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WO2004093919A2 - Cible pour la therapie du cancer et la decouverte de medicaments - Google Patents

Cible pour la therapie du cancer et la decouverte de medicaments Download PDF

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WO2004093919A2
WO2004093919A2 PCT/GB2004/001743 GB2004001743W WO2004093919A2 WO 2004093919 A2 WO2004093919 A2 WO 2004093919A2 GB 2004001743 W GB2004001743 W GB 2004001743W WO 2004093919 A2 WO2004093919 A2 WO 2004093919A2
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fbx7
inhibitor
cancer
treatment
fbxv
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WO2004093919A3 (fr
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Heike Laman
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University College London
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11022—Cyclin-dependent kinase (2.7.11.22)
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    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the invention relates to the treatment of cancers.
  • the invention also relates to methods for the identification of substances useful in the treatment of cancers.
  • Cyclins are activators of a large family of cyclin-dependent kmases (Cdk), whose phase specific activity drive cell division.
  • D-type cyclins are expressed early in Gl phase and their activity is mitogen-dependent. They have a half-life of approximately 15 - 30 minutes. Thus continual mitogenic stimulation is needed to accumulate sufficient levels of active D- type cyclin/Cdk complexes, and later cyclin E/Cdk2 complexes, to traverse a restriction point in Gl phase, when continued passage through the cell cycle becomes mitogen-independent.
  • the short-lived nature of D-type cyclins enables them to act as sensors to the suitability of the external environment to cell proliferation.
  • D-type cyclins There are three cellular D-type cyclins (Dl, D2, and D3) and each can activate either Cdk4 or Cdk6. These kinases phosphorylate proteins from the retinoblastoma family, pRb, pi 07 and pi 30. pRb is inactivated by multiple phosphorylation events by D-type cyclin/Cdk4 or Cdk6 and cyclin E/Cdk2 complexes. Cyclin/Cdk complexes are further regulated by two families of cyclin- dependent kinase inhibitors (CDKI), the Cip/Kip, which includes p21, p27, and p57, as well as the INK4a family, which includes pi 5, pi 6, pi 8, and pi 9.
  • CDKI cyclin- dependent kinase inhibitors
  • cyclin D/pRb/INK4a pathway This signalling pathway is referred to as the cyclin D/pRb/INK4a pathway as numerous mutations are found along it to either inactivate the tumour suppressors (pi 6 or pRb), or to enhance D-type cyclin/Cdk activity, by over-expression of either the cyclin or the kinase subunit.
  • D-type cyclins are considered proto-oncogenes, and in fact, cyclin Dl has been established as a proto-oncogene in multiple types of cancer, including breast cancer, parathyroid adenoma, and some lymphomas and leukemias. In some cases, the over-expression of cyclin Dl has been linked to amplification of the locus or translocation adjacent to a strong enhancer. Also, cyclins D2 and D3 are suspected proto-oncogenes in several types of lymphomas, myelomas, and gonadal cancers.
  • Protein abundance is regulated both at the level of rates of synthesis and of degradation.
  • the poly-ubiquitination through lysine 68 of ubiquitm is a cellular signal for marking proteins for delivery to and degradation by the 26s proteasome.
  • poly-ubiquitination through lysine 63 has proteasome-independent functions.
  • the ligation of 76 amino acid ubiquitin peptides onto lysine residues of proteins is catalyzed by a cascade of enzymes. These enzymes are involved in ubiquitin activation (El), conjugation (E2), and ligation (E3) onto substrates.
  • the best characterised E3 activities include the anaphase-promoting complex (APC), the HECT domain proteins, and the SCF complexes.
  • SCF E3 ubiquitin ligases are comprised of multiple subunits, including Skpl, a cullin protein (Cull/CDC53), a RING finger protein (ROCl/Rbxl), and an F box protein.
  • F box proteins are characterised by the presence of an F box motif, which binds the Skp 1 subunit.
  • the F box protein subunit of SCF ubiquitin ligases is thought to determine the specificity of the E3 ubiquitin ligase for the phosphorylated target protein.
  • D-type cyclins are poly-ubiquitinated proteins. Furthermore, they have a short half-life, although the proteins regulating their turnover have not yet been identified. Summary of the invention
  • This invention is based on the identification of an F box protein, Fbx7, which is part of an E3 ubiquitin ligase.
  • Fbx7 which is part of an E3 ubiquitin ligase.
  • the inventor has shown that SCF Fbx7 specifically regulates cyclin/cdk ⁇ -cornplexes and that over-expression of Fbx7 causes an invasive phenotype in otherwise non-invasive cells.
  • Fbx7 interacts with cellular and viral D-type cyclins, but distinguishes between cdk4 and cdk6, interacting specifically with cdk6. Knockdown of Fbx7 does not affect cyclin or cdk6 abundance but decreases levels of cdk6 associated with D-type cyclins. Conversely, over-expression of Fbx7 increases cyclin D/cdk6 association. Expression of Fbx7 in NIH3T3 cells renders them transformed and tumorigenic in mice. Fbx7 transformation is dependent on cdk6, as knockdown by siRNA abrogates these phenotypes.
  • Fbx7 is highly expressed in epithelial and hematological tumors, but is absent in corresponding normal tissues, indicating Fbx7 represents a target for the treatment of cancers in humans. According to the invention there is thus provided use of an inhibitor of Fbx7 in the manufacture of a medicament for use in the treatment of a cancer.
  • the invention also provides:
  • composition comprising an inhibitor of Fbx7 and a pharmaceutically acceptable carrier or diluent;
  • a method for the treatment of a host suffering from a cancer which method comprises administering to the host a therapeutically effective amount of an inhibitor ofFbx7;
  • a method for the treatment of a host suffering from a cancer which method comprises inhibiting Fbx7 in the host;
  • a method for identifying a substance suitable for use in the treatment of a cancer comprises determining whether or not a test substance is an inhibitor of Fbx7, thereby to determine whether or not the test substance is suitable for use in the treatment of a cancer; - an inhibitor identified by such a method;
  • an inhibitor of the invention for use in a method of treatment of a cancer - an inhibitor of the invention for use in a method of treatment of a cancer. - use of an inhibitor of the invention in the manufacture of a medicament for use in the treatment of a cancer;
  • a pharmaceutical composition comprising an inhibitor of the invention and a pharmaceutically acceptable carrier or diluent; - a method for the treatment of a host suffering from a cancer, which method comprises administering to the host a therapeutically effective amount of an inhibitor of the invention;
  • a method for the preparation of a pharmaceutical composition comprises: (a) identifying a substance suitable for use in the treatment of a cancer by a method as set out above; and (b) formulating the inhibitor identified in (a) with a pharmaceutically acceptable carrier or diluent; and
  • Fbx7 interacts with viral cyclin in a yeast two hybrid screen.
  • A pGAD-Fbx7 interacts specifically with HVS bait. Yeast, co-transformed with the indicated pGBD bait plasmids and pGAD clones, are grown on media selecting for the plasmids (SC-ura-leu) and on media selecting for activation of reporter genes (SC-ura-leu-his-ade).
  • B Schematic diagram of Fbx7. Location of the F box motif (black), C-terminal proline rich domain (dark grey), the Ubl motif (light grey) are indicated. Fbx7 sequences contained within the GAD-Fbx7 fusion protein are shown below.
  • Fbx7 interacts with D-type cyclins and specifically with cdk6.
  • A Fbx7 co-immunoprecipitates with viral and cellular D-type cyclins.
  • U20S cells were co-transfected with flag-tagged cyclins and a T7-tagged allele of Fbx7 (129-398) that overlaps the pGAD-Fbx7 clone.
  • Fbx7 was present in the anti-flag immunoprecipitates of the various cyclins as evidence by immuno-blotting for the T7 epitope.
  • B Fbx7 co-immunoprecipitates with endogenous cdk6, but not cdk4 or cdk2.
  • U20S cells were transfected with T7-tagged Fbx7. Fbx7 was detected in immunoprecipitates of these lysates only with the cdk6 antibody.
  • C GST-cdk6 fusion proteins purified from bacterial extracts pull down endogenous Fbx7 from lysates of U20S cells. Arrow indicates the GST-cdk6 fusion protein.
  • D Mapping cdk6-interaction domains on Fbx7 using a panel of 3D-PSSM-designed Fbx7 constructs.
