WO2000055202A1 - Membre de la famille frzb, de frazzled - Google Patents

Membre de la famille frzb, de frazzled Download PDF

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Publication number
WO2000055202A1
WO2000055202A1 PCT/US2000/006820 US0006820W WO0055202A1 WO 2000055202 A1 WO2000055202 A1 WO 2000055202A1 US 0006820 W US0006820 W US 0006820W WO 0055202 A1 WO0055202 A1 WO 0055202A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
sequence
polynucleotide
frazzled
Prior art date
Application number
PCT/US2000/006820
Other languages
English (en)
Inventor
Ian Edward James
Michael William Lark
Tania Tamson Testa
Original Assignee
Smithkline Beecham Corporation
Smithkline Beecham Plc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation, Smithkline Beecham Plc filed Critical Smithkline Beecham Corporation
Priority to EP00917976A priority Critical patent/EP1165608A4/fr
Publication of WO2000055202A1 publication Critical patent/WO2000055202A1/fr

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one Frazzled activity
  • R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof.
  • R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right, bound to R3.
  • any stretch of nucleic acid residues denoted by either R and/or R 2 , where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double- stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary
  • m and/or n is an integer between 1 and 1000.
  • Other prefe ⁇ ed embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500
  • Polynucleotides that are identical, or are substantially identical to a nucleotide sequence of SEQ ID NO.1 may be used as hybridization probes for cDNA and genomic DNA or as pnmers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than Bos taurus) that have a high sequence identity to SEQ ID NO.1 Typically these nucleotide sequences are 95% identical to that of the referent.
  • PCR nucleic acid amplification
  • This invention further relates to a method of producing transgemc animals, preferably Bos taurus, under-expressing or regulatably expressing Frazzled, which method may compnse the introduction of a weak promoter or a regulatable promoter (e.g. , an mducible or repressible promoter) respectively, expressibly linked to the polynucleotide sequence of SEQ ID NO:l into the cells of a Bos taurus embryo at an early stage.
  • a weak promoter or a regulatable promoter e.g. , an mducible or repressible promoter
  • This invention also relates to transgemc animals, characte ⁇ zed m that they are obtained by a method, as defined above
  • Any technique known in the art may be used to introduce a Bos taurus Frazzled transgene into animals to produce a founder line of animals.
  • Such techniques include, but are not limited to: pronuclear micromjection (U.S. Patent No. 4,873,191); retrovirus mediated gene transfer into germ lines (Nan der Putten, et al , Proc Natl. Acad. Sci , USA 82: 6148-6152 (1985); gene targeting in embryonic stem cells (Thompson, et al., Cell 56: 313-321 (1989); electropolation of embryos (Lo, Mol. CellBiol 3: 1803-1814 (1983); and sperm-mediated gene transfer (Lavitrano, et al, Cell 57: 717-723 (1989); etc.
  • This invention further relates to a method of producing "knock-out" animals, preferably mice, no longer expressing Frazzled.
  • a Bos taurus Frazzled cD ⁇ A SEQ ID NO: 1
  • hbra ⁇ es hbra ⁇ es
  • the method used to create a knockout mouse is charactenzed in that: a suitable mutation is produced in the polynucleotide sequence of the munne Frazzled genomic clone, which inhibits the expression of a gene encoding munne Frazzled, or inhibits the activity of the gene product; said modified munne Frazzled polynucleotide is introduced into a homologous segment of munne genomic DNA, combined with an appropnate marker, so as to obtain a labeled sequence compnsmg said modified munne genomic DNA, said modified munne genomic DNA comp ⁇ smg the modified polynucleotide is transfected into embryonic stem cells and co ⁇ ectly targeted events selected in vitro; then said stem cells are remjected into a mouse embryo; then said embryo is implanted into a female recipient and brought to term as a chimera which transmits said mutation through the germlme; and homozygous re
  • the mutation may constitute an insertion, deletion, substitution, or combination thereof.
  • the DNA construct can be introduced into cells by, for example, calcium-phosphate DNA co-precipitation It is prefe ⁇ ed that a mutation be introduced into cells using electroporation, micromjection, virus infection, hgand-DNA conjugation, virus-hgand-DNA conjugation, or lrposomes
  • osteoporosis e.g , osteoporosis
  • cancer e g., lymphoprohferative disorders
  • atherosclerosis Alzheimer's disease
  • retmitis pigmentosa e.g., macular degeneration
  • macular degeneration e.g., macular degeneration
  • screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide of the present mvention, to form a mixture, measuring Bos taurus Frazzled activity in the mixture, and comparing a Bos taurus Frazzled activity of the mixture to a control mixture which contains no candidate compound.
  • Polypeptides of the present invention may be employed in conventional low capacity screening methods and also high-throughput screening (HTS) formats.
  • HTS formats include not only the well-established use of 96- and, more recently, 384-well microtiter plates but also emerging methods such as the nanowell method described by Schullek, et al , Anal Biochem., 246, 20-29, (1997).
  • polypeptide antagonists examples include antibodies or, in some cases, ohgopeptides or proteins that are closely related to hgands, substrates, receptors, enzymes, etc , as the case may be, of a Frazzled polypeptide, eg , a fragment of a hgand, substrate, receptor, enzyme, etc. ; or small molecules which bind to a Frazzled polypeptide but do not elicit a response, so that an activity of a Frazzled polypeptide is prevented
  • the present invention relates to a screening kit for identifying agonists, antagonists, inhibitors, hgands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which compounds comprise a member selected from the group consisting of: (a) a polypeptide of the present invention,
  • polypeptide of the present mvention may also be used m a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by:
  • the present invention relates to the use of Bos taurus Frazzled polypeptides, polynucleotides, and recombmant materials thereof in selection screens to identify compounds which are neither agonists nor antagonist/inhibitors of Bos taurus Frazzled.
  • the data from such a selection screen is expected to provide in vitro and in vivo comparisons and to predict oral absorption, pharmacokmetics in humans. The ability to make such a comparison of data will enhance formulation design through the identification of compounds with optimal development characte ⁇ stics, i e , high oral bioavailabihty, UID (once a day) dosmg, reduced drug interactions, reduced variability, and reduced food effects, among others
  • GAP aligns two sequences, finding a "maximum similarity", according to the algo ⁇ thm of Neddleman and Wunsch (J Mo I Biol , 48, 443-453, 1970). GAP is more suited to companng sequences that are approximately the same length and an alignment is expected over the entire length.
  • the parameters "Gap Weight” and “Length Weight” used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
  • NCBI National Center for Biotechnology Information
  • NCBI National Center for Biotechnology Information
  • FASTA Pearson W R and Lipman D.J., Proc Nat Acad Sci USA, 85. 2444-2448 (1988) (available as part of the Wisconsin Sequence Analysis Package)
  • BLOSUM62 ammo acid substitution matnx Hemkoff S. and Henikoff J G.
  • point mutations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups withm the reference sequence.
  • a polynucleotide sequence having at least 95% identity to a reference polynucleotide sequence up to 5% of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descnbed.
  • % identities such as 96%, 97%, 98%, 99% and 100%.
  • a polypeptide sequence having, for example, at least 95% identity to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include up to five point mutations per each 100 ammo acids of the reference sequence.
  • Such point mutations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These point mutations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence.
  • “Knock-out” refers to partial or complete suppression of the expression of a protein encoded by an endogenous DNA sequence in a cell
  • the “knock-out” can be affected by targeted deletion of the whole or part of a gene encoding a protein, m an embryonic stem cell. As a result, the deletion may prevent or reduce the expression of the protein in any cell in the whole animal in which it is normally expressed.
  • polynucleotide refers to tnple-stranded regions comprising RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs comprising one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tntylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found m nature, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often refe ⁇ ed to as ohgonucleotides.
  • Modifications include acetylation, acylation, ADP- ⁇ bosylation, 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 hpid derivative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitmation (see, for instance, PRO

