least one secretory signal sequence.
  • Secretory Signal Sequence A DNA sequence encoding a secretory peptide.
  • a secretory peptide is an amino acid sequence that acts to direct the secretion of a mature polypeptide or protein from a cell.
  • Secretory peptides are generally characterized by a core of hydrophobic amino acids and are typically (but not exclusively) found at the amino termini of newly synthesized proteins. Very often the secretory peptide is cleaved from the mature protein during secretion. Such secretory peptides contain processing sites that allow cleavage of the secretory peptides from mature proteins as they pass through the secretory pathway.
  • Processing sites may be encoded within the secretory peptide or may be added to the secretory peptide by, for example, jln vitro mutagenesis.
  • Certain secretory peptides may be used in concert to direct the secretion of polypeptides and proteins.
  • One such secretory peptide that may be used in combination with other secretory peptides is the third domain of the yeast Barrier protein (disclosed in U.S. patent application Serial No. 07/270,933, which is incorporated herein by reference) .
  • the present invention provides isolated DNA molecules encoding human galanin, a 30-amino acid sympathetic neurotransmitter that is believed to mediate hormone secretion by pancreatic islet cells.
  • galanin opens ATP-sensitive K + channels, inhibits adenylate cyclase and directly interferes with exocytosis. These effects may individually and collectively inhibit insulin secretion.
  • Recent studies by Dunning et al. (Diabetes 39 (Suppl.): 135A, 1990) suggest that galanin is indeed a regulator of insulin secretion in humans. These DNA molecules are therefore useful in generating galanin antagonists that may be used to release pancreatic islet cells from hormonal inhibition of insulin secretion, thereby providing a more direct treatment for type II diabetes.
  • an object of the present invention is to provide methods for detecting galanin antagonists using recombinant methods and host cells.
  • the present invention provides the ability to produce galanin analogs from transformed or transfected host cells.
  • the analogs are exposed, in the presence of native galanin, to a galanin receptor coupled to a response pathway.
  • a reduction in the inhibition of the response pathway as compared to the inhibition obtained using native galanin alone is indicative of the presence of a galanin antagonist.
  • the reduced inhibition of a response pathway is seen as, for example, increased secretion of insulin or somatostatin, or increased cAMP production as compared to galanin-treated cells.
  • Galanin analogs produced according to the present invention may be screened in high through ⁇ put antagonist screens.
  • the present invention also provides a method for screening pools of galanin analogs within such high through-put screens to identify galanin antagonists.
  • the present invention also provides methods for directly producing galanin antagonists through the use of recombinant host cells.
  • the present invention provides methods for producing large numbers of galanin analogs through the use of pools of DNA sequences encoding such analogs.
  • Galanin coding sequences may be produced synthetically using standard techniques or may be cloned from, for example, pheochromocytoma cells (e.g. Bauer et al., J. Clin. Endocrinol. Metab. 63: 1372-1378, 1986), using standard cloning methods such as those described by Maniatis et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, 1982; which is incorporated herein by reference), Sambrook et al. (Molecular Cloning: A Laboratory Manual. Second Edition.
  • RNA template was annealed to a synthetic oligonucleotide primer, first strand cDNA was synthesized, and galanin sequences were amplified.
  • a clone encoding 27 amino acids of mature galanin and part of the C-terminal propeptide was identified.
  • Such partial clones are extended according to conventional methods (e.g., the RACE procedure of Froh an et al., Proc. Natl. Acad. Sci. USA 85: 8998-9002, 1988, which is incorporated herein by reference) to obtain full-length clones.
  • a genomic galanin clone may also be obtained by polymerase chain reaction amplification.
  • Pools of DNA sequences encoding galanin analogs may be generated by saturation mutagenesis of a DNA sequence encoding galanin (using, for example, the methods described by Little [Gene 88.:113-115, 1990] or Hermes et al. [Gene 88.:143-151, 1989]), or segment-directed mutagenesis (as described, for example, by Shortle et al. , rProc. Natl. Acad. Sci. USA 77:5375-5379, 1980]).
  • pools of galanin analogs may be generated by forced nucleotide misincorporation as described by, for example, Liao and Wise (Gene 88.:107-111, 1990).
  • Liao and Wise describe a method for introducing random point mutations into cloned DNA fragments via the forced misincorporation of deoxynucleoside triphosphates by either a reverse transcriptase or a mutant T7 DNA polymerase.
  • these two polymerases which lack proofreading activity, incorporate incorrect nucleotides into the primed sequence and provide a wide spectrum of random mutations in a given sequence.
  • oligonucleotides encoding galanin analogs be synthesized to form adapters upon hybridization such that the galanin analog coding sequences are flanked by adhesive ends. It may be particularly preferred to add a sequence encoding a bridging region which allows the in-frame fusion of sequences encoding a secretory signal sequence and a galanin coding sequence.
  • Galanin analog coding sequences are preferably synthesized on an oligonucleotide synthesizer by cross contaminating the reagent bottles that normally contain pure phosphoramidites corresponding to the bases A, G, C, and T at low levels with each of the other bases.
  • Cross contamination of the reagent bottles may be achieved by adding between .01% and 14% of each incorrect base, with a cross contamination of between 0.8% and 2% being preferred, and 1% being particularly preferred. A 1% cross contamination with each incorrect base will theoretically lead to approximately 2.5 base changes per molecule.
  • the galanin analog coding sequences are inserted into a suitable expression vector to produce a library that is in turn introduced by transfection or transformation into a host cell.
  • Expression vectors for use in carrying out the present invention will comprise a promoter capable of directing the transcription of a cloned DNA and a transcriptional terminator.
  • Host cells for use in practicing the present invention include mammalian, avian, plant, insect, bacterial and fungal cells. Fungal cells, including species of yeast (e.g., Saccharomyces spp. , Schizosaccharomvces spp. , Kluyveromyces spp.) or filamentous fungi (e.g., Aspergillus spp. , Neurospora spp.) may be used as host cells within the present invention. Strains of the yeast Saccharomyces cerevisiae are particularly preferred.
  • galanin analogs in eukaryotic host cells that can secrete the analogs into the culture media.
  • at least one secretory signal sequence is operably linked to the galanin analog DNA sequence.
  • Preferred secretory signals include the galanin secretory signal (pre-pro sequence) , the alpha factor signal sequence (pre- pro sequence; Kurjan and Herskowitz, Cell 30:933-943 , 1982; Kurjan et al., U.S. Patent No.
  • the PH05 signal sequence (Beck et al., WO 86/00637) , the BAR1 secretory signal sequence (MacKay et al., U.S. Patent No. 4,613,572; MacKay, WO 87/002670), the SUC2 signal sequence (Carlson et al., Mol. Cell. Biol. 2:439-447, 1983), the ⁇ -1-antitrypsin signal sequence (Kurachi et al., Proc. Natl. Acad. Sci. USA 7_8:6826-6830, 1981) , the ⁇ -2 plasmin inhibitor signal sequence (Tone et al., J.
  • a secretory signal sequence may be synthesized according to the rules established, for example, by von Heinje (Eur. J. Biochem. 133.:17-21, 1983; J. Mol. Biol. 184:99-105, 1985; Nuc. Acids Res. 14:4683-4690. 1986).
  • Secretory signal sequences may be used singly or may be combined.
  • a first secretory signal sequence may be used singly or in combination with a sequence encoding the third domain of the Saccharomyces cerevisiae Barrier protein (described in U.S. Patent Application Serial No. 07/270,933, which is incorporated by reference herein in its entirety) .
  • the third domain of Barrier may be positioned in proper reading frame 2 ' of the galanin analog DNA sequence or 5' to the DNA sequence and in proper reading frame with both the secretory signal sequence and the galanin analog DNA sequence.
  • Suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039. 1978), YEpl3 (Broach et al. , Gene 8.:121-133, 1979), POT vectors (Kawasaki et al, U.S. Patent No. 4,931,373, which is incorporated by reference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof.
  • Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.
  • selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include LEU2 (Broach et al., ibid.), URA3 (Botstein et al., Gene 8.:17, 1979), HIS3 (Struhl et al., ibid.) or P0T1 (Kawasaki et al., ibid.).
  • Another suitable selectable marker is the CAT gene, which confers chloramphenicol resistance on yeast cells.
  • promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-12080. 1980; Alber and Kawasaki, J. Mol. Appl. Genet.1:419-434. 1982; Kawasaki, U.S. Patent No. 4,599,311) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals. Hollaender et al., (eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzvmol. .10.1:192-201, 1983).