  • Immunoprecipitates were conducted as in B from lysates from U20S cells that were transfected with Fbx7 deletion constructs, as indicated. Upper panels show the expression of each of the Fbx7 deletion mutants; bottom panels show corresponding anti-cdk6 immunoprecipitations.
  • E Deletion of the F box domain within Fbx7 abrogates binding to endogenous Skpl. Co-immunoprecipitation of endogenous Skpl with Fbx7 is dependent upon the presence of the F box domain in Fbx7.
  • F Schematic summarizing the data mapping the domains on Fbx7 that interact with cdk6. Figure 3. Fbx7 increases cyclin/cdk ⁇ levels.
  • A Total cell lysates from U20S cells with endogenous or reduced Fbx7 levels were analyzed by Western blotting for cell cycle proteins, as indicated. Knockdown of Fbx7 protein correlates with a reduction in the levels of cdk ⁇ , p21, and p27 protein, while D-cyclin and cdk4 levels are unchanged.
  • B Knock down of Fbx7 reduces endogenous cyclin Dl association with cdk6 levels.
  • U20S cells were transfected with dsRNA that was either a non- silencing control (C) or directed against Fbx7 (F) sequences.
  • Lysates were immunoprecipitated with antibodies to cdk6 or an isotype (iso) control and endogenous cyclin Dl was detected by immunoblotting.
  • C Schematic timeline of experiments conducted in figures D and E.
  • D Reduction or over-expression of Fbx7 affects the levels of cdk6 associated with cyclin D3. After dsRNA transfection,
  • U20S cells were transfected with flag-tagged cyclin D3.
  • Fbx7 was co-transfected in lane 3. Immunoprecipitates using the flag-epitope were analyzed by immunoblotting for cdk4 and cdk6. Levels of cdk6 protein associated with cyclin correlate with an increase or decrease in Fbx7 levels, while cdk4 protein is not significantly changed. * indicates a non-specific protein band which serves as an internal loading control.
  • E Decreasing Fbx7 levels reduces the amount of cdk6 assembled de novo with cellular and viral cyclins. Experiments were conducted as outlined for Figure 3D.
  • Figure 4. NIH3T3 cells are transformed by stable Fbx7 over-expression.
  • A Western analysis for the T7 epitope on Fbx7 in total lysates from either 3T3/vec cells or 3T3/Fbx7 cells.
  • B Cells over-expressing Fbx7 have increased amounts of cyclin/cdk6 complexes. Cyclins Dl and D3 transfected into 3T3/vec or 3T3/Fbx7 cells were assayed for co-immunoprecipitation of cdk4 and cdk6.
  • C Light microscopy of 3T3/vec and 3T3/Fbx7 cells show that Fbx7 expressing cells have an altered morphology.
  • Fbx7 over-expression causes disorganization of F-actin and a loss of stress fibers (indicated by white arrows).
  • Cells were fixed and stained with rhodamine conjugated to phalloidin, and visualized by confocal microscopy.
  • E 3T3/vec and 3T3/Fbx7 cells were assayed for their ability to invade a reconstituted extra-cellular membrane matrix.
  • NIH3T3 and H1299 cells were used as a negative and positive control, respectively.
  • Membranes were stained 72 hours after seeding, and cells that invaded through the ECM appear violet.
  • F 3T3/Fbx7 cells undergo anchorage-independent growth.
  • Soft agar assays show a five-fold increase in the number of colonies formed by 3T3/Fbx7 cells compared to 3T3/vec control cells. Experiments were conducted in triplicate and repeated four times. A representative experimental result is shown.
  • H&E hematoxylin and eosin
  • IHC immunohistochemistry
  • FIG. 5 Knockdown of cdk6 reverses the transformed phenotypes of 3T3/Fbx7 cells.
  • 5x105 cells of the indicated cells were plated into 60mm dishes. After 10 days, cells were fixed in methanol and stained with Giemsa and photographed. D: 3T3/Fbx7 cells with reduced cdk6 lost their invasive phenotype. Cells were assayed for the ability to invade a reconstituted
  • ECM as described in Figure 4E.
  • E The morphology of 3T3/Fbx7 cells with reduced cdk6 levels resembled that of the parental cells. Cells were visualized by phase contrast light microscopy, and photographed.
  • Fbx7 is expressed in normal proliferating and cancerous cells, but not in normal resting cells.
  • B Fbx7 expression was induced by culturing primary CD3+ cells in vitro. Cells were freshly harvested, and half were lysed immediately, while the remainder was cultured. Lysates were analyzed by immunoblotting for the presence of indicated proteins.
  • Lung cancer cell lines exhibit variability in their Gl cdk profiles. Fbx7 was detected in all cell lines tested. However, some lines had either cdk4 (L47) or cdk6 (L23R, H1299) or both kinases (H61P, U1810).
  • SEQ ID NO: 1 sets out the nucleic acid sequence of the coding region of the human Fbx7 gene.
  • SEQ ID NO: 2 sets out the amino acid sequence of the human Fbx7 polypeptide.
  • SEQ ID NO: 3 sets out the sequence of a primer used to amplify a sequence encoding amino acids 129-381 of Fbx7 from the two-hybrid pGAD-Fbx7 (VTP30) clone.
  • SEQ ID NO: 4 sets out the sequence of a primer used to amplify a sequence encoding amino acids 129-381 of Fbx7 from the two-hybrid pGAD-Fbx7 (VTP30) clone.
  • SEQ ID NO: 5 sets out the sequence of a primer used to amplify a sequence encoding amino acids 239-522 of Fbx7.
  • SEQ ID NO: 6 sets out the sequence of a primer used to amplify a sequence encoding the 5' region (amino acids 1 to 237) of Fbx7.
  • SEQ ID NO: 7 sets out the sequence of a primer used to amplify a sequence encoding the 5' region (amino acids 1 to 237) of Fbx7.
  • SEQ ID NO: 8 sets out the sequence of a primer used to amplify the Rbxl coding sequence.
  • SEQ ID NO: 9 sets out the sequence of a primer used to amplify the Rbxl coding sequence.
  • SEQ ID NO: 10 sets out the sequence of a primer used to amplify a sequence encoding an F-box deletion of Fbx7.
  • SEQ ID NO: 11 sets out the sequence of a primer used to amplify a sequence encoding an F-box deletion of Fbx7.
  • SEQ ID NO: 12 sets out the sequence of a primer used to amplify a sequence encoding amino acids 169-398 of Fbx7.
  • SEQ ID NO: 13 sets out the sequence of a primer used to amplify a sequence encoding amino acids 169-398 of Fbx7.
  • SEQ ID NO: 14 sets out the sequence of a primer used to amplify a sequence encoding amino acids 239-381 of Fbx7.
  • SEQ ID NO: 15 sets out the sequence of a primer used to amplify a sequence encoding amino acids 239-381 of Fbx7.
  • SEQ ID NO: 16 sets out the sequence of a C-terminal peptide used to generate anti-Fbx7 antibodies.
  • SEQ ID NO: 17 sets out the sequence of a primer used to amplify a full- length Fbx7 clone for stable integration.
  • SEQ ID NO: 18 sets out the sequence of an RNAi molecule used to target the Fbx7 gene.
  • SEQ ID NO: 19 sets out the sequence of a control RNAi molecule which has no homology with the human genome.
  • SEQ ID NO: 20 sets out the sequence of an RNAi molecule used to target the cdk6 gene.
  • D-type cyclins are considered proto-oncogenes, as their over-expression is associated with many different cancers, including head and neck and breast cancers.
  • D-type cyclins activate the Gl kinases, cdk4 and cdk6, which phosphorylate the retinoblastoma protein, permitting entry into the cell cycle.
  • the inventor has now shown that Fbx7 distinguishes between the Gl cdks and specifically enhances the levels of cyclin/cdk6 complexes. Cells engineered to over-express Fbx7 become transformed, exhibiting many of the phenotypes of cancerous cells, including anchorage independent growth and invasiveness.
  • Fbx7 over-expression is detected in lung, colon, and hematological cancers, supporting the conclusion that Fbx7 is a novel oncoprotein which acts by way of cyclinD/cdk6.
  • the present invention thus relates to the use of an inhibitor of the F box gene, Fbx7, in the treatment of a cancer.