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des polypeptides et polynucléotides de Bos taurus Frazzled, ainsi qu'une méthode de production de ces polypeptides par des techniques de recombinaison. L'invention concerne également des méthodes de criblage de composés agonistes ou antagonistes de Bos taurus Frazzled. Ces composés sont supposés utiles dans le traitement de maladies humaines, y compris, de manière non exhaustive : les inflammations chroniques et aiguës, l'arthrite, l'arthrite rhumatoïde, l'ostéoarthrite, la septicémie, les maladies auto-immunes (par exemple, les maladies intestinales inflammatoires, le psoriasis), le rejet des transplantations, la réaction du greffon contre l'hôte, les infections, l'apoplexie, l'ischémie, le syndrome de l'atteinte respiratoire aiguë, les troubles rénaux, la resténose, les lésions cérébrales, le SIDA, les troubles métaboliques et autres troubles osseux (par exemple l'ostéoporose), le cancer (par exemple les affections lymphoprolifératives), l'athérosclérose, la maladie d'Alzheimer, la rétinite pigmentaire, la dégénérescence maculaire et toute autre affection dégénérative des yeux.
PCT/US2000/006820 1999-03-18 2000-03-15 Membre de la famille frzb, de frazzled WO2000055202A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP00917976A EP1165608A4 (fr) 1999-03-18 2000-03-15 Membre de la famille frzb, frazzled

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US12503899P 1999-03-18 1999-03-18
US60/125,038 1999-03-18
US51939700A 2000-03-03 2000-03-03
US09/519,397 2000-03-03

Publications (1)

Publication Number Publication Date
WO2000055202A1 true WO2000055202A1 (fr) 2000-09-21

Family

ID=26823210

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/006820 WO2000055202A1 (fr) 1999-03-18 2000-03-15 Membre de la famille frzb, de frazzled

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EP (1) EP1165608A4 (fr)
WO (1) WO2000055202A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0879881A1 (fr) * 1997-05-23 1998-11-25 Smithkline Beecham Corporation Gene humain similair à proteine secreté frizb (ATG-1639)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2230971A1 (fr) * 1997-05-13 1998-11-13 Smithkline Beecham Corporation Recepteur couple a la proteine-g (hofnh30)
US6057126A (en) * 1997-12-24 2000-05-02 Allelix Biopharmaceuticals, Inc. Mammalian EDG-5 receptor homologs
AU1797200A (en) * 1998-12-11 2000-07-03 Takeda Chemical Industries Ltd. Novel g protein-coupled receptor protein and dna thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0879881A1 (fr) * 1997-05-23 1998-11-25 Smithkline Beecham Corporation Gene humain similair à proteine secreté frizb (ATG-1639)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1165608A4 *

Also Published As

Publication number Publication date
EP1165608A4 (fr) 2002-09-04
EP1165608A1 (fr) 2002-01-02

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