  • promoters are the TPI1 promoter (Kawasaki, U.S. Patent No. 4,599,311, 1986) and the ADH2-4— promoter (Russell et al., Nature 304:652- 654, 1983; Irani and Kilgore, European Patent Office Publication EP 284,044, which is incorporated herein by reference) .
  • the expression units may also include a transcriptional terminator.
  • a preferred transcriptional terminator is the TPI1 terminator (Alber and Kawasaki, ibid.) .
  • galanin analogs can be expressed in filamentous fungi, for example, strains of the fungi Aspergillus (McKnight et al., U.S. Patent No. 4,935,349, which is incorporated herein by reference).
  • useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4:2093-2099. 1985) and the tpiA promoter.
  • An example of a suitable terminator is the ADH3 terminator.
  • the expression units utilizing such components are cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus.
  • the genotype of the host cell will generally contain a genetic defect that is complemented by the selectable marker present on the expression vector. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art. To optimize production of heterologous proteins, it is preferred that the host strain carry a mutation, such as the yeast pep4 mutation (Jones, Genetics 85:23-33. 1977), which results in reduced proteolytic activity.
  • cultured mammalian cells may be used as host cells within the present invention.
  • Preferred cultured mammalian cells for use in the present invention include the COS-1 (ATCC CRL 1650) , BHK, and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol.
  • a preferred BHK cell line is the BHK 570 cell line (deposited with the American Type
  • NCTC 1469 ATCC CCL 9.1
  • DUKX cells Urlaub and Chasin, Proc. Natl. Acad. Sci USA77: 4216-4220, 1980.
  • Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA.
  • Preferred promoters include viral promoters and cellular promoters.
  • Viral promoters include the immediate early cytomegalovirus promoter (Boshart et al., Cell 41:521-530, 1985) and the SV40 promoter (Subramani et al. , Mol. Cell. Biol. 1:854-864. 1981).
  • Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al. , U.S. Patent No.
  • telomeres a promoter that promotes the telomere telomere telomere telomere telomere telomere telomere s telomere s telomere s telomere s telomere s telomere s telomere s telomere s telomere s telomere s telomere s telomere s telomereactive telomere sequence a mouse VH promoter (Loh et al. , Cell ____:85-93, 1983).
  • a particularly preferred promoter is the major late promoter from Adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2.:1304-13199, 1982) .
  • Such expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence to be expressed.
  • RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes.
  • a polyadenylation signal located downstream of the coding sequence of interest. Suitable polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. £:3719-3730, 1981) .
  • the expression vectors may include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites.
  • Preferred vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse ⁇ enhancer (Gillies, Cell 3_3.:717-728, 1983). Expression vectors may also include sequences encoding the adenovirus VA RNAs.
  • enhancer sequences such as the SV40 enhancer and the mouse ⁇ enhancer (Gillies, Cell 3_3.:717-728, 1983).
  • Expression vectors may also include sequences encoding the adenovirus VA RNAs.
  • Cloned DNA sequences may be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell .14.:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 2:603, 1981; Graham and Van der Eb, Virology 52:456. 1973.)
  • Other techniques for introducing cloned DNA sequences into mammalian cells such as electroporation (Neumann et al., EMBO J. 1:841-845. 1982), may also be used.
  • a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.
  • Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.
  • the selectable marker may be an amplifiable selectable marker.
  • a preferred amplifiable selectable marker is the DHFR gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology. Butterworth Publishers, Stoneham, MA, which is incorporated herein by reference) . The choice of selectable markers is well within the level of ordinary skill in the art.
  • Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Patent No. 4,713,339). It may also be advantageous to add additional DNA, known as "carrier DNA" to the mixture which is introduced into the cells.
  • Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker, the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.
  • Preferred prokaryotic host cells for use in carrying out the present invention are strains of the bacteria Escherichia coli, although Bacillus and other genera are also useful. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known , in the art (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982; which is incorporated herein by reference, or Sambrook et al., Molecular Cloning: A
  • Vectors used for expressing cloned DNA sequences in bacterial hosts will generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter that functions in the host cell.
  • a selectable marker such as a gene for antibiotic resistance
  • Appropriate promoters include the trp (Nichols and Yanofsky, Meth. Enzymol. 101:155-164. 1983), lac (Casadaban et al., J. Bacteriol. 143:971-980, 1980), and phage ⁇ (Queen, J. Mol. Appl. Genet. 2 . :1-10, 1983) promoter systems.
  • Plasmids useful for transforming bacteria include pBR322 (Bolivar et al., Gene 2.-95-113, 1977), the pUC plasmids (Messing, Meth. Enzymol. 101:20- 78, 1983; Vieira and Messing, Gene 9.:259-268, 1982), pCQV2 (Queen, ibid.), and derivatives thereof. Plasmids may contain both viral and bacterial elements.
  • promoters, terminators and methods for introducing expression vectors encoding galanin analogs of the present invention into plant, avian and insect cells would be evident to those of skill in the art.
  • the use of baculoviruses, for example, as vectors for expressing heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 2 ⁇ :215-224,1990).
  • the use of Agrobacterium rhizogenes as vectors for expressing genes in plant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangalore) 11:47-58. 1987) .
  • transformants or transfectants expressing the galanin analogs are then cloned.
  • individual transformants may be picked onto selective media using sterile toothpicks.
  • individual transfectants may be isolated by cylinder cloning into multi-well culture plates.
  • the cloned cells are then used in assays that will generally include the steps of (a) growing host cells containing a DNA construct capable of directing the expression of a galanin analog, the construct comprising the following operably linked elements: a transcriptional promoter, a DNA sequence encoding a galanin analog, and a transcriptional terminator, under growth conditions suitable for the expression of the galanin analog; (b) isolating the galanin analog encoded by the DNA sequence from the host cells; (c) exposing the isolated galanin analogs in the presence of native galanin to a galanin receptor coupled to a response pathway under conditions and for a time sufficient to allow binding of the galanin analog to the receptor and an associated response through the pathway; and (d) detecting a reduction in the inhibition of the response pathway resulting from the binding of the galanin analog to the galanin receptor relative to the inhibition of the response pathway by native galanin, and therefrom determining the presence of a galanin antagonist.
  • the transfected or transformed cells are grown according to standard methods in a growth medium containing nutrients required for growth of the particular host cells.
  • suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.
  • the growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.
  • Suitable growth conditions for yeast cells include culturing in a medium comprising a nitrogen source (e.g. yeast extract or nitrogen-containing salts) , inorganic salts, vitamins and essential amino acid supplements as necessary at a temperature between 4°C and
  • the pH of the medium is preferably maintained at a pH greater than 2 and less than 8, more preferably pH 5-6.
  • Methods for maintaining a stable pH include buffering and constant pH control, preferably through the addition of sodium hydroxide.
  • Preferred buffering agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, MO).
  • Cultured mammalian cells are generally cultured in commercially available serum-containing or serum-free media. Selection of a medium appropriate for the particular cell line used and determination of suitable growth condition are within the level of ordinary skill in the art.
  • Galanin analogs expressed by the host cells are then isolated from the cells. If the analogs are secreted by the cells, it will generally be sufficient to remove the cells from the culture media (e.g. by centrifugation or filtration) and assay media samples as described herein. If the galanin analogs are retained within the host cells it will be necessary to lyse the cells and recover the analog(s) from the lysate.
  • Conditions and times sufficient for the binding of the galanin analog to the receptor will vary with the source of the receptor and the particular assay used; however, conditions suitable for binding will generally be between 4°C and 55°C, preferably 30°C-40°C, under physiological conditions.
  • physiological conditions indicates conditions approximating the normal environment of a cell-associated galanin receptor, and includes cell culture media and buffered, low-salt solutions within a pH range of between 5 and 9, preferably between 6.8 and 8. Sufficient time for the binding and response will be between 5 and 60 minutes after exposure, with 15-30 minutes being particularly preferred.
  • antagonists are capable of binding to a cellular receptor of the corresponding native ligand, but either are incapable of affecting a response pathway or exhibit a reduced effect on a response pathway as compared to " the native ligand.
  • galanin antagonists are identified through their ability to bind to a cellular galanin receptor and their inability or reduced ability to inhibit the adenylate cyclase response pathway that is stimulated by forskolin or gastric inhibitory polypeptide (GIP) .
  • GIP gastric inhibitory polypeptide
  • galanin antagonists are identified through their ability to reduce galanin inhibition of insulin release or somatostatin release.