  • An inhibitor of Fbx7 is a substance which reduces/attenuates/decreases or eliminates expression and/or activity of that gene or the polypeptide product thereof.
  • Expression of the Fbx7 gene in this context is used to refer to any of the steps of transcription and translation.
  • Activity of the polypeptide product of the Fbx7 gene in this context is used to refer to enzymatic activity of that polypeptide.
  • An inhibitor of Fbx7 may also be a substance which reduces/attenuates/decreases or eliminates Fbx7 activity by disrupting the the ability of Fbx7 to interact with one or more of its targets and/or substrates.
  • Such an inhibitor may disrupt the ability of Fbx7 to interact with one or more of its targets and/or substrates by, for example, breaking down the target and/or substrate, by binding to the target and or substrate such that Fbx 7 is unable to bind to it or by binding to Fbx7 so that it cannot bind to the target and/or substrate.
  • an inhibitor suitable for use in the invention may exert inhibition via any mechanism.
  • Any suitable inhibitor of expression and/or activity of Fbx7 may be employed in the present invention.
  • a suitable inhibitor will be capable of specifically inhibiting Fbx7.
  • Specific inhibitors of Fbx7 are inhibitors which inhibit Fbx7 to a substantially greater degree than any other gene/gene product, in particular other F box genes/gene products.
  • a specific inhibitor will inhibit Fbx7 to a degree of about 2 to 1000 times, for example about 10 to 500 times, in particular about 50 to 100 times that to which it inhibits any other F box gene/gene product.
  • a specific inhibitor may be one which inhibits Fbx7, but which substantially does not inhibit any other F box gene/gene product or indeed any other gene/gene product.
  • an inhibitor suitable for use in the invention is one which is capable of inhibiting a mammalian Fbx7, in particular human Fbx7.
  • the amino acid sequence of human Fbx7 is set out in Figure 1 and in SEQ ID NO: 2.
  • the coding sequence of human Fbx7 is set out in SEQ ID NO: 1.
  • the Genbank accession number for human Fbx7 is NM_012179.
  • An inhibitor of the expression of Fbx7 may act by binding directly to the promoter of the Fbx7 gene, thus preventing the initiation of transcription.
  • the inhibitor could bind to a polypeptide/polypeptide complex which is associated with the promoter and is required for transcription. This may result in reduced levels of transcription.
  • An inhibitor may inhibit expression of Fbx7 by binding directly to the ' untranslated region of the Fbx7 mRNA. This may prevent the initiation of translation.
  • an inhibitor may bind to a polypeptide/polypeptide complex associated with the untranslated region and prevent that polypeptide/polypeptide associating with the untranslated region.
  • An inhibitor of Fbx7 may act by binding to the Fbx7 gene product and inhibiting its enzymatic activity in that way. Such inhibition may be reversible or irreversible.
  • An irreversible inhibitor dissociates very slowly from its target enzyme because it becomes very tightly bound to the enzyme, either covalently or non- covalently. Reversible inhibition, in contrast with irreversible inhibition, is characterised by a rapid dissociation of the enzyme-inhibitor complex.
  • the inhibitor may be a competitive inhibitor.
  • the enzyme can bind substrate (forming an enzyme-substrate complex) or inhibitor
  • a competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.
  • the inhibitor may be a non-competitive inhibitor. In non-competitive inhibition, which is also reversible, the inhibitor and substrate can bind simultaneously to an enzyme molecule. This means that their binding sites do not overlap.
  • a non-competitive inhibitor acts by decreasing the turnover number of an enzyme rather than by diminishing the proportion of enzyme molecules that are bound to substrate.
  • the inhibitor can be a mixed inhibitor.
  • Mixed inhibition occurs when an inhibitor affects both the binding of substrate and alters the turnover number of the enzyme.
  • An inhibitor of Fbx7 may act by binding to its substrate or to one of more the polypeptides with which it forms a complex.
  • the substance may itself catalyze a reaction of the substrate or one or more polypeptides with which Fbx7 forms a complex, so that the substrate or other polypeptide(s) are not available to Fbx7.
  • the inhibitor may simply prevent the substrate or one or more polypeptides with which Fbx7 forms a complex binding to Fbx7.
  • inhibitors of Fbx7 suitable for use in the invention may reduce or eliminate the interaction between Fbx7 and one or more of the other components of the E3 ubiquitin ligase holoenzyme, i.e. cullinl or Skpl.
  • a suitable inhibitor may reduce or eliminate the interaction between Fbx7 and cdk6 and/or cdk6-associated protein.
  • the F box motif (from about amino acid 335 to about amino acid 367 to 372 of SEQ NO: 2) is required for interaction of Fbx7 with Skpl and thus an inhibitor suitable for use in the invention may inhibit Fbx7 by specifically reducing or eliminating interaction between the F box and Skpl .
  • An inhibitor suitable for use in the invention may be capable of disrupting (i.e. inhibiting) the interaction between Fbx7 and a Gl kinase, in particular cdk6, and/or a D-type cyclin. Such an inhibitor may also be capable of disrupting (i.e. inhibiting) the interaction of Fbx7 with a D-type cyclin
  • Two non-contiguous domains in Fbx7 have been shown to interact with cdk6.
  • the two domains are a small domain of about 40 amino acids (from about amino acid 129 to about amino acid 169 of SEQ ID NO: 2) and a proline rich domain (from about amino acid 398 to about amino acid 522 of SEQ ID NO: 2).
  • An inhibitor suitable for use in the invention may thus reduce or eliminate the interaction of Fbx7 with cdk6 by targetting one, or preferably both of those domains.
  • An inhibitor may target all or part of one or both of the domains set out above.
  • Suitable inhibitors may be antibody products (for example, monoclonal or polyclonal antibodies, single chain antibodies, chimaeric antibodies, CDR-grafted or humanised antibodies) which are, for example, specific to Fbx7. " Defined chemical entities, peptide and peptide mimetics, oligonucleotides or natural products may also be suitable inhibitors.
  • a suitable inhibitor may be a chemical compound, for example a small molecule.
  • An inhibitor of Fbx7 may act via an antisense mechanism or via an RNA interference mechanism (RNAi).
  • RNAi RNA interference mechanism
  • An inhibitor of Fbx7 which acts via an antisense mechanism may comprise a polynucleotide which has substantial complementarity to all or part of the mRNA of Fbx7.
  • a polynucleotide which has substantial sequence complementarity to all or part of the mRNA of Fbx7 is typically one which is capable of hybridizing to that mRNA. If the inhibitor has substantial complementarity to a part of the mRNA of Fbx7, it generally has substantial complementarity to a contiguous set of nucleotides within that mRNA.
  • a vector is used which allows for the expression of a polynucleotide which has substantial sequence complementarity to all or part of the mRNA of Fbx7 (i.e. a polynucleotide which can hybridize to that mRNA). This results in the formation of an RNA-RNA duplex which may result in the direct inhibition of translation and/or the destabilization of the target message, by rendering it susceptibility to nucleases, for example.
  • the vector will typically allow the expression of a polynucleotide which hybridizes to the ribosome binding region and/or the coding region of the Fbx7 mRNA.
  • an oligonucleotide may be delivered which is capable of hybridizing to the Fbx7 mRNA.
  • Antisense ohgonucleotides are postulated to inhibit target gene expression by interfering with one or more aspects of RNA metabolism, for example processing, translation or metabolic turnover.
  • Chemically modified ohgonucleotides may be used and may enhance resistance to nucleases and or cell permeability.
  • the vector is capable of expressing a polynucleotide which has substantial sequence complementarity to all of part of the Fbx7 mRNA.
  • a polynucleotide will be capable of hybridizing to the mRNA.
  • such a polynucleotide will be an RNA molecule.
  • Such a polynucleotide may hybridize to all or part of the Fbx7 mRNA.
  • the polynucleotide will be complementary to all of or part of such an mRNA.
  • the polynucleotide may be the exact complement of such an mRNA.
  • absolute complementarity is not required and preferred polynucleotides which have sufficient complementarity (i.e.
  • the polynucleotide may be a polynucleotide which hybridises to the Fbx7 mRNA under conditions of medium to high stringency, such as 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C.
  • the polynucleotide hybridizes to a coding region of the Fbx7 mRNA.
