+

WO2005105064A2 - Modulateurs de l'interaction frat-lrp dans le traitement des maladies osseuses - Google Patents

Modulateurs de l'interaction frat-lrp dans le traitement des maladies osseuses Download PDF

Info

Publication number
WO2005105064A2
WO2005105064A2 PCT/IB2005/001878 IB2005001878W WO2005105064A2 WO 2005105064 A2 WO2005105064 A2 WO 2005105064A2 IB 2005001878 W IB2005001878 W IB 2005001878W WO 2005105064 A2 WO2005105064 A2 WO 2005105064A2
Authority
WO
WIPO (PCT)
Prior art keywords
fratl
lrp5
bone
disorders
interaction
Prior art date
Application number
PCT/IB2005/001878
Other languages
English (en)
Other versions
WO2005105064A3 (fr
Inventor
Chi Faucheu
Eric Hay
Georges Rawadi
Sergio Roman-Roman
Original Assignee
Proskelia Pharmaceuticals
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Proskelia Pharmaceuticals filed Critical Proskelia Pharmaceuticals
Publication of WO2005105064A2 publication Critical patent/WO2005105064A2/fr
Publication of WO2005105064A3 publication Critical patent/WO2005105064A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/502—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 non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/5044—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 involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • Wnts are cysteine-rich secreted proteins involved in a wide range of developmental processes such as embryonic axis specification and organogenesis [Wodarz, 1998]. Wnts appear to activate a variety of signaling pathways both in vertebrates and in invertebrates. In the so- called « canonical » Wnt/R-catenin pathway, the interaction between Wnt and frizzled receptors leads to inactivation of the kinase GSK-3 ⁇ . Genetic epistasis experiments suggest that disheveled lies upstream and represses the activity of GSK-3 ⁇ . As a consequence, ⁇ -catenin is stabilized in the cytoplasm and then forms a complex with TCF/LEF to activate transcription of target genes.
  • Frizzled proteins have been shown to function as Wnt receptors [Bhanot, 1996] and they constitute a large family of seven transmembrane receptors with at least ten members in mammals [Fredriksson, 2003;Bhanot, 1996]. All frizzled receptors have a conserved extracellular cysteine-rich domain (CRD) followed by seven putative transmembrane segments. Their cytoplasmic regions differ in length and sequence. Functional analyses in Drosophila and Xenopus embryos indicate that frizzled proteins have distinct functions in Wnt/ ⁇ -catenin signaling [Sheldahl, 1999].
  • LRP low-density lipoprotein receptor-related protein family
  • FIG. 1 Fratl interacts with LRP5.
  • COS-7 cells were co-transfected with Frat1-Flag and either LRP5tail-Myc, LRP5tail ⁇ 28-Myc or LRP5tail ⁇ 78- Myc. Control were carried out by transfecting each of the expression construct alone 18 hours after transfection media was replaced by fresh culture media and cultured for additional 24 hours.
  • Immunoprecipitation (IP) was performed on total cell lysates using anti-Myc antibody.
  • Total cell lysates and immunoprecipitates were analyzed by Western blotting using either anti-Flag or anti-Myc antibody. Arrows indicate the expected band size.
  • FIG. 2 effect of LRP5C truncated mutant forms on Fratl activity.
  • COS-7 cells were transiently co-transfected with TCF-1 expression construct, TOPflash, pTK-Renilla. Where indicated, Fratl expressing vector was added with either empty vector (Vector), LRP5C, LRP5 ⁇ 28, LRP5 ⁇ 47 or LRP5 ⁇ 78. Control experiments were carried out by performing transfection with each of the used constructs alone. 18 hours after transfection media was replaced by fresh culture media and 24h later luciferase activity was determined in cell lysates and normalized to renilla signal. All experiments were performed in triplicate and repeated three times. Data ⁇ S.D. from one representative experiment are presented (p ⁇ 0.01).
  • COS-7 cells were transfected with control vector (I), LRP5C (II) LRP5 ⁇ 78 (III), Fratl (IV), LRP5C and Fratl (V) or LRP5 ⁇ 78 and Fratl (VI). 18 hours after transfection media was replaced by fresh culture media and cultured for additional 24 hours. Cells were immuno-stained for Fratl expression using a mouse anti-Flag antibody and revealed by a goat anti-mouse antibody conjugated to rhodamine (red fluorescence). Fluorescent cells were visualized under confocal microscopy. Experiment was repeated three times and photos from one representative experiment are shown.
  • LRP5 expression is necessary for Fratl activity.
