EP1054966A1 - Ammaid, un recepteur couple a une proteine g - Google Patents

Ammaid, un recepteur couple a une proteine g

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Publication number
EP1054966A1
EP1054966A1 EP99908250A EP99908250A EP1054966A1 EP 1054966 A1 EP1054966 A1 EP 1054966A1 EP 99908250 A EP99908250 A EP 99908250A EP 99908250 A EP99908250 A EP 99908250A EP 1054966 A1 EP1054966 A1 EP 1054966A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
identity
seq
polypφtide
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99908250A
Other languages
German (de)
English (en)
Other versions
EP1054966A4 (fr
Inventor
Ping Tsui
Nabil A. Elshourbagy
Lisa Vawter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
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Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Publication of EP1054966A1 publication Critical patent/EP1054966A1/fr
Publication of EP1054966A4 publication Critical patent/EP1054966A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • AMMAID A G protein Coupled Receptor
  • This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agonists, antagonists and /or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
  • proteins participating in signal transduction pathways that involve G-proteins and or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354).
  • these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins.
  • Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl Acad.
  • G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
  • effector proteins e.g., phospholipase C, adenyl cyclase, and phosphodiesterase
  • actuator proteins e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
  • the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell.
  • Enzyme activation by hormones is dependent on the presence of the nucleotide GTP.
  • GTP also influences hormone binding.
  • a G-protein connects the hormone receptor to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by a hormone - 2 -
  • G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
  • G-protein coupled receptors (otherwise known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops.
  • the G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders.
  • members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, l ⁇ nin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors.
  • G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure.
  • the 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7.
  • TM3 has been implicated in signal transduction.
  • Phosphorylation and lipidation (palmitylation or femesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors.
  • Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus.
  • G-protein coupled receptors such as the ⁇ -adrenoreceptor
  • phosphorylation by protein kinase A and or specific receptor kinases mediates receptor desensitization.
  • the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protein coupled receptors.
  • the hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form polar ligand binding site.
  • TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue.
  • TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding. - 3 -
  • G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331)
  • Different G-protein ⁇ -subunits preferentially stimulate particular effectors to modulate various biological functions in a cell.
  • Phosphorylation of cytoplasmic residues of G-protein coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors.
  • G-protein coupled receptors are found in numerous sites within a mammalian host. Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market.
  • the present invention relates to AmMaid, in particular AmMaid polypeptides and AmMaid polynucleotides, recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HIV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, hereinafter referred to as "the Diseases",
  • the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with AmMaid imbalance with the identified compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate AmMaid activity or levels.
  • the present invention relates to AmMaid polypeptides.
  • Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • Such polypeptides include those comprising the amino acid of SEQ ID NO:2.
  • peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2.
  • polypeptides include the polypeptide of SEQ ID NO:2.
  • peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO:l.
  • Polypeptides of the present invention are believed to be members of the G protein coupled receptor family of polypeptides. They are therefore of interest because G protein-coupled receptors are the basis of much of cell-to-cell communication in our bodies. As such, they have been the basis of action of more pharmaceutical drugs than any other gene family. These properties are hereinafter referred to as "AmMaid activity” or “AmMaid polypeptide activity” or "biological activity of AmMaid”.
  • a polypeptide of the present invention exhibits at least one biological activity of AmMaid.
  • polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • the present invention also includes include variants of the aforementioned polyp ⁇ tides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
  • Polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally c x.urring polyp ⁇ tides, recombinantiy produced polyp ⁇ tides, synthetically produced polyp ⁇ tides, or polyp ⁇ tides produced by a combination of these methods. Means for pr ⁇ aring such polypeptides are well understood in the art.
  • the present invention relates to AmMaid polynucleotides.
  • Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polyp ⁇ tide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
  • polyp ⁇ tides which have at least 97% identity are highly preferred, whilst - 5 -
  • polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding the polyp ⁇ tide of SEQ ID NO:2.
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region.
  • polynucleotides which have at least 97%> identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.
  • polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
  • polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identify are more highly preferred, and those with at least 99% identity are most highly preferred.
  • Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 as well as the polynucleotide of SEQ ID NO: 1.
  • the invention also provides polynucleotides which are complementary to all the above described polynucleotides.
  • the nucleotide sequence of SEQ ID NO:l shows homology with GPR35 (O'Dowd B.F. et al., Genomics 47, 310-313 (1998)).
  • the nucleotide sequence of SEQ ID NO:l is a cDNA sequence and comprises a polyp ⁇ tide encoding sequence (nucleotidel to 957) encoding a polyp ⁇ tide of 319 amino acids, the polyp ⁇ tide of SEQ ID NO:2.
  • the nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO: 1 or it may be a sequence other than the one contained in SEQ ID NO: 1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polyp ⁇ tide of the SEQ ID NO:2 is structurally related to other proteins of the G protein coupled rec ⁇ tor family, having homology and/or structural similarity with GPR35 (O'Dowd B.F. et al., Genomics 47, 310-313 (1998)).
  • Preferred polyp ⁇ tides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polyp ⁇ tides and polynucleotides. Furthermore, preferred polyp ⁇ tides and polynucleotides of the present invention have at least one AmMaid activity.
  • Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of human fetal lung, using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252:1651-1656; Adams, M.D. et al, Nature, (1992) 355:632-634; Adams, M.D., et al, Nature (1995) 377 Supp:3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
  • EST expressed sequence tag
  • the polynucleotide may include the coding sequence for the mature polyp ⁇ tide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or pr ⁇ ro- protein sequence, or other fusion p ⁇ tide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine p ⁇ tide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al. , Proc NatlAcad Sci USA (1989) 86:821-824, or is an HA tag.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • polynucleotides encoding polyp ⁇ tide variants which comprise the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, __rnino acid residues are substituted, deleted or added, in any combination.
  • Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polyp ⁇ tides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to SEQ ID NOT .
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95 % identical to that of the referent.
  • the probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.
