WO2004061123A2 - Mbcat servant de modificateurs de la voie de beta-catenine et leurs procedes d'utilisation - Google Patents

Mbcat servant de modificateurs de la voie de beta-catenine et leurs procedes d'utilisation Download PDF

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WO2004061123A2
WO2004061123A2 PCT/US2003/041404 US0341404W WO2004061123A2 WO 2004061123 A2 WO2004061123 A2 WO 2004061123A2 US 0341404 W US0341404 W US 0341404W WO 2004061123 A2 WO2004061123 A2 WO 2004061123A2
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mbcat
beta
assay
catenin
agent
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PCT/US2003/041404
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WO2004061123A3 (fr
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Helen Francis-Lang
Christopher G. Winter
Richard Benn Abegania Ventura
Kim Lickteig
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Exelixis, Inc.
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Publication of WO2004061123A3 publication Critical patent/WO2004061123A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • beta -catenin levels are tightly regulated by a complex containing APC, Axin, and GSK3 beta /SGG/ZW3 (Peifer et al. (1994) Development 120: 369-380).
  • the Wingless/ beta -catenin signaling pathway is frequently mutated in human cancers, particularly those of the colon. Mutations in the tumor suppressor gene APC, as well as point mutations in beta -catenin itself lead to the stabilization of the beta -catenin protein and inappropriate activation of this pathway.
  • the ability to manipulate the genomes of model organisms such as Drosophila provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms.
  • a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a "genetic entry point") that yields a visible phenotype. Additional genes are mutated in a random or targeted manner.
  • a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a "modifier" involved in the same or overlapping pathway as the genetic entry point.
  • modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
  • modifiers of beta-catenin MBCAT
  • the invention provides methods for utilizing these beta-catenin modifier genes and polypeptides to identify MBCAT-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired beta-catenin function and/or MBCAT function.
  • Preferred MBCAT- modulating agents specifically bind to MBCAT polypeptides and restore beta-catenin function.
  • MBCAT-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress MBCAT gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
  • nucleic acid modulators such as antisense oligomers and RNAi that repress MBCAT gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
  • MBCAT modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with an MBCAT polypeptide or nucleic acid.
  • candidate MBCAT modulating agents are tested with an assay system comprising a MBCAT polypeptide or nucleic acid.
  • Agents that produce a change in the activity of the assay system relative to controls are identified as candidate beta-catenin modulating agents.
  • the assay system may be cell-based or cell-free.
  • MBCAT- modulating agents include MBCAT related proteins (e.g.
  • a small molecule modulator is identified using a binding assay.
  • the screening assay system is selected from an apoptosis assay, a cell proliferation assay, an angiogenesis assay, and a hypoxic induction assay.
  • candidate beta-catenin pathway modulating agents are further tested using a second assay system that detects changes in the beta-catenin pathway, such as angiogenic, apoptotic, or cell proliferation changes produced by the originally identified candidate agent or an agent derived from the original agent.
  • the second assay system may use cultured cells or non-human animals.
  • the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the beta-catenin pathway, such as an angiogenic, apoptotic, or cell proliferation disorder (e.g. cancer).
  • the invention further provides methods for modulating the MBCAT function and/or the beta-catenin pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a MBCAT polypeptide or nucleic acid.
  • the agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be administered to a mammalian animal predetermined to have a pathology associated with the beta-catenin pathway.
  • Table 1 (Example II) lists the modifiers and their orthologs.
  • Modulation of the MBCAT or their respective binding partners is useful for understanding the association of the beta-catenin pathway and its members in normal and disease conditions and for developing diagnostics and therapeutic modalities for beta-catenin related pathologies.
  • MBCAT-modulating agents that act by inhibiting or enhancing MBCAT expression, directly or indirectly, for example, by affecting an MBCAT function such as enzymatic (e.g., catalytic) or binding activity, can be identified using methods provided herein.
  • MBCAT modulating agents are useful in diagnosis, therapy and pharmaceutical development.
  • Nucleic acids and polypeptides of the invention Sequences related to MBCAT nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) or RefSeq number), shown in Table 1 and in the appended sequence listing.
  • preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of an MBCAT. In further preferred embodiments, the fragment comprises the entire functionally active domain.
  • MBCAT nucleic acid refers to a DNA or RNA molecule that encodes a MBCAT polypeptide.
  • the MBCAT polypeptide or nucleic acid or fragment thereof is from a human, but can also be an ortholog, or derivative thereof with at least 70% sequence identity, preferably at least 80%, more preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with human MBCAT.
  • Methods of identifying orthlogs are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3- dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences.
  • Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al, Genome Research (2000) 10:1204-1210).
  • Programs for multiple sequence alignment such as CLUSTAL (Thompson JD et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees.
  • orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species.
  • Structural threading or other analysis of protein folding e.g., using software by ProCeryon, Biosciences, Salzburg, Austria
  • a gene duplication event follows speciation, a single gene in one species, such as Drosophila, may correspond to multiple genes (paralogs) in another, such as human.