  • Primary fiboblastics cells from wild type mouse (lrp5 ⁇ ), heterozygote (Irp5 ⁇ , black box) or homozygote (Irp5 y ) were transiently co-transfected with TCF-1 expression construct, TOPflash, pTK-Renilla. Empty control vector or Fratl expression construct was added to the transfection mix. 18 hours after transfection media was replaced by fresh culture media. Cells tranfected with empty vector were either left untreated (CTRL) or treated with Wnt3a-CM (Wnt3a). 24h later luciferase activity was determined in cell lysates and normalized to renilla signal. All experiments were performed in triplicate and repeated three times. Data ⁇ S.D. from one representative experiment are presented (p ⁇ 0.01 ).
  • FIG. 4 Fratl is required for LRP5C activity.
  • COS-7 cells were transiently cotransfected with TCF-1 expression construct, TOPflash, pTK- Renilla. Where indicated either empty vector (Vector), LRP5C, Fratl , Fratl ⁇ N, LRP5C and Fratl or LRP5C and Fratl ⁇ N expression constrcuts were added to the transfection mix. 18 hours after transfection media was replaced by fresh culture media and 24h later luciferase activity was determined in cell lysates and normalized to renilla signal. All experiments were performed in triplicate and repeated three times. Data ⁇ S.D. from one representative experiment are presented (p ⁇ 0.01).
  • FIG. 5 Cellular localization of Fratl .
  • A C3H10T1/2 cells were transfected with Fratl -Flag tagged then either left untreated (I) or treated with Wnt3a-CM (II).
  • C3H10T1/2 cells were transfected with Frat1-Flag and one of the following expression constructs: LRP5C (III), LRP5C ⁇ 28 (IV), LRP5C ⁇ 36 (V), LRP5C ⁇ 47 (VI) or LRP5C ⁇ 78(VI).
  • Fratl localization was immunodetected by mouse anti-Flag antibody and revealed by a goat anti- mouse antibody conjugated to rhodamine (red fluorescence). This assay was performed three time and representative photos are shown.
  • B The percentage (average of three experiments) of Fratl localized at the membrane, in the cytoplasm or in the nucleus.
  • FIG. 6 Effect of disheveled dominant negative mutant on Fratl /LRP5C activity and association.
  • C3H10T1/2 cells were transfected with control vector, Fratl , LRP5C, LRP5C ⁇ 28 or LRP5C ⁇ 78 in the presence or absence of in absence or in presence of disheveled dominant negative (Xdd). Transfection mix systematically included TCF1 plasmid. When indicated cells were treated with Wnt3a-CM (Wnt3a).
  • B Cells were cotransfected with Fratl -Flag and LRP5 or LRP5C-Myc in absence or presence of Xdd expression construct.
  • IP Immunoprecipitation
  • COS-7 cells were transiently co-transfected with TCF-1 expression construct, TOPflash, pTK-Renilla. Where indicated either empty vector (Vector), Wnt3a, Fratl , LRP5C or Fratl and LRP5C expression constrcuts were added to the transfection mix.
  • Cos-7 cells were transiently transfected with Axin-Myc expression vector with either Fratl -Myc, LRP5C-GFP or both expression constructs.
  • Cells lysates were immunoprecipitated (IP) with an anti-Flag antibody (left lanes) or an anti- GFP antibody (right lanes).
  • Immunoprecipitates were analyzed by Western blotting using anti-GFP, anti-Myc or anti-Flag antibodies.
  • total cell lysates were analyzed by Western blotting using anti-Myc antibody (lower box). Arrows indicate the size of expected band.
  • FIG. 8 A conceptual model for Wnt signalling through LRP5 receptor. Following activation by Wnt, conformational modification of the LRP5 cytoplasmic domain occurs allowing the recruitment of Fratl to the membrane. The activated status of LRP5 receptor is also able to translocate Axin to the membrane where it interacts with Fratl. Axin recruitment to the membrane prevents it from enhancing the degradation of ⁇ -catenin. In the absence of Wnts, Axin is found within a protein complex that includes APC, GSK3 ⁇ and ⁇ -catenin. The recruitment of Axin by LRP5 in the presence of Wnts will bring GSK3 ⁇ close to Fratl .
  • LRP6 a close homologue of LRP5
  • Disease means herein an alteration of the health of a mammal, due to internal and/or external causes, said alteration becoming apparent through symptoms and resulting in an impairment of one or more biological functions, such as metabolic functions, and/or in one or more lesions in said mammal.
  • disorder it is meant herein a pathological modification of an organ or of a physical or psychological function in a mammal.
  • bone-related disease refers to a disorder directly or indirectly affecting bone cells, that gives rise to a condition of clinical relevance for skeletal health.
  • the mechanisms that give rise to such a disease are diverse and may be mediated by primary pathology affecting bone cells (an example is Paget's disease of bone), or indirectly.