  • Galanin receptors have been reported in a number of cells and tissues, for example pancreatic 0-cells, insulinoma cells, gut cells, pituitary tissue and brain tissue.
  • Adenylate cyclase activity assays or insulin secretion assays may be carried out using, for example, the method described by Amiranoff et al. (Eur. J. Biochem. 177: 147-152, 1988; incorporated herein by reference) . Briefly, RINm5 cells, preferably RINSmAH T 2 B (RIN5) , cells are incubated in culture media in the presence of 0.1 mM forskolin or 0.1 ⁇ m GIP for 30 minutes at 37°C to stimulate cAMP production.
  • cAMP production in the control cells is compared to that in cells cultured in the presence of forskolin or GIP plus galanin or galanin and a galanin analog.
  • Cyclic AMP is extracted by adding 1 M perchloric acid, succinylated, and assayed by radioimmunoassa . It is generally preferred that cAMP production be measured using a commercially available kit (e.g. the Scintillation Proximity Assay manufactured by Amersham Corp. , Arlington Heights, IL) . Using such a kit, the production of cAMP is determined by competition of iodinated-cAMP with anti-cAMP antibodies.
  • Insulin secretion is assayed in forskolin- or GIP- stimulated RIN5 cells by radioimmunoassay as disclosed by Amiranoff et al. (ibid.) or Vallar et al. (J. Biol. Chem. 262: 5049-5056, 1987) or may be assayed using a commercially available kit (e.g. the Scintillation Proximity Assay manufactured by Amersham Corp. , Arlington Heights, IL) . Briefly, target cells are incubated in medium containing an insulin secretagogue (e.g. forskolin or GIP) with or without galanin and galanin antagonists for 30-60 minutes at 37°C.
  • an insulin secretagogue e.g. forskolin or GIP
  • galanin and galanin antagonists for 30-60 minutes at 37°C.
  • the medium is then collected, and insulin secretion is quantitated.
  • An insulin RIA kit is available from Novo Nordisk (Bagsvaerd, Denmark) or insuling may be assayed using the Scintillation Proximity Assay (Amersham Corp.). Somatostatin release is assayed as disclosed by Amiranoff et al. (Eur. J. Pharmaco1. 191: 401-405, 1990) by measuring the inhibition of basal somatostatin release by RIN5 cells incubated in the presence of galani •n (10— • 1 L ⁇ U -10—7'M) .
  • Assays may also be carried out on other cell types, such as hamster pancreatic S-cell tumor cells (Amiranoff et al., Eur. J. Biochem. 159: 353, 1986; Amiranoff et al., Endocrinology 121: 284-289, 1987) or other cells having galanin receptors (e.g. cultured human pancreatic islet cells) or on isolated J-cell membranes.
  • cell types such as hamster pancreatic S-cell tumor cells (Amiranoff et al., Eur. J. Biochem. 159: 353, 1986; Amiranoff et al., Endocrinology 121: 284-289, 1987) or other cells having galanin receptors (e.g. cultured human pancreatic islet cells) or on isolated J-cell membranes.
  • rat galanin analog library is constructed and tested as generally described above.
  • the rat galanin sequence is disclosed by Kaplan et al. fProc. Natl. Acad. Sci. USA 85: 1065-1069, 1988) and Vrontakis et al. (J. Biol. Chem. 262: 16755-16758) , which are incorporated herein by reference.
  • Amino acid changes found to produce rat galanin antagonists are then used to design corresponding human analogs, which are tested for antagonist activity.
  • human galanin analogs showing antagonist activity on cultured cells or isolated cell membranes may be confirmed by jLn vivo testing of substantially pure galanin antagonists, such as by systemic administration or using a whole animal infused pancreas model or in an isolated pancreas model.
  • substantially pure human galanin analogs are those that are of at least about 50% purity, at least about 70-80% more preferred, and 95-99% or more homogeneity most preferred.
  • Methods for assaying galanin in a whole animal infused pancreas model are described in more detail herein and, in addition, are disclosed by Dunning et al. (Am. J. Phvsiol. 251 (Endocrinol. Metab.
  • the expression vector may be isolated and the insert (antagonist coding sequence) sequenced to confirm the presence of a mutation.
  • Antagonist coding sequences may be tranferred to other expression vectors as desired and strains or cell lines expressing the antagonist are scaled up for production. Sequence information from different galanin antagonists facilitates the design and construction of additional sequences encoding antagonists with multiple amino acid differences.
  • the galanin antagonists of the present invention may be purified by ion-exchange and partition chromatography as described by, for example, Coy et al. (Peptides Structure and Function, Pierce Chemical Company, Rockford, IL, pp. 369-372, 1983), by high performance liquid chromatography as described, for example, by Lagny- Pour ir et al. (Peptides 10: 757-761, 1989) or Fisone et al. (Proc. Natl. Acad. Sci. USA 86: 9588-9591, 1989). Additional purification may be achieved by conventional chemical purification means, such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others.
  • galanin antagonists identified using the screening methods described herein may be synthesized following any suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution addition.
  • Synthetic galanin antagonsists of the present invention may be prepared by hand synthesis or using a suitable peptide synthesizer.
  • the polypeptides are synthesized on an Applied Biosystems Model 431A peptide synthesizer.
  • Solid phase peptide synthesis is the preferred method for preparing polypeptides of the present invention.
  • a particularly preferred method for peptide synthesis is by the Fmoc method essentially described by Carpino and Han (J. A er. Chem. Soc.
  • solid phase synthesis proceeds from the C-terminus of the peptide to the N-terminus and begins with the coupling of an activated, Fmoc-protected, ⁇ -amino acid to a suitable resin.
  • suitable resins have been described, for example, by Wang et al. (J. Am. Chem. Var: 1328-1333, 1973), Rink (Tetrahedron Lett. 28: 3787-3790, 1987), Breipohl et al. (Tetrahedron Lett. 28: 5651-5654, 1987), Funakoshi et al., J. Am. Soc.
  • HMP-resin 4-hydroxymethylphenoxymethylcopolystyrene-l% divinylbenzene resin
  • Fmoc-protected ⁇ -amino acids are activated using any of a variety of suitable activation methods known in the art, such as conventional N,N'-dicyclohexylcarbodiimide (DCC) activation, symmetric anhydride activation and 1- hydroxybenzotriazole (HOBt) /DCC activation.
  • DCC N,N'-dicyclohexylcarbodiimide
  • HOBt 1- hydroxybenzotriazole
  • a particularly preferred method of activation of the first amino acid of the polypeptide is the symmetric anhydride method.
  • Subsequent ⁇ -amino acids are preferably activated using the HOBt/DCC activation method essentially described by Konig and Geiger (Chem. Ber. 103: 788-798, 1970).
  • the Fmoc protecting group is removed by treatment with 20% piperidine followed by a wash with N-methylpyrrolidone (NMP) .
  • NMP N-methylpyrrolidone
  • activated ⁇ -amino acids preferably as HOBt active esters, are added in the desired order in cycles consisting of activated ⁇ -amino acid addition followed by piperidine deprotection.
  • An alternative to adding amino acids to the ⁇ polypeptide in sequential order, is the addition of amino acids that have been coupled prior to addition to the growing polypeptide.
  • the polypeptide may be removed from the resin, following deprotection of the terminal amino acid, by cleavage with, for example, trifluoroacetic acid.
  • the peptide may be purified by reverse-phase high-pressure liquid chromatography.
  • galanin analogs may be chemically synthesized, such as by the solid-phase method of Barany and Merrifield
  • Substantially pure galanin antagonists of at least about 50% purity are preferred, at least about 70- 80% more preferred, and 95-99% or more homogeneity most preferred, particularly for pharmaceutical uses.
  • the recombinant galanin analogs may be used therapeutically.
  • the antagonists of the present invention are administered by injection (intravenous or subcutaneous) , inhalation or infusion to patients suffering from inter alia, type II diabetes or stress-related hyperglycemia.
  • the antagonists of the present invention may be present as free bases or as acid salts. Suitable salts will be pharmaceutically acceptable and include metal salts, including alkali and alkaline earth metal salts such as potassium or sodium salts.
  • compositions may be formulated in aqueous isotonic solutions of between pH 5.6 and 7.4. Suitable isotonic solutions include sodium chloride, dextrose, boric acid, sodium tartrate, and propylene glycol solutions.
  • Therapeutic doses of antagonists of the present invention may be administered simultaneously with insulin either in the same composition or in separate compositions.