  • a polynucleotide encoding a polyp ⁇ tide of the present invention may be obtained by a process which comprises the st ⁇ s of screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ED NO: 1 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • stringent hybridization conditions include overnight incubation at 42°C in a solution - 7 -
  • the present invention also includes polynucleotides obtainable by screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof.
  • an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA. This is a consequence of reverse transcriptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis.
  • cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end.
  • Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers.
  • the PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal vwthin the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence).
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
  • Recombinant polyp ⁇ tides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polyp ⁇ tides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. - 8 -
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • R ⁇ resentative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and
  • Aspergillus cells insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
  • chromosomal, ⁇ isomal and virus- derived systems e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast ⁇ isomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polyp ⁇ tide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).
  • Appropriate secretion signals may be incorporated into the desired polyp ⁇ tide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polyp ⁇ tide or they may be heterologous signals.
  • a polyp ⁇ tide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell.
  • the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polyp ⁇ tide is denatured during isolation and or purification.
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene characterized by the polynucleotide of SEQ ID NO:l which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susc ⁇ tibility to a disease, which results from under-expression, over-expression or altered expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled AmMaid nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
  • DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (e.g., Myers et al. , Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al. , Proc Natl Acad Sci USA (1985) 85: 4397-4401).
  • an array of oUgonucleotides probes comprising AmMaid nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
  • the diagnostic assays offer a process for diagnosing or determining a susc ⁇ tibility to the Diseases through detection of mutation in the AmMaid gene by the methods described.
  • diseases may be diagnosed by methods comprising deterrnining from a sample derived from a subject an - 10 -
  • abnormally decreased or increased level of polypeptide or mRNA can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein, such as a polyp ⁇ tide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the present invention relates to a diagonostic kit which comprises:
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ;
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or
  • kits may comprise a substantial component.
  • a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HTV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic d ⁇ ression, d ⁇ ression, delirium, dementia, and severe mental retardation; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, amongst others.
  • the nucleotide sequences of the present invention are also valuable for chromosome
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical - 11 -
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be deteirnined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • polyp ⁇ tides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for polyp ⁇ tides of the present invention.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polyp ⁇ tides in the prior art.
  • Antibodies generated against polyp ⁇ tides of the present invention may be obtained by administering the polyp ⁇ tides or ⁇ itope-bearing fragments, analogs or cells to an animal, preferably a non-human .tnirnal, using routine protocols. For pr ⁇ aration of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used.
  • Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B- cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polyp ⁇ tide or to purify the polyp ⁇ tides by affinity chromatography.
  • Antibodies against polyp ⁇ tides of the present invention may also be employed to treat the Diseases, amongst others.
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood - 12 -
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy.
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914.
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned, amongst others.
  • Yet .mother aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • a further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention.
  • the vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably actministered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • Polyp ⁇ tides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polyp ⁇ tide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to - 13 -
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned.
  • Compounds may be identified from a variety of sources, for example, cells, cell-free pr ⁇ arations, chemical libraries, and natural product mixtures.
  • Such agonists, antagonists or inhibitors so- identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of the polyp ⁇ tide; or may be structural or functional mimetics thereof (see Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve competition with a labeled competitor.
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • Constitutively active polypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring AmMaid activity in the mixture, and comparing the AmMaid activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and AmMaid polyp ⁇ tide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D.
  • One screening technique includes the use of cells which express rec ⁇ tors of this invention (for example, transfected CHO cells) in a system which measures extracellular pH or intracellular calcium changes caused by rec ⁇ tor activation.
  • rec ⁇ tors of this invention for example, transfected CHO cells
  • compounds may be contacted with cells expressing a rec ⁇ tor polyp ⁇ tide of the present invention.
  • a second messenger response e.g., signal transduction, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the rec ⁇ tor.
  • Another method involves screening for rec ⁇ tor inhibitors by determining inhibition or stimulation of rec ⁇ tor-mediated cAMP and/or adenylate cyclase accumulation.
  • Such a method involves transfecting a eukaryotic cell with the rec ⁇ tor of this invention to express the rec ⁇ tor on the cell surface. The cell is then - 14 -
  • Another method for detecting agonists or antagonists for the rec ⁇ tor of the present invention is the yeast based technology as described in U.S. Patent No. 5,482,83 .
  • polynucleotides, polyp ⁇ tides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells.
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polyp ⁇ tide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the polyp ⁇ tide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.
  • polyp ⁇ tide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the hgands, substrates, rec ⁇ tors, enzymes, etc., as the case may be, of the polyp ⁇ tide, e.g., a fragment of the hgands, substrates, rec ⁇ tors, enzymes, etc.; or small molecules which bind to the polyp ⁇ tide of the present invention but do not elicit a response, so that the activity of the polyp ⁇ tide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises:
  • polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by: (a) dete ⁇ nining in the first instance the three-dimensional structure of the polypeptide;
  • the present invention provides methods of treating abnormal conditions such as, for instance, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HTV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Paridnson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic d ⁇ ression, d ⁇ ression, delirium, dementia, and severe mental retardation; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome, related to either an excess o ⁇ or an under-expression of, AmMaid polyp ⁇ tide activity.
  • infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HTV-2
  • pain cancers
  • One approach comprises admii ⁇ stering to a subject in need thereof an inhibitor compound (antagonist) as hereinabove described, optionally in combination with a pharmaceutically acc ⁇ table carrier, in an amount effective to inhibit the function of the polyp ⁇ tide, such as, for example, by blocking the binding of hgands, substrates, rec ⁇ tors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnornial condition.