  • Indirect mechanisms include the effects of abnormal endocrine secretion of major calcium and skeletal regulating hormones, including sex hormones (estrogen, androgen, progesterone, and the like). Examples include post-menopausal osteoporosis, primary hyper parathyroid ism and Cushing's disease. Bone disease may also arise from the local or systemic effects of cytokines such as in multiple myeloma, periodontal disease. Intrinsic bone disease may be genetic (e.g., epiphyseal dysplasia) or acquired (e.g., osteomyelitis). However, in practice, as knowledge of pathophysiology advances, the distinction between intrinsic and metabolic bone diseases becomes increasingly blurred.
  • bone-related disease encompasses at least disorders of mineral metabolism, disorders of parathyroid hormone (PTH) secretion and/or activity, metabolic bone disorders comprising osteoporosis, vitamin D-related disorders, renal bone diseases, hypophosphatasia, dysplastic disorders, infiltrative disorders, extra- skeletal calcification and ossification, miscellaneous disorders, and the like (for a literature reference, see Baron et al.).
  • PTH parathyroid hormone
  • disorders of mineral metabolism it is meant herein at least hypercalcaemia of diverse causes, hypocalcaemia of diverse causes, hyperphosphataemia, hypophosphataemia, hypermagnesaemia, hypomagnesaemia, and the like.
  • disorders of PTH secretion and/or activity are included for instance hyperparathyroidism, hypoparathyroidism, pseudohypoparathyroidism, and the like.
  • Major disorders encompass at least medullary carcinoma, skeletal toxicity syndromes (e.g., aluminium, iron>cadmium, fluorosis), alveolar bone resorption, non-union and fracture repair, bone reconstruction, ischaemic disorders, osteonecrosis, and the like.
  • a “metabolic bone disorder” includes at least osteoporosis, which may be for instance postmenauposal, involutional, secondary; as well as hypo-remodelling syndromes; and the like.
  • vitamin D-related disorders relates at least to nutritional, resistance, secondary hyperparathyroidism, ectopic 1-alpha-hydroxylase activity, oncogenic, and the like.
  • renal bone disease it is meant for instance osteitis fibrosa, osteomalacia, osteosclerosis, osteoporosis, adynamic bone disease, and the like.
  • “Hypophosphatasia” refers to, for example, hyperphosphatasia,
  • “Dysplastic disorders” may be for instance sclerosing bone dysplasias and osteoporosis, fibrous dysplasia, mucopolysaccharidoses, periostoses, ankylosing spondylarthritis, osteochond roses, osteophytosis,
  • DISH Diffuse Osteopathic Skeletal Hyperostosis
  • Infiltrative disorders include at least primary skeletal neoplasms, secondary skeletal neoplasms, systemic mastocytosis and histiocytosis, sarcoidosis, oxalosis, and the like.
  • Extra-skeletal calcification and ossification may be for example renal bone disease, fibrodysplasia ossificans progressiva, nephroiithiasis, and the like.
  • a "bone-related disease” is osteoporosis.
  • the term “mammals” encompasses animals and humans.
  • a “mammal” is a human.
  • a “compound” herein refers to any type of molecule, biological or chemical, natural, recombinant or synthetic.
  • such a compound may be a nucleic acid (e.g., an antisense or sense oligonucleotide including an antisense RNA), a protein, a fatty acid, an antibody, a polysaccharide, a steroid, a purine, a pyrimidine, an organic molecule, a chemical moiety, and the like.
  • the term “compound” is preferably used herein to refer to a compound which exhibits the function of interest, i.e., the ability to modulate Frat-LRP interaction.
  • a "molecule” is of any type, biological or chemical, natural, recombinant or synthetic.
  • a molecule may be a nucleic acid (e.g., an antisense or sense oligonucleotide including an antisense RNA), a protein, a fatty acid, an antibody, a polysaccharide, a steroid, a purine, a pyrimidine, an organic molecule, a chemical moiety, and the like.
  • molecule and “compound” thus refer to the same structures. However, as used herein, these terms are not equivalent, since a “compound” is, as defined above, capable of modulating Frat-LRP interaction, whereas a “molecule” either displays a biological function, which is thus different than the ability to modulate Frat-LRP interaction, or it is inert, i.e., it does not have any biological function.
  • activity and “active”, and “function” and “functional” are synonymous, respectively.
  • biological activity biological function
  • activity and “function” are also synonymous.
  • Frat herein refers to all Frat (for frequently rearranged in advanced T-cell lymphomas) proteins and homologs thereto, preferably from mammals.
