WO2010020647A2 - New method for identifying compounds useful for treating and/or preventing disease-associated bone loss - Google Patents

New method for identifying compounds useful for treating and/or preventing disease-associated bone loss Download PDF

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WO2010020647A2
WO2010020647A2 PCT/EP2009/060691 EP2009060691W WO2010020647A2 WO 2010020647 A2 WO2010020647 A2 WO 2010020647A2 EP 2009060691 W EP2009060691 W EP 2009060691W WO 2010020647 A2 WO2010020647 A2 WO 2010020647A2
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rac
dock5
methyl
protein
phenyl
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PCT/EP2009/060691
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French (fr)
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WO2010020647A3 (en
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Anne Blangy
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Centre National De La Recherche Scientifique (Cnrs)
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Priority to US13/059,446 priority Critical patent/US9091678B2/en
Priority to CA2734678A priority patent/CA2734678C/en
Priority to EP09781968.4A priority patent/EP2331967B1/en
Priority to JP2011523415A priority patent/JP5539356B2/en
Publication of WO2010020647A2 publication Critical patent/WO2010020647A2/en
Publication of WO2010020647A3 publication Critical patent/WO2010020647A3/en

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    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
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    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • the invention relates to the field of diseases associated with bone loss, and more specifically to a new method for identifying compounds useful for treating and/or preventing diseases associated with bone loss.
  • Bone is a dynamic tissue that is continually remodeled throughout life depending on factors such as nutrition and the load the bone must carry. Normal bone formation depends on the delicate balance between new bone addition and old bone resorption. Bone formation is based on the deposition of bone matrix by osteoblasts and bone resorption and more specifically mineralized tissue, chiefly calcium carbonate and calcium phosphate resorption in vertebrates is achieved by osteoclasts. Typically, in a normal adult, about 5- 10% of bone is replaced by these processes annually.
  • osteoclasts are multinucleated cells of up to 400 ⁇ m related to macrophage and other cells that develop from monocyte cells, which are actively motile cells that migrate along the surface of bone.
  • macrophage osteoclasts are derived from haematopoietic progenitor cells.
  • the bone resorption is initiated when an osteoclast attaches to the surface of mineralized bone, forms a tight "sealing zone" and secretes necessary acids and proteases that initiate the resorption of mineralized tissue from the bone.
  • the osteoclast detaches from the bone, leaving a pit on the bone surface. Under normal conditions, the pit is a target for osteoblasts, which deposit a material that ultimately becomes new bone.
  • Bone loss can result when the bone resorptive process is dominant over the bone formative process.
  • Diseases associated with bone loss are usually accompanied by increased osteoclast activation.
  • Such diseases include any bone loss resulting notably from an estrogen deficiency after the menopause but not only and comprise osteoporosis, osteopenia due to bone metastases, periarticular erosions in rheumatoid arthritis, primary hyperparathyroidism, hypercalcemia of malignancy, Paget's disease of bone, periodontal disease, immobilization induced osteopenia, and glucocorticoid treatment.
  • the aim of the present invention is to elaborate new screening methods which can be useful for treating and/or preventing bone loss diseases, and to use such compounds to prepare a drug for treating and/or preventing bone loss diseases.
  • DOCK5 corresponds to a new therapeutic target for treating and/or preventing bone loss diseases.
  • yeast exchange assay for identifying inhibitors of DOCK5, which inhibitors can be useful for treating and/or preventing bone loss diseases.
  • the present invention is directed to a method for identifying a compound which inhibits the activation of RAC GTP ase, more specifically RAC 1/2 GTPase, by DOCK5 protein comprising the steps of:
  • the selected compound is useful for treating disease associated with bone loss.
  • the inventors have established that the conversion of inactive RAC to active RAC by DOCK5 is associated with the sealing zone formation.
  • RAC 1/2" means "RACl and/or RAC2".
  • the inhibition of the activation of RACl GTP ase and/or of RAC2 GTP ase give rise to the same kind of results, while both RACl and RAC2 are involved in (and thus necessary for) the osteoclast differenciation and resorption functions.
  • the present invention is directed to a method for identifying a compound which inhibits the activation of RAC 1/2 GTP ase and which is useful for treating disease associated with bone loss by DOCK5 protein comprising the steps of:
  • DOCK5 protein induces the conversion of inactive RAC, which inactive RAC is bound to GDP, to active RAC, which active RAC is bound to GTP.
  • osteoclasts corresponding to the testing of mineralised matrix resorption by osteoclasts, by the selected compounds.
  • disease associated with bone loss one can cites menopause, osteoporosis, osteopenia due to bone metastases, periarticular erosions in rheumatoid arthritis, primary hyperparathyroidism, hypercalcemia of malignancy, Paget's disease of bone, periodontal disease, immobilization induced osteopenia, or in glucocorticoid treatment.
  • said disease associated with bone loss is osteoporosis.
  • results from the cellular and bone resorption assay systems used herein are widely accepted in the art as predictive of in vivo effects.
  • the bone resorption assay uses material that includes bone marrow isolated cells, it is an ex vivo assay.
  • the showing that the inhibition of RAC activation by DOCK5 inhibits bone resorption in these assays is evidence of the clinical utility of inhibitors of this specific activation for treating osteoporosis.
  • the method of the invention further comprises the step of testing the inhibition of bone resorption by the selected compound.
  • the method of the invention includes a further step of comparing the conversion of inactive RAC to active RAC in presence of the tested compound and in the absence of said compound.
  • Said inhibition of bone resorption can be simply tested by method well known from the skilled person, such as the one disclosed in the examples, wherein mineralised matrix resorption by osteoclasts is tested by culturing said osteoclasts on calcium phosphate substrates and mineralised matrix resorption is determined by VON KOSSA staining.
  • the term "compound” refers to a natural or synthetic compound, suchemical or peptidic compound.
  • the compounds are chosen in the group consisting in:
  • the compounds are chosen in the group consisting in:
  • DOCK5 protein refers to a polypeptide comprising at least the DHR2 domain of the protein DOCK5 corresponding to the amino acid 1 132 to 1661 of the DOCK5 protein from Mus musculus SEQ ID NO:1 and derivatives thereof.
  • the present invention is directed to a method for identifying a compound which inhibits the activation of RAC GTP ase, more specifically RAC 1/2 GTP ase, by DOCK5 protein comprising the steps of:
  • DOCK5 and the RAC proteins in a cell wherein said polypeptide induces the conversion of inactive RAC, which inactive RAC is bound to GDP, to active RAC, which active RAC is bound to GTP,
  • the full length DockS protein has an aminoterminal SH3 domain, between aminoacids Kl 1 and E68, followed by the DHRl domain, between aminoacids G440 and E682, and the DHR2 domain between aminoacids Ml 132 and Y1661 ( Figure 3E).
  • said DOCK5 protein corresponds to SEQ ID NO:1.
  • said DOCK5 protein corresponds to SEQ ID NO:4 corresponding to Homo sapiens DOCK5 protein.
  • RAC protein refers to SEQ ID NO:2 and derivatives thereof.
  • said cell is an eukaryotic cell, preferably a yeast cell.
  • said method comprises the expression of any protein, capable to interact with the active RAC protein and not with inactive RAC protein.
  • any protein capable to interact with the active RAC protein and not with inactive RAC protein.
  • the protein capable to interact with the active RAC protein is chosen in the group comprising PAKl protein.
  • PAKl protein refers to the SEQ ID NO:3 and derivatives thereof.
  • derivatives' refer to a polypeptide having a percentage of identity of at least 80% with amino acid 1132 to 1661 of SEQ ID NO: 1, SEQ ID NO: 2, EQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:9, or orthologs thereof, preferably of at least 90%, as an example of at least 95%, and more preferably of at least 99%.
  • percentage of identity between two amino acids sequences or two nucleic sequences, means the percentage of identical amino-acids or nucleotides, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the amino acids sequences.
  • best alignment or “optimal alignment” means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity.
  • the identity percentage between two sequences of amino acids two nucleic sequence is determined by comparing these two sequences optimally aligned, the amino acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.