  • a polynucleotide may be employed which hybridises to all or part of the 5'- or 3 '-untranslated region of such an mRNA.
  • the polynucleotide will typically be at least 40, for example at least 60 or at least 80, nucleotides in length and up to 100, 200, 300, 400, 500, 600 or 700 nucleotides in length or even up to a few nucleotides, such as five or ten nucleotides, shorter than the Fbx7 full-length mRNA.
  • the polynucleotide may be expressed in a cell from a suitable vector.
  • a suitable vector is typically a recombinant replicable vector comprising a sequence which, when transcribed, gives rise to the polynucleotide (typically an RNA).
  • the sequence encoding the polynucleotide is operably linked to a control sequence which is capable of providing for the transcription of the sequence giving rise to the polynucleotide.
  • control sequence "operably linked" to a sequence giving rise to an antisense RNA is ligated in such a way that transcription of the sequence is achieved under conditions compatible with the control sequences.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for transcription to occur and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • Vectors may be used in vitro, for example for the production of antisense RNA, or used to transfect or transform a host cell.
  • the vector may also be adapted for used in vivo, for example in a method of gene therapy. Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.
  • mammalian promoters such as b-actin promoters
  • Viral promoters may also be used, for example the Moloney murine leukaemia vims long terminal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalo virus (CMV) IE promoter, herpes simplex vims promoters or adenovirus promoters. All these promoters are readily available in the art.
  • Preferred promoters are tissue specific promoters, for example promoters driving expression specifically within vascular tissue.
  • Vectors may further include additional sequences, flanking the sequence giving rise to the antisense polynucleotide, which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination.
  • retroviruses examples include retroviruses, including lentiviruses, adeno viruses, adeno-associated viruses and herpes simplex viruses. Gene transfer techniques using such viruses are will known to those skilled in the art. Retrovirus vectors, for example, may be used to stably integrate the polynucleotide giving rise to the antisense RNA into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression. In the antisense oligonucleotide approach, a suitable oligonucleotide will typically have a sequence such that it will bind to the Fbx7 mRNA.
  • a suitable oligonucleotide will typically have substantial complementarity to a contiguous set of nucleotides within the Fbx7 mRNA.
  • An antisense oligonucleotide will generally be from about 6 to about 40 nucleotides in length. Preferably it will be from 12 to 20 nucleotides in length.
  • the oligonucleotide used will have a sequence that is absolutely complementary to the target sequence. However, absolute complementarity may not be required and in general any oligonucleotide having sufficient complementarity (i.e. substantial complementarity) to form a stable duplex (or triple helix as the case may be) with the target nucleic acid is considered to be suitable.
  • the stability of a duplex (or triplex) will depend inter alia on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity between the antisense oligonucleotide and the target sequence. The system can tolerate less complementarity when longer oligonucleotides are used.
  • ohgonucleotides especially oligonucleotides of from 6 to 40 nucleotides in length, which have sufficient complementarity to from a duplex having a melting temperature of greater than 40oC under physiological conditions are particularly suitable for use in the present invention.
  • the polynucleotide may be a polynucleotide which hybridises to under conditions of medium to high stringency such as 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C.
  • Antisense oligonucleotides may be chemically modified. For example, phosphorothioate oligonucleotides may be used.
  • deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3T5'- phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-0-alkyl analogs and 2'-0-methylribonucleotide methylphosphonates.
  • mixed backbone oligonucleotides MBOs may be used. MBOs contain segments of phosphothioate oligodeoxynucleotides and appropriately placed segments of modified ohgodeoxy- or oligoribonucleotides.
  • MBOs have segments of phosphorothioate linkages and other segments of other modified oligonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or 2'- O-alkyloligoribonucleotides.
  • An inhibitor suitable for use in the invention may act via an RNA interference (RNAi) mechanism.
  • RNAi RNA interference
  • Such an inhibitor is typically a double-stranded RNA and has a sequence substantially similar to part of the Fbx7 mRNA.
  • Preferred inhibitors of this type are typically short, for example 15mers to 25mers, in particular l ⁇ mers to 23mers.
  • a particularly preferred inhibitor has the sequence :
  • inhibitors of the type described above are preferred because such inhibitors do not appear to trigger viral defence mechanisms of higher organisms.
  • Such inhibitors can be used to inhibit translation of the mRNA.
  • small fragments of sequence encoding the Fbx7 gene product are preferred because such inhibitors do not appear to trigger viral defence mechanisms of higher organisms.
  • Such inhibitors can be used to inhibit translation of the mRNA.
  • RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi . RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNA RNA RNA RNA RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNAi RNA RNA
  • the invention provides methods for the identification of substances which are inhibitors of Fbx7 (as defined above). Such substances may be used in the treatment of a cancer. Thus, the invention also provides methods for identifying a substance for use in the treatment of a cancer. Such a method typically comprises determining whether or not a test substance is an inhibitor of FbxV (as defined above). Typically, a method of the invention will comprise determining whether or not the test substance is an inhibitor of the expression and/or activity of Fbx7.
  • a suitable method of the invention comprises:
  • a functional equivalent of a promoter of the Fbx7 gene is a promoter having a sequence similar to that of the wild-type Fbx7 promoter and which retains ability to drive transcription.
  • the functional equivalent may be a promoter from a homolog or ortholog.
  • the coding sequence encodes a reporter polypeptide.
  • the reporter polypeptide may be any suitable reporter polypeptide, for example GUS, the lacZ gene product or a GFP
  • any suitable assay format may be used for carrying out such a method.
  • the method is adapted for use in a high-throughput screen.
  • the method can be carried out in a single well of a microtitre plate.
  • a cell harbouring a promote ⁇ reporter construct is used as follows: - a defined number of cells are inoculated, in for example lOO ⁇ l of growth medium, into the wells of a plastics micro-titre plate in the presence of a test substance;
  • OD optical density
  • micro-titre plates are covered and incubated at 37°C in the dark;
  • the OD is read again and expression of the reporter polypeptide assayed at convenient time intervals.
  • GUS expression may assayed by measuring the hydrolysis of a suitable substrate, for example 5-bromo-4-chloro-3- indolyl- ⁇ -D-glucoronic acid (X-gluc)or 4-methylumbelliferyl- ⁇ -glucuronide (MUG).
  • X-gluc 5-bromo-4-chloro-3- indolyl- ⁇ -D-glucoronic acid
  • MUG 4-methylumbelliferyl- ⁇ -glucuronide
  • the hydrolysis of MUG yields a product which can be measured fluorometrically.
  • GFP is quantified by measuring fluorescence at 590nm after excitation at 494nm.
  • the coding sequence may be the Fbx7 coding sequence itself.
  • the expression of Fbx7 may be followed by, for example, Northern/RNA blotting, Western/antibody blotting or biochemical assay.
  • the invention provides a further method for identifying a substance for use in the treatment of a cancer, which method comprises:
  • Activity of the polypeptide or fragment may be determined by, for example, the ability of the polypeptide or fragment to coprecipitate with Skpl and/or cdk6.
  • a polypeptide substantially similar substantial to the Fbx7 polypeptide is one which shares sequence similarity with that enzyme and also retains similar enzymatic activity thereto.
  • a fragment suitable for use in the assay will also retain similar enzymatic activity to the Fbx7 polypeptide (or a polypeptide substantially similar thereto) and preferably ability to coprecipitate with Skpl and/or cdk6.
  • Fragments of Fbx7 suitable for use in the assay described above may comprise from about amino acid 129 to about amino acid of 169 of SEQ ID NO: 2, from about amino acid 398 to about amino acid 522 of SEQ ID NO: 2, the F-box (from about amino acid 335 - to about amino acid 367 to 372 of SEQ ID NO: 2), a sequence substantially similar to any thereto or a combination of any of the above sequences.
  • the invention further provides a further method for identifying an inhibitor of
  • Fbx 7, and thus a substance suitable for use in the treatment of a cancer which method comprises: providing, as a first component, a polypeptide encoded by the Fbx7 gene, a polypeptide substantially similar thereto or a fragment of either thereof; providing, as a second component, cdk6, a polypeptide substantially similar thereto or a fragment of either thereof; contacting the two components with a test substance under conditions that, in the absence of the test substance, would permit the two components to interact; and determining whether the test substance is capable of inhibiting the interaction between the first and second components.
