EP1165608A1 - Frazzled, ein mitglied der frzb familie - Google Patents
Frazzled, ein mitglied der frzb familieInfo
- Publication number
- EP1165608A1 EP1165608A1 EP00917976A EP00917976A EP1165608A1 EP 1165608 A1 EP1165608 A1 EP 1165608A1 EP 00917976 A EP00917976 A EP 00917976A EP 00917976 A EP00917976 A EP 00917976A EP 1165608 A1 EP1165608 A1 EP 1165608A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- seq
- sequence
- polynucleotide
- frazzled
- 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.)
- Pending
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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
- This mvention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in identifying compounds that may be agonists and/or antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
- SFRPs secreted f ⁇ zzled-related proteins
- FrzB-1 SFRPs
- CCD cysteme- ⁇ ch domain
- SFRP homologs such as the SARPs (secreted apoptosis-related proteins) have the ability to either sensitize cells to apoptosis or inhibit the apoptotic response (Melkonyan, et al , Proc Natl Acad Set USA,
- the present invention relates to Bos taurus Frazzled, in particular Bos taurus Frazzled polypeptides and Bos taurus Frazzled polynucleotides, recombmant mate ⁇ als and methods for their production.
- the invention relates to methods for identifying agonists and antagonists/inhibitors of the Bos taurus Frazzled gene
- This invention further relates to the generation of in vitro and in vivo comparison data relating to the polynucleotides and polypeptides m order to predict oral absorption and pharmacokmetics in man of compounds that either agonize or antagonize the biological activity of such polynucleotides or polypeptides.
- Such a comparison of data will enable the selection of drugs with optimal pharmacokmetics in man, i e., good oral bioavailabihty, blood-bram barrier penetration, plasma half life, and minimum drug interaction.
- the present invention further relates to methods for creating transgemc animals, which overexpress or underexpress or have regulatable expression of a Frazzled gene and "knock-out" animals, preferably mice, in which an animal no longer expresses a Frazzled gene. Furthermore, this invention relates to transgemc and knock-out animals obtained by using these methods. Such animal models are expected to provide valuable insight into the potential pharmacological and toxicological effects in humans of compounds that are discovered by the aforementioned screening methods as well as other methods.
- Bos taurus Frazzled gene functions are expected to provide an insight into treating and preventing human diseases including, but not limited to chronic and acute inflammation, arthritis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, septicemia, autoimmune diseases (e g , inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
- human diseases including, but not limited to chronic and acute inflammation, arthritis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, septicemia, autoimmune diseases (e g , inflammatory bowel disease, pso ⁇ asis), transplant rejection, graft vs.
- the Diseases any other degenerative eye diseases, hereinafter referred to as "the Diseases,” amongst others.
- the present invention relates to Bos taurus Frazzled polypeptides
- polypeptides include isolated polypeptides comprising an ammo acid sequence having at least a 95% identity, most preferably at least a 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO.2.
- polypeptides include those comprising the ammo acid of SEQ ID NO:2 (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1;
- Polypeptides of the present invention are believed to be members of the f zzled-related proteins family of polypeptides. They are, therefore, of interest, because numerous studies suggest that members of the frizzled family play key roles in cartilage and bone morphogenesis. However, it is unclear what role, if any, these proteins play in the maintenance of adult bone and/or cartilage. Consistent with a potential role in mature tissues, Frzb was originally isolated from calf articular cartilage. Furthermore, it has been proposed that at sites of active bone and cartilage remodeling, exemplified by osteoarthritis and fracture callus healing [Hughes, et. al., J. Bone Miner. Res.
- polypeptides of the present invention can be used to establish assays to predict oral absorbtion and pharmacokmetics m man and thus enhance compound and formulation design, among others.
- Bos taurus Frazzled activity or “Bos taurus Frazzled polypeptide activity” or “biological activity of Frazzled.”
- a polypeptide of the present invention exhibits at least one biological activity of Bos taurus Frazzled
- Polypeptides of the present mvention also include va ⁇ ants of the aforementioned polypeptides, including alleles and splice va ⁇ ants.
- Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative.
- Particularly preferred va ⁇ ants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination.
- Particularly preferred p ⁇ mers will have between 20 and 25 nucleotides.
- Preferred fragments of polypeptides of the present mvention include an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO 2, or an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO.2.