  • the term “Frat” may, depending on the context, also refer to the nucleic acids corresponding to all Frat proteins and homologs thereto.
  • LRP encompasses all LRPs (for tow- density lipoprotein-receptor-related p_roteins) and homologs thereto, preferably from mammals.
  • LRP may, depending on the context, also refer to the nucleic acids corresponding to all LRP proteins and homologs thereto. This definition includes, for instance, LRP5 (Kim et al., 1998; Hey et al., 1998) and LRP6 (Brown et al., 1998; Tamai et al., 2000; Pinson et al., 2000), as well as functional (i.e., capable of behaving and acting like LRP) fragments and derivatives thereof.
  • a "fragment” include a part of a Frat or a LRP of reference, which retains the biological activity of said Frat or LRP of reference.
  • a functional "derivative" of a Frat or LRP of reference may be, for example and without any limitation, a protein, a peptide, an hormone, an antibody, either natural or synthetic, as soon as it may be derived structurally from said Frat or LRP of reference and it exhibits the biological function thereof.
  • the expression "Frat-LRP interaction” refers not only to the structural cooperation between the two partners Frat and LRP, but also the functional cooperation therebetween, as illustrated in the Examples.
  • modulating Frat-LRP interaction it is meant that said interaction is either positively or negatively affected by acting directly or indirectly on the structural and/or functional cooperation between Frat and LRP.
  • the interaction is “positively affected”, it is meant that the interaction is induced, promoted, activated, enhanced, increased.
  • the interaction when the interaction is "negatively affected”, it is meant that the interaction is suppressed, inhibited, reduced, decreased.
  • modulating effects are encompassed by this definition: - stabilizing (positive) or destabilizing (negative) one partner (Frat or LRP), both partners (Frat and LRP), or the two-partner complex; - activating (positive) or inhibiting (negative) inducers of one or both Frat and LRP, or of the complex; - inhibiting (positive) or activating (negative) inhibitors of one or both Frat and LRP, or of the complex; - increasing (positive) or decreasing (negative) the expression and/or the cell turnover and/or the availability of one or both Frat and LRP so that the two-partner complex cannot be readily formed.
  • a "pharmaceutical composition” is equivalent to a pharmaceutical composition
  • pharmaceutical preparation both referring to a “drug” as commonly understood by the skilled artisan in the field of the invention. More precisely, said “pharmaceutical composition” or “pharmaceutical preparation” or “drug” comprises a pharmaceutically acceptable amount of one or more compounds and, optionally, one or more molecules, all of them being generally associated to, or contained in, at least one pharmaceutically acceptable carrier.
  • pharmaceutically effective amount of an active compound is the amount of said compound that results in amelioration of symptoms in a mammal.
  • the present invention relates to a method for preventing and/or treating a bone-related disease in a mammal in need of such treatment, wherein said method comprises: administering to said mammal an effective amount of a pharmaceutical composition comprising at least one compound capable of modulating Frat-LRP interaction, so that GSK-3 is inhibited and, thus, ⁇ -catenin is stabilized, i.e., it is not submitted to phosphorylation by GSK-3.
  • Such a pharmaceutical composition comprises one or more different compounds and, optionally, one or more molecules which, as defined above, do not exhibit the ability to modulate Frat-LRP interaction.
  • these molecules may only act as adjuvants or carriers, such as polylactic acid, polyglycolic acid, polydioxanone, collagen, albumin, detergent (e.g., polyoxyethylenesorbitan), and the like.
  • Other useful molecules may have a biological function (hereafter referred to as "biologically-active molecules”), different that the one of compounds capable of modulating Frat-LRP interaction, but the association of which may be of interest regarding bone formation and protection.
  • biologically-active molecules may be vitamins.
  • Other useful biologically-active molecules may be molecules that promote tissue growth or infiltration, including bone morphogenic proteins such those described in U.S. Patent No. 4,761 ,471 and PCT Publication
  • biologically-active molecules may be targeting molecules, i.e., molecules that bind to (have affinity with) the tissue of interest.