  • test substance is an inhibitor of FbxV and therefore whether or not it is suitable for use in the treatment of a cancer.
  • cdk The sequence of cdk is well know to those skilled in the art for use in the invention: the Genbank accession number for human FbxV is NM_001259.
  • a polypeptide which is substantially similar to cdk6 is one which shares sequence similarity with that enzyme and also retains similar enzymatic activity thereto.
  • a fragment suitable for use in the assay described above will also retain similar enzymatic activity to the cdk6 polypeptide (or a polypeptide substantially similar thereto) and preferably ability to coprecipitate with FbxV.
  • Polypeptides substantially similar to FbxV or cdk6 may be used in the assays of the invention. Such polypeptides will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98 or at least 99% sequence identity with Fbx7 or cdk ⁇ , calculated over the full length of those sequences. However, the identity may be calculated over a shorter length, for example 20, 50, 100 or more amino acids. If identity is calculated over a shorter length, it is typically done so over a contiguous length of amino acids.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
  • the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
  • Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • a functional variant may be a naturally occurring sequence, such as an allelic variant of Fbx7 or cdk6.
  • An allelic variant will generally be of human or non-human mammal, for example bovine or porcine, origin.
  • a polypeptide which is substantially similar to Fbx7 or cdk ⁇ may have a non-naturally occurring sequence.
  • a non-naturally occurring polypeptide which is substantially similar to Fbx7 or cdk ⁇ may be a modified version of one of those polypeptide, obtained by, for example, amino acid substitution, deletion or addition. Up to 1, up to 5, up to 10, up to 50 or up to 100 amino acid substitutions or deletions or additions, for example, may be made.
  • a fragment of FbxV or cdk6 may be used in an assay of the invention.
  • substitutions will be conservative substitutions, for example according to the following Table.
  • Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
  • Deletions are preferably deletions of amino acids from one or both ends of FbxV or cdk6.
  • deletions are of regions not involved in the interaction between FbxV or cdk ⁇ , i.e. not the regions at from about amino acid 129 to about amino acid 169 of SEQ ID NO: 2 or from about amino acid 398 to about amino acid 522 of SEQ ID NO: 2 (in the case of FbxV).
  • polypeptides or fragments thereof for use in the assays described above can be obtained, for example, recombinantly by any method known to those skilled in the art.
  • polypeptides may be chemically synthesized.
  • Synthetic techniques such as a solid-phase Merrifield-type synthesis, may be preferred for reasons of purity, antigenic specificity, freedom from unwanted side products and ease of production.
  • Suitable techniques for solid-phase peptide synthesis are well known to those skilled in the art (see for example, Merrifield et al., 1969, Adv. Enzymol 32, 221-96 and Fields et al., 1990, Int. J. Peptide Protein Res, 35, 161-214).
  • solid-phase synthesis methods comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain.
  • Polypeptides (or fragments or variants thereof) suitable for use in the invention may be fused to a carrier polypeptide.
  • additional amino acid residues may be provided at, for example, one or both termini of FbxV and/or cdk6 or a functional variant of either thereof for the purpose of providing a carrier polypeptide, by which the polypeptide can be, for example, affixed to a label, solid matrix or carrier.
  • a polypeptide for use in a method of the invention may be in the form of a fusion polypeptide which comprises heterologous sequences. Indeed, in practice it may often be convenient to use fusion polypeptides.
  • fusion polypeptides may be easily and cheaply produced in recombinant cell lines, for example recombinant bacterial or insect cell lines.
  • fusion polypeptides may be easy to identify and isolate.
  • fusion polypeptides will comprise a polypeptide sequence as described above and a carrier or linker sequence.
  • the carrier or linker sequence will typically be derived from a non-human, preferably a non-mammalian source, for example a bacterial source. This is to minimize the occurrence of non-specific interactions between FbxV and cdk6 if a two component assay is to be used.
  • Polypeptides may be modified by, for example, addition of histidine residues, a TV tag or glutathione S-transferase, to assist in their isolation.
  • the carrier polypeptide may, for example, promote secretion of the polypeptide from a cell or target expression of the polypeptide to the cell membrane.
  • Amino acids carriers can be from 1 to 400 amino acids in length or more typically from 5 to 200 residues in length.
  • the polypeptide may be linked to a carrier polypeptide directly or via an intervening linker sequence. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic acid or aspartic acid.
  • Suitable polypeptides for use in the methods of the invention may be chemically modified, for example, post translationally modified. For example they may be glycosylated or comprise modified amino acid residues.
  • Polypeptides can be in a variety of forms of polypeptide derivatives, including amides and conjugates with polypeptides i.e. FbxV and/or cdk6 or polypeptides substantially similar to either thereof may be so-modified.
  • Chemically modified polypeptides also include those having one or more residues chemically derivatized by reaction of a functional side group.
  • Such derivatized side groups include those which have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups and formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine.
  • the invention provides a further method for identifying a substance for use in the treatment of a cancer, which method comprises:
  • determining whether or not the cell has an invasive phenotype comprises determining whether or nor the cell is capable of invading a basement membrane, migrating through pores in a polycarbonate membrane and adhering to the underside of the membrane.
  • This assay is illustrated in detail in the Examples.
  • control experiments can be carried out, for example in which the test substance is omitted.
  • substances so-identified may be tested with other known promoters (to exclude the possibility that the test substance is a general inhibitor of gene expression) and other known polypeptides (to exclude the possibility that the test substance is a general inhibitor of enzyme activity, for example a protease).
  • the reaction mixtures may contain a suitable buffer.
  • a suitable buffer includes any suitable biological buffer that can provide buffering capability at a pH conducive to the reaction requirements of the enzyme in question.
  • the assays may be adapted so that they can be carried out in a single reaction vessel and more preferably can be carried out in a single well of a plastics microtitre plate and thus can be adapted for high through-put screening.
  • Suitable test substances include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimaeric antibodies, CDR- grafted antibodies and humanised antibodies) which are specific for the FbxV gene product.
  • antibody products for example, monoclonal and polyclonal antibodies, single chain antibodies, chimaeric antibodies, CDR- grafted antibodies and humanised antibodies
  • combinatorial libraries, defined chemical entities, peptide and peptide mimetics, oligonucleotides and natural product libraries may be screened for activity as inhibitors of FbxV in assays such as those described below.
  • the candidate substances may be chemical compounds.
  • the candidate substances may be used in an initial screen often, for example, test substances per reaction, and the test substance of those batches which show inhibition may then be tested individually.
  • a substance suitable for use in the treatment of a cancer is one which produces a measurable reduction in expression and/or activity of FbxV in an assay described above.
  • Preferred substances are those which inhibit expression and/or activity of FbxV by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%> at a concentration of the inhibitor of l ⁇ g ml-1, lO ⁇ g ml-1, lOO ⁇ g ml-1, 500 ⁇ g ml-1, lm ml-1, lOmg ml-1, lOOmg ml-l.
  • the percentage inhibition represents the percentage decrease in expression activity in a comparison of assays in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage inhibition and concentration of inhibitor may be used to define a substance suitable for use in the treatment of a cancer or other proliferative disorder. Substances having greater inhibition at lower concentrations being preferred.
  • Candidate substances suitable for use in the treatment of a cancer i.e. candidate inhibitors of FbxV which show activity in assays such as those described above, can be tested on mammalian cell lines for the ability to inhibit the activity and/or expression of FbxV and/or an ability to inhibit invasiveness.
  • Candidate inhibitors could be tested for their ability to inhibit expression and/or activity of FbxV or to inhibit invasion in a cancer cell line.
  • Inhibitors of FbxV including those identified according to a method as set out above (ie. substances identified by a method of the invention), in particular inhibitors specific for Fbx7, may be used in a method of treatment of the human or animal body by therapy.
  • the invention provides an inhibitor of Fbx7, for example an inhibitor identified in a method of the invention, for use in a method of treatment of the human or animal body by therapy.
  • the invention also provides use of such inhibitors in the manufacture of a medicament for use in the treatment of a cancer.
  • the invention also provides a method of treatment of a cancer in a host, which method comprises the step of administering to the host an effective amount of an inhibitor of FbxV, for example an inhibitor identified in a method of the invention.
  • the host may be a human or an animal.
  • the condition of a patient suffering from a cancer can be improved by administration of inhibitor of FbxV, for example an inhibitor identified in a method of the invention.