- biologically active fragments which are those fragments that mediate activities of Frazzled, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigemc or lmmunogenic m an animal, especially in a human. Particularly preferred are fragments comp ⁇ smg receptors or domains of enzymes that confer a function essential for viability of Bos taurus or the ability to initiate, or maintain cause the Diseases in an individual, particularly a human.
- polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the invention.
- the polypeptides of the present invention may be in the form of a "mature" protein or may be a part of a larger protein such as a fusion protein It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance, multiple histidine residues, or an additional sequence for stability during recombmant production.
- the present invention also includes va ⁇ ants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characte ⁇ stics.
- Typical substitutions are among Ala, Nal, Leu and lie; 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 va ⁇ ants in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combmation
- Polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occur ⁇ ng polypeptides, recombmantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for prepa ⁇ ng such polypeptides are well understood in the art. In a further aspect, the present invention relates to Bos taurus Frazzled polynucleotides.
- polynucleotides include isolated polynucleotides comp ⁇ sing a nucleotide sequence encodmg a polypeptide having at least a 95% identity, to the ammo acid sequence of SEQ ID NO .2, over the entire length of SEQ ID NO 2
- polypeptides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred.
- polynucleotides include a polynucleotide compnsmg the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO:2.
- polynucleotides of the present invention include isolated polynucleotides compnsmg a nucleotide sequence having at least a 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region.
- polynucleotides which have at least a 97% identity are highly prefe ⁇ ed, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred.
- polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least a 95% identity, to SEQ ID NO: l over the entire length of SEQ ID NO.1.
- polynucleotides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identify are more highly preferred, and those with at least a 99% identity are most highly preferred.
- Such polynucleotides include a polynucleotide compnsmg the polynucleotide of SEQ ID NO: 1 , as well as the polynucleotide of SEQ ID NO: 1.
- the mvention also provides polynucleotides which are complementary to all the above described polynucleotides.
- the nucleotide sequence of SEQ ID NO:l shows homology with Homo sapiens f ⁇ zzled-related protein [frpHE].
- the nucleotide sequence of SEQ ID NO:l is a cDNA sequence and comp ⁇ ses a polypeptide encoding sequence (nucleotide 1 to 1038) encoding a polypeptide of 346 ammo acids, the polypeptide of SEQ ID NO.2.
- the nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO:l or it may be a sequence other than SEQ ED NO: 1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
- the polypeptide of SEQ ID NO:2 is structurally related to other proteins of the f ⁇ zzled-related proteins family, having homology and/or structural sirmla ⁇ ty with
- frpHE Homo sapiens f ⁇ zzled-related protein
- 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
- Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of Bos taurus bovme heart, spleen, lung and kidney, 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 hbranes 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 polypeptide, 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 prepro- protem sequence, or other fusion peptide portions.
- a marker sequence that facilitates purification of the fused polypeptide can be encoded.
- the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and desc ⁇ bed in Gentz, et al , Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
- the polynucleotide may also comp ⁇ se non-coding 5 ' and 3 ' sequences, such as transcnbed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA.
- polypeptide vanants that compnse the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 1 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination.
- Particularly preferred probes will have between 30 and 50 nucleotides, but may have between 100 and 200 contiguous nucleotides of the polynucleotide of SEQ ID NO: 1
- a preferred embodiment of the invention is a polynucleotide of consisting of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1038 set forth in SEQ ID NO:l of Table 1, both of which encode a Frazzled polypeptide
- the invention also includes a polynucleotide consisting of or comp ⁇ smg a polynucleotide of the formula.
- 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
- Prefe ⁇ ed probes or pnmers will generally compnse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides, and may even have at least 100 nucleotides. Particularly prefe ⁇ ed pnmers will have between 20 and 25 nucleotides.
- a polynucleotide encoding a polypeptide of the present invention, mcludmg homologs and orthologs from a species other than Bos taurus, may be obtained by a process compnsmg the steps of screening an appropnate library under stnngent hybridization conditions with a labeled probe having the sequence of SEQ ID NO.1 or a fragment thereof, preferably of at least 15 nucleotides m length, and isolating full-length cDNA and genomic clones compnsmg said polynucleotide sequence.
- Such hyb ⁇ dization techniques are well known to the skilled artisan.