  • the term "orthologs" encompasses paralogs.
  • percent (%) sequence identity with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0al9 (Altschul et al., J. Mol. Biol. (1997) 215:403-410) with all the search parameters set to default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched.
  • a % identity value is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. "Percent (%) amino acid sequence similarity" is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
  • Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
  • an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489; database: European Bioinformatics Institute; Smith and Waterman, 1981, J. of Molec.Biol., 147:195-197; Nicholas et al., 1998, "A tutorial on Searching Sequence Databases and Sequence Scoring Methods” (www.psc.edu) and references cited therein.; W.R. Pearson, 1991, Genomics 11:635-650).
  • This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.
  • the transgenic animal is a "knock-out" animal having a heterozygous or homozygous alteration in the sequence of an endogenous MBCAT gene that results in a decrease of MBCAT function, preferably such that MBCAT expression is undetectable or insignificant.
  • Knock-out animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it.
  • the transgene can be a human gene (e.g., from a human genomic clone) but more preferably is an ortholog of the human gene derived from the transgenic host species.
  • a mouse MBCAT gene is used to construct a homologous recombination vector suitable for altering an endogenous MBCAT gene in the mouse genome.
  • homologous recombination vector suitable for altering an endogenous MBCAT gene in the mouse genome.
  • Detailed methodologies for homologous recombination in mice are available (see Capecchi, Science (1989) 244: 1288-1292; Joyner et al, Nature (1989)
  • knock-out animals such as mice harboring a knockout of a specific gene, may be used to produce antibodies against the human counterpart of the gene that has been knocked out (Claesson MH et al., (1994) Scan J Immunol 40:257-264; Declerck PJ et al., (1995) J Biol Chem. 270:8397-400).
  • the transgenic animal is a "knock-in" animal having an alteration in its genome that results in altered expression (e.g., increased (including ectopic) or decreased expression) of the MBCAT gene, e.g., by introduction of additional copies of MBCAT, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the MBCAT gene.
  • a regulatory sequence include inducible, tissue-specific, and constitutive promoters and enhancer elements.
  • the knock-in can be homozygous or heterozygous.
  • Transgenic nonhuman animals can also be produced that contain selected systems allowing for regulated expression of the transgene.
  • a system that may be produced is the cre/loxP recombinase system of bacteriophage PI (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182).
  • both Cre-LoxP and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun X et al (2000) Nat Genet 25:83-6).
  • the genetically modified animals can be used in genetic studies to further elucidate the beta-catenin pathway, as animal models of disease and disorders implicating defective beta-catenin function, and for in vivo testing of candidate therapeutic agents, such as those identified in screens described below.
  • the candidate therapeutic agents are administered to a genetically modified animal having altered MBCAT function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered MBCAT expression that receive candidate therapeutic agent.
  • animal models having defective beta-catenin function can be used in the methods of the present invention.
  • a beta-catenin knockout mouse can be used to assess, in vivo, the activity of a candidate beta-catenin modulating agent identified in one of the in vitro assays described below.
  • the candidate beta-catenin modulating agent when administered to a model system with cells defective in beta-catenin function, produces a detectable phenotypic change in the model system indicating that the beta-catenin function is restored, i.e., the cells exhibit normal cell cycle progression.
  • the invention provides methods to identify agents that interact with and/or modulate the function of MBCAT and/or the beta-catenin pathway. Modulating agents identified by the methods are also part of the invention. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the beta-catenin pathway, as well as in further analysis of the MBCAT protein and its contribution to the beta-catenin pathway. Accordingly, the invention also provides methods for modulating the beta- catenin pathway comprising the step of specifically modulating MBCAT activity by administering a MBCAT-interacting or -modulating agent.
  • the MBCAT- modulating agent is a modulator of the beta-catenin pathway (e.g. it restores and/or upregulates beta-catenin function) and thus is also a beta-catenin- modulating agent.
  • Preferred MBCAT-modulating agents include small molecule compounds; MBCAT-interacting proteins, including antibodies and other biotherapeutics; and nucleic acid modulators such as antisense and RNA inhibitors.
  • the modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in "Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, PA, 19 th edition.
  • Small molecule modulators Small molecules are often preferred to modulate function of proteins with enzymatic function, and/or containing protein interaction domains.
  • Chemical agents referred to in the art as "small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight up to 10,000, preferably up to 5,000, more preferably up to 1,000, and most preferably up to 500 daltons.
  • This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the MBCAT protein or may be identified by screening compound libraries.
  • modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for MBCAT-modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151:1947-1948).
  • Small molecule modulators identified from screening assays can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in treating pathologies associated with the beta-catenin pathway.
  • the activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing.
  • candidate clinical compounds are generated with specific regard to clinical and pharmacological properties.