  • bone-targeting molecules include tetracyclines; calcein; biphosphonates; polyaspartic acid; polyglutamic acid; aminophosphosugars; peptides known to be associated with the mineral phase of bone such as osteonectin, bone sialoprotein and osteopontin; bone specific antibodies; proteins with bone mineral binding domains; and the like (for example, see Bentz et al. in EP 0512844 and Murakami et al. in EP 0341961 ).
  • toxicity and therapeutic efficacy of said compounds can be determined by standard pharmaceutical procedures in cell cultures (in vitro) or in experimental animals (in vivo).
  • the LD50 (the dose lethal to 50% of the population), as well as the ED50 (the dose therapeutically effective in 50% of the population) can be determined using methods known in the art. Accordingly, the data obtained from cell culture assays (in vitro) and/or animal model studies (in vivo) can be used in formulating a range of dosage of these compounds which lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • administration of a drug may be performed via any route such as locally, orally, systemically, intravenously, intramuscularly, mucosally, using a patch, using encapsulating or embedding liposomes, microparticles, microcapsules, and the like.
  • the present invention is related to a method for selecting a compound useful for preventing and/or treating a bone-related disease in a mammal in need of such treatment, wherein said method comprises: a) testing the ability of a candidate compound to modulate Frat-LRP interaction in vitro and/or in vivo; and b) if said candidate compound modulates Frat-LRP interaction, selecting said compound.
  • this method further comprises purifying the selected compound.
  • Methods for detecting a modulating effect on Frat-LRP interaction include both in vitro and in vivo procedures (e.g., protein-protein binding assays, biochemical screening assays, immunoassays, cell-based assays, animal model experiments, which are well-characterized in the art).
  • in vitro and in vivo procedures e.g., protein-protein binding assays, biochemical screening assays, immunoassays, cell-based assays, animal model experiments, which are well-characterized in the art.
  • the person skilled in the art may use only one in vitro and/or one in vivo selection technique. However, in order to strengthen the validity and reproducibility of the results, this person may prefer to use at least two in vitro and/or at least two in vivo selection methods.
  • Appropriate examples of conventional procedures for showing a modulating effect on Frat-LRP interaction are FRET, BRET, any method for detecting an energy transfer between the Frat and LRP, double-hybrid assays, ELISA- like methods, and the like...
  • the present invention also encompasses: - the compounds themselves and/or for use as medicaments ; and - the use of at least one compound as defined above for the manufacture of a pharmaceutical composition for preventing and/or treating bone-related diseases in a mammal. Summary of the Examples
  • LDL receptor-related protein 5 has been identified as a Wnt co receptor involved in the activation of the R-catenin signaling pathway.
  • LRP5 LDL receptor-related protein 5
  • the MATCHMAKER yeast two-hybrid (Y2H) system and the mouse 11- day and 19-day embryo cDNA libraries were purchased from Clonetech.
  • the bait for library screening was the intracellular domain of LRP5 (1419- 1615 residues).
  • Y2H screening was carried out as suggested by the manufacturer.
  • the interaction of the target proteins was determined phenotypically by growing of yeast clones on His-deficient media and measuring ⁇ -galactosidase activity.
  • bait and prey constructs were retrieved from yeast clone of interest (as described by the manufacturer), re-introduced again in the same yeast strain and confirmed for their ability to grow on His-deficient media and to induce ⁇ -galactosidase activity.
  • bait and prey cDNAs were removed from their original plasmid and recloned using appropriate restriction sites in prey and bait plasmids, respectively, re- introduced again in the same yeast strain and confirmed for their ability to grow on His-deficient media and to induce ⁇ -galactosidase activity.
  • a potential target will be considered for further analysis if passed those two assays.
  • Wnt3a-conditioned media was prepared as described by Shibamoto et al. [Shibamoto, 1998]. Briefly, to collect the conditioned medium from cultures of Wnt-3aproducing L cells, we seeded these cells were seeded at a density of 6x10 6 cells in a 125 cm 2 flask containing DMEM with 10% FCS. 24 hours after seeding, medium was changed to DMEM with 2% FCS and cultured them for 3 days. Then Wnt3a-CM was harvested, centrifuged at 1000 g for 10 min, and filtered through a nitrocellulose membrane.