- Prefe ⁇ ed stringent hybndization conditions include overnight incubation at 42°C m a solution compnsmg: 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters 0 lx SSC at about 65°C
- the present invention also includes isolated polynucleotides, preferably of at least 100 nucleotides in length, obtained by screening an appropriate library under stnngent hybndization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides
- an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide 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
- PCR PCR is then earned out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide pnmers
- the PCR reaction is then repeated using 'nested' primers, that is, pnmers designed to anneal withm the amplified product (typically an adaptor specific primer that anneals further 3' m the adaptor sequence and a gene specific pnmer that anneals further 5' m 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' pnmer
- Recombmant polypeptides of the present invention may be prepared by processes well known m the art from genetically engineered host cells compnsmg expression systems Accordingly, in a further aspect, the present invention relates to expression systems compnsmg a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombmant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the present invention
- 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 desc ⁇ bed 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 Sp ⁇ ng
- Prefe ⁇ ed methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection
- Representative examples of approp ⁇ ate hosts include bactenal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosoph ⁇ a S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
- a great vanety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bactenal plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids.
- the expression systems may comp ⁇ se control regions that regulate as well as engender expression
- any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
- the approp ⁇ ate nucleotide sequence may be mserted into an expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth in Sambrook, et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).
- a polypeptide of the present invention is to be expressed for use screening assays, it is generally prefe ⁇ ed that the polypeptide be produced at the surface of the cell.
- the cells may be harvested pnor to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered m order to recover and purify the polypeptide If produced lntracellularly, the cells must first be lysed before the polypeptide is recovered.
- Polypeptides of the present invention can be recovered and punfied from recombmant 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 pu ⁇ fication. Well known techniques for refolding protems may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and/or punfication.
- Bos taurus Frazzled gene products can be expressed in transgemc animals Animals of any species, including, but not limited to mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human p ⁇ mates, e g , baboons, monkeys, chimpanzees, may be used to generate Frazzled transgemc animals
- This invention further relates to a method of producing transgemc animals, preferably Bos taurus, over-expressing Frazzled, which method may comp ⁇ se the introduction of several copies of a segment compnsmg at least the polynucleotide sequence encoding SEQ ID NO:2 with a suitable promoter into the cells of a Bos taurus embryo, or the cells of another species, at an early stage.
- 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.
- a further aspect of the present invention involves gene targeting by homologous recombination m embryonic stem cells to produce a transgemc animal with a mutation in a Frazzled gene ("knock-out" mutation).
- knock-out animals
- there is inactivation of the Frazzled gene or altered gene expression such that the animals are useful to study the function of the Frazzled gene, thus providing animals models of human disease, which are otherwise not readily available through spontaneous, chemical or i ⁇ adiation mutagenesis.
- Another aspect of the present invention involves the generation of so-called "knock-m” animals m which a portion of a wild-type gene is fused to the cD ⁇ A of a heterologous gene.
- 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
- Va ⁇ ous methods for producing mutations in non-human animals are contemplated and well known in the art.
- a mutation is generated in a munne Frazzled allele by the introduction of a DNA construct compnsmg DNA of a gene encoding munne Frazzled, which munne gene contains the mutation.
- the mutation is targeted to the allele by way of the DNA construct.
- the DNA of the gene encodmg munne Frazzled compnsed in the construct may be foreign to the species of which the recipient is a member, may be native to the species and foreign only to the individual recipient, may be a construct compnsed of synthetic or natural genetic components, or a mixture of these.
- 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
- mice obtained by a method of producing recombmant mice as defined above, among others
- Another aspect of this invention provides for in vitro Frazzled "knock-outs", i e , tissue cultures
- Animals of any species including, but not limited to mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human pnmates, e g , baboons, monkeys, chimpanzees, may be used to generate in vitro Frazzled "knock-outs”
- Methods for "knocking out" genes in vitro are desc ⁇ bed in Galh-Tahadoros, et al , Journal of Immunological Methods 181" 1-15 (1995)
- Transgemc "knock-m”, and “knock-out” animals, as defined above, are a particularly advantageous model, from a physiological point of view, for studying f ⁇ zzled-related proteins Such animals will be valuable tools to study the functions of a Frazzled gene. Moreover, such animal models are expected to provide information about potential toxicological effects m humans of any compounds discovered by an aforementioned screening method, among others An understanding of how a Bos taurus Frazzled gene functions in these animal models is expected to provide an msight into treating and preventing human diseases including, but not limited to.
- 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
- Polypeptides of the present invention are responsible for many biological functions, including many disease states, m particular the Diseases mentioned herein. It is, therefore, an aspect of the invention to devise screening methods to identify compounds that stimulate (agonists) or that inhibit
- the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function of the polypeptide.