  • the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
  • MBCAT-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MBCAT polypeptides.
  • an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MBCAT polypeptides.
  • ELISA enzyme-linked immunosorbant assay
  • Other assays such as radioimmunoassays or fluorescent assays might also be used.
  • Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used.
  • the vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential.
  • the antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml to aboutlO mg/ml.
  • an MBCAT-interacting protein may have biotherapeutic applications.
  • Biotherapeutic agents formulated in pharmaceutically acceptable carriers and dosages may be used to activate or inhibit signal transduction pathways. This modulation may be accomplished by binding a ligand, thus inhibiting the activity of the pathway; or by binding a receptor, either to inhibit activation of, or to activate, the receptor.
  • the biotherapeutic may itself be a ligand capable of activating or inhibiting a receptor. Biotherapeutic agents and methods of producing them are described in detail in U.S. Pat. No. 6,146,628.
  • the MBCAT When the MBCAT is a ligand, it may be used as a biotherapeutic agent to activate or inhibit its natural receptor. Alternatively, antibodies against MBCAT, as described in the previous section, may be used as biotherapeutic agents.
  • the MBCAT When the MBCAT is a receptor, its ligand(s), antibodies to the ligand(s) or the MBCAT itself may be used as biotherapeutics to modulate the activity of MBCAT in the beta-catenin pathway.
  • MBCAT-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit MBCAT activity.
  • Preferred nucleic acid modulators interfere with the function of the MBCAT nucleic acid such as DNA replication, transcription, translocation of the MBCAT RNA to the site of protein translation, translation of protein from the MBCAT RNA, splicing of the MBCAT RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the MBCAT RNA.
  • the antisense oligomer is an oligonucleotide that is sufficiently complementary to an MBCAT mRNA to bind to and prevent translation, preferably by binding to the 5' untranslated region.
  • MBCAT-specific antisense oligonucleotides preferably range from at least 6 to about 200 nucleotides. In some embodiments the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length.
  • the antisense oligomer is a phosphothioate morpholino oligomer (PMO).
  • PMOs are assembled from four different morpholino subunits, each of which contain one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate intersubunit linkages. Details of how to make and use PMOs and other antisense oligomers are well known in the art (e.g. see WO99/18193; Probst JC, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods.
  • RNAi double-stranded RNA species mediating RNA interference (RNAi).
  • RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Methods relating to the use of RNAi to silence genes in C.
  • the screening method comprises contacting a suitable assay system comprising an MBCAT polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e.g. kinase activity), which is based on the particular molecular event the screening method detects.
  • a reference activity e.g. kinase activity
  • a statistically significant difference between the agent- biased activity and the reference activity indicates that the candidate agent modulates MBCAT activity, and hence the beta-catenin pathway.
  • the MBCAT polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above.
  • binding equilibrium constants usually at least about more preferably at least about 10 9 M "1
  • immunogenicity e.g. ability to elicit MBCAT specific antibody in a heterologous host such as a mouse, rat, goat or rabbit.
  • binding may be assayed by, respectively, substrate and ligand processing.
  • the screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of a MBCAT polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein.
  • the MBCAT polypeptide can be full length or a fragment thereof that retains functional MBCAT activity.
  • the MBCAT polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag.
  • the MBCAT polypeptide is preferably human MBCAT, or is an ortholog or derivative thereof as described above.
  • the screening assay detects candidate agent-based modulation of MBCAT interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has MBCAT -specific binding activity, and can be used to assess normal MBCAT gene function.
  • a binding target such as an endogenous or exogenous protein or other substrate that has MBCAT -specific binding activity
  • screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes PB, Curr Opin Chem Biol (1998) 2:597-603; Sundberg SA, Curr Opin Biotechnol 2000, 11:47-53).
  • screening assays uses fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer.
  • a variety of suitable assay systems may be used to identify candidate MBCAT and Wingless/beta-catenin pathway modulators (e.g. U.S. Pat. Nos. 5,550,019 and 6,133,437 (apoptosis assays); U.S. Pat. No. 6,114,132 (phosphatase and protease assays), U.S. Pat. Nos. 5,976,782, 6,225,118 and 6,444,434 (angiogenesis assays), among others). Specific preferred assays are described in more detail below.
  • Protein phosophatases catalyze the removal of a gamma phosphate from a serine, threonine or tyrosine residue in a protein substrate. Since phosphatases act in opposition to kinases, appropriate assays measure the same parameters as kinase assays. In one example, the dephosphorylation of a fluorescently labeled peptide substrate allows trypsin cleavage of the substrate, which in turn renders the cleaved substrate significantly more fluorescent (Nishikata M et al, Biochem J (1999) 343:35-391).
  • fluorescence polarization a solution-based, homogeneous technique requiring no immobilization or separation of reaction components
  • HTS high throughput screening
  • Transporter proteins carry a range of substrates, including nutrients, ions, amino acids, and drugs, across cell membranes.