  • plaques in the brains of Alzheimer's patients are associated with neuronal hyperplasia. This hyperplasia leads to an overproduction of galanin at these sites.
  • the galanin antagonists of the present invention are thus useful tools for studying the pathology of this disease.
  • Galanin may also be an important diagnostic marker for tumors, particularly pheochromocytomas.
  • the human galanin DNA sequence is thus useful as a probe in DNA-based diagnostic tests, and recombinant galanin may be used to generate antibodies for immunoassay diagnostic tests.
  • Pheochromocytoma specimens were obtained from multiple patients and pooled.
  • the tissue was fragmented by mortar and pestle in liquid nitrogen and solubilized in extraction buffer (4 M guanidinium thiocyanate, 0.1 M Tris-HCl, pH 7.5, 1% .-mercaptoethanol, 0.5% sodium lauryl sarcosinate) .
  • the fragmented tissue was homogenized for 20 seconds using a tissue homogenizer. Phenol:chloroform:isoamyl alcohol (50:48:2) was added and the mixture was vortexed and centrifuged. Nucleic acids were precipitated with isopropanol, and the pellet was resuspended in extraction buffer and precipitated again with isopropanol.
  • RNA pellet was sequentially washed with 75% and 100% ethanol.
  • Poly (A) + RNA was purified using oligo d(T)-cellulose column chromatography as described by Sambrook et al. , eds. (Molecular Cloning: A Laboratory Manual, vol. 1, 7.26-7.29, Cold Spring Harbor Laboratory Press, 1989) .
  • First strand cDNA was synthesized from the poly (A) + RNA by first incubating 1.0 ⁇ g of poly(A) + RNA at 65°C for 3 minutes in 5 mM Tris-HCl pH 7.6, 0.05 mM EDTA.
  • RNA was cooled on ice, and the cDNA synthesis reaction was primed with 5 pmol of oligonucleotide ZC2487 (Table 1, Sequence ID Number 4) in a 10 ⁇ l reaction volume containing 50 mM Tris-HCl pH 8.3, 75 mM KC1, 3 mM MgCl 2 , 10 mM dithiothreitol, 0.5 mM each deoxynucleotide triphosphate, and 200 units of MMLV (RNase H ⁇ ) reverse transcriptase (GIBCO-BRL, Gaithersburg, MD) .
  • the reaction mixture was incubated at 45°C for 1 hour, following which the mixture was diluted with 180 ⁇ l of 10 mM Tris-HCl pH 7.6, 1 mM EDTA and stored at 4°C.
  • ZC2488 (Sequence ID Number 5) GAC TCG AGT CGA CAT CGA TCA GCC CCC CCC CC
  • Galanin cDNA sequences were amplified using degenerate primers encoding galanin DNA sequences using the polymerase chain reaction (PCR) method (Mullis et al., ibid.) Each of these primers also contained a 5' tail of 10 nucleotides, which provided convenient restriction enzyme sites for subcloning.
  • PCR polymerase chain reaction
  • Inserts were detected by agarose gel analysis of PCR reactions performed on selected transfected cells using oligonucleotide pools ZC3518 (Sequence ID Number 7) and ZC3520 (Sequence ID Number 8) (Table 1) .
  • the reactions were carried out by inoculating a reaction mixture with the selected transformant under the conditions set forth above, and the samples were preheated for five minutes at 94°C. After 30 cycles (one minute at 94°C, one minute at 48°C, one minute at 72°C) , aliquots of the reactions were electrophoresed in an agarose gel. The presence of an approximately 250 bp fragment indicated that the transformants contained an insert.
  • galanin-specific first strand cDNA was prepared as described above using 5 pmol of ZC3520 (Sequence ID Number 8) as the galanin-specific primer.
  • the reaction was terminated by the addition of KOH to a concentration of 0.5 M and EDTA to a concentration of 50 mM followed by an incubation at 65°C for 5 minutes. After incubation, 1 ml of 50 mM KOH, 0.1 mM EDTA was added to the reaction, and the material was centrifuged through a Centricon 100 concentrator in a Sorvall SS-34 rotor at 6000 rpm for 5 minutes.
  • the cDNA was d(G)- tailed by incubation at 35°C for 10 minutes with 0.1 M potassium cacodylate pH 7.2, 2 mM C0CI2, 0.2 mM dithiothreitol, 0.1 mM dGTP and 19 units of terminal deoxynuceotidyl transferase (Pharmacia) .
  • the reaction was terminated by the addition of EDTA to 50 mM, and the cDNA was precipitated by ammonium acetate and ethanol in the presence of 2 ⁇ g of oyster glycogen.
  • the cDNA was pelleted, washed with 75% ethanol, dried briefly and resuspended in 20 ⁇ l water.
  • Second strand cDNA synthesis was performed in a 100 ⁇ l reaction volume containing 5 pmol ZC2488 (Table 1; Sequence ID Number 5) , 50 mM NaCl, 10 mM Tris-HCl pH 8.3, 2 mM MgCl 2 , 0.2 mM each deoxynucleotide triphosphate, and 0.01% gelatin.
  • the mixture was heated to 94°C for 3.5 minutes, then 4.5 units of Taq DNA Polymerase was added, and the mixture was overlaid with 50 ⁇ l of mineral oil.
  • the temperature of the reaction mixture was dropped to 40°C for 5 minutes and then increased to 72°C for 15 minutes.
  • the reaction was terminated by the addition of EDTA to a final concentration of 2.5 mM, and the reaction mixture was extracted with chloroform.
  • the cDNA was precipitated with sodium acetate and ethanol in the presence of 5 ⁇ g oyster glycogen.
  • the cDNA was pelleted, washed with 75% ethanol, dried, resuspended in 100 ⁇ l of water and stored at 4°C.
  • the reaction was terminated with an incubation at 72°C for ten minutes. An aliquot of the reaction was analyzed by gel electrophoresis.
  • the PCR fragments were isolated, digested with Sal I and Bam HI, then ligated to pUC19 plasmid DNA which was previously digested with Sal I and Bam HI.
  • the ligation mixture was precipitated in the presence of 2 ⁇ g of oyster glycogen, and the DNA was resuspended in 3 ⁇ l of water, electroporated into E. coli DH10B cells and plated as described above.
  • the DNA is amplified for 5 cycles of (one minute at 94°C, two minutes at 56°C, three minutes at 72°C) , followed by 35 cycles of (one minute at 94°C, two minutes at 65°C, three minutes at 72°C) .
  • the reaction is terminated with a 10 minute incubation at 72°C.
  • An aliquot of the reaction mixture is analyzed by agarose gel electrophoresis and the prominent band is isolated, digested with Eco RI and Bam HI, and ligated to pUC19 plasmid DNA previously digested with Eco RI and Bam HI.
  • the ligation mixture is precipitated in the presence of 2 ⁇ g of oyster glycogen.
  • the DNA pellet is resuspended in water, electroporated into E. coli DH10B cells and plated as described above. Selected transformants are picked and the presence of cDNA inserts is detected by PCR as described above using oligonucleotides ZC3518 and ZC3757 (Table 1; Sequence ID Nos. 7 and 9) . Template DNA is prepared and the DNA sequences of PCR products are determined.
  • Plasmid pEAS102 comprising portions of the yeast vectors YIp5 and pJDB207, was constructed as follows. Plasmid pJDB207 (Beggs, Proceedings of Alfred Benzon Symposium 16: 383-389, "Molecular Genetics in Yeast," Copenhagen, Denmark, 1981), a derivative of pJDB219 (Beggs, ibid., 1978), was digested with Bam HI and Pst I to isolate the 4.4 kb fragment comprising the leu2-d gene, 2 micron plasmid and pBR322 sequences. Plasmid YIp5 (Struhl et al.
  • Hind III sites in plasmid pEAS102 was destroyed by first digesting pEAS102 with Hind III to completion. The linearized plasmid was then incubated with the DNA polymerase I (Klenow fragment) in the presence of nucleotide triphosphates, recircularized by treatment with T4 DNA ligase and transformed into E. coli strain HB101. DNA prepared from the resulting transformants was screened for those plasmids which could no longer be linearized by digestion with Hind III.
  • the promoter and terminator regions from the Saccharomyces cerevisiae TPI1 gene along with the alpha factor prepro sequence were inserted into the pEAS102 derivative described above.
  • the TPI1 promoter and alpha factor prepro sequence were obtained from plasmid pTGF ⁇ m.
  • the plasmid pTGF ⁇ m was derived from plasmid pB12, which contained the TPI1 promoter, the MF ⁇ l signal sequence, PDGF-BB sequence, the TPI1 terminator and pIC19R vector sequences.