  • Wnt3a-CM The activity of Wnt3a-CM was assayed on normal L cells by examining increase in R-catenin as described by Willert et al. [Willert, 1999]. Wnt3a-CM was added to cells at 20% final concentration in all subsequent experiments.
  • COS-7 monkey kidney cells (ATCC) and, mouse fibroblast L cells (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • Mesenchymal mouse C3H10T1/2 cells were grown in ⁇ -modified Eagle's medium supplemented with 10% FBS.
  • Cells were plated in 24-well plates at 4x10 cells/well 24h prior to transfection, then transiently transfected with the indicated construct (1 ⁇ g total DNA) using DNA-lipid complex Fugene 6 (Boehringer Mannheim) according to the manufacturer's protocol. 16 h after transfection, cells were washed, and cultured in medium at 2% fetal calf serum for an additional 48h under indicated conditions.
  • luciferase assays were then collected for luciferase assays, immunoprecipitation or Western blotting. Controls were carried out by replacing expression constructs by empty vector.
  • pRL-TK Promega
  • Luciferase assays were performed with the Dual Luciferase Assay Kit (Promega) according to the manufacturer's instructions.
  • IP Western analysis and immunoprecipitation
  • transiently transfected cells were harvested and lysed with M-PER Mammalian Protein Extraction Reagent (Pierce).
  • Total protein content in cell lysates was estimated using the BCA-Protein assay (Pierce).
  • BCA-Protein assay Pierce
  • 10 ⁇ g of total protein were loaded on Nu-PAGE 4-12% (Invitrogen).
  • proteins were transferred on Hybond-ECL nitrocellulose membrane (Amersham-biosciences) and revealed with either HRP anti-flag (monoclonal mouse anti body, Sigma), HRP anti-c- Myc (Boehringer Mannheim) or HRP anti-GFP (polyclonal goat anti body, Abeam).
  • Immunoreactive proteins were visualized by enhanced chemiluminescence detection according to the manufacturer (ECL+ Amersham-biosciences). IP was the carried out on 500 ⁇ g of total protein. Total protein were incubated with 50 ⁇ l of ⁇ Macsprotein G microbeads (Miltenyi Biotec) and 2 ⁇ g of indicated anti-epitope anti-body at room temperature for 1h. The magnetically labeled immune complex is passed over a microcolumn placed in magnetic field. This complex is bound to the column where other proteins are washed-away. Immunoprecipitated proteins are eluted from the microcolumn with hot denaturant gel loading buffer, then subjected to Western blotting as described above.
  • C3H10T1/2T1/2 cells are plated at 40 000 cell per wells in 6 wells plate, each well contain a sterile micro cover glass which can bee removed for observation. 48 hours after transfection or treatment, cell are fixed with 3.7% formaldehyde (sigma) for 10 minute, watched tow time with PBS. Cell are also permeabilized by PBS/0.025% triton 100x (sigma) for 5minut.es, and blocked in PBS/3% BSA for 15minutes.
  • Calvarias were collected in 15 ml tube and washed tow time with Phosphate buffered saline with 0.1 % of Penicilline/Streptomycine. Calvarias were treated with collagenase type IA (sigma) and dyspase type II (Boehringer) ⁇ MEM medium (Gibco) for 1 minutes at 37° c under agitation.
  • LRP5 interacts with Fratl It has been clearly established that LRP5/6 receptor is required for Wnt to induce the canonical R-catenin signaling pathway. Interestingly, in the absence of Wnt, overexpression of LRP5 lacking either the extracellular or both the transmembrane and extracellular domains leads to the activation of the canonical signaling pathway (data not shown, [Mao, 2001]).
  • LRP5 cytoplasmic domain was used as bait to screen for potential partners in mouse embryo cDNA libraries (11 and 19 days). From each library, around 20 million clones were screened; clones showing interaction with LRP5 bait in the primary screen were confirmed in secondary screen (see material and methods). Prey from confirmed clones were selected for the specificity of interaction with LRP5 cytoplasmic tail by testing for interaction with two other unrelated baits (i.e., Id3 and Sprouty2).
  • Myc-LRP5tail and flag-Frat were co- expressed in COS-7 cells and found to co-immunoprecipitate (Fig. 1).
  • Flag- Fratl was shown to co-immunoprecipitate with the Myc- LRP5 full length protein (figure 1).
  • Co-expression of Flag-Frat1 with two other unrelated Myc-tagged proteins did not allow co- immunoprecipitation (data not show).
  • LRP5 lacking the extra cellular domain acts as a constitutive active receptor and triggers R-catenin pathway in the absence of Wnt proteins (Fig.2A and [Mao, 2001]).
  • LRP5C deleted of its last 78 amino acids failed to activate the TCF transcriptional activity whereas LRP5C ⁇ 28 was as active as LRP5C.