  • Assays for modulators of transporters may use labeled substrates.
  • exemplary high throughput screens to identify compounds that interact with different peptide and anion transporters both use fluorescently labeled substrates; the assay for peptide transport additionally uses multiscreen filtration plates (Blevitt JM et al., J Biomol Screen 1999, 4:87-91; Cihlar T and Ho ES, Anal Biochem 2000, 283:49-55).
  • Apoptosis assays may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay.
  • TUNEL terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling
  • the TUNEL assay is used to measure nuclear DNA fragmentation characteristic of apoptosis ( Lazebnik et al, 1994, Nature 371, 346), by following the incorporation of fluorescein-dUTP (Yonehara et al, 1989, J. Exp. Med. 169, 1747).
  • Apoptosis may further be assayed by acridine orange staining of tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41).
  • cell-based apoptosis assays include the caspase-3/7 assay and the cell death nucleosome ELISA assay.
  • the caspase 3/7 assay is based on the activation of the caspase cleavage activity as part of a cascade of events that occur during programmed cell death in many apoptotic pathways.
  • the caspase 3/7 assay commercially available Apo- ONETM Homogeneous Caspase-3/7 assay from Promega, cat# 67790
  • lysis buffer and caspase substrate are mixed and added to cells.
  • the caspase substrate becomes fluorescent when cleaved by active caspase 3/7.
  • the nucleosome ELISA assay is a general cell death assay known to those skilled in the art, and available commercially (Roche, Cat# 1774425). This assay is a quantitative sandwich-enzyme-immunoassay which uses monoclonal antibodies directed against DNA and histones respectively, thus specifically determining amount of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Mono and oligonucleosomes are enriched in the cytoplasm during apoptosis due to the fact that DNA fragmentation occurs several hours before the plasma membrane breaks down, allowing for accumalation in the cytoplasm.
  • Nucleosomes are not present in the cytoplasmic fraction of cells that are not undergoing apoptosis.
  • An apoptosis assay system may comprise a cell that expresses an MBCAT, and that optionally has defective beta-catenin function (e.g. beta-catenin is over-expressed or under-expressed relative to wild-type cells).
  • a test agent can be added to the apoptosis assay system and changes in induction of apoptosis relative to controls where no test agent is added, identify candidate beta-catenin modulating agents.
  • an apoptosis assay may be used as a secondary assay to test a candidate beta-catenin modulating agents that is initially identified using a cell-free assay system.
  • An apoptosis assay may also be used to test whether MBCAT function plays a direct role in apoptosis.
  • an apoptosis assay may be performed on cells that over- or under-express MBCAT relative to wild type cells. Differences in apoptotic response compared to wild type cells suggests that the MBCAT plays a direct role in the apoptotic response.
  • Apoptosis assays are described further in US Pat. No. 6,133,437.
  • Cell proliferation and cell cycle assays may be assayed via bromodeoxyuridine (BRDU) incorporation.
  • BRDU bromodeoxyuridine
  • This assay identifies a cell population undergoing DNA synthesis by incorporation of BRDU into newly-synthesized DNA. Newly-synthesized DNA may then be detected using an anti-BRDU antibody (Hoshino et al, 1986, Int. J. Cancer 38, 369; Campana et al, 1988, J. Immunol. Meth. 107, 79), or by other means.
  • This assay allows for quantitative characterization of S-phase DNA syntheses.
  • cells synthesizing DNA will inco ⁇ orate [ 3 H]-thymidine into newly synthesized DNA. Inco ⁇ oration can then be measured by standard techniques such as by counting of radioisotope in a scintillation counter (e.g., Beckman LS 3800 Liquid Scintillation Counter).
  • a scintillation counter e.g., Beckman LS 3800 Liquid Scintillation Counter.
  • Another proliferation assay uses the dye Alamar Blue (available from Biosource International), which fluoresces when reduced in living cells and provides an indirect measurement of cell number (Voytik-Harbin SL et al., 1998, In Vitro Cell Dev Biol Anim 34:239-46).
  • Cell proliferation may also be assayed by measuring ATP levels as indicator of metabolically active cells.
  • assays are commercially available, for example Cell Titer-GloTM, which is a luminescent homogeneous assay available from Promega.
  • Involvement of a gene in the cell cycle may be assayed by flow cytometry (Gray JW et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49:237-55).
  • Cells transfected with an MBCAT may be stained with propidium iodide and evaluated in a flow cytometer (available from Becton Dickinson), which indicates accumulation of cells in different stages of the cell cycle.
  • an angiogenesis assay system may comprise a cell that expresses an MBCAT, and that optionally has defective beta-catenin function (e.g. beta-catenin is over-expressed or under-expressed relative to wild-type cells).
  • a test agent can be added to the angiogenesis assay system and changes in angiogenesis relative to controls where no test agent is added, identify candidate beta- catenin modulating agents.