  • the construction of pB12 is disclosed by Murray et al. (U.S. Patent No. 4,766,073, which is incorporated herein by reference) .
  • the MF ⁇ l signal sequence and PDGF-BB sequence were subcloned as an Eco RI- Xba I fragment into M13.
  • the Sst I site present in the MF ⁇ l signal sequence was changed to a Hind III site by in vitro mutagenesis using the method described by Kunkel et al. (U.S. Patent Number 4,873,192) and oligonucleotide ZC1159 (Table 1, Sequence ID No. 3) .
  • a clone having a Hind III site in place of the Sst I site was identified.
  • a fragment containing the MF ⁇ l signal sequence was isolated as an Eco RI-Hind III fragment.
  • the Eco RI-Hind III fragment containing the MF ⁇ l signal sequence and a Hind III-Xba I fragment containing a synthesized transforming growth factor ⁇ (TGF ⁇ ) coding sequence were ligated with Eco Rl-Xba I linearized pUC13.
  • the resultant plasmid, designated ⁇ fTGF ⁇ /pUC13, was digested with Eco RI and Xba I to isolate the MF ⁇ l-TGF ⁇ insert, which was cloned into pl70CB/pBR.
  • the construction of plasmid pl70CB/pBR is described by Murray (U.S. Patent Application Serial No.
  • Plasmid pB170CB/pBR was digested with Eco Rl-Xba I to isolate the fragment containing the TPIl promoter, pBR322 vector sequence and the TPIl terminator.
  • the Eco Rl-Xba I pB170CB/pBR fragment and the Eco Rl-Xba I MF ⁇ l-TGF ⁇ fragment were ligated.
  • TGF ⁇ CB The resulting plasmid, designated TGF ⁇ CB, was digested with Cla I and Bam HI to isolate the expression unit, which was then subcloned into the yeast expression vector pMP0T2 (a yeast 2 micron-based plasmid containing yeast and bacterial replication origins, ampicillin resistance gene and P0T1 selectable marker; deposited with American Type Culture Collection, Rockville, MD. under accession number 67788) to construct pTGF ⁇ m. Plasmid pTGF ⁇ m was digested with Bgl II and Hind III to isolate the 1236 base pair fragment containing the TPIl promoter and MF ⁇ l signal sequence.
  • pMP0T2 a yeast 2 micron-based plasmid containing yeast and bacterial replication origins, ampicillin resistance gene and P0T1 selectable marker; deposited with American Type Culture Collection, Rockville, MD. under accession number 67788
  • the Saccharomyces cerevisiae TPIl terminator fragment was obtained from plasmid pFGl (Alber and Kawasaki, ibid.). It encompassed the region from the penultimate amino acid codon of the TPIl gene to the Eco RI site approximately 700 base pairs downstream.
  • a Bam HI site was substituted for the unique Eco RI site of pFGl by first digesting the plasmid with Eco RI, then blunting the adhesive ends with DNA polymerase I (Klenow fragment) , adding synthetic Bam HI linkers (CGGATCCA) , and re- ligating to produce plasmid pl36. The TPIl terminator was then excised from pl36 as an Xba I-Bam HI fragment.
  • p270 This fragment was ligated into YEpl3 (Broach et al., ibid.), which had been linearized with Xba I and Bam HI.
  • the resulting plasmid is known as p213.
  • the Hind III site was then removed from the TPIl terminator region of p213 by digesting the plasmid with Hind III, blunting the resultant termini with DNA polymerase I (Klenow fragment) , and recircularizing the linear molecule using T4 DNA ligase.
  • the resulting plasmid was designated p270.
  • p270 may be constructed by digesting plasmid pM220 (deposited with American Type Culture Collection as an E.
  • the TPIl terminator was removed from plasmid p270 as an Xba I-Bam HI fragment. This fragment was cloned into pUC19 along with another fragment containing the TPIl promoter joined to the CAT (chloramphenicol acetyl transferase) gene to obtain a TPIl terminator fragment with an Eco RV end. The resultant plasmid was designated pCAT. The TPIl terminator was then removed from pCAT as an Eco RV-Bam HI fragment and cloned into pIC19H (Marsh et al., ibid.), which had been linearized with the same enzymes, to obtain plasmid pTTI. Plasmid pTTI was then digested with Hind III and Sal I to isolate the 718 bp TPIl terminator fragment.
  • the 1236 base pair Bgl II-Hind III TPIl promoter-MF ⁇ l fragment and the 718 base pair Hind Ill-Sal I TPIl terminator fragment were ligated with the pEAS102 derivative that have been linearized by digestion with Bam HI and Sal I.
  • the ligation mixture was transformed into E. coli strain HB101, and plasmid DNA prepared from selected transformants was screened by restriction analysis to identify a clone bearing a plasmid of the correct structure.
  • a positive clone was designated pBS114 ( Figure 2) .
  • Example 3 Construction Of Yeast Strains Secreting Rat And Human Galanin Plasmids useful in the secreted expression of galanin from yeast were constructed by inserting pairs of synthetic oligonucleotides (Sequence ID Numbers 10 and 11; Table 2) into pBS114.
  • the oligonuceotides contain a galanin coding sequence flanked by bridge sequences which correctly link the galanin coding sequence to the expression and secretion elements within pBS114.
  • the coding region for galanin was designed to utilize nucleotide triplets corresponding to codons which occur in highly expressed yeast genes.
  • ZC3762 (Sequence ID Number 10) 5' AGC TTA GAT AAG AGA GGT TGG ACC TTG AAC TCT GCA GGT TAC TTG TTG GGT CCA CAC GCT ATC GAT AAC CAC CGT TCT TTC TCT GAT AAG CAC GGT TTG ACC GGT TGA
  • ZC3763 (Sequence ID Number 11) 5' GAT CTG AAT TCA ACC GGT CAA ACC GTG CTT ATC AGA GAA AGA ACG GTG GTT ATC GAT AGC GTG TGG ACC CAA
  • ZC3842 (Sequence ID Number 12) 5' AGC TTA GAT AAG AGA GGT TGG ACC TTG AAC TCT GCA
  • the oligonucleotides were annealed to form 119 a
  • the first 15 bases of oligonucleotide ZC3762 (Sequence ID Number 10) include codons for the amino acid sequence LYS-ARG, which provides a site for the action of the product of the yeast KEX2 gene so that the alpha factor leader will be removed from galanin produced by yeast.
  • the ligation mixture was transformed into E. coli, and plasmid DNA prepared from selected transformants was analyzed by restriction analysis. A plasmid having the correct rat galanin insert was chosen to transform into Saccharomyces cerevisiae.
  • the yeast-produced rat galanin is not amidated at its carboxy terminus, and is therefore a 30 amino acid polypeptide terminating in a glycine residue.
  • oligonucleotides were synthezied for the construction of an expression vector containing human galanin.
  • Oligonucleotides ZC3842 and ZC3843 (Table 2; Sequence ID Nos. 12 and 13), which were designed to encode human galanin using yeast- preferred codons, were annealed to form a 109 base pair duplex DNA with four-base 2 ' overhangs at either end.
  • the annealed oligonucleotides were ligated with pBS114 that had been linearized by digestion with Hind III and Bgl II, and the ligation mixtures were transformed into E. coli.
  • the annealed oligonucleotides contained six mismatched base pairs which are noted as the underlined bases in ZC3843 (Sequence ID Number 13) .
  • plasmid DNA prepared from selected transformants was analyzed by restriction analysis and sequence analysis.
  • a plasmid having the an insert having a sequence corresponding to ZC3842 (Sequence ID Number 12) the correct sequence for human galanin was chosen to transform into Saccharomyces cerevisiae.
  • Plasmids containing rat or human galanin coding sequences were transformed into Saccharomyces cerevisiae strain ZY100 (ade2-101 leu2-3 leu2-112 ura3-52 suc2-A9 ga!2 pep4: :TPIlp-CAT) . Transformants were selected on -URADS plates (Table 3) and then restreaked for individual colonies on -LEUD plates (Table 3) . These colonies were assayed for. galanin secretion using the method described in Example 5.
  • oligonucleotides were synthesized using phophoramidite solutions that have been purposely cross-contaminated such that the solutions that normally correspond to the four bases A, G, C, and T each contain small amounts of the other three phosphoramidites. After the four phosphoramidites have been dissolved at the concentration used for synthesis (0.13 M) , three 60 ⁇ l aliquots were removed from each 10 ml supply bottle of the four phosphoramidite solutions.