  • said cell further comprises a reporter gene under the control of a promoter sequence, and said RAC and PAKl proteins are each fused either with a transactivation domain or with a DNA binding domain specific of said promoter sequence, wherein the interaction of RAC with PAKl results in the induction of expression of the reporter gene.
  • the method corresponds to the Yeast Exchange Assay (YEA) as disclosed in DE TOLEDO et al. (FEBS, vol.480, p:287-292, 200) and International Patent application PCT WO 2005/064007 using the DOCK5 and the RAC protein.
  • YEA Yeast Exchange Assay
  • reporter gene is well known from the skilled person and can correspond to an auxotrophic marker or to a gene coding for a protein which can be simply detected such as GFP, luciferase or ⁇ -Gal.
  • the determination of the conversion of inactive RAC to active RAC is done by determining the expression of the reporter gene.
  • the inhibition of the expression of the reporter gene corresponding to an inhibition of the conversion of inactive RAC to active RAC.
  • the present invention provides a method for the selection of compounds, which permit to decrease the level of expression of a DOCK5 gene (SEQ ID N 0 IO) in diseases associated with bone loss comprising the step of: a) contacting a test compound with an host cell expressing a reporter nucleic acid comprising a nucleic acid sequence coding for a reporter placed under the control of a promoter, which promoter comprises all or part of the promoter sequence of DOCK5 gene or a derivative thereof, and
  • derivatives' refer to a nucleic sequence having a percentage of identity of at least 80% with the sequence of DOCK5 promoter, preferably of at least 90%, as an example of at least 95%, and more preferably of at least 99%.
  • the percentage of identity is as defined above.
  • compound or “test compound”
  • test compound compounds of different nature, structure and origin, particularly biological compounds, nuclear factors, cofactors, and the like, chemical, synthetic compounds and the like, which are tested for their capacity of enhancing the level of expression of said gene implicated in antimicrobial defence.
  • the concentration of said test compound can be adjusted by the skilled person according to the characteristics of said compound (its toxicity, ability to penetrate cells, etc.), the number of cells, the length of the incubation period, etc. Generally, the cells are exposed to concentrations of test compounds ranging from 1 nM to 1 mM. Of course it is possible to test other concentrations without deviating from the invention, and also to test simultaneously different test compound concentrations.
  • Different adjuvants and/or vectors and/or products facilitating the penetration of the test compounds into the host cell such as liposomes, cationic lipids or polymers can also be used, when necessary.
  • DOCK5 gene By “decreasing the level of expression of a DOCK5 gene”, one should understand that the expression level of DOCK5 gene is diminished or inhibited compared to a control level.
  • said expression level of the DOCK5 gene is correlated to the expression level of the reporter gene in the method of the invention.
  • a test compound can decrease the expression level of the DOCK5 gene from the capacity of said compound to obtain an diminished expression level of the reporter gene in the method of the invention.
  • the control level can be determined, by example, by measuring the expression level of the reporter gene in the absence of the test compound.
  • the method according to the invention further comprises a step c) of comparing the level of expression of the reporter gene as measured in step b) with the level of expression of the reporter gene in the absence of said test compound.
  • the present invention provides a method for identifying a compound which inhibits the activation of RAC 1/2 GTPaseby inhibiting the binding of ELMOl protein (SEQ ID N°9) to the SH3 domain of DOCK5 comprising the steps of:
  • ELMOl protein refers to SEQ ID N°9 and derivatives thereof.
  • the binding between said ELMOl protein and the tested compound can be measured by methods well known from one skilled in the art.
  • the compound is an inhibitor of the binding of ELMOl and the SH3 domain of D0CK5, and that this compound is useful to inhibit the conversion of inactive RACl/2 to active RACl/2.
  • said method can include a further step after step b) of contacting a polypeptide comprising at least the SH3 domain of D0CK5 or the derivative thereof with said test compound and ELMOl protein, and comparing the binding between said ELMOl protein and said polypeptide in the presence or in the absence of said compound.
  • the present invention provides a method for identifying a compound which inhibits the activation of RAC 1/2 GTP ase by inhibiting the binding of ELMOl to the SH3 domain of DOCK5 comprising the steps of:
  • the binding between said ELMOl protein and said polypeptide can be measured by methods well known from one skilled in the art. If the binding between said ELMOl protein and said polypeptide in the presence of the tested compound is lower than the one measured in absence of said compound, it can thus be conclude that the compound is an inhibitor of the binding of ELMOl to the SH3 domain of DOCK5, and that this compound is useful to inhibit the conversion of inactive RAC 1/2 to active RAC 1/2.
  • the compounds as described above are coupled with a bisphosphonate radical.
  • the bisphosphonate radical permits a fast incorporation of the compound after its administration.
  • Another object of the present invention is a compound as described above for treating and/or preventing bone loss diseases in a subject in need thereof.
  • the present invention relates to the use of at least one compound as described above in preparing a drug for treating and/or preventing bone loss disease in a subject in need thereof.
  • Another object of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound as described above and, optionally, a pharmaceutically acceptable support for treating and/or preventing bone loss diseases in a subject in need thereof.
  • the present invention relates to the use of a pharmaceutical composition comprising at least one compound as described above in preparing a drug for treating and/or preventing bone loss diseases in a subject in need thereof.
  • the composition can include emulsions, microemulsions, oil in water emulsions, anhydrous lipids and water in oil emulsions or other types of emulsions.
  • the inventive composition can further include one or more additives such as diluents, excipients, stabilizers and preservatives.
  • additives are well known to those skilled in the art and are described notably in "Ullmann's Encyclopedia of Industrial Chemistry, 6 th Ed.” (various editors, 1989-1998, Marcel Dekker) and in “Pharmaceutical Dosage Forms and Drug Delivery Systems” (ANSEL et al, 1994, WILLIAMS & WILKINS).
  • the term “subject” refers to a mammal such as a rodent, cat, dog, primate or human, preferably said subject is a human.
  • Another object of the invention relates to a therapeutic method for treating a subject and/or preventing bone loss diseases, comprising the administration of a therapeutically effective quantity of a pharmaceutical composition as described above.
  • a "therapeutically effective quantity” means a quantity that inhibits or reduces the osteoclats activation. Those skilled in the art will be able to determine said therapeutically effective quantity based on their general knowledge and on the methods described in the examples.
  • the compounds can be administered by any mode of administration such as, for example, by intramuscular, intravenous or oral route, etc.
  • the inventive compounds preferably will be administered at a concentration chosen by those skilled in the art according to the state of advancement of the disease and the targeting mode used, the age and the weight of the subject.
  • the compound will be administrated at a concentration of between 5 and 200 ⁇ M, preferably at a concentration comprised between 10 and 100 ⁇ M.
  • RNA isolation kit (ROCHE DIAGNOSTICS).
  • RNA was primed with 10-mer random primers and reverse transcription catalysed using SUPERSCRIPT II reverse transcriptase (INVITROGEN).
  • Quantitative PCR was performed with a Light Cycler (ROCHE DIAGNOSTICS) or a Mx3000 ⁇ PCR system (STRATAGENE) using the PLATINIUM Taq DNA polymerase (INVITROGEN) and SYBR GREEN I (BIOWITAKKER) as in described in COELHO et al. (Proc. Natl. Acad.
  • figure IA and B show the expression of Dock5 in different mouse tissues In figure IA, said expression has been normalised according to Dock5 osteoclasts' expression (i.e., Dock5 osteoclasts' expression corresponding to 100% level).
  • mammary gland at 10.5 days of embryo's development (GM 10.5), mammary gland at 13.5 days of embryo's development (GM 13.5), mammary gland at 15.5 days of embryo's development (GM 15.5), mammary gland at 18.5 days of embryo's development (GM 18.5), mammary gland of juvenile mouse (GM j), mammary gland at lactation (GM 1), brain (Br), kidney (Kd), uterus (Ut), liver (Lv), macrophage (Mac), Testis 1 (Tl), Testis 2 (T2), spleen (Sp), colon (Co), bone marrow (Bm), placenta at 13.5 days of embryo's development (Pl 13.5), placenta at 15.5 days of embryo's development (GM 15.5), and osteoclasts (Os).