  • the angiogenesis assay may be used as a secondary assay to test a candidate beta-catenin modulating agents that is initially identified using another assay system.
  • An angiogenesis assay may also be used to test whether MBCAT function plays a direct role in cell proliferation.
  • an angiogenesis assay may be performed on cells that over- or under-express MBCAT relative to wild type cells. Differences in angiogenesis compared to wild type cells suggests that the MBCAT plays a direct role in angiogenesis.
  • hypoxia inducible factor-1 The alpha subunit of the transcription factor, hypoxia inducible factor-1 (HIF-1), is upregulated in tumor cells following exposure to hypoxia in vitro.
  • HIF-1 hypoxia inducible factor-1
  • hypoxia inducible factor-1 stimulates the expression of genes known to be important in tumour cell survival, such as those encoding glyolytic enzymes and VEGF.
  • Induction of such genes by hypoxic conditions may be assayed by growing cells transfected with MBCAT in hypoxic conditions (such as with 0.1% O2, 5% CO2, and balance N2, generated in a Napco 7001 incubator (Precision Scientific)) and normoxic conditions, followed by assessment of gene activity or expression by Taqman®.
  • a hypoxic induction assay system may comprise a cell that expresses an MBCAT, and that optionally has defective beta-catenin function (e.g. beta-catenin is over- expressed or under-expressed relative to wild-type cells).
  • a test agent can be added to the hypoxic induction assay system and changes in hypoxic response relative to controls where no test agent is added, identify candidate beta-catenin modulating agents.
  • the hypoxic induction assay may be used as a secondary assay to test a candidate beta-catenin modulating agents that is initially identified using another assay system.
  • a hypoxic induction assay may also be used to test whether MBCAT function plays a direct role in the hypoxic response.
  • hypoxic induction assay may be performed on cells that over- or under-express MBCAT relative to wild type cells. Differences in hypoxic response compared to wild type cells suggests that the MBCAT plays a direct role in hypoxic induction.
  • Cell adhesion assays measure adhesion of cells to purified adhesion proteins, or adhesion of cells to each other, in presence or absence of candidate modulating agents.
  • Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2.5g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2x final test concentration and added to the blocked, coated wells. Cells are then added to the wells, and the unbound cells are washed off.
  • High-throughput cell adhesion assays have also been described.
  • small molecule ligands and peptides are bound to the surface of microscope slides using a microarray spotter, intact cells are then contacted with the slides, and unbound cells are washed off.
  • this assay not only the binding specificity of the peptides and modulators against cell lines are determined, but also the functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip is measured (Falsey JR et al., Bioconjug Chem. 2001 May-Jun;12(3):346-53).
  • Tubulogenesis assays monitor the ability of cultured cells, generally endothelial cells, to form tubular structures on a matrix substrate, which generally simulates the environment of the extracellular matrix.
  • exemplary substrates include MatrigelTM (Becton Dickinson), an extract of basement membrane proteins containing laminin, collagen IV, and heparin sulfate proteoglycan, which is liquid at 4°C and forms a solid gel at 37° C.
  • Other suitable matrices comprise extracellular components such as collagen, fibronectin, and/or fibrin. Cells are stimulated with a pro-angiogenic stimulant, and their ability to form tubules is detected by imaging.
  • a tubulogenesis assay system comprises testing an MBCAT's response to a variety of factors, such as FGF, VEGF, phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.
  • factors such as FGF, VEGF, phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.
  • An invasion/migration assay tests the ability of cells to overcome a physical barrier and to migrate towards pro-angiogenic signals.
  • Migration assays are known in the art (e.g., Paik JH et al., 2001, J Biol Chem 276: 11830-11837).
  • cultured endothelial cells are seeded onto a matrix-coated porous lamina, with pore sizes generally smaller than typical cell size.
  • the matrix generally simulates the environment of the extracellular matrix, as described above.
  • the lamina is typically a membrane, such as the transwell polycarbonate membrane (Corning Costar Co ⁇ oration, Cambridge, MA), and is generally part of an upper chamber that is in fluid contact with a lower chamber containing pro-angiogenic stimuli. Migration is generally assayed after an overnight incubation with stimuli, but longer or shorter time frames may also be used. Migration is assessed as the number of cells that crossed the lamina, and may be detected by staining cells with hemotoxylin solution (VWR Scientific, South San Francisco, CA), or by any other method for determining cell number. In another exemplary set up, cells are fluorescently labeled and migration is detected using fluorescent readings, for instance using the Falcon HTS FluoroBlok (Becton Dickinson).
  • a preferred assay system for migration/invasion assays comprises testing an MBCAT's response to a variety of pro-angiogenic factors, including tumor angiogenic and inflammatory angiogenic agents, and culturing the cells in serum free medium.
  • spheroid embedded in a collagen gel-based matrix.
  • the spheroid can serve as a starting point for the sprouting of capillary-like structures by invasion into the extracellular matrix (termed “cell sprouting") and the subsequent formation of complex anastomosing networks (Korff and Augustin, 1999, J Cell Sci 112:3249-58).