  • each oligonucleotide pool begins with a 2 ' base coupled to a support resin and as such cannot be conveniently mutagenized, breakpoints in the galanin sequences were chosen to result in 2 ' ends that were in the third position of codons in which all four bases result in the same amino acid insertion. Mutagenesis was also limited to avoid certain codons, such as chain termination codons. Sites were also chosen to provide seven base cohesive 3' overhanging ends after complementary pairs of oligonucleotides are annealed. To mutagenize rat galanin, the oligonucleotide sequences shown in Table 4 (Sequence ID Nos. 14, 15, 16, 17, 18 and 19) were used as the starting sequence.
  • the starting sequence of the generation of human galanin nalaogs were the oligonucleotide sequences shown in Table 4 (Sequence ID Nos. 14, 19, 21, 22 and 23) . Uncontaminated phosphoramidites were used for the bases shown bold text of Table 4. Doped positions for the oligonucleotides shown in Table 4 are underlined.
  • ZC4153 (Sequence ID Number 14) AGCTTAGATA AGAGAGGTTG GACCTTGAAC TCTGCAGGTT AC
  • ZC4162 (Sequence ID Number 23) GATCTGAATT CAAGAGGTCA AACCGTTCTT ATCAGAGAAA
  • Oligonucleotide pools ZC4153, ZC4154, and ZC4301 (Sequence ID Nos. 14, 15 and 16; Table 4) form the sense strand of the rat galanin insert; and ZC4156, ZC4157, and
  • ZC4158 (Sequence ID Nos. 17, 18 and 19; Table 4) form the antisense strand respectively in the 5' to 3' direction for each strand.
  • ZC4153, ZC4159, ZC4161, ZC4162, ZC4160 and ZC4158 (Sequence ID Nos. 14, 20, 22, 23, 21 and
  • Oligonucleotide pools that contained 5' ends that were internal to each 113 base pair insert sequence (i.e. ZC4154, ZC4301, ZC4157, ZC4159, ZC4161, ZC4160, and ZC4158; Sequence ID Nos. 15, 16, 18, 20, 22, 21 and 19, respectiively; Table 4) were treated with polynuleotide kinase in the presense of 1 mM ATP at 37°C for one hour to add the 5' phosphate groups required for T4 DNA ligase activity. External 5' ends were not treated in order to prevent tandem inserts from forming.
  • the mixtures were annealed by heating the mixtures to 85°C and allowing them to cool slowly to room temperature.
  • the mixtures were ligated in the presence of ATP and T4 DNA ligase, and the ligation mixtures electrophoresed on a 1.2% agarose gel.
  • the 113 base pair fragments were gel purified, and the fragments were each ligated with with pBS114 that had been linearized by digestion with Bgl II and Hind III.
  • the ligation mixtures were transformed into E. coli strain DH10B by electroporation, and twelve individual transformant colonies were selected from each library for DNA sequence analysis.
  • the remaining approximately 10,000 colonies in the rat library are pooled, as are approximately 10,000 human library clones, and both pools are used to prepare plasmid DNA for yeast transfomation.
  • Saccharomyces cerevisiae strain ZY100 fade2-101 leu2-3 leu2-112 ura3-52 suc2- ⁇ 9 ga!2 pep4: :TPIlp-CAT) are transformed with the pooled plasmid DNA from the DH10B transformants, and transformant colonies are selected on - URADS plates (Table 3) .
  • Individual URA + colonies are streaked on -LEUD plates (Table 3) and one colony from each streak is saved as a unique clone expressing one of the galanin library sequences.
  • Screening assays for galanin antagonists are performed on conditioned medium from cultures of individual transformant colonies derived from a library of yeast strains producing mutagenized galanin analogs.
  • Conditioned medium is obtained by using each of the patched individual colonies from master plates to innoculate 200-300 microliters of liquid -LEUD medium
  • Clarified conditioned medium is obtained by centrifuging the microtiter plates for 5 minutes at 1000 X g and the medium is stored at 4°C for up to three days.
  • RIN5 cells are seeded into the wells of a 96- well microtiter plate at a density of 4 x 10 4 cells/well in 100 ⁇ l RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MC) supplemented with 10% fetal bovine serum, 20 mM HEPES (Sigma, St. Louis, MO) 2 mM L-glutamine (GIBCO-BRL) , 100 units/ml penicillin (GIBCO-BRL) and 100 ⁇ g/ml streptomycin (GIBCO-BRL) and grown at 37°C, 5% C0 2 atmosphere. Following one day of growth, the medium is removed from each well and replaced with 100 ⁇ l fresh medium.
  • the cells are washed three-times with 100 ⁇ l DMEM (GIBCO-BRL) supplemented with 2% BSA and 10 ⁇ g/ml aprotinin, leaving 50 ⁇ l of the medium in each well after the final wash.
  • Clarified conditioned from the yeast cultures is diluted ten-fold with RIN5 cell stimulation medium (DMEM
  • insulin assay buffer 150 mM NaCl, 50 mM Na 2 HP0 4 , 25 mM EDTA, pH 7.4.
  • Each diluted sample is assayed for insulin content by radioimmunoassay.
  • a 75 ⁇ l aliquot of each sample is transferred to individual wells of a 96-well LKB T-tray.
  • standard solutions containing known amounds of rat insulin (0, 0.25, 0.5, l, 2, 5 and 10 ng/ l) are added, in duplicate, to control wells.
  • To each well 75 ⁇ l of insulin assay buffer containing approximately 5 nCi of (3-[ 1 5 I]iodotyrosyl A14) human insulin (Amersham, Arlington Heights, IL) is added, followed by 75 ⁇ l of dilusted guinea pig anti-rat insulin serum (Linco Research, Inc., St.
  • Yeast strains corresponding to the highest insulin values are selected for rescreening. The strains are grown and assayed in triplicate as described above. Galanin analogs that consistantly show higher levels of insulin secretion in the stimulation assay relative to a control strain making no analog are designated as galanin antagonists. Plasmid DNA recovered from strains producing galanin antagonists is subjected to DNA sequence analysis to determine the polypeptide sequence of potential galanin antagonists.
  • triplicate assays were set up by culturing RIN5 cells in standard six-well tissue culture plates in RPMI 1640 medium (GIBCO-BRL) supplemented with 10% (v/v) heat-inactivated fetal calf serum (GIBCO-BRL) , 24 mM NaHCC- 3 , 20 mM Hepes, 2 mM L-glutamine, 100 units/ml penicillin and 100 ⁇ l/ml streptomycin under conditions essentially described by Karlsen et al. (ibid.). Three days after plating, the medium was replaced with fresh medium. One or two days before the cells were assayed, the medium was removed and the cells in each well were washed with KRBB.
  • the cells were incubated for two hours with 2 ml of KRBB.
  • KRBB the cells were exposed to the galanin peptide by incubating the cells for one hour in 1 ml KRBB with amino acids (lx essential and non-essential minimun essential medium, GIBCO-BRL) in the presence of 5.6 mM glucose and in the presence of dilutions containing between 10 ⁇ to 10 ⁇ 6 M of each species of galanin.
  • amino acids lx essential and non-essential minimun essential medium, GIBCO-BRL
  • Plasmid DNAs prepared from the yeast cells that are shown in Example 5 to secrete galanin antagonists are sequenced to determine the nucleotide sequence and deduced amino acid sequence of the galanin antagonists.
  • Galanin antagonists are then synthesized from the deduced amino acid sequences on an Applied Biosystems Model 43IA peptide synthesizer, and the peptides are purified by reverse- phase high-pressure liquid chromatography.
  • the synthetic galanin antagonists are tested for the ability to reduce galanin-induced inhibition of insulin secretion from the right (duodenal) lobe of a dog pancreas essentially as described by Dunning et al. (Am. J. Phvsiol. 251 fEndocrinol. Metab. 14): E127-E133, 1986, which is incorporated herein by reference in its entirety) .
  • a synthetic galanin antagonist is infused for 30 minutes via the femoral vein at 2.5 or 25 pmol/kg/min in a pentobarbital-anesthetized, laparotomized dog (24-26 kg) .
  • human or rat galanin is infused intravenously at doses needed to produce a 20%, 50% or 80% inhibition of insulin secretion (corresponding to rates of 0.25 pmol/kg/min, 2.5 pmol/kg/min, or 25 pmol/kg/min, respectively) .