  • RNA of bone marrow macrophages (ND), induced for osteoclastic differentitation (OC) or dendritic cell differentiation (DC) and from mensenchymal stem cells (MSC JO) induced for osteoblastic differentiation (MSC J4) were extracted and level of Dock5 mRNA relative to Gapdh mRNA was determined by RT-PCR.
  • a rabbit polyclonal antibody was raised to a mouse DOCK5 C-terminus peptide corresponding to amino acids 1658-1869 from mouse DOCK5 and purified by immunoaff ⁇ nity.
  • the amino acids sequences significantly differ between the differents members of the subgroup DOCK-A.
  • Osteoclastogenesis was induced by RANKL-stimulation in purified mouse bone marrow macrophages were purified and in RAW264.7 cell line as described in BRAZIER et al. (abovementioned, 2006), which cells were maintained in culture. At 0, 3 or 5 days of stimulation, the cells were subjected to SDS-PAGE and blotted on polyvinyl difluoride membrane (MILLIPORE IMMOBILON-P pore size 0.45 ⁇ m).
  • the membrane was incubated in TBS-T (Tris buffered saline containing 0.1% TWEEN) with 2% skim millk at room temperature for 30 min and then with rabbit antisera diluted 1 : 1000 in TBS-T overnight at 4°C.
  • TBS-T Tris buffered saline containing 0.1% TWEEN
  • rabbit antisera diluted 1 : 1000 in TBS-T overnight at 4°C.
  • the bound antibodies were detected by peroxidase labelled anti-rabbit immunoglobulin chemoluminescence system (AMERSHAM) and LAS-1000 image analyser (FUJI FILM).
  • AMERSHAM peroxidase labelled anti-rabbit immunoglobulin chemoluminescence system
  • FUJI FILM LAS-1000 image analyser
  • the membrane was further incubated with GAPDH antibodies, the bound antibodies being detected as previously.
  • the Figure 2 A and B show the expression of DOCK5 and GAPDH proteins in purified mouse bone marrow macrophages at 0, 3 and 5 days from the RANKL-stimulated osteoclastogenesis .
  • the results established that a protein of 215 kDa was induced during RANKL- stimulated osteoclastogenesis of purified mouse bone marrow macrophages (figure 2) and of RAW264.7 cell line (data not shown). This size is compatible with the size of the DOCK5 protein deduced from its mRNA.
  • total proteins were extracted from mouse tissues and subjected to western blot with antibodies against Dock5 and against tubulin for normalization.
  • ShRNA target sequences were selected in mouse Docks open reading frames, and the 65-mer sense and antisense strands of DNA oligonucleotides were designed according to the CLONTECH BIOINFORMATICS DATA server and are described in BRAZIER et al (abovementioned, 2006).
  • the oligonucleotide was then synthetised by INVITROGEN annealed and cloned in pSINREN-RETROQ vector containing a puromycin resistance selection marker according to the manufacturer's instructions (CLONTECH).
  • the pSIREN-RETROQ-Luc vector (CLONTECH) targeting firefly luciferase was used as a control.
  • Retrovirus packaging was done by co-transfection of pSIREN-RETROQ vectors, the Friend MLV-based Gag-Pol expression vector pC57GP (LASSAUX et al, J. Virol, vol.79, p:6560-6564, 2005), and the VSV-G envelope glycoprotein expression vector pCSIG (BATTINI et al, Proc. Natl Acad. ScL, vol.96, p:1385-1390, 1999) into 293T cells using Jet PI (QBIOGEN) according to manufacturer's instructions. Viral supernatants were harvested 3 days after transfection and filtered through a 0.45 ⁇ m pore size filter.
  • RAW264.7 cells were plated at 2.10 5 cells per 6-cm dish. The next day, the medium was replaced for 4h with 1.5 ml of viral supernatant and 0.5 ml of growth medium containing 8 ⁇ g/ml polybrene. Cells were left to recover in growth medium for 24 h, and infected cells were selected by addition of puromycin (3 ⁇ g/ml) for another 24h. Infected RAW264.7 were scrapped and reseeded in growth medium at 5.10 4 cells/well of a 6-well plate for RANKL-stimulated osteoclastogenesis as described in BRAZIER et al. (abovementioned, 2006).
  • the Figure 2 A shows the expression of DOCK5 and GAPDH proteins in RAW264.7 cell lines infected with retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5) at 0, 3 and 5 days from the RANKL- stimulated osteoclastogenesis.
  • DOCK5 protein and more specifically its DHR2 domain, could activate small GTPases of the Rho-family -i.e., RACl/2 and cdc42-.
  • 293 -T cells were transfected in six- wells plates with a vector coding for the GFP fusion protein comprising the DHR2 domain of DOCK5 (DHR2) or with a vector coding for GFP (GFP). 48 hours after transfection, cells were lysed in MLB buffer (25mM
  • the figure 3 B shows the expression levels of Rac, cdc42, GFP-DHR2 and GFP proteins in total cell lysates (total) and the protein detected after GTP -trapping.
  • 293 -T cells were cotransfected as described previously with a vector coding for the ELMOl protein or deleted from the C-terminus ( ⁇ T625) - (GUMIENNY et al, Cell, vol.107, p:27-41, 2001) and a vector coding GFP fusion proteins comprising the Full length DOCK5 protein (FL), the DHR2 domain, the DOCK5 protein sequence deleted from (i) the amino acids 1 to 559 of its N-terminus extremity ( ⁇ Nter), including the SH3 domain and half of the DHRl domain, or the DOCK5 protein sequence deleted from (ii) the amino acids 1 to 82 comprising the SH3 domain ( ⁇ SH3) (see figure 3 E).
  • the clarified lysates were immunoprecipitated with anti-GFP antibody and the bound ELMOl protein was detected by immunoblotting. Equal amount of input lysate were analysed by immunoblotting to verify the expression levels of ELMOl protein.
  • the figure 3C and 3F show the expression levels of ELMOl protein in total cell lysates (total) and after immunopreciptation with anti-GFOP antibody (IP GFP), in cells cotransfeted with a vector coding for ELMOl protein and full length DOCK5 (FL), the DHR2 domain (DHR2), DOCK5 deleted from its SH3 domain ( ⁇ SH3) or from its N-term domain ( ⁇ Nter).
  • FL full length DOCK5
  • DHR2 domain DHR2 domain
  • ⁇ SH3 DOCK5 deleted from its SH3 domain
  • ⁇ Nter N-term domain
  • N-terminal domain binds Elmol C-terminus.
  • the figure 3D shows the expression levels of Rac in total cell lysates (total) and the
  • GFP GFP protein
  • DHR2 DHR2 domain of DOCK5
  • ⁇ SH3 DOCK5 protein deleted from its SH3 domain
  • FL DOCK protein
  • DOCK5 is a major activator of Rac in osteoclats.
  • RAW264.7 cell lines stimulated with RANKL were infected as described previously with a retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5).
  • the figure 4 shows the average of three independent experiments with active Rac levels set to 1 in control shLuc and Dock5 +/+ osteoclasts. Error bars : SD.
  • the figure 4A show the expression levels of Rac in total cell lysates (total Rac) and the RAC-GTP protein detected after GTP trapping in the cells infected with a retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5).
  • the figure 4B shows that Dock5 " ⁇ osteoclasts have reduced active Rac levels compared to the control level of Dock5 +/+ osteoclasts.
  • DOCK5 is an essential exchange factor of RAC in osteoclasts.
  • DOCK5 is necessary for mineralised matrix resorption
  • RAW264.7 cell lines were infected as described previously with a retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5), and then osteoclastogenesis was stimulated with RANKL. The obtained cells were then cultured on calcium phosphate substrates to induce the formation of the actin ring. After 48 hours, cells were fixed and stained for actin using rhodamine-labeled Phalloidin to reveal the sealing zone (figure 5).
  • the figure 6 shows the polymerisation of actin in RAW264.7 cell lines stimulated with RANKL which have been infected with a retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5) and the mineralised matrix resorption in the presence of said osteoclasts.
  • the results show that in the osteoclasts, the DOCK5 protein is associated with the podosome and with the sealing zone (data not shown).
  • the osteoclasts wherein DOCK5 expression was inhibited show a default of contraction and of sealing zone formation.