  • cell sprouting the extracellular matrix
  • spheroids are prepared by pipetting 400 human umbilical vein endothelial cells into individual wells of a nonadhesive 96-well plates to allow overnight spheroidal aggregation (Korff and Augustin: J Cell Biol 143: 1341-52, 1998).
  • Spheroids are harvested and seeded in 900 ⁇ l of methocel-collagen solution and pipetted into individual wells of a 24 well plate to allow collagen gel polymerization. Test agents are added after 30 min by pipetting 100 ⁇ l of 10-fold concentrated working dilution of the test substances on top of the gel. Plates are incubated at 37°C for 24h. Dishes are fixed at the end of the experimental incubation period by addition of paraformaldehyde. Sprouting intensity of endothelial cells can be quantitated by an automated image analysis system to determine the cumulative sprout length per spheroid.
  • ELISA enzyme-linked immunosorbant assay
  • screening assays described for small molecule modulators may also be used to test antibody modulators.
  • primary assays may test the ability of the nucleic acid modulator to inhibit or enhance MBCAT gene expression, preferably mRNA expression.
  • expression analysis comprises comparing MBCAT expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express MBCAT) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art.
  • MBCAT mRNA expression is reduced in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al, Biotechniques (1999) 26: 112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm DH and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47).
  • the nucleic acid modulator e.g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al, Biotechniques (1999) 26: 112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm DH and Guiseppi-Elie, A Curr
  • Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the MBCAT protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra).
  • screening assays described for small molecule modulators may also be used to test nucleic acid modulators.
  • Secondary assays may be used to further assess the activity of MBCAT- modulating agent identified by any of the above methods to confirm that the modulating agent affects MBCAT in a manner relevant to the beta-catenin pathway.
  • MBCAT-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a modulating agent on a particular genetic or biochemical pathway or to test the specificity of the modulating agent's interaction with MBCAT.
  • Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express MBCAT) in the presence and absence of the candidate modulator.
  • such assays test whether treatment of cells or animals with a candidate MBCAT-modulating agent results in changes in the beta-catenin pathway in comparison to untreated (or mock- or placebo- treated) cells or animals.
  • Certain assays use "sensitized genetic backgrounds", which, as used herein, describe cells or animals engineered for altered expression of genes in the beta-catenin or interacting pathways.
  • Cell based assays may detect endogenous beta-catenin pathway activity or may rely on recombinant expression of beta-catenin pathway components. Any of the aforementioned assays may be used in this cell-based format.
  • Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.
  • mice Female athymic nude mice (Taconic, Germantown, ⁇ Y) to support an intense vascular response.
  • Mice with Matrigel® pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous (IN) routes with the candidate modulator. Mice are euthanized 5 - 12 days post-injection, and the Matrigel® pellet is harvested for hemoglobin analysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel is found to correlate the degree of neovascularization in the gel.
  • Candidate modulator treatment is initiated on the day the mean tumor weight reaches 100 mg.
  • Candidate modulator is delivered IV, SC, IP, or PO by bolus administration.
  • dosing can be performed multiple times per day.
  • the tumor weight is assessed by measuring pe ⁇ endicular diameters with a caliper and calculated by multiplying the measurements of diameters in two dimensions.
  • the excised tumors maybe utilized for biomarker identification or further analyses.
  • xenograft tumors are fixed in 4% paraformaldehyde,
  • Tumorogenicity and modulator efficacy may be evaluated by assaying the quantity of viable cells present in the macrocapsule, which can be determined by tests known in the art, for example, MTT dye conversion assay, neutral red dye uptake, trypan blue staining, viable cell counts, the number of colonies formed in soft agar, the capacity of the cells to recover and replicate in vitro, etc.
  • a tumorogenicity assay use a transgenic animal, usually a mouse, carrying a dominant oncogene or tumor suppressor gene knockout under the control of tissue specific regulatory sequences; these assays are generally referred to as transgenic tumor assays.
  • tumor development in the transgenic model is well characterized or is controlled.
  • the "RIPl-Tag2" transgene comprising the SV40 large T-antigen oncogene under control of the insulin gene regulatory regions is expressed in pancreatic beta cells and results in islet cell carcinomas (Hanahan D, 1985, Nature 315:115-122; Parangi S et al, 1996, Proc Natl Acad Sci USA 93: 2002-2007; Bergers G et al, 1999, Science 284:808-812).
  • the RIP1-TAG2 mice die by age 14 weeks.
  • Candidate modulators may be administered at a variety of stages, including just prior to the angiogenic switch (e.g., for a model of tumor prevention), during the growth of small tumors (e.g., for a model of intervention), or during the growth of large and/or invasive tumors (e.g., for a model of regression).