  • LRP5C ⁇ 47 displays some activity but significantly minor than LRP5C (Fig. 2A). Then, we tested the effect of overexpression of Fratl on the activity of LRP5C and its derivative mutants. Overexpression of Fratl alone is able to induce TCF1 activation (Fig.2A, and [Li, 1999]). As depicted in Fig.
  • LRP5C ⁇ 28 did not cooperate with Fratl to induce TCF1 activation, and surprisingly Fratl activity was found to be inhibited by LRP5C ⁇ 78 (Fig. 2A).
  • overexpression of either LRP5C or Fratl induced R-catenin nuclear translocation Fig. 2B panel II and IV.
  • LRP5C ⁇ 78 had no effect on R-catenin localization (Fig.2B, panel III), but it completely blocked Fratl induced-R-catenin nuclear translocation (Fig.2B, panel XVI).
  • Fratl has been shown to be able to localize into different cell compartments [Franca-Koh, 2002; Freemantle, 2002].
  • LRP5C LRP5C-induced Fratl cell distribution by immunohistochemistry using confocal microscopy. Overexpressed Fratl predominantly localized in the cytoplasm, yet a significant fraction ( ⁇ 25%) was also found on the cell membrane (Fig. 5A panel I; Fig. 5B).
  • Stimulation of cells with Wnt3a induced a shift in Fratl cell localization from cytoplasm to the membrane, where over 80% of Fratl could be localized (Fig. 5A panel II; Fig. 5B).
  • LRP5C ⁇ 78 which we have shown to inhibit Fratl activity, induces the majority of Fratl to be localized into the nucleus (Fig. 5A panel VII). The percentage of membrane, cytoplasm or nucleus location of Fratl was determined and data are shown in figure 5B.
  • data presented herein strongly suggest that the interaction of LRP5 with Fratl modulate Wnt/LRP5 signaling by regulating Fratl cellular localization.
  • Fratl has been shown to interact with several other components of Wnt/ ⁇ - catenin pathway, including dishevelled (Dvl) and GSK3 [Farr, 2000; Li, 1999]. We have investigated how Dvl might be involved in LRP5/Frat1 interaction and its ability to transduce signal.
  • Dvl dishevelled
  • GSK3 GSK3
  • Axin is known to play an important role in the regulation of ⁇ -catenin phosphorylation and degradation.
  • Several authors have already demonstrated that Axin directly binds LRP5 and LRP5C [Tolwinski, 2003; Mao, 2001].
  • Axin overexpression inhibits Wnt3a (data not shown) or Fratl inducing TCF-1 transcriptional activity (Fig. 7A, [Thomas, 1999]).
  • Our data obtained with truncated LRP5 mutants see Fig. 1 and results reported by Mao et al. [Mao, 2001] suggest that Fratl and axin might interact with LRP5 within close motifs.
  • LRP5/6 is a major actor in the activation of the Wnt canonical pathway (for review [Nusse, 2001])
  • the precise molecular mechanisms by which LRP5/6 participate in this important cascade remain to be elucidated.
  • the cytoplasmic tail of LRP5 displays no NPXY motif that was identified to interact with the adaptor protein Dab-1 (Gotthardt et al., 2000).
  • LRP5/6 cytoplasmic tail is rich in praline residues suggests that it might be able to bind SH3-motif-like but nobody has investigated such a possibility.
  • Today the only protein shown to be able to interact with LRP5 cytoplasmic domain is axin [Mao, 2001; Tolwinski, 2003].
  • Fratl also named GBP
  • GBP GBP
  • LRP5 cytoplasmic domain Following Wnt activation, conformational modification of the LRP5 cytoplasmic domain occurs allowing the recruitment of Fratl to the membrane.
  • the activated status of LRP5 receptor is also able to translocate Axin to the membrane where it interacts with Fratl.
  • Axin recruitment to the membrane prevent it from enhancing the degradation of ⁇ -catenin.
  • Axin is found within a protein complex that includes APC, GSK3 ⁇ and ⁇ -catenin.
  • TSH is a negative regulator of skeletal remodeling. Cell 115, 151-162.
  • the G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.
  • Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391, 357-362.
  • LDL receptor-related protein 5 affects bone accrual and eye development. Cell 107, 513-523. Holmen, S. L., Salic, A., Zylstra, C. R., Kirschner, M. W., and Williams, B. O. (2002).
  • a novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate beta -catenin-dependent signaling.
  • LDL-receptor-related protein 6 is a receptor for Dickkopf proteins.
  • Murine frizzled-1 behaves as an antagonist of the canonical Wnt/beta -catenin signaling. J Biol Chem 24,
  • Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity.
  • Murine Wnt10a and Wnt10b cloning and expression in developing limbs, face and skin of embryos and in adults. Oncogene 13, 1 ⁇ 37-1 ⁇ 44.
  • the dishevelled protein is modified by wingless signaling in