  • the measure of mineralised matrix resorption surface by VON KOSSA staining shows a strong decrease of the resorption by osteoclasts wherein D0CK5 expression was decreased.
  • BMMs bone marrow macrophages isolated from Dock5 + + and Dock5 ⁇ mice were differentiated into osteoclasts in the presence of 100 ng/ml RANKL and 10 ng/ml M-CSF.
  • TCL total cell extracts
  • Osteoclasts derived from Dock5 v ⁇ BMMs express Dock5 truncated after aminoacid 1115, between DHRl and DHR2 domains, and fused to a ⁇ -geo cassette (Figure 7A). Furthermore, the differentiated osteoclasts were fixed and stained with TRAP and
  • the levels of indicated gene mRNAs relative to Gapdh mRNA were determined by RT-PCR.
  • Preosteoclasts prepared from Dock5 +/+ and Dock5 " ⁇ BMMs were stimulated with M- CSF or RANKL for the inducated amount of time.
  • the levels of ERK, ⁇ 38 and Akt phosphorylation in TCL were determined by western blot.
  • DOCK5 is a new therapeutic target for limiting bone loss in menopause, osteoporosis, osteopenia due to bone metastases, periarticular erosions in rheumatoid arthritis, primary hyperparathyroidism, hypercalcemia of malignancy, Paget's disease of bone, periodontal disease, immobilization induced osteopenia, or in glucocorticoid treatment. Because of the specific osteoclasts DOCK5 expression, the targeting of DOCK5 may limit side effects such as the ones observed with drugs for treating bone loss.
  • DOCK5 inhibitors which inhibitors can be useful for treating bone loss associated disease
  • YEA Yeast Exchange Assay
  • the transformed yeast is contacted with several chemical or peptidic molecules, and the chemical or peptidic molecules inhibiting the expression of reporter genes ⁇ -Gal and His3 are selected for further testing in the bone loss model disclosed in 8 and then in bone loss diseases models.
  • the yeast strain TAT7 was used to identify DOCK5 inhibitors.
  • the strain was seeded, in a 96-well culture plate in a selective medium devoid of histidine or in a non selective medium where histidine is added.
  • 2560 compounds were screened to select the ones which inhibit the growth of the strains in a selective medium without having effect on the growth in a non selective medium.
  • DMSO was used as a control.
  • the compounds were tested at a concentration of 200 ⁇ M in presence of 1% DMSO.
  • the inhibiting compounds were defined as follows:
  • the growth derivative Cr (medium) (OD600Tn-OD600T2)/Tn-T2 in test medium (-HIS) and in toxic medium (+HIS).
  • the selected compounds are those showing a ratio I(compound) ⁇ 0.9 at each time.
  • RAW264.7 cells used as osteoclasts inhibitors were allowed to grow for 72 hours with 10 to lOO ⁇ M of compound. The growth of the cells was compared to control cells which were grown with 0.5% DMSO.
  • the identified compounds were used at the same concentration as defined above on osteoclats seeded on minerOalised matrix resorption surface of calcium phosphate (Osteologic Biocoat Clontech Reference 354609) during 72 hours. Then the mineralised matrix was coloured with silver nitrate in order to show the resorbed areas. The compounds were classified in 3 categories:
  • the compounds of the resorption categories (+/-) and (-) represent new inhibitors of the bone resorption. They were used at a concentration of 10 to 100 ⁇ M.

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Abstract

The present invention concerns a method for identifying a compound which inhibits the activation of RAC GTPase by DOCK5 protein comprising the steps of (i) coexpressing the DOCK5 and the RAC proteins in a cell, wherein said D0CK5 protein induces the conversion of inactive RAC, which inactive RAC is bound to GDP, to active RAC, which active RAC is bound to GTP, (ii) contacting or not said cell with said compound, (iii) determining the conversion of inactive RAC to active RAC in the presence or absence of said compound, and (iv) selecting the compound inhibiting the conversion of inactive RAC to active RAC. Said compound is useful for treating disease-associated bone loss.

Description

NEW METHOD FOR IDENTIFYING COMPOUNDS USEFUL FOR TREATING AND/OR PREVENTING DISEASE - ASSOCIATED BONE LOSS
Field of the invention
The invention relates to the field of diseases associated with bone loss, and more specifically to a new method for identifying compounds useful for treating and/or preventing diseases associated with bone loss.
Background of the Invention
Bone is a dynamic tissue that is continually remodeled throughout life depending on factors such as nutrition and the load the bone must carry. Normal bone formation depends on the delicate balance between new bone addition and old bone resorption. Bone formation is based on the deposition of bone matrix by osteoblasts and bone resorption and more specifically mineralized tissue, chiefly calcium carbonate and calcium phosphate resorption in vertebrates is achieved by osteoclasts. Typically, in a normal adult, about 5- 10% of bone is replaced by these processes annually.
These osteoclasts are multinucleated cells of up to 400μm related to macrophage and other cells that develop from monocyte cells, which are actively motile cells that migrate along the surface of bone. Like macrophage, osteoclasts are derived from haematopoietic progenitor cells. The bone resorption is initiated when an osteoclast attaches to the surface of mineralized bone, forms a tight "sealing zone" and secretes necessary acids and proteases that initiate the resorption of mineralized tissue from the bone. After a period of several hours to days, the osteoclast detaches from the bone, leaving a pit on the bone surface. Under normal conditions, the pit is a target for osteoblasts, which deposit a material that ultimately becomes new bone.
Bone loss can result when the bone resorptive process is dominant over the bone formative process. Diseases associated with bone loss are usually accompanied by increased osteoclast activation. Such diseases include any bone loss resulting notably from an estrogen deficiency after the menopause but not only and comprise osteoporosis, osteopenia due to bone metastases, periarticular erosions in rheumatoid arthritis, primary hyperparathyroidism, hypercalcemia of malignancy, Paget's disease of bone, periodontal disease, immobilization induced osteopenia, and glucocorticoid treatment.
As an example, there are currently 20 million people with detectable fractures of the vertebrae due to osteoporosis in the United States. In addition, there are 250,000 hip fractures per year attributed to osteoporosis. This clinical situation is associated with a 12% mortality rate within the first two years, while 30% of the patients require nursing home care after the fracture.
Since diseases of bone loss are associated with increased activity of osteoclast, it is important to understand the mechanisms by which osteoclasts are activated in these disease states, and to devise rational and therapeutic means to inhibit or reduce this activation.
Thus, the aim of the present invention is to elaborate new screening methods which can be useful for treating and/or preventing bone loss diseases, and to use such compounds to prepare a drug for treating and/or preventing bone loss diseases.
Description of the invention
The inventors have presently identified the DOCKS protein is implicated in sealing zone formation and consequently in bone resorption. Thus, DOCK5 corresponds to a new therapeutic target for treating and/or preventing bone loss diseases. Finally, the inventors have used yeast exchange assay (YEA) for identifying inhibitors of DOCK5, which inhibitors can be useful for treating and/or preventing bone loss diseases.
Thus, in a first object, the present invention is directed to a method for identifying a compound which inhibits the activation of RAC GTP ase, more specifically RAC 1/2 GTPase, by DOCK5 protein comprising the steps of:
- coexpressing the DOCK5 and the RAC proteins in a cell, wherein said DOCK5 protein induces the conversion of inactive RAC, which inactive RAC is bound to GDP, to active RAC, which active RAC is bound to GTP,
contacting or not said cell with said compound, - determining the conversion of inactive RAC to active RAC, more specifically the conversion of inactive RAC 1/2 to active RAC 1/2, in the presence or absence of said compound, and
selecting the compound inhibiting the conversion of inactive RAC to active RAC, more specifically the conversion of inactive RAC 1/2 to active RAC 1/2.
The selected compound is useful for treating disease associated with bone loss. In fact, the inventors have established that the conversion of inactive RAC to active RAC by DOCK5 is associated with the sealing zone formation.
According to the present invention "RAC 1/2" means "RACl and/or RAC2". In fact, the inhibition of the activation of RACl GTP ase and/or of RAC2 GTP ase give rise to the same kind of results, while both RACl and RAC2 are involved in (and thus necessary for) the osteoclast differenciation and resorption functions.