  • Blood is collected simultaneously from the femoral artery and the superior pancreaticoduodenal vein (SPVD) after surgical exclusion of venous drainage from the duodenum. Pancreatic venous blood flow is measured with an electromagnetic flow probe placed in an extracorporeal shunt from the SPVD to the portal vein.
  • the synthetic galanin may be infused directly into the pancreatic artery via a Clear- Cath ® 22-G x 1 catherter (Abbott Hospital, Inc., North Chicago, IL) .
  • the results are compared with insulin level before the galanin infusion (basal level) and are expressed as percent of change from the basal level.
  • the ability to prevent or reduce the 20%, 50%, and 80% galanin-induced inhibition of insulin secretion marks galanin antagonists that are active in the in vivo system.
  • the synthetic galanin antagonists are tested for their ability to reduce galanin-induced inhibition of insulin secretion in chronically catheterized rats essentially as described by Dunning and Taborsky (Diabetologia 33: 125-126, 1990, which is incorporated herein by reference in its entirety) .
  • conscious, non-fasted rats bearing indwelling jugular cannulae are infused with a synthetic galanin antagonist at three doses, each one log unit apart, either in the presence or absence of galanin doses that would normally impair insulin secretion by 20%, 50% or 80. Insulin secretion is measured as described above.
  • Galanin antagonists capable of preventing the galanin-induced inhibition or reduction of insulin secretion at the 20%, 50% and 80% levels are considered active antagonists in the in vivo assay.
  • a galanin antagonist is able to prevent circulating hepatic galanin from inhibiting insulin release when given before hepatic nerve stimulation
  • the galanin antagonist is administered to overnight-fasted adult mongrel dogs (25-30 kg) that are anesthetized using 30 mg/kg intravenous thiamylal sodium induction (Surital, Park-Davis, Morris Plains, NJ) followed by 1% halothane (Ayerst Laboratories, Inc., New York, NY) in 100% oxygen via mechanical ventilator.
  • the femoral artery is cannulated for blood sampling and blood pressure measurement.
  • the hepatic artery is catheterized with a Swan-Ganz catheter inserted into the femoral vein, passed up into the inferior vena cava and threaded into a main branch of a hepatic vein under radiographic guidance. A midline laparotomy is performed and a sampling catheter is placed in the portal vein within five centimeters of the porta hepatis. Ultrasonic probes (Transionic Inc., Ithica, NY) are placed around the hepatic artery and portal vein for measurement of blood flows into and out of the gut and liver.
  • Hepatic nerve stimulation is achieved by separating the neural sheath surrounding the hepatic artery from the vessel and placing the sheath in a bipolar electrode.
  • a ganglionic blockade with hexamethonium bromide (Sigma Chemical, St. Louis, MO) and atropine is used.
  • Intravenous boluses of 0.1 mg/kg of hexamethonium bromide are administered until the mean arterial pressure drops 10 mm Hg and remains at that level. Hexamethonium is then infused at 0.7 ⁇ g/kg/min x number of boluses required for the 10 mm Hg reduction of the mean arterial pressure.
  • a 0.25 mg/kg atropine (Elkin- Sinn, Inc. , Cherry Hill NJ) bolus is administered followed by an infusion of 0.4 ⁇ g/kg/min.
  • the galanin antagonist is administered at 2.5 or 25 pmol/kg/min for fifteen minutes before and during the hepatic nerve stimulation.
  • the hepatic nerve is stimulated for 10 minutes with square wave pulses of 8 Hz frequency, 10 mA current and 1 msec duration using a model S-44 Stimulator (Grass Instruments, Quincy, MA) .
  • Blood samples are drawn simultaneously from the femoral artery, the portal vein and the hepatic vein before and during the antagonist infusion at five-15 minute intervals and aliqoted into tubes containing EDTA for anti-coagulation, and kept on ice until centrifuged. The plasma is then pipetted off and frozen for later analysis. Mean arterial pressure, hepatic arterial blood flow, portal vein blood flows and hematocrit are monitored throughout the experiments. Insulin levels are determined using the method described above.
  • antagonists are administered to adult mongrel dogs using the method essentially described by Dunning et al. (Am. J. Physiol. 258 (Endocrinol. Metab. 2_1) : E436-E444, 1990, which is incorporated herein by reference in its entirety) . Briefly, overnight-fasted, adult mongrel dogs (19-37 kg) are anesthetized with 30 mg/kg intravenous thiamylal sodium (SURITAL, Parke Davis, Morris Plains, NJ) .
  • SURITAL intravenous thiamylal sodium
  • halothane (Ayerst Laboratories, Inc., New York, NY) administered from a calibrated vaporizer (Draeger, FRG) by mechanical ventilation in 100% oxygen.
  • a laparotomy is performed on the anesthetized animal and an extracorporeal shunt containing a sampling port and an electromagnetic flow probe (Zepeda Instruments, Seattle, WA) is introduced between the superior pancreaticoduodenal vein and the portal vein.
  • the femoral artery and vein are cannulated for arterial blood sampling and intravenous infusions of saline or the synthetic galanin antagonist.
  • Splanchnic nerves are dissected after bilateral thoracotomies are performed at the seventh intercostal space.
  • the sympathetic trunks are dissected from the surrounding tissue along the dorsal rib cage, and bipolar electrodes (Harvard Apparatus, South Natick, MA) are placed on each nerve at the level of approximately T ⁇ g.
  • the nerve trunks are then severed anterior to the electrodes. A sixty minute stabilization period follows the surgical procedures before experimentation.
  • the synthetic galanin antagonist is administered at 2.5 or 25 pmol/kg/min prior to the splanchnic nerve stimulation.
  • the thoracic splanchnic nerves are stimulated by electrically stimulating the sympathetic trunks for ten minutes with square wave pulses of 1-ms duration and 10 mA current at a frequency of 8 Hz.
  • the stimulation is performed with a model S-44 stimulator coupled to a PSIU6 stimulus isolation unit (Grass Instruments, Quincy, MA) .
  • Stimulation parameters are monitored with an oscilloscope. Mean arterial blood pressure and superior pancreaticoduodenal vein blood flow is monitored continuously and he atocrit is determined at regular intervals.
  • the inhibition of insulin secretion in the presence of antagonist is expressed as percentage change from basal baseline and is compared to that in the absence of antagonist. Significantly less inhibition of insulin release during the antagonist period is interpreted as successful reversal of the insulin inhibitory effect of neurally-released galanin.
  • Galanin antagonists capable of preventing the decrease of basal insulin normally seen during mixed pancreatic nerve stimulation are identified using the method essentially described by Dunning et al. (Am. J. Phvsiol. 256 (Endocrinol. Metab. 19): E191-E198, 1989; which is incorporated herein by reference in its entirety) . Briefly, overnight-fasted adult male mongrel dogs are anesthetized as described above first with thiamylol and then with halothane. The femoral artery and vein are cannulated for blood sampling, blood pressure recording and continuous saline infusion. A midline laparotomy is performed to expose the duodenum and the associated lobe of the pancreas.
  • An extracorporeal shunt containing a sampling port and an electromagnetic flow probe is then introduced between the superior pancreaticoduodenal vein and the portal vein.
  • the autonomic pancreatic nerves which course in the sheath of connective tissue surrounding the superior pancreaticoduodenal artery are isolated at their entrance into the pancreatic parenchyma and placed in a bipolar electrode (Harvard Apparatus, South Natick, MA) . After a one hour stabilization period, baseline superior pancreaticoduodenal vein and femoral artery blood samples are obtained.
  • the pancreatic nerve is stimulated by electrically stimulating the sympathetic trunks for ten minutes with square wave pulses of 1-ms duration and 10 mA current at a frequency of 8 Hz.
  • the stimulation is performed with a model S-44 stimulator coupled to a PSIU6 stimulus isolation unit (Grass Instruments, Quincy, MA) . Stimulation parameters are monitored with an oscilloscope. Blood sample are collected at five-15 minute intervals and aliqoted into tubes containing EDTA for anti-coagulation, and kept on ice until centrifuged. The plasma is then pipetted off and frozen for later analysis. The inhibition of insulin secretion in the presence of antagonist is expressed as percentage change from basal baseline and is compared to that in the absence of antagonist. Significantly less inhibition of insulin release during the antagonist period is interpreted as successful reversal of the insulin inhibitory effect of neurally-released galanin.