- agonists or antagonists may be employed for therapeutic and prophylactic purposes for the Diseases mentioned herein mentioned
- Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures
- Such agonists and antagonists so-identified may be natural or modified substrates, gands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Co gan, et al , CURRENT PROTOCOLS IN IMMUNOLOGY 1(2)- Chapter 5 (1991))
- the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes beanng the polypeptide, or a fusion protein thereof
- a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the polypeptide with a labeled competitor (e g , agonist or antagonist). Further, screening methods may test whether the candidate compound results in a signal generated by an agonist or antagonist of the polypeptide, using detection systems appropriate to cells bearing the polypeptide. Antagonists are generally assayed in the presence of a known agonist and an effect on activation by the agonist by the presence of the candidate compound is observed.
- 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).
- Fusion proteins such as those made from Fc portion and Bos taurus Frazzled polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists of antagonists of the polypeptide of the present mvention (see D. Bennett, et al, J Mol Recognition, 8:52-58 (1995); and K Johanson, et al , J Bwl Chem., 270(16):9459-9471 (1995)).
- Bos Taurus Frazzled cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of Bos taurus Frazzled mRNA and protein m cells
- an ELISA may be constructed for measu ⁇ ng secreted or cell associated levels of Bos taurus Frazzled protein using monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents (i.e.
- Bos taurus Frazzled protein may be used to identify membrane bound or soluble hgand or receptors through standard hgand/receptor binding techniques known in the art. These include, but are not limited to, hgand binding and crosslinkmg assays in which the Bos taurus Frazzled is labeled with a radioactive isotope (e.g. 125 I), chemically modified (e.g.
- a radioactive isotope e.g. 125 I
- chemically modified e.g.
- binding assays can be used to identify agonists and antagonists of Bos taurus Frazzled which compete with the binding of Bos taurus Frazzled to its receptors or hgands.
- the above binding assays can be used to identify cell which respond biologically to Bos taurus Frazzled.
- Cells which respond to Bos taurus Frazzled may show changes m mtracellular signal transduction pathways and in gene expression. These changes can be used m screens for agonists or antagonists which mimic or inhibit the action of Bos taurus Frazzled respectively
- 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 invention (c) a cell membrane expressing a polypeptide of the present invention, which polypeptide is preferably that of SEQ ID NO:2.
- 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
- Allele refers to one or more alternative forms of a gene occurnng at a given locus in the genome.
- “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. “Fragment” of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1.
- 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 immunoglobulm molecules together with another human protein or part thereof.
- employing an immunoglobulm Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g., EP-A 0232 262]
- “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined. Falling withm this genenc term are the terms, "ortholog", and “paralog”. "Ortholog” refers to polynucleotides/genes or polypeptide that are homologs via speciation, that is closely related and assumed to have commend descent based on structural and functional considerations. "Paralog” refers to polynucleotides/genes or polypeptide that are homologs via gene duplication, for instance, duplicated vanants within a genome.
- Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences In general, identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid co ⁇ espondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared. For sequences where there is not an exact co ⁇ espondence, a "% identity" may be determined.
- the two sequences to be compared are aligned to give a maximum co ⁇ elation between the sequences This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment
- a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length
- Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences
- similarity means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact co ⁇ espondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact co ⁇ espondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated 'score' from which the "% simila ⁇ ty" of the two sequences can then be determined.
- 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.
- % identities and similanties are determined when the two sequences being compared are optimally aligned
- 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.
- the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described.
- a polynucleotide sequence having, for example, at least 95% identity to a reference polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference sequence.
- Such point mutations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
- 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.
- a sequence polypeptide sequence having at least 95% identity to a reference polypeptide sequence up to 5% of the ammo acids of the m the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descnbed.
- Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1 , wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, 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 m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO.1 by the integer defining the percent identity divided by 100 and then
- n n is the number of nucleotide alterations
- x n is the total number of nucleotides m SEQ ID NO: l
- y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
- • is the symbol for the multiplication operator, and wherein any non-mteger product of x n and y is rounded down to the nearest integer pnor 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.
- Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO.2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids SEQ ID NO.2, or
- Isolated means altered “by the hand of man” from its natural state, i e., if it occurs in nature, it has been changed or removed from its ongmal environment, or both.
- a polynucleotide or a polypeptide naturally present in a living organism is not “isolated ' but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is “isolated”, as the term is employed herein.
- a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombmant method is “isolated” even if it is still present in said organism, which organism may be living or non-living.