Advantageously, the present invention is directed to a method for identifying a compound which inhibits the activation of RAC 1/2 GTP ase and which is useful for treating disease associated with bone loss by DOCK5 protein comprising the steps of:
- coexpressing the DOCK5 and the RAC proteins in a cell, wherein said DOCK5 protein induces the conversion of inactive RAC, which inactive RAC is bound to GDP, to active RAC, which active RAC is bound to GTP.
contacting or not said cell with said compound,
- determining the conversion of inactive RAC to active RAC in the presence or absence of said compound,
- selecting the compound inhibiting the conversion of inactive RAC to active RAC since this conversion is associated with the sealing zone formation, and
- testing the inhibition of bone resorption, corresponding to the testing of mineralised matrix resorption by osteoclasts, by the selected compounds. As an example of disease associated with bone loss, one can cites menopause, osteoporosis, osteopenia due to bone metastases, periarticular erosions in rheumatoid arthritis, primary hyperparathyroidism, hypercalcemia of malignancy, Paget's disease of bone, periodontal disease, immobilization induced osteopenia, or in glucocorticoid treatment. Preferably, said disease associated with bone loss is osteoporosis.
Results from the cellular and bone resorption assay systems used herein are widely accepted in the art as predictive of in vivo effects. As the bone resorption assay uses material that includes bone marrow isolated cells, it is an ex vivo assay. Thus, the showing that the inhibition of RAC activation by DOCK5 inhibits bone resorption in these assays is evidence of the clinical utility of inhibitors of this specific activation for treating osteoporosis. Various scientific publications, such as Carano et al. (1990); Blair & Schlesinger (1992); Schlesinger & Blair (1992); Vaananen et al., 1990; all support the fact that such assays are accepted as being predictive of in vivo activity.
Methods for determining the conversion of inactive RAC to active RAC are well known from the skilled person. As an example of such methods, one can cites the methods disclosed in the examples and in COTE & VUORI (J Cell. ScL, vol.115, p: 4901-4913, 2002).
In a preferred embodiment, the method of the invention further comprises the step of testing the inhibition of bone resorption by the selected compound.
In another preferred embodiment, the method of the invention includes a further step of comparing the conversion of inactive RAC to active RAC in presence of the tested compound and in the absence of said compound. Said inhibition of bone resorption can be simply tested by method well known from the skilled person, such as the one disclosed in the examples, wherein mineralised matrix resorption by osteoclasts is tested by culturing said osteoclasts on calcium phosphate substrates and mineralised matrix resorption is determined by VON KOSSA staining. As used herein, the term "compound" refers to a natural or synthetic compound, suchemical or peptidic compound.
Preferably, the compounds are chosen in the group consisting in:
- 4-[5-(4-bromophenyl)-3 -(4-nitrophenyl)-4,5-dihydro- 1 H-pyrazol- 1 -yl]-4- oxobutanoic acid;
- 2,2,2-trichloro-N-(l,l-dioxido-2,3-dihydro-3-thienyl)-N-(4- methylphenyl)acetamide;
- 3-(3-chlorophenyl)-7-methyl-4-methylene-3,4-dihydro-2(lH)-quinazolinone;
- 3-[4-(3-bromobenzylidene)-3-methyl-5-oxo-4,5-dihydro- 1 H-pyrazol- 1 -yljbenzoic acid;
- N-2, 1 ,3-benzothiadiazol-4-yl-5-bromo-2-furamide;
- 1 -acetyl -4-(2-chloro-4-nitrophenyl)-2-methylpiperazine;
- 3-(3-methoxybenzylidene)-5-(4-methylphenyl)-2(3H)-furanone;
- 3-[5-(3,4-dichlorophenyl)-2-furyl]acrylic acid;
- (2-chloro-4- {[5-(2-chlorophenyl)-6-(ethoxycarbonyl)-7-methyl-3-oxo-5H-
[l,3]thiazolo[3,2-a]pyrimidin-2(3H)-ylidene]methyl}-6-methoxyphenoxy)acetic acid;
- 4- { [4-(diphenylmethyl)- 1 -piperazinyl] sulfonyl } -2, 1 ,3-benzothiadiazole;
- 4-[4-phenyl-5-(2-thienyl)-lH-imidazol-2-yl]-l,2-benzenediol;
- N-(3 ,4-dimethoxyphenyl)-4-[methyl(phenylsulfonyl)amino]benzamide;
- l-[(2-hydroxyphenyl)carbonothioyl]-3-phenyl-5-(trifluoromethyl)-4,5-dihydro-lH- pyrazol-5-ol;
- 2-methoxyethyl 4-[(4-tert-butylbenzoyl)amino]benzoate;
- N-(2,3-dichlorophenyl)-3-(5-methyl-2-furyl)acrylamide; - N-(4-fluorophenyl)-3-[3-(trifluoromethyl)phenyl]acrylamide;
- 3-(2-fixrylmethyl)-2-(2-hydroxyphenyl)-2,3-dihydro-4(lH)-quinazolinone;
- N-(4-ethoxyphenyl)-2- {[5-(4-methoxyphenyl)-l ,3,4-oxadiazol-2- yl] thio } acetamide;
- 5-(4-nitrobenzylidene)-2-thioxo-3-[3-(trifluoromethyl)phenyl]- 1 ,3-thiazolidin-4- one;
- (3,5-dichlorophenyl)[(phenylsulfonyl)carbonyl]amine;
- N-(2-bromophenyl)-3 -(5-methyl-2-furyl)acrylamide;
- 2-(2-chlorophenoxy)-N-[2-chloro-5-(trifluoromethyl)phenyl]acetamide;
- N- [4-(4-acetyl- 1 -piperazinyl)phenyl]propanamide;
- 8-[(dimethylamino)methyl]-9-hydroxy-2-methyl-4H-pyrido[ 1 ,2-a]pyrimidin-4-one;
- 4-tert-butyl-N-[ 1 - {[(2-methoxyphenyl)amino]carbonyl}-2-(2- thienyl)vinyl]benzamide;
- 2-chloro-N-(3-chloro-4-methoxyphenyl)benzamide;
- 2,6-di-tert-butyl-4-(2,3-dihydro-lH-perimidin-2-yl)phenol;
- 3-benzyl-2-(2,6-dichlorophenyl)-2,3-dihydro-4(lH)-quinazolinone;
- 1 -(3 ,4-dichlorobenzyl)- 1 H-indole-3 -carbaldehyde;
- N-[5-(l-adamantyl)-l,3,4-thiadiazol-2-yl]-N'-phenylurea;
- N-(3,4-dichlorophenyl)-N'-{5-[(4-methylphenoxy)methyl]-l,3,4-thiadiazol-2- yljurea;
- N-(2,3-dihydro-l ,4-benzodioxin-6-yl)-2-(l -naphthyloxy)acetamide;
- N-[4-(4-acetyl- 1 -piperazinyl)phenyl]-4-ethoxy-3 -nitrobenzamide;
- N-(2-chlorophenyl)-3 -(4-fluorophenyl)acrylamide; - l-[(dimethyl-lambda~4 — sulfanylidene)amino]-2-methoxy-4-nitrobenzene;
- 5-benzylidene-l-(2-chlorophenyl)-2,4,6(lH,3H,5H)-pyrimidinetrione;
- 4-ethyl-5,6-dimethyl-2-phenylpyrimidine;
- 2-(3-chlorobenzylidene)-lH-indene-l ,3(2H)-dione;
- 5- {5-[(3-methyl-5-oxo-l -phenyl- l,5-dihydro-4H-pyrazol-4-ylidene)methyl]-2- furyl } - 1 H-isoindole- 1 ,3 (2H)-dione;
- N-(2,5-dimethylphenyl)-3-(4-methoxyphenyl)acrylamide;
- 2-({2-[(4-nitrophenyl)amino]ethyl}amino)ethanol;
- N-(3 -methoxyphenyl)-4-propoxybenzamide;
- 2-(4-hydroxyphenyl)-3-phenyl-2,3-dihydro-4(lH)-quinazolinone;
- 4-methyl- 1 -(2-nitrobenzoyl)piperidine;
- 2-hydroxy-N'-[(2-methylphenyl)sulfonyl]benzohydrazide;
- 4-(l,3-benzothiazol-2-yl)butanoic acid;
- 4-(3-methylbenzylidene)-l-phenyl-3,5-pyrazolidinedione;
- 4-(2,4-dichlorophenoxy)-N-(2-ethoxyphenyl)butanamide;
- N-(2-methoxyphenyl)-N'-(phenylsulfonyl)benzenecarboximidamide;
- N-[2-(2-chloro-5-iodophenyl)-l,3-benzoxazol-5-yl]-2-methylpropanamide;
- 5-(4-butoxyphenyl)-3-cyclohexyl- 1 ,2,4-oxadiazole;
- N-(3,4-dichloroρhenyl)-N'-4H-l,2,4-triazol-4-yl urea;
- 6-chloro-4-phenyl-3-[3-(3,4,5-trimethoxyphenyl)acryloyl]-2(lH)-quinolinone;
- 6-bromo-4-phenyl-3-[3-(3,4,5-trimethoxyphenyl)acryloyl]-2(lH)-quinolinone; and - N-(IH- 1 ,2,3-benzotriazol- 1 -ylmethyl)-4-nitro- 1 ,2,5-oxadiazol-3-amine.