  • a therapeutically effective amount of an inhibitor of FbxV, for example an inhibitor identified in a method of the invention may be given to a patient in need thereof.
  • cancers examples include primary and secondary cancers.
  • the cancer may be, for example, a leukaemia, a lymphoma, a sarcoma, a carcinoma, or an adenocarcinoma.
  • Specific types of cancer that may be treated according to the invention include breast, colon, brain, lung, ovarian, pancreatic, stomach, skin, testicular, head, neck and tongue cancers.
  • cancers which result from the over-expression of Dl -type cyclin may be treated according to the invention.
  • Such cancers include breast cancers, B and T cell leukaemias and lymphomas, head and neck cancers and parthyroid adenoma (PRAD).
  • cancers that may be treated according to the invention are an epithelial or haematological tumor or a lymphoma.
  • An inhibitor of FbxV is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g.
  • disaggregating agents e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginte, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • a suitable inhibitor is administered to a patient.
  • the dose of a suitable inhibitor may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical dose is from about 0.1 to 500 mg.
  • Appropriate dosages may depend on a variety of factors, for example, body weight, according to the activity of the specific antagonist, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration.
  • a dose may be given, for example, once only, or more than once for example 2, 3, 4 or 5 times.
  • the dose may be given, for example daily, every other day, weekly or monthly.
  • the antisense oligonucleotides or RNA interference (RNAi) molecules described above may be administered by direct injection into the site to be treated.
  • the antisense oligonucleotides or RNAi molecules are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition.
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • the dose at which an antisense oligonucleotide or RNAi molecule is administered to a patient will depend upon a variety of factors such as the age, weight and general condition of the patient, the cancer that is being treated and the stage which the cancer or proliferative condition has reached, and the particular antisense oligonucleotide or RNAi molecule that is being administered.
  • a suitable dose may, however, be from 0.1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight.
  • a polynucleotide having substantial sequence complementarity to all or part of an mRNA of FbxV or a vector capable of expressing such a polynucleotide may be administered directly as a naked nucleic acid construct.
  • nucleic acid constructs Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
  • transfection agents include cationic agents (for example calcium phosphate and DEAE- dextran) and lipofectants (for example lipofectamTM and transfectamTM).
  • cationic agents for example calcium phosphate and DEAE- dextran
  • lipofectants for example lipofectamTM and transfectamTM.
  • nucleic acid constructs are mixed with the transfection agent to produce a composition.
  • polynucleotide, vector or composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition.
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
  • the pharmaceutical composition is administered in such a way that the polynucleotide or vector can be incorporated into cells at an appropriate area.
  • the polynucleotide of the invention is delivered to cells by a viral vector, the amount
  • 4 8 5 of virus administered is in the range of from 10 to 10 pfu, preferably from 10 to
  • Such a system may, for example, involve administering the antibiotic tetracycline, to activate gene expression via its effect on the tet repressor/VPl 6 fusion protein.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • several neurological disorders are due to aberrant expression of particular gene products in only a small subset of cells. It will be advantageous to be able express therapeutic genes in only the relevant affected cell types, especially where such genes are toxic when expressed in other cell types.
  • the routes of administration and dosages described above are intended only as a guide since a skilled physician will be able to determine readily the optimum route of administration and dosage for any particular patient and condition.
  • the invention also provides a method for the diagnosis of a cancer in a host, which method comprises determining the level of expression and/or activity of FbxV in the host or in a sample from the host, thereby to determine whether the host is suffering from a cancer.
  • the host may be a human or an animal. Abnormally high/elevated levels of FbxV (in comparison to the level in equivalent non-cancerous tissue) may be indicative of a cancer.
  • the sample may be a tissue sample, for example a sample obtained in a biopsy.
  • the invention also provides a method for determining the effectiveness of a treatment for a cancer, which method comprises determining the level of expression and/or activity of FbxV in the host or in a sample from the host, thereby to determine the effectiveness of the treatment for a cancer.
  • the host may be a human or an animal.
  • the level of expression and/or activity of FbxV in the host or in a sample from the host may be monitored during the course of treatment, in order to determine whether or not that course of treatment is having any effect.
  • the method may be used to monitor a host after a course of treatment has finished, for example during a remission period, in order to monitor potential recurrence of the cancer.
  • the invention further provides a method for determining whether a cancer will be susceptible to treatment with an inhibitor of FbxV. In such a method, the Gl kinase status of the tumor is determined. Tumors which express cdk ⁇ will be susceptible to treatment with an inhibitor of FbxV. Tumors which do not express cdk6 may not be susceptible to treatment with an inhibitor of FbxV.
  • PBMCs Peripheral blood mononuclear cells
  • NIH3T3 -derived cells were made by stable integration of FbxV cloned into pOPRSVI (Strategene). This construct or the empty parental vector was transfected into NIH3T3 cells by the calcium phosphate method, as previously described (Swanton et al, Nature 390, 184-V 199V). Clones were selected by growth in media containing 600 ⁇ g ml "1 of G418 sulfate (Cat. #11811-064, Gibco), and screened for expression by RT-PCR and Western analysis.
  • Anchorage-independent growth was assayed by growth in soft agar, essentially as previously described (Radkov et al, 2000). Cell lines were stained with phalloidin-conjugated to rhodamine (Molecular Probes), and visualised by fluorescence microscopy. Invasion assays (Cat. #ECM550, Chemicon International) were done exactly to manufacturer's instructions.
  • VTP30 The two-hybrid pGAD-FbxV (VIP30) clone was fully sequenced and found to contain a 38bp deletion from base pair 1144 - 1183. Thus the fusion protein made contained only amino acids 129 - 381 of FbxV.
  • VTP30 was used as a template for amplification with primers, including an EcoRI site
  • the 5 'sequence of FbxV was amplified from a Hela cDNA library using primers to PCR from the start site 5' CGGAATTCTTATGAGGCTGCGGGTGCGGCTTCTG 3' (SEQ ID NO: 6), and to an internal Pstl site 5' TGTACTGCAGCTTATACACCCCGCTCAACT 3' (SEQ ID NO: V). These sequences were subcloned into pBluescript KS (Stratagene) to generate Hlpl029.
  • the full-length FbxV clone was constructed by a three-way ligation using EcoRI - Pstl 5' fragment from Hip 1029, Pstl - Xhol 3' fragment from Hip 1034, which were ligated into apcDNA3 (Invitrogen) modified vector, which has either a flag epitope or a TV epitope subcloned into the Hindlll and BamHI sites. This yielded a full- length FbxV clone N-terminally tagged with either the flag (HlplOVl) or TV (Hip 1036) epitope. These clones were fully sequenced to ensure the epitope tags were in frame and that the coding sequence was not mutated during the PCR reactions. Both clones behaved identically and were used interchangeably throughout these experiments.
  • the his-tagged Cullinl clone in a pCDNA3 vector was a gift from M. Pagano.
  • the TV-Rbxl clone was made by PCR amplification from a human foetal cDNA library, a gift from H. Wang, using primers to introduce an EcoRI site 5' GCGCGGATCCATGGCGGCAGCGATGGATCTGG 3 ' (SEQ ID NO: 8) and an Xhol site
  • PCR was used to generate various deletion constructs, the clones were all fully sequenced to ensure that no mutations were introduced.
  • the F box deletion was made using the EcoRI primer above and 5 ' CCGTCGACCCACTCCTGTGGAGGTTTTTATATCTG 3 ' (SEQ ID NO: 10) which amplified the 5' of the gene from base pair 1 - 1003 and introduced a Sail site.
  • the 3 '.of the gene from base pair 1102 - 1569 was amplified with the 3 'Xhol primer and
  • FbxV (169-398) was made by PCR amplification with the following primers: 5' GGAATTCTAATGCTCTGTAGTGAATCGGTGGAA 3' (SEQ ID NO: 12); and 5' CCGCTCGAGTCAATCATTTACTTCCTGTACAGTTCTTTCCAATC 3' (SEQ ID NO: 13). The PCR fragment was subcloned as an EcoRI/XhoI fragment into pCDNA3 bearing a TV epitope tag, resulting in clone Hip 1046.