  • Tumorogenicity and modulator efficacy can be evaluating life-span extension and/or tumor characteristics, including number of tumors, tumor size, tumor mo ⁇ hology, vessel density, apoptotic index, etc.
  • the invention also provides methods for modulating the beta-catenin pathway in a cell, preferably a cell pre-determined to have defective or impaired beta-catenin function (e.g. due to overexpression, underexpression, or misexpression of beta-catenin, or due to gene mutations), comprising the step of administering an agent to the cell that specifically modulates MBCAT activity.
  • the modulating agent produces a detectable phenotypic change in the cell indicating that the beta-catenin function is restored.
  • the phrase "function is restored", and equivalents, as used herein, means that the desired phenotype is achieved, or is brought closer to normal compared to untreated cells. For example, with restored beta-catenin function, cell proliferation and/or progression through cell cycle may normalize, or be brought closer to normal relative to untreated cells.
  • the invention also provides methods for treating disorders or disease associated with impaired beta-catenin function by administering a therapeutically effective amount of an MBCAT -modulating agent that modulates the beta-catenin pathway.
  • the invention further provides methods for modulating MBCAT function in a cell, preferably a cell pre-determined to have defective or impaired MBCAT function, by administering an MBCAT -modulating agent. Additionally, the invention provides a method for treating disorders or disease associated with impaired MBCAT function by administering a therapeutically effective amount of an MBCAT -modulating agent.
  • Various expression analysis methods can be used to diagnose whether MBCAT expression occurs in a particular sample, including Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR, and microarray analysis, (e.g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al, Biotechniques (1999) 26:112-125; Kallioniemi OP, Ann Med 2001, 33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001, 12:41-47).
  • Tissues having a disease or disorder implicating defective beta-catenin signaling that express an MBCAT are identified as amenable to treatment with an MBCAT modulating agent.
  • the beta-catenin defective tissue overexpresses an MBCAT relative to normal tissue.
  • a Northern blot analysis of mRNA from tumor and normal cell lines, or from tumor and matching normal tissue samples from the same patient, using full or partial MBCAT cDNA sequences as probes can determine whether particular tumors express or overexpress MBCAT.
  • the TaqMan® is used for quantitative RT-PCR analysis of MBCAT expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).
  • reagents such as the MBCAT oligonucleotides, and antibodies directed against an MBCAT, as described above for: (1) the detection of the presence of MBCAT gene mutations, or the detection of either over- or under-expression of MBCAT mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of MBCAT gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the signal transduction pathway mediated by MBCAT.
  • the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in MBCAT expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for MBCAT expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of the disease or disorder.
  • the disease is cancer.
  • the probe may be either DNA or protein, including an antibody.
  • Drosophila beta-catenin screen Two dominant loss of function screens were carried out in Drosophila to identify genes that interact with the Wg cell signaling molecule, beta -catenin (Riggleman et al. (1990) Cell 63:549-560; Peifer et al. (1991) Development 111:1029-1043). Late stage activation of the pathway in the developing Drosophila eye leads to apoptosis (Freeman and Bienz (2001) EMBO reports 2: 157-162), whereas early stage activation leads to an overgrowth phenotype. We discovered that ectopic expression of the activated protein in the wing results in changes of cell fate into ectopic bristles and wing veins.
  • transgene was carried in a separate fly stock: Stocks and genotypes were as follows: eye overgrowth transgene: isow; P ⁇ 3.5 eyeless-Gal4 ⁇ ; P ⁇ arm(S56F)-pExp- UAS) ⁇ /TM6b; eye apoptosis transgene: y w; P ⁇ arm(S56F)-pExp-GMR ⁇ /CyO; and wing transgene: P ⁇ arm( ⁇ N)-pExp-VgMQ ⁇ /FM7c In the first dominant loss of function screen, females of each of these three transgenes were crossed to a collection of males containing genomic deficiencies.
  • Resulting progeny containing the transgene and the deficiency were then scored for the effect of the deficiency on the eye apoptosis, eye overgrowth, and wing phenotypes, i.e., whether the deficiency enhanced, suppressed, or had no effect on their respective phenotypes. All data was recorded and all modifiers were retested with a repeat of the original cross. Modifying deficiencies of the phenotypes were then prioritized according to how they modified each of the three phenotypes.
  • Transposons contained within the prioritized deficiencies were then screened as described. Females of each of the three transgenes were crossed to a collection of 4 types of transposons (3 piggyBac-based and 1 P-element-based). The resulting progeny containing the transgene and the transposon were scored for the effect of the transposon on their respective phenotypes. All data was recorded and all modifiers were retested with a repeat of the original cross. Modifiers of the phenotypes were identified as either members of the Wg pathway, components of apoptotic related pathways, components of cell cycle related pathways, or cell adhesion related proteins.
  • MBCATs as available from National Center for Biology Information (NCBI), MBCAT protein Genbank identifier number (GI#), MBCAT name, and MBCAT description, all available from Genbank, respectively.