- Knock-m refers to the fusion of a portion of a wild-type gene to the cDNA of a heterologous gene
- “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.
- Splice Nanant refers to cD ⁇ A molecules produced from R ⁇ A molecules initially transc ⁇ bed from the same genomic D ⁇ A sequence but which have undergone alternative R ⁇ A splicing.
- Alternative R ⁇ A splicing occurs when a primary R ⁇ A transcnpt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mR A molecule each of that may encode different ammo acid sequences.
- the term splice variant also refers to the proteins encoded by the above cD ⁇ A molecules.
- Transgemc animal refers to an animal to which exogenous D ⁇ A has been introduced while the animal is still in its embryonic stage
- the transgemc approach aims at specific modifications of the genome, e g , by introducing whole transc ⁇ ptional units into the genome, or by up- or down-regulating pre-existing cellular genes.
- the targeted character of certain of these procedures sets transgemc technologies apart from expenmental methods in which random mutations are confe ⁇ ed to the germlme, such as administration of chemical mutagens or treatment with ionizing solution.
- Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeoxnbonucleotide, which may be unmodified R ⁇ A or D ⁇ A or modified R ⁇ A or D ⁇ A
- Polynucleotides include, without limitation, single- and double-stranded D ⁇ A, D ⁇ A that is a mixture of single- and double- stranded regions, single- and double-stranded R ⁇ A, and R ⁇ A 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 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.
- Polypeptide refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e , peptide isosteres
- Polypeptide refers to both short chains, commonly refe ⁇ ed to as peptides, ohgopeptides or oligomers, and to longer chains, generally refe ⁇ ed to as proteins.
- Polypeptides may comprise ammo 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.
- Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo 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 comprise many types of modifications. Polypeptides may be branched as a result of ubiquitmation, 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- ⁇ 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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US12503899P | 1999-03-18 | 1999-03-18 | |
US125038P | 1999-03-18 | ||
US51939700A | 2000-03-03 | 2000-03-03 | |
US519397 | 2000-03-03 | ||
PCT/US2000/006820 WO2000055202A1 (en) | 1999-03-18 | 2000-03-15 | A member of the frzb family, frazzled |
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EP1138693A1 (de) * | 1998-12-11 | 2001-10-04 | Takeda Chemical Industries, Ltd. | G-protein-gekoppeltes rezeptor protein und dessen dns |
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CA2230971A1 (en) * | 1997-05-13 | 1998-11-13 | Smithkline Beecham Corporation | G-protein coupled receptor (hofnh30) |
EP0879881A1 (de) * | 1997-05-23 | 1998-11-25 | Smithkline Beecham Corporation | Ein menschliches Gen ähnlich einem sekretiertem Protein frizb (ATG-1639) |
US6057126A (en) * | 1997-12-24 | 2000-05-02 | Allelix Biopharmaceuticals, Inc. | Mammalian EDG-5 receptor homologs |
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Non-Patent Citations (5)
Title |
---|
DATABASE EMBL [Online] EBI; 21 October 1999 (1999-10-21) MARRA ET AL: "Human Q926333 lysophosphatidic acid receptor mRNA" Database accession no. AW107032 XP002199180 * |
DATABASE GENSEQ [Online] EBI; 13 September 1999 (1999-09-13) CHUN ET AL: "Murine EDG-5 receptor alpha subclone nucleotide sequence" Database accession no. AAX88748 XP002199181 -& DATABASE GENSEQ [Online] EBI; 13 September 1999 (1999-09-13) CHUN ET ALL: "Murine EDG-5 receptor alpha subclone protein" Database accession no. AAY24243 XP002199182 -& WO 99 33972 A (ALLELIX BIOPHARMA ;GUPTA ASHWANI K (CA); MUNROE DONALD G (CA); VYA) 8 July 1999 (1999-07-08) * |
DATABASE GENSEQ [Online] EBI; 26 February 1999 (1999-02-26) CLINKENBEARD: "HOFNH30" Database accession no. AAW86801 XP002199179 -& EP 0 878 479 A (SMITHKLINE BEECHAM PLC ;SMITHKLINE BEECHAM CORP(US)) 18 November 1998 (1998-11-18) * |
RATTNER AT AL: "A family of secreted proteins contains homology to the cystein-rich ligand-binding domain of frizzled receptors" PMAS USA, vol. 94, April 1997 (1997-04), pages 2859-2863, XP002199178 * |
See also references of WO0055202A1 * |
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