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Endocrinology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des méthodes de prévention et/ou de traitement des maladies osseuses dans lesquelles on utilise des composés agissant en tant que modulateurs de l'interaction Frat-LRP . Cette invention se rapporte également à des méthodes de sélection de tels composés.
PCT/IB2005/001878 2004-05-05 2005-05-04 Modulateurs de l'interaction frat-lrp dans le traitement des maladies osseuses WO2005105064A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US56781804P 2004-05-05 2004-05-05
US60/567,818 2004-05-05

Publications (2)

Publication Number Publication Date
WO2005105064A2 true WO2005105064A2 (fr) 2005-11-10
WO2005105064A3 WO2005105064A3 (fr) 2006-02-16

Family

ID=34982497

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2005/001878 WO2005105064A2 (fr) 2004-05-05 2005-05-04 Modulateurs de l'interaction frat-lrp dans le traitement des maladies osseuses

Country Status (1)

Country Link
WO (1) WO2005105064A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7049291B2 (en) * 1996-10-11 2006-05-23 The United States Of America As Represented By The Department Of Health And Human Services Isolation and method of using tissue growth-inducing Frzb protein
EP1313753A4 (fr) * 2000-08-18 2004-09-15 Proskelia Gene regulateur et systeme utilise dans le diagnostic et le traitement de l'osteoporose
US20040265808A1 (en) * 2001-04-05 2004-12-30 Teresa Garcia Genes involved in osteogenesis, and methods of use

Also Published As

Publication number Publication date
WO2005105064A3 (fr) 2006-02-16

Similar Documents

Publication Publication Date Title
Wang et al. Wnt and the Wnt signaling pathway in bone development and disease
Kim et al. R-Spondin family members regulate the Wnt pathway by a common mechanism
Tang et al. Roles of N-glycosylation and lipidation in Wg secretion and signaling
Dorwart et al. The solute carrier 26 family of proteins in epithelial ion transport
US7541431B2 (en) Cristin/R-spondin ligands active in the Wnt signaling pathway and methods, compositions and kits relating thereto
Doubravska et al. Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling
Schipani et al. PTHrP, PTH, and the PTH/PTHrP receptor in endochondral bone development
Hay et al. Interaction between LRP5 and Frat1 mediates the activation of the Wnt canonical pathway
US20140044713A1 (en) Compounds
Bompard et al. Membrane targeting of protein tyrosine phosphatase PTPL1 through its FERM domain via binding to phosphatidylinositol 4, 5-biphosphate
Miranda et al. Identification of the WNT1 residues required for palmitoylation by Porcupine
Verkaar et al. A model for signaling specificity of Wnt/Frizzled combinations through co-receptor recruitment
Haeseleer Interaction and colocalization of CaBP4 and Unc119 (MRG4) in photoreceptors
Sakurai et al. Interactions between the L1 cell adhesion molecule and ezrin support traction‐force generation and can be regulated by tyrosine phosphorylation
Tasma et al. Pharmacology of PACAP and VIP receptors in the spinal cord highlights the importance of the PAC1 receptor
Serrano et al. GTPase of the immune-associated nucleotide protein 5 regulates the lysosomal calcium compartment in T lymphocytes
Kim et al. D2 dopamine receptor expression and trafficking is regulated through direct interactions with ZIP
WO2005105064A2 (fr) Modulateurs de l'interaction frat-lrp dans le traitement des maladies osseuses
Halford et al. The RYK receptor family
M Larrayoz et al. Is the cytoskeleton an intracellular receptor for adrenomedullin and PAMP?
Routledge Extracellular trafficking of Wnt signals in gastric cancer
Kotova Molecular Mechanisms Underlying Regulation and Function of Neuronal Gap Junction Proteins: Connexin 36 and Connexin 27.5.
Li Hox proteins as BMP downstream transcription factors
van der List Comparing placental phenotype similarities between Neuropathy Target Esterase and the Canonical Wnt Signaling Pathway
Eapen DMP1 activates osteolytic cycle in a tumor environment

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: IN PCT GAZETTE 45/2005 UNDER (30) REPLACE "60/657,818" BY "60/567,818"

NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载