More preferably, the compounds are chosen in the group consisting in:
- 4-[5-(4-bromophenyl)-3-(4-nitrophenyl)-4,5-dihydro-lH-pyrazol-l-yl]-4- oxobutanoic acid
- 2,2,2-trichloro-N-(l,l-dioxido-2,3-dihydro-3-thienyl)-N-(4- methylphenyl)acetamide
- 3-(3-chlorophenyl)-7-methyl-4-methylene-3,4-dihydro-2(lH)-quinazolinone
- 3-[4-(3-bromobenzylidene)-3-methyl-5-oxo-4,5-dihydro-lH-pyrazol-l-yl]benzoic acid
- N-2,l,3-benzothiadiazol-4-yl-5-bromo-2-furamide
- 1 -acetyl-4-(2-chloro-4-nitrophenyl)-2-methylpiperazine
- 3-(3-methoxybenzylidene)-5-(4-methylphenyl)-2(3H)-ftιranone
- 3-[5-(3,4-dichlorophenyl)-2-furyl]acrylic acid
- (2-chloro-4-{[5-(2-chlorophenyl)-6-(ethoxycarbonyl)-7-methyl-3-oxo-5H- [l,3]thiazolo[3,2-a]pyrimidin-2(3H)-ylidene]methyl}-6-methoxyphenoxy)acetic acid
- 4- { [4-(diphenylmethyl)- 1 -piperazinyl] sulfonyl } -2, 1 ,3 -benzothiadiazole
- 4-[4-phenyl-5-(2-thienyl)- 1 H-imidazol-2-yl]- 1 ,2-benzenediol
- N-(3,4-dimethoxyphenyl)-4-[methyl(phenylsulfonyl)amino]benzamide
- l-[(2-hydroxyphenyl)carbonothioyl]-3-phenyl-5-(trifluoromethyl)-4,5-dihydro-lH- pyrazol-5-ol
- 2-methoxyethyl 4-[(4-tert-butylbenzoyl)amino]benzoate
- N-(2,3-dichlorophenyl)-3-(5-methyl-2-furyl)acrylamide - N-(4-fluorophenyl)-3-[3-(trifluoromethyl)phenyl]acrylamide
- 3-(2-fiirylniethyl)-2-(2-hydroxyphenyl)-2,3-dihydro-4(lH)-quinazolinone
- 2,6-di-tert-butyl-4-(2,3-dihydro- 1 H-perimidin-2-yl)phenol
- 3-benzyl-2-(2,6-dichlorophenyl)-2,3-dihydro-4(lH)-quinazolinone
- l-(3,4-dichlorobenzyl)-lH-indole-3-carbaldehyde
- N-(4-ethoxyphenyl)-2-{[5-(4-methoxyphenyl)-l,3,4-oxadiazol-2-yl]thio}acetamide
- 5-(4-nitrobenzylidene)-2-thioxo-3-[3-(trifluoromethyl)phenyl]-l,3-thiazolidin-4- one
- (3,5-dichlorophenyl)[(phenylsulfonyl)carbonyl]amine
- N-(2-bromophenyl)-3-(5-methyl-2-furyl)acrylamide
- 2-(2-chlorophenoxy)-N-[2-chloro-5-(trifluoromethyl)phenyl]acetamide
- N-[4-(4-acetyl- 1 -piperazinyl)phenyl]propanamide
- 8-[(dimethylamino)methyl]-9-hydroxy-2-methyl-4H-pyrido[l,2-a]pyrimidin-4-one
- 4-tert-butyl-N-[l-{[(2-methoxyphenyl)amino]carbonyl}-2-(2- thienyl)vinyl]benzamide
- 2-chloro-N-(3 -chloro-4-methoxyphenyl)benzamide
- N-[5-(l-adamantyl)-l,3,4-thiadiazol-2-yl]-N'-phenylurea
- N-(3,4-dichlorophenyl)-N'-{5-[(4-methylphenoxy)methyl]-l,3,4-thiadiazol-2- yl}urea
- N-(2,3-dihydro-l ,4-benzodioxin-6-yl)-2-(l -naphthyloxy)acetamide
- N-[4-(4-acetyl- 1 -piperazinyl)phenyl]-4-ethoxy-3-nitrobenzamide
- N-(2-chlorophenyl)-3 -(4-fluorophenyl)acrylamide - l-[(dimethyl-lambda~4~-sulfanylidene)amino]-2-methoxy-4-nitrobenzene
- 5-benzylidene-l-(2-chlorophenyl)-2,4,6(lH,3H,5H)-pyrimidinetrione; and
- 4-ethyl-5,6-dimethyl-2-phenylpyrimidine.
As used herein, the expression "DOCK5 protein" refers to a polypeptide comprising at least the DHR2 domain of the protein DOCK5 corresponding to the amino acid 1 132 to 1661 of the DOCK5 protein from Mus musculus SEQ ID NO:1 and derivatives thereof.
Therefore, the present invention is directed to a method for identifying a compound which inhibits the activation of RAC GTP ase, more specifically RAC 1/2 GTP ase, by DOCK5 protein comprising the steps of:
- coexpressing a polypeptide comprising at least the DHR2 domain of the protein
DOCK5 and the RAC proteins in a cell, wherein said polypeptide induces the conversion of inactive RAC, which inactive RAC is bound to GDP, to active RAC, which active RAC is bound to GTP,
- contacting or not said cell with said compound,
- determining the conversion of inactive RAC to active RAC, more specifically the conversion of inactive RAC 1/2 to active RAC 1/2, in the presence or absence of said compound, and
- selecting the compound inhibiting the conversion of inactive RAC to active RAC, more specifically the conversion of inactive RACl/2 to active RACl/2.
The full length DockS protein has an aminoterminal SH3 domain, between aminoacids Kl 1 and E68, followed by the DHRl domain, between aminoacids G440 and E682, and the DHR2 domain between aminoacids Ml 132 and Y1661 (Figure 3E).
Preferably, said DOCK5 protein corresponds to SEQ ID NO:1.
Again preferably, said DOCK5 protein corresponds to SEQ ID NO:4 corresponding to Homo sapiens DOCK5 protein. As used herein, the expression "RAC protein" refers to SEQ ID NO:2 and derivatives thereof.
According to a preferred embodiment, said cell is an eukaryotic cell, preferably a yeast cell.
Advantageously, said method comprises the expression of any protein, capable to interact with the active RAC protein and not with inactive RAC protein. One skilled in the art knows such protein known as a GTPase effector. According to a preferred embodiment, the protein capable to interact with the active RAC protein is chosen in the group comprising PAKl protein.
As used herein, the expression "PAKl protein" refers to the SEQ ID NO:3 and derivatives thereof.
As used herein, the term "derivatives'" refer to a polypeptide having a percentage of identity of at least 80% with amino acid 1132 to 1661 of SEQ ID NO: 1, SEQ ID NO: 2, EQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:9, or orthologs thereof, preferably of at least 90%, as an example of at least 95%, and more preferably of at least 99%.