  • an inhibitor compound as hereinabove described
  • a pharmaceutically acc ⁇ table carrier in an amount effective to inhibit the function of the polyp ⁇ tide, such as, for example, by blocking the binding of hgands, substrates, rec ⁇ tors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnornial condition.
  • expression of the gene encoding endogenous AmMaid polypeptide can be inhibited usmg expression blocking techniques
  • Known such techniques mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • ohgonucleotides which form t ⁇ ple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360)
  • These oligomers can be administered per se or the relevant oligomers can be expressed in vivo
  • a polynucleotide of the mvention may be engineered for expression m a replication defective retro viral vector, as discussed above
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containmg RNA encoding a polyp ⁇ tide of the present invention such that the packagmg cell now produces infectious viral particles containmg the gene of interest
  • producer cells may be administered to a subject for engineering cells n vivo and expression of the polyp ⁇ t
  • the present mvention provides for pharmaceutical compositions compnsing a therapeutically effective amount of a polyp ⁇ tide, such as the soluble form of a polyp ⁇ tide of the present mvention, agonist/antagonist p ⁇ tide or small molecule compound, in combination with a pharmaceutically acc ⁇ table earner or excipient
  • a polyp ⁇ tide such as the soluble form of a polyp ⁇ tide of the present mvention, agonist/antagonist p ⁇ tide or small molecule compound
  • Such earners mclude, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof
  • the mvention further relates to pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients - 17 -
  • compositions of the invention are of the aforementioned compositions of the invention.
  • Polyp ⁇ tides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • composition will be adapted to the route of ⁇ idministratiori, for instance by a systemic or an oral route.
  • Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible. Acbrninistration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.
  • Suitable dosages are in the range of 0.1 - 100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
  • Polyp ⁇ tides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polyp ⁇ tide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • a polynucleotide such as a DNA or RNA
  • Polynucleotide and polyp ⁇ tide sequences form a valuable information resource with which to identify further sequences of similar homology. This is most easily facilitated by storing the sequence in a computer readable medium and then using the stored data to search a sequence database using well known searching tools, such as GCC. Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1 and/or a polypeptide sequence encoded thereby.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
  • Isolated means altered “by the hand of man” from the natural state. If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene- encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many - 19 -
  • Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, PROTEINS - STR
  • Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. - 20 -
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol.
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NUT Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990).
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following:
  • a program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI.
  • the aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
  • Preferred parameters for polynucleotide comparison include the following:
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NOT, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence.
  • Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and trans version, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the numerical percent of the respective percent identity(divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NOT, or:
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NOT
  • y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%.
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference - 22 -
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity(divided by 100) and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: n a ⁇ x a - (x a • y),
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO:2
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non- integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • Fusion protein refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262].
  • Examples An EST N78034 showed homology to the 7TM superfamily sequences. Oligonucleotides from these regions were designed to obtain the 5' 'coding region using the Marathon PCR technology. Fragments of approximately 1000 bp were obtained. The fragment was subcloned into PCR 2.1 vector (Invitrogen) and then sequenced. Sequence analysis showed an open reading frame start with (ATG) and with (TGA). The coding region is 319 amino acids in length.
  • Example 1 Mammalian Cell Expression
  • the rec ⁇ tors of the present invention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells.
  • HEK293 human embryonic kidney 293
  • adherent dhfr CHO cells typically all 5' and 3' untranslated regions (UTRs) are removed from the rec ⁇ tor cDNA prior to insertion into a pCDN or pCDNA3 vector.
  • the cells are transfected with individual rec ⁇ tor cDNAs by lipofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis.
  • HEK293 or CHO cells transfected with the vector alone serve as negative controls.
  • Example 2 Ligand bank for binding and functional assays.
  • a bank of over 200 putative rec ⁇ tor hgands has been assembled for screening.
  • the bank comprises: transmitters, hormones and chemokines known to act via a human seven transmembrane (7TM) rec ⁇ tor; naturally occurring compounds which may be putative agonists for a human 7TM rec ⁇ tor, non- marnmaHan, biologically active p ⁇ tides for which a mammahan counterpart has not yet been identified; and compounds not found in nature, but which activate 7TM rec ⁇ tors with unknown natural hgands.
  • This bank is used to initially screen the rec ⁇ tor for known hgands, using both functional (i.e . calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays.
  • Ligand binding assays provide a direct method for ascertaining rec ⁇ tor pharmacology and are adaptable to a high throughput format.
  • the purified hgand for a rec ⁇ tor is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the hgand towards its rec ⁇ tor.
  • Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell rec ⁇ tor sources.
  • specific rec ⁇ tor binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing hgand. Where possible, more than one competing hgand is used to define residual nonspecific binding.
  • RNA transcripts from linearized plasmid templates encoding the rec ⁇ tor cDNAs of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures.
  • In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml.
  • Ovarian lobes are removed from adult female toads, Stage V defolhculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a microinjection apparatus.
  • Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response to agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature.
  • the Xenopus system can be used to screen known hgands and tissue/cell extracts for activating hgands .
  • Example 5 Microphysiometric Assays - 24 -
  • Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell.
  • the acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process.
  • the pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, CA).
  • the CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing intracellular signaling pathway such as the G-protein coupled rec ⁇ tor of the present invention.
  • the 7TM rec ⁇ tor of the invention is also functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural hgands. Extracts that produce positive functional responses can be sequencially subfractionated until an activating hgand is isolated identified.
  • Example 8 Calcium and cAMP Functional Assays 7TM rec ⁇ tors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimuation or inhibition. Basal calcium levels in the HEK 293 cells in rec ⁇ tor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant rec ⁇ tors are loaded with fura 2 and in a single day > 150 selected hgands or tissue/cell extracts are evaluated for agonist induced calcium mobilization.
  • HEK 293 cells expressing recombinant rec ⁇ tors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP flucuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing rec ⁇ tor.
  • SEQ ID NO:2 S14-Ctg.pep Length: 319..