  • GGC CTC ACC AGC AAG CGG GAG CTG CGG CCC GAA GAT GAC ATG AAA CCA 144 Gly Leu Thr Ser Lys Arg Glu Leu Arg Pro Glu Asp Asp Met Lys Pro 35 40 45

Abstract

Molécules ADN codant la galanine humaine. Les molécules sont utilisées dans le cadre de méthodes de détection des antagonistes de la galanine, avec des techniques basées sur les ADN recombinés. En termes succincts, des séquences ADN codant des analogues de la galanine sont générées et exprimées dans des cellules hôtes appropriées. Les analogues sont exposés à un récepteur de la galanine couplé à une voie d'accès de réponse en présence de galanine native. Une réduction de l'inhibition de la voie d'accès de réponse résultant de la liaison de l'analogue galanine au récepteur galanine, par rapport à l'inhibition de la voie d'accès de réponse par la galanine native seule, révèle la présence d'un antagoniste de la galanine. L'invention s'étend également aux antagonistes de la galanine identifiés et isolés par ces méthodes.
PCT/US1992/001469 1991-02-25 1992-02-25 Methodes de detection des antagonistes de la galanine WO1992015015A1 (fr)

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FR2716205A1 (fr) * 1994-02-17 1995-08-18 Rhone Poulenc Rorer Sa Récepteur galanine, acides nucléiques, cellules transformées et utilisations.
EP0819167A2 (fr) * 1996-01-24 1998-01-21 Synaptic Pharmaceutical Corporation Adn codant les recepteurs galr2 de la galanine et ses utilisations
WO1998003059A1 (fr) * 1996-07-24 1998-01-29 University Of Bristol Galanine
WO1998003548A1 (fr) * 1996-07-19 1998-01-29 Astra Pharma Inc. Nouveau recepteur de galanine
US5756460A (en) * 1991-03-06 1998-05-26 Garvan Institute Of Medical Research Human galanin, CDNA clones encoding human galanin and a method of producing human galanin
US5972624A (en) * 1996-01-24 1999-10-26 Synaptic Pharmaceutical Corporation Method of identifying ligands which bind recombinant galanin receptor (GALR2)
US6287788B1 (en) 1996-10-09 2001-09-11 Synaptic Pharmaceutical Corporation DNA encoding galanin GALR3 receptors and uses thereof
WO2003093295A2 (fr) * 2002-04-30 2003-11-13 University Of North Carolina At Chapel Hill Vecteurs de signal de sécrétion
EP1421214A2 (fr) * 2001-08-27 2004-05-26 Tularik Inc. Oncogenes amplifies et leur implication dans le cancer
WO2007057691A2 (fr) 2005-11-18 2007-05-24 Hunter-Fleming Limited Utilisations therapeutiques de composes steroides
US7582673B2 (en) 2004-10-21 2009-09-01 High Point Pharmaceuticals, Llc Bissulfonamide compounds as agonists of GalR1, compositions, and methods of use
US7628989B2 (en) 2001-04-10 2009-12-08 Agensys, Inc. Methods of inducing an immune response
US7927597B2 (en) 2001-04-10 2011-04-19 Agensys, Inc. Methods to inhibit cell growth
CN113512592A (zh) * 2021-04-23 2021-10-19 中国科学院深圳先进技术研究院 用于检测甘丙肽分泌细胞的引物、探针以及试剂盒

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US5756460A (en) * 1991-03-06 1998-05-26 Garvan Institute Of Medical Research Human galanin, CDNA clones encoding human galanin and a method of producing human galanin
EP0587571A4 (fr) * 1991-03-06 1995-03-15 Garvan Inst Med Res GALANINE HUMAINE, DES CLONES ADNc CODANT POUR LA GALANINE HUMAINE ET PROCEDE DE PRODUCTION DE GALANINE HUMAINE.
EP0587571A1 (fr) * 1991-03-06 1994-03-23 Garvan Institute Of Medical Research GALANINE HUMAINE, DES CLONES ADNc CODANT POUR LA GALANINE HUMAINE ET PROCEDE DE PRODUCTION DE GALANINE HUMAINE
FR2716205A1 (fr) * 1994-02-17 1995-08-18 Rhone Poulenc Rorer Sa Récepteur galanine, acides nucléiques, cellules transformées et utilisations.
WO1995022608A1 (fr) * 1994-02-17 1995-08-24 Rhone-Poulenc Rorer S.A. Recepteur galanine, acides nucleiques, cellules transformees et utilisations
US6447996B1 (en) 1994-02-17 2002-09-10 Aventis Pharma S.A. Galanin receptors, nucleic acids, transformed cells and uses thereof
US6586191B2 (en) 1996-01-24 2003-07-01 Synaptic Pharmaceutical Corporation Method of identifying compounds that bind galanin receptor (GALR2)
US7132248B2 (en) 1996-01-24 2006-11-07 H. Lundbeck A/S Uses of galanin GALR2 receptors
US6790656B1 (en) 1996-01-24 2004-09-14 Synaptic Pharmaceutical Corporation DNA encoding galanin GALR2 receptors
US5972624A (en) * 1996-01-24 1999-10-26 Synaptic Pharmaceutical Corporation Method of identifying ligands which bind recombinant galanin receptor (GALR2)
US7060449B2 (en) 1996-01-24 2006-06-13 H. Lundbeck A/S Method of preparing GALR2 receptors composition
EP0819167A2 (fr) * 1996-01-24 1998-01-21 Synaptic Pharmaceutical Corporation Adn codant les recepteurs galr2 de la galanine et ses utilisations
EP0819167A4 (fr) * 1996-01-24 2002-06-12 Synaptic Pharma Corp Adn codant les recepteurs galr2 de la galanine et ses utilisations
US7510846B2 (en) 1996-07-19 2009-03-31 National Research Council Of Canada Assay employing human or rat GAL-R2 galanin receptor
US7375207B2 (en) 1996-07-19 2008-05-20 Astrazeneca A.B. Galanin receptor 2 proteins and nucleic acids
US7407761B2 (en) 1996-07-19 2008-08-05 National Research Council Of Canada Methods for assaying expression of novel galanin receptors
US6562945B1 (en) 1996-07-19 2003-05-13 Astrazeneca Canada Inc. Galanin receptor
WO1998003548A1 (fr) * 1996-07-19 1998-01-29 Astra Pharma Inc. Nouveau recepteur de galanine
US7528229B2 (en) 1996-07-19 2009-05-05 National Research Council Of Canada Isolated human and rat GAL-R2 galanin receptors
GB2331301B (en) * 1996-07-24 2001-02-14 Univ Bristol Galanin
EP1342410A3 (fr) * 1996-07-24 2003-12-10 Neurotargets Limited Animaux transgéniques ayant une déficience dans le gène de la galanine
WO1998003059A1 (fr) * 1996-07-24 1998-01-29 University Of Bristol Galanine
GB2331301A (en) * 1996-07-24 1999-05-19 Univ Bristol Galanin
US6368812B1 (en) 1996-10-09 2002-04-09 Synaptic Pharmaceutical Corporation Process for determining the agonist or antagonist of galanin receptor (GALR3)
US7022489B2 (en) 1996-10-09 2006-04-04 H. Lundbeck A/S Method of using cells expressing galanin receptor 3 (GALR3)
US6287788B1 (en) 1996-10-09 2001-09-11 Synaptic Pharmaceutical Corporation DNA encoding galanin GALR3 receptors and uses thereof
US6329197B2 (en) 1996-10-09 2001-12-11 Synaptic Pharmaceutical Corporation DNA encoding galanin GALR3 receptors and uses thereof
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US7951375B2 (en) 2001-04-10 2011-05-31 Agensys, Inc. Methods of inducing an immune response
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers
US7628989B2 (en) 2001-04-10 2009-12-08 Agensys, Inc. Methods of inducing an immune response
EP1421214A4 (fr) * 2001-08-27 2005-11-02 Tularik Inc Oncogenes amplifies et leur implication dans le cancer
EP1421214A2 (fr) * 2001-08-27 2004-05-26 Tularik Inc. Oncogenes amplifies et leur implication dans le cancer
WO2003093295A3 (fr) * 2002-04-30 2005-07-28 Univ North Carolina Vecteurs de signal de sécrétion
WO2003093295A2 (fr) * 2002-04-30 2003-11-13 University Of North Carolina At Chapel Hill Vecteurs de signal de sécrétion
US7071172B2 (en) * 2002-04-30 2006-07-04 The University Of North Carolina At Chapel Hill Secretion signal vectors
US7582673B2 (en) 2004-10-21 2009-09-01 High Point Pharmaceuticals, Llc Bissulfonamide compounds as agonists of GalR1, compositions, and methods of use
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