  • FbxV (129-398) used made by replacing the Bglll/Xhol fragment from Hlpl046 into Hlpl034, creating P GEX(KG)-Fbx7 (129-398). The EcoRI/XhoI fragment was subcloned into pCDNA3 bearing a TV epitope tag, resulting in clone Hlpl066.
  • FbxV (239-381) made by PCR amplification with the following primers :
  • Double-stranded RNA 5'r(UACCCGAUUUACAAUUACA)dTT3 targeting FbxV sequences bpl2V-149 and control non-silencing dsRNA (Cat. #80-11310-A) targeting sequence 5' AATTCTCCGAACGTGTCACGT 3' (SEQ ID NO: 19) which has no homology in the human genome were purchased from Xeragon, and transfected in cells using Effectene (Qiagen).
  • a double-stranded oligo, CDK6-4 which created a hairpin siRNA against cdk ⁇ sequence 5' GACTTGACCACf TACTTGG 3' (SEQ ID NO: 20) was cloned into pRETROSUPER (Brummelkamp et al., 2002). Either the empty vector or the CDK6-4 construct was transfected into a BOSC packaging cell line to make retroviruses to infect FbxV expressing NIH3T3 cell lines with selection in 2 ⁇ g/ml puromycin. Sub-cellular fractionation
  • the nuclear pellet was lysed in RIPA buffer (140mM NaCL, 50mM Tris, pH 8.0, 0.5% TritonX-100, 0.2% IGEPAL, 10% glycerol) with protease inhibitors.
  • the cytoplasmic fraction was centrifuged at 13000rpm for 15 minutes at 4°C.
  • the (Cyto-low) pellet was dissolved in RIPA, and the supernatant was further centrifuge at 55,000rpm for one hour at 4°C.
  • the (Cyto-high) pellet was dissolved in RIPA. The remaining supernatant is the cytosolic fraction. Fractionated lysates were analysed by Western analysis as indicated.
  • Cells were typically seeded the day prior to transfection with l ⁇ g of plasmid DNA using Fugene transfection reagent, as per manufacturer's instructions (Cat. #1814443, Roche). Immunoprecipitations for cell cycle proteins that associated with FbxV were done as previously described (Diehl et al., Mol. Cell Biol. IV, V362-V3V4, 199V), and immunoprecipitations for cyclin/cdk complexes were carried out essentially as previously described (Mann et al, Embo J 18, 654-63, 1999).
  • cdk2 (SC163, Santa Cruz Biotechnology, Inc.), cdk4 (SC260, Santa Cruz Biotechnology, Inc.), cdk ⁇ (SC1V7, Santa Cruz Biotechnology, Inc.), cyclin Dl (SC246, Santa Cruz Biotechnology, Inc and 28V.4, G. Peters, LRI Antibody
  • the antibody detected a protein at the predicted size of VOkD, and which was specifically competed by the immunogen (data not shown).
  • Tissue immunostaining using HLA3 at a concentration of 1/400 on paraffin embedded sections that were microwave pre- treated was performed essentially as previously described (Ye et al, Am. J. Pathol. 15V, 1147-1154, 2000).
  • cdk2 (SCI 63, Santa Cruz Biotechnology, Inc.), cdk4 (SC260, Santa Cruz Biotechnology, Inc.), cdk6 (SCI 77, Santa Cruz Biotechnology, Inc.), cyclin Dl (SC246, Santa Cruz Biotechnology, Inc and 287.4, G.
  • cyclin D2 (SCI 81, Santa Cruz Biotechnology, Inc., and Ab4, Neomarkers, Inc.), cyclin D3 (SCI 82, Santa Cruz Biotechnology, Inc.), cyclin A (SC751, Santa Cruz Biotechnology, Inc.), PCNA (SC56, Santa Cruz Biotechnology, Inc.), Skpl (SCV163, Santa Cruz Biotechnology, Inc.), M2 flag epitope (F3165, Sigma Chemicals), TV epitope (Cat. # 69522-3, Novagen), His epitope (Cat. # 8916-1, Clontech) and HA epitope (12CA5, LRI Antibody Production).
  • a Kpnl site and T7 site were introduced using the primers 5'GGGGTACCCACCATGGCTAGCATGACTGGTGGACAG 3' (SEQ ID NO: 17) and the 3' Xhol primer to amplify full-length FbxV from Hip 1036 (see below).
  • the PCR product was subcloned into pOPRSVI (Strategene) to create Hip 1049.
  • This construct or the empty parental vector were transfected into NIH3T3 cells by the calcium phosphate method, as previously described (Swanton et al., 1997, supra).
  • Cells were selected for stable integration of the T7-FbxV construct by growth in media containing 600 ⁇ g/ml of G418 sulfate (Gibco Cat. #11811-064) in 0.1M Hepes, pH 7.5, and screened for expression by Western analysis for the T7 epitope to generate a polyclonal line (HML36) or a derived clonal line (HML37), as well as a polyclonal line of the empty vector (HML35). These cell lines were stained with Hoechst 33342 and phalloidin-conjugated to rhodamine (Molecular Probes, Eugene, OR), and visualised by fluorescence microscopy. Invasion assays were performed with 300,000 cells using a cell invasion assay kit (Cat. #ECM550) according to the manufacturer's instructions (Chemicon International, Temecula, CA).
  • Fbx7 an F box protein, interacts with a viral cyclin
  • the cyclins encoded by oncogenic herpesviruses are functionally homologous to the proto-oncogenic family of D-type cyclins.
  • To discover cellular proteins important in viral or D-type cyclin-mediated oncogenesis we conducted a yeast two-hybrid screen of a human B and T cell cDNA library (Laman et al., Curr Opin Genet Dev 10, V0-V4, 2001). Using the cyclin homologue encoded by
  • Herpesvirus saimiri as bait, a single clone encoding amino acids 129 to 393 of FbxV fused with Gal4 activation domain (GAD) was isolated. Specificity for the pGAD-FbxV interaction with the HVS cyclin bait plasmid was tested by assaying for activation of the HIS3 and ADE2 reporter genes. Yeast co-transformed with the HVS cyclin bait plasmid and pGAD-FbxV, grew on media lacking histidine and adenine, whereas pGAD-FbxV in combination with either the Gal4 binding domain alone or fused to cyclin E were unable to grow (Figure 1 A).
  • FbxV was also identified in screens for proteins containing F box domains, which mediate Skpl binding, in SCF-type E3 ubiquitin ligases (Ilyin et al., Genomics 67, 40-4V, 2000; Winston et al., Curr. Biol. 9, 1180-1182, 1999; Cenciarelli et al., Curr. Biol. 9, 1177-1179, 1999).
  • Fbx7 is a 522 amino acid protein, with an F box domain at 335-372, and a C-terminal proline rich domain.
  • N-terminal Ubl motifs are found in a variety of proteins, including Parkin and Rad23, and are responsible for direct binding to proteasomal subunits (Sakata et al, EMBO Rep. 4, 301-306, 2003; Elsasser et al., Nat. Cell Biol. 4, 725- 730, 2002; Kleijnen et al., Mol. Cell 6, 409-419, 2000; Schauber et al., Nature 391, 715-718, 1998).
  • FbxV homologues Database searches for human FbxV homologues revealed that other F box- containing proteins, such as Fbx9 and Fbxl 1, have homology to FbxV, but only within their F box domains (data not shown). Searches for FbxV homologues in other species revealed that several vertebrate organisms, including M. museums, X. laevis, and R. rattus, have proteins similar to FbxV ( Figure 1C). In addition, A. thaliana has three partial homologues to FbxV (data not shown), however, none were found in C. elegans, D. melanogaster, or in S. cerevisiae.
  • Fbx7 interacts with viral and cellular D-type cyclins and specifically with cdk ⁇
  • human U20S cells were transfected with an FbxV clone spanning amino acids 129-398, and tagged with a TV epitope at the N-terminus.
  • Flag-tagged HVS cyclin and cellular D- type cyclins (Dl and D3) were tested for their ability to co-immunoprecipitate with FbxV.
  • Both cellular D-type cyclins and the HVS cyclin were able to bind to FbxV (129-398), indicating that this interaction occurred in human cells, was not unique to viral cyclins, and was not restricted to a particular cellular D-type cyclin (Figure 2A).