As used herein, "percentage of identity" between two amino acids sequences or two nucleic sequences, means the percentage of identical amino-acids or nucleotides, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the amino acids sequences. As used herein, "best alignment" or "optimal alignment", means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity. The best sequences alignment to perform comparison can be realized, beside by a manual way, by using the global homology algorithm developed by SMITH and WATERMAN (Ad. App. Math., vol.2, p:482, 1981), by using the local homology algorithm developed by NEDDLEMAN and WUNSCH (J MoI. Biol, vol.48, p:443, 1970), by using the method of similarities developed by PEARSON and LIPMAN (Proc. Natl. Acd. ScL USA, vol.85, p:2444, 1988), by using computer softwares using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA), by using the MUSCLE multiple alignment algorithms (Edgar, Robert C, Nucleic Acids Research, vol. 32, p:1792, 2004 ). To get the best local alignment, one can preferably used BLAST software, with the BLOSUM 62 matrix, or the PAM 30 matrix. The identity percentage between two sequences of amino acids two nucleic sequence is determined by comparing these two sequences optimally aligned, the amino acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.
Advantageously, said cell further comprises a reporter gene under the control of a promoter sequence, and said RAC and PAKl proteins are each fused either with a transactivation domain or with a DNA binding domain specific of said promoter sequence, wherein the interaction of RAC with PAKl results in the induction of expression of the reporter gene.
The method corresponds to the Yeast Exchange Assay (YEA) as disclosed in DE TOLEDO et al. (FEBS, vol.480, p:287-292, 200) and International Patent application PCT WO 2005/064007 using the DOCK5 and the RAC protein.
Thus, the disclosure of YEA in Patent application PCT WO 2005/064007 (page 6, "description de l'invention" paragraph, to page 23) are incorporated herein by reference.
The term "reporter gene" is well known from the skilled person and can correspond to an auxotrophic marker or to a gene coding for a protein which can be simply detected such as GFP, luciferase or β-Gal.
In this embodiment, the determination of the conversion of inactive RAC to active RAC is done by determining the expression of the reporter gene. The inhibition of the expression of the reporter gene corresponding to an inhibition of the conversion of inactive RAC to active RAC.
In another embodiment, the present invention provides a method for the selection of compounds, which permit to decrease the level of expression of a DOCK5 gene (SEQ ID N0IO) in diseases associated with bone loss comprising the step of: a) contacting a test compound with an host cell expressing a reporter nucleic acid comprising a nucleic acid sequence coding for a reporter placed under the control of a promoter, which promoter comprises all or part of the promoter sequence of DOCK5 gene or a derivative thereof, and
b) measuring the level of expression of the reporter.
As used herein, the term "derivatives'" refer to a nucleic sequence having a percentage of identity of at least 80% with the sequence of DOCK5 promoter, preferably of at least 90%, as an example of at least 95%, and more preferably of at least 99%. The percentage of identity is as defined above.
By "compound" or "test compound", one should understand compounds of different nature, structure and origin, particularly biological compounds, nuclear factors, cofactors, and the like, chemical, synthetic compounds and the like, which are tested for their capacity of enhancing the level of expression of said gene implicated in antimicrobial defence.
The concentration of said test compound can be adjusted by the skilled person according to the characteristics of said compound (its toxicity, ability to penetrate cells, etc.), the number of cells, the length of the incubation period, etc. Generally, the cells are exposed to concentrations of test compounds ranging from 1 nM to 1 mM. Of course it is possible to test other concentrations without deviating from the invention, and also to test simultaneously different test compound concentrations.
Different adjuvants and/or vectors and/or products facilitating the penetration of the test compounds into the host cell such as liposomes, cationic lipids or polymers can also be used, when necessary.
By "decreasing the level of expression of a DOCK5 gene", one should understand that the expression level of DOCK5 gene is diminished or inhibited compared to a control level.
It should be noticed that said expression level of the DOCK5 gene is correlated to the expression level of the reporter gene in the method of the invention. In fact, one of skilled in the art can deduce that a test compound can decrease the expression level of the DOCK5 gene from the capacity of said compound to obtain an diminished expression level of the reporter gene in the method of the invention. In the present invention, the control level can be determined, by example, by measuring the expression level of the reporter gene in the absence of the test compound.
Thus, in a preferred embodiment, the method according to the invention further comprises a step c) of comparing the level of expression of the reporter gene as measured in step b) with the level of expression of the reporter gene in the absence of said test compound.
In another embodiment, the present invention provides a method for identifying a compound which inhibits the activation of RAC 1/2 GTPaseby inhibiting the binding of ELMOl protein (SEQ ID N°9) to the SH3 domain of DOCK5 comprising the steps of:
a) contacting a test compound with the ELMOl protein or a derivative thereof; b) determining the possible binding of said test compound to the ELMOl protein or the derivative thereof; and optionally c) selecting the compound inhibiting the conversion of inactive RAC 1/2 to active RAC 1/2.
As used herein, the expression "ELMOl protein" refers to SEQ ID N°9 and derivatives thereof.
The binding between said ELMOl protein and the tested compound can be measured by methods well known from one skilled in the art.
If the binding between said ELMOl protein and said test compound is observed, it can thus be conclude that the compound is an inhibitor of the binding of ELMOl and the SH3 domain of D0CK5, and that this compound is useful to inhibit the conversion of inactive RACl/2 to active RACl/2.
Optionally, said method can include a further step after step b) of contacting a polypeptide comprising at least the SH3 domain of D0CK5 or the derivative thereof with said test compound and ELMOl protein, and comparing the binding between said ELMOl protein and said polypeptide in the presence or in the absence of said compound. Alternatively, the present invention provides a method for identifying a compound which inhibits the activation of RAC 1/2 GTP ase by inhibiting the binding of ELMOl to the SH3 domain of DOCK5 comprising the steps of:
a) contacting a test compound with the ELMOl protein or the derivative thereof and a polypeptide comprising at least the SH3 domain of DOCK5 or the derivative thereof; b) measuring the binding between said ELMOl protein and said polypeptide in the presence or in the absence of said compound; and optionally c) selecting the compound inhibiting the conversion of inactive RAC 1/2 to active RAC 1/2.
The binding between said ELMOl protein and said polypeptide can be measured by methods well known from one skilled in the art. If the binding between said ELMOl protein and said polypeptide in the presence of the tested compound is lower than the one measured in absence of said compound, it can thus be conclude that the compound is an inhibitor of the binding of ELMOl to the SH3 domain of DOCK5, and that this compound is useful to inhibit the conversion of inactive RAC 1/2 to active RAC 1/2.
Optionally, the compounds as described above are coupled with a bisphosphonate radical. The bisphosphonate radical permits a fast incorporation of the compound after its administration.
Another object of the present invention is a compound as described above for treating and/or preventing bone loss diseases in a subject in need thereof.
Therefore, the present invention relates to the use of at least one compound as described above in preparing a drug for treating and/or preventing bone loss disease in a subject in need thereof.
Another object of the present invention is a pharmaceutical composition comprising at least one compound as described above and, optionally, a pharmaceutically acceptable support for treating and/or preventing bone loss diseases in a subject in need thereof.
Therefore, the present invention relates to the use of a pharmaceutical composition comprising at least one compound as described above in preparing a drug for treating and/or preventing bone loss diseases in a subject in need thereof. As examples of pharmaceutically acceptable supports, the composition can include emulsions, microemulsions, oil in water emulsions, anhydrous lipids and water in oil emulsions or other types of emulsions.
The inventive composition can further include one or more additives such as diluents, excipients, stabilizers and preservatives. Such additives are well known to those skilled in the art and are described notably in "Ullmann's Encyclopedia of Industrial Chemistry, 6th Ed." (various editors, 1989-1998, Marcel Dekker) and in "Pharmaceutical Dosage Forms and Drug Delivery Systems" (ANSEL et al, 1994, WILLIAMS & WILKINS). As used in the present application, the term "subject" refers to a mammal such as a rodent, cat, dog, primate or human, preferably said subject is a human.
Another object of the invention relates to a therapeutic method for treating a subject and/or preventing bone loss diseases, comprising the administration of a therapeutically effective quantity of a pharmaceutical composition as described above. A "therapeutically effective quantity" means a quantity that inhibits or reduces the osteoclats activation. Those skilled in the art will be able to determine said therapeutically effective quantity based on their general knowledge and on the methods described in the examples.