  • NCBI National Center for Biology Information
  • GI# MBCAT protein Genbank identifier number
  • MBCAT name MBCAT name
  • MBCAT description all available from Genbank, respectively.
  • the length of each amino acid is in the "MBCAT Protein Length" column. 5 Names and Protein sequences of Drosophila modifiers of beta-catenin from screen
  • Example I are represented in the "Modifier Name” and “Modifier GI_AA” column by GI#, respectively.
  • Fluorescently-labeled MBCAT peptide/substrate are added to each well of a 96- well microtiter plate, along with a test agent in a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Changes in fluorescence polarization, determined by using a Fluorolite FPM-2 Fluorescence Polarization Microtiter System (Dynatech Laboratories, Inc), relative to control values indicates the test compound is a candidate modifier of MBCAT activity.
  • 33 P-labeled MBCAT peptide is added in an assay buffer (100 mM KC1, 20 mM HEPES pH 7.6, 1 mM MgCl 2 , 1% glycerol, 0.5% NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors) along with a test agent to the wells of a Neutralite-avidin coated assay plate and incubated at 25°C for 1 hour. Biotinylated substrate is then added to each well and incubated for 1 hour. Reactions are stopped by washing with PBS, and counted in a scintillation counter. Test agents that cause a difference in activity relative to control without test agent are identified as candidate beta- catenin modulating agents.
  • NCI National Cancer Institute
  • ATCC American Type Culture Collection, Manassas, VA 20110-2209
  • Normal and tumor tissues are obtained from Impath, UC Davis, Clontech, Stratagene, Ardais, Genome Collaborative, and Ambion.
  • TaqMan® analysis is used to assess expression levels of the disclosed genes in various samples.
  • Primers for expression analysis using TaqMan® assay are prepared according to the TaqMan® protocols, and the following criteria: a) primer pairs are designed to span introns to eliminate genomic contamination, and b) each primer pair produced only one product. Expression analysis is performed using a 7900HT instrument.
  • TaqMan® reactions are carried out following manufacturer's protocols, in 25 ⁇ l total volume for 96-well plates and 10 ⁇ l total volume for 384-well plates, using 300nM primer and 250 nM probe, and approximately 25ng of cDNA.
  • the standard curve for result analysis is prepared using a universal pool of human cDNA samples, which is a mixture of cDNAs from a wide variety of tissues so that the chance that a target will be present in appreciable amounts is good.
  • the raw data are normalized using 18S rRNA (universally expressed in all tissues and cells). For each expression analysis, tumor tissue samples are compared with matched normal tissues from the same patient.
  • a gene is considered overexpressed in a tumor when the level of expression of the gene is 2 fold or higher in the tumor compared with its matched normal sample.
  • a universal pool of cDNA samples is used instead.
  • a gene is considered overexpressed in a tumor sample when the difference of expression levels between a tumor sample and the average of all normal samples from the same tissue type is greater than 2 times the standard deviation of all normal samples (i.e., Tumor - average(all normal samples) > 2 x STDEV(all normal samples) ).
  • a modulator identified by an assay described herein can be further validated for therapeutic effect by administration to a tumor in which the gene is overexpressed. A decrease in tumor growth confirms therapeutic utility of the modulator.
  • the likelihood that the patient will respond to treatment can be diagnosed by obtaining a tumor sample from the patient, and assaying for expression of the gene targeted by the modulator.
  • the expression data for the gene(s) can also be used as a diagnostic marker for disease progression.
  • the assay can be performed by expression analysis as described above, by antibody directed to the gene target, or by any other available detection method.

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Abstract

L'invention concerne des gènes MBCAT identifiés comme modulateurs de la voie de la béta-caténine, et se prêtant par conséquent comme cibles thérapeutiques pour les troubles associés au fonctionnement défectueux de la béta-caténine. Font également l'objet de cette invention, des procédés d'identification de modulateurs de la béta-caténine, consistant à cribler les agents modulant l'activité de MBCAT.
PCT/US2003/041404 2002-12-30 2003-12-29 Mbcat servant de modificateurs de la voie de beta-catenine et leurs procedes d'utilisation WO2004061123A2 (fr)

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WO2008107900A2 (fr) * 2007-03-07 2008-09-12 Yeda Research And Development Co. Ltd. Compositions et procédés permettant de moduler une migration cellulaire

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LESORT M. ET AL: 'Glycogen synthase kinase-3ß, ß-catenin, and tau in postmortem bipolar brain' JOURNAL OF NEURAL TRANSMISSION vol. 106, no. 11-12, 1999, pages 1217 - 1222, XP002981251 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008107900A2 (fr) * 2007-03-07 2008-09-12 Yeda Research And Development Co. Ltd. Compositions et procédés permettant de moduler une migration cellulaire
WO2008107900A3 (fr) * 2007-03-07 2009-03-12 Yeda Res & Dev Compositions et procédés permettant de moduler une migration cellulaire

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