  • Fbx7 facilitates D-cvclin association with cdk ⁇ specifically
  • FbxV Interaction of FbxV with D-type cyclins and cellular cdk ⁇ suggested two models: either FbxV affects these proteins or FbxV is a cyclin/cdk substrate, similar to Skp2 phosphorylation by cyclinA/cdk2 complexes (Yam et al, Mol. Cell Biol. 19, 635-645, 1999). FbxV has a potential cyclin/cdk serine phosphoacceptor site located at amino acid 406 ( Figure IB).
  • FbxV is a substrate for phosphorylation by cyclin/cdk complexes
  • a GST-FbxV fusion protein was used in kinase assays as a substrate for viral and cellular D/cdk6, cyclin E/cdk2 and cyclinA cdk2 complexes.
  • Fbx7 affects D-type cyclins and/or cellular cdk ⁇ .
  • Fbx7 may function as part of an SCF-type E3 ubiquitin ligase to ubiquitinate these proteins.
  • One consequence of protein ubiquitination is degradation by the 26S proteasome.
  • reducing Fbx7 levels should increase the abundance of D-type cyclins or cdk6.
  • transient transfection of dsRNA targeting Fbx7 was used to reduce its protein levels.
  • Analysis of equal amounts of protein lysates from U20S cells, transfected with dsRNA against Fbx7 or a non-silencing control showed that endogenous Fbx7 levels were reduced >90% after 48 hours (Figure 3 A).
  • Cyclin D3 co- immunoprecipitates predominantly with cdk ⁇ , but also with cdk4, in U20S lysates, (lane 2, Figure 3D).
  • cdk ⁇ immunoprecipitated by cyclin D3 in the absence of Fbx7
  • the amount of cdk4 remained constant (lanes 2 and 5, Figure 3D).
  • Fbx7 levels were increased, greater amounts of cdk ⁇ co-immunoprecipitated with cyclin D3, whereas the level of associated cdk4 was unchanged (lanes 2 and 3, Figure 3D).
  • Viral cyclins predominantly bind and activate cellular cdk ⁇ , extending its range of substrates to include those of cyclin/cdk2 complexes (Mittnacht and Boshoff, Rev Med Virol 10, 175-84, 2000; Laman et al., Curr Opin Genet Dev 10, 70-74, 2000).
  • Viral cyclin/cdk6 complexes are exceptional in that they are not bound by p21 or p27 proteins and are therefore resistant to their inhibitory activities and are likely to be independent of their assembly function (Swanton et al., Nature 390, 184- 7, 1997).
  • D type cyclins are proto-oncogenes, and their sustained over-expression transforms NIH3T3 cells (Alt et al, Genes Dev. 14, 3102-3114, 2000). Since FbxV augmented the levels of D-cyclin associated with cdk ⁇ , the over-expression of FbxV was tested for its transforming ability.
  • NIH3T3 cells were engineered to stably over- express a TV-tagged human FbxV. Polyclonal 3T3/FbxV and 3T3/vector only cell lines were created, and a clonal line derived. Both 3T3/FbxV polyclonal and clonal cell lines were used in parallel and behaved similarly in all assays.
  • 3T3/FbxV cells had lost stress fibers and had a disorganized actin cytoskeleton when compared to 3T3/vec cells (Figure 4D), characteristics that correlate with a migratory and invasive phenotype (Ridley et al, Biochem. Soc. Symp. 65, 111-123, 1999; Nobes and Hall, Cell 81, 53-62, 1995).
  • Retroviruses were used to infect 3T3/vec and 3T3/FbxV polyclonal lines and stable lines were selected. Three independent pools of each of the cell lines were created and assayed for the expression of cdk ⁇ . Infection with pRS-cdk6 retroviruses causes a substantial reduction in the protein levels (Figure 5A).
  • NIH3T3 cells over-expressing FbxV have an increased capacity to associate D-type cyclins with cdk ⁇ . This suggests that FbxV-mediated transformation is by way of the pRb/cyclin/cdk/LNK4a pathway. This is supported by data showing the reversal of FbxV-mediated transformed phenotypes upon knockdown of cdk ⁇ protein.
  • Fbx7 is highly expressed in hematolosical and epithelial malignancies As FbxV was cloned from a B and T cell library, its expression in normal tonsil sections was analyzed. Immunostaining of normal tonsils showed that FbxV predominantly stained the cells in the germinal centres (GC), where lymphocytes are activated, while those in the surrounding mantle (MZ) and marginal zones (MGZ) were weakly stained or negative (Figure 6A). These data suggested that Fbx7 is not expressed in resting lymphocytes, but is up-regulated when cells are proliferating and activated. In order to test this more directly, peripheral blood mononuclear cells were freshly harvested from six healthy donors.
  • CD3+ T lymphocytes were purified by MACS separation and were either immediately lysed or cultured for 96 hours in the presence of 20% FBS and IL-2 prior to lysis. Equal amounts of protein from these lysates were analyzed by Western blotting for Fbx7 expression and for other cell cycle proteins. The results from one sample are shown in Figure 6B, although similar results were seen for all six samples. Though absent in resting cells, Fbx7 protein was up-regulated, along with PCNA, D-cyclins, cdk ⁇ , and cyclin A, when placed in culture, whereas p27, which is highly expressed in quiescent lymphocytes decreased upon culturing in vitro. Thus the up-regulation of Fbx7 protein correlates with that of other proteins that promote cell cycle progression.
  • the F box protein, Skp2 targets cell cycle regulators for ubiquitin-mediated degradation, and its over-expression in cancers suggests it has an oncogenic role (Pagano and Benmaamar, Cancer Cell 4, 251-256, 2003).
  • the transforming effects of Fbx7 over-expression suggest it may be similarly oncogenic.
  • Immunohistochemistry on tissue sections from several solid or hematological tumors was undertaken to determine the profile of Fbx7 expression in cancers. Sections from two different types of lymphoma were stained.
  • LLBCL diffuse large B cell lymphomas
  • Fbx7 in D-cyclin/cdk ⁇ association and cell cycle progression, which is supported by the finding that Fbx7 is not detected in resting cells, but is expressed in proliferating lymphocytes. Its expression also correlates with that of other cell cycle regulators.
  • the presence of Fbx7 in lung and colon cancers and in proliferative hematological cancers implies that over-expression of Fbx7 may be significant in human cancers.
  • Human osteosarcoma cells were transfected with either double-stranded RNA that targeted Fbx7 or a non-silencing control. 48 hours later, cells were transfected with HA-tagged human cdk4 or cdk6. 24 hours later cells were fixed with 4% paraformaldehyde and stained with antibodies against the HA epitope. Anti-mouse antibodies conjugated to FITC were used to visualize the sub-cellular localisation of ckd4 and cdk ⁇ by confocal microscopy.
  • Lung cancer cell lines exhibit variability in the Gl cdk profiles
  • Equal amounts of protein lysates from various lung cancer cells were assayed for the presence of Fbx7 and the cell cycle regulators as cdk4 and cdk ⁇ .
  • Fbx 7 was detected in all cell lines tested ( Figure 8). However, some cell lines had either cdk4 (L47) or cdk ⁇ (L23R, H1299) or both kinases (H61P, U1810).
  • Fbx7 specifically regulates cdk ⁇ , diagnosing the presence of Fbx7 as well as the Gl cdk status will indicate which tumors are more likely to benefit from anti-Fbx therapies.
  • Fbx7 which uniquely distinguishes between the Gl cdks. Its function specifically enhances the levels of D-cyclin/cdk ⁇ complexes, and it is transforming by way of cdk ⁇ . Intriguingly although ubiquitinated substrates of Fbx7 have not yet been identified, our data suggest that Fbx7 substrates will be linked in their regulation with that of cyclin cdk ⁇ . In this way, Fbx7 might coordinate its substrates for ubiquitination with cell cycle progression.
  • Fbx7 -transformed cells cause tumors in thymic nude mice, and Fbx7 protein is highly expressed in several different human cancers suggesting that it is a novel, putative oncoprotein acting along the pRb/cyclin/cdk/LNK4a pathway.

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L'invention concerne l'utilisation d'un inhibiteur de Fbx7 lors de la production d'un médicament destiné à être utilisé pour le traitement d'un cancer.
PCT/GB2004/001743 2003-04-22 2004-04-22 Cible pour la therapie du cancer et la decouverte de medicaments Ceased WO2004093919A2 (fr)

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