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Abstract

La présente invention concerne des polypeptides et des polynucléotides AmMaid ainsi que leurs procédés de fabrication utilisant des techniques de recombinaison. Cette invention concerne par ailleurs des procédés d'utilisation de ces polypeptides et polynucléotides AmMaid dans des méthodes thérapeutiques et diagnostiques.
EP19990908250 1998-02-19 1999-02-19 Ammaid, un recepteur couple a une proteine g Withdrawn EP1054966A4 (fr)

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AUPP438598A0 (en) 1998-06-29 1998-07-23 Garvan Institute Of Medical Research NPY-Y7 receptor gene
WO2000023588A2 (fr) * 1998-10-16 2000-04-27 Millennium Pharmaceuticals, Inc. Recepteurs couples a la proteine g
EP1208196B1 (fr) 1999-08-03 2006-10-18 Millennium Pharmaceuticals, Inc. Nouvelle molecule 15571 de type gpcr de la famille secretine et utilisations de cette derniere

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WO2000023588A2 (fr) * 1998-10-16 2000-04-27 Millennium Pharmaceuticals, Inc. Recepteurs couples a la proteine g
WO2001075067A2 (fr) * 2000-03-31 2001-10-11 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides

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WO2000023588A2 (fr) * 1998-10-16 2000-04-27 Millennium Pharmaceuticals, Inc. Recepteurs couples a la proteine g
WO2001075067A2 (fr) * 2000-03-31 2001-10-11 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE [Online] EBI; 26 August 1996 (1996-08-26) "zi05b04.s1 Soares fetal liver spleen 1NFLS S1 Homo sapiens cDNA clone" retrieved from EMBL Database accession no. AA034005 XP002215810 *
See also references of WO9942582A1 *

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