The compounds can be administered by any mode of administration such as, for example, by intramuscular, intravenous or oral route, etc.
The inventive compounds preferably will be administered at a concentration chosen by those skilled in the art according to the state of advancement of the disease and the targeting mode used, the age and the weight of the subject. Preferably, the compound will be administrated at a concentration of between 5 and 200μM, preferably at a concentration comprised between 10 and 100 μM.
In the following, the invention is described in more detail with reference to amino acid sequences, nucleic acid sequences and the examples. Yet, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.
EXAMPLES 1) Dock 5 niRNA expression
The expression of Dock5 was established in different mouse tissue. For this, DNasel- treated total RNA was extracted using the High pure RNA isolation kit (ROCHE DIAGNOSTICS). To generate cDNA, RNA was primed with 10-mer random primers and reverse transcription catalysed using SUPERSCRIPT II reverse transcriptase (INVITROGEN). Quantitative PCR was performed with a Light Cycler (ROCHE DIAGNOSTICS) or a Mx3000ρ PCR system (STRATAGENE) using the PLATINIUM Taq DNA polymerase (INVITROGEN) and SYBR GREEN I (BIOWITAKKER) as in described in COELHO et al. (Proc. Natl. Acad. ScL U.S.A., vol.102, p:l 1917-11922, 2005) with the primers Dock5-Up (TGGTGACACAGGGACAGTGG, SEQ ID NO:5) and Dock5-Do (CACCCCAACTAGCACGTGG, SEQ ID NO: 6) for Dock5, and Gapdh-Up (ACAGTCCATGCCATCACTGCC, SEQ ID NO: 7) and Gapdh-Do (GCCTGCTTCACCACCTTCTT, SEQ ID NO: 8) for Gapdh as a control.
The specificity was assessed by purification and sequencing of the PCR product. All real-time PCR measures to quantify cDNA were done in triplicate, and the 95% confidence limits of the ratios to Gapdh were determined by Student's t-test.The figure IA and B show the expression of Dock5 in different mouse tissues In figure IA, said expression has been normalised according to Dock5 osteoclasts' expression (i.e., Dock5 osteoclasts' expression corresponding to 100% level).
The analysed tissues of figure IA are as follow: Muscle 1 (Ml), Muscle 2 (M2), heart
(H), mammary gland at 10.5 days of embryo's development (GM 10.5), mammary gland at 13.5 days of embryo's development (GM 13.5), mammary gland at 15.5 days of embryo's development (GM 15.5), mammary gland at 18.5 days of embryo's development (GM 18.5), mammary gland of juvenile mouse (GM j), mammary gland at lactation (GM 1), brain (Br), kidney (Kd), uterus (Ut), liver (Lv), macrophage (Mac), Testis 1 (Tl), Testis 2 (T2), spleen (Sp), colon (Co), bone marrow (Bm), placenta at 13.5 days of embryo's development (Pl 13.5), placenta at 15.5 days of embryo's development (GM 15.5), and osteoclasts (Os).
Furthermore, total RNA of bone marrow macrophages (ND), induced for osteoclastic differentitation (OC) or dendritic cell differentiation (DC) and from mensenchymal stem cells (MSC JO) induced for osteoblastic differentiation (MSC J4) were extracted and level of Dock5 mRNA relative to Gapdh mRNA was determined by RT-PCR.
The results of figure 1 B show that Dock5 mRNA is not expressed in dendritic cells and osteoblasts.
The results show that Dock5 is predominantly expressed in osteoclats, but an important expression of Dock5 is also found in placenta (i.e., nearly 50%) and testis. The expression of Dock5 is reduced in bone marrow, colon, spleen and testis compared to osteoclasts (i.e., nearly 20%), whereas its expression in the other tested tissues is fewer (i.e., nearly 10%). Thus, the results established that the expression of Dock5 is very specific from the osteoclats.
2) obtaining of DOCK5 polyclonal antibody
A rabbit polyclonal antibody was raised to a mouse DOCK5 C-terminus peptide corresponding to amino acids 1658-1869 from mouse DOCK5 and purified by immunoaffϊnity. In fact, the amino acids sequences significantly differ between the differents members of the subgroup DOCK-A.
Osteoclastogenesis was induced by RANKL-stimulation in purified mouse bone marrow macrophages were purified and in RAW264.7 cell line as described in BRAZIER et al. (abovementioned, 2006), which cells were maintained in culture. At 0, 3 or 5 days of stimulation, the cells were subjected to SDS-PAGE and blotted on polyvinyl difluoride membrane (MILLIPORE IMMOBILON-P pore size 0.45 μm). After transfer, the membrane was incubated in TBS-T (Tris buffered saline containing 0.1% TWEEN) with 2% skim millk at room temperature for 30 min and then with rabbit antisera diluted 1 : 1000 in TBS-T overnight at 4°C. The bound antibodies were detected by peroxidase labelled anti-rabbit immunoglobulin chemoluminescence system (AMERSHAM) and LAS-1000 image analyser (FUJI FILM). As a control, the membrane was further incubated with GAPDH antibodies, the bound antibodies being detected as previously.
The Figure 2 A and B show the expression of DOCK5 and GAPDH proteins in purified mouse bone marrow macrophages at 0, 3 and 5 days from the RANKL-stimulated osteoclastogenesis . The results established that a protein of 215 kDa was induced during RANKL- stimulated osteoclastogenesis of purified mouse bone marrow macrophages (figure 2) and of RAW264.7 cell line (data not shown). This size is compatible with the size of the DOCK5 protein deduced from its mRNA.
Furthermore, total proteins were extracted from mouse tissues and subjected to western blot with antibodies against Dock5 and against tubulin for normalization.
The analysed tissues of figure 2C are as follow Ey: Eye, Sp: Spleen, St: Stomac, Te: Testis, Pl: Placenta, Lu: Lung, Br: Brain, He: Heart, Li: Liver, Ki: Kidney; Mu: Muscle.
The results of figure 2A confirm that Dock 5 is predominantly expressed in osteoclasts, testis and placenta.
3) DOCK5 polyclonal antibody specificity
ShRNA target sequences were selected in mouse Docks open reading frames, and the 65-mer sense and antisense strands of DNA oligonucleotides were designed according to the CLONTECH BIOINFORMATICS DATA server and are described in BRAZIER et al (abovementioned, 2006). The oligonucleotide was then synthetised by INVITROGEN annealed and cloned in pSINREN-RETROQ vector containing a puromycin resistance selection marker according to the manufacturer's instructions (CLONTECH). The pSIREN-RETROQ-Luc vector (CLONTECH) targeting firefly luciferase was used as a control. Retrovirus packaging was done by co-transfection of pSIREN-RETROQ vectors, the Friend MLV-based Gag-Pol expression vector pC57GP (LASSAUX et al, J. Virol, vol.79, p:6560-6564, 2005), and the VSV-G envelope glycoprotein expression vector pCSIG (BATTINI et al, Proc. Natl Acad. ScL, vol.96, p:1385-1390, 1999) into 293T cells using Jet PI (QBIOGEN) according to manufacturer's instructions. Viral supernatants were harvested 3 days after transfection and filtered through a 0.45 μm pore size filter.
For infections, RAW264.7 cells were plated at 2.105 cells per 6-cm dish. The next day, the medium was replaced for 4h with 1.5 ml of viral supernatant and 0.5 ml of growth medium containing 8μg/ml polybrene. Cells were left to recover in growth medium for 24 h, and infected cells were selected by addition of puromycin (3 μg/ml) for another 24h. Infected RAW264.7 were scrapped and reseeded in growth medium at 5.104 cells/well of a 6-well plate for RANKL-stimulated osteoclastogenesis as described in BRAZIER et al. (abovementioned, 2006).
Then, the detection of the DOCK5 protein was realized with the rabbit polyclonal anti-DOCK5 as described previously.
The Figure 2 A shows the expression of DOCK5 and GAPDH proteins in RAW264.7 cell lines infected with retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5) at 0, 3 and 5 days from the RANKL- stimulated osteoclastogenesis.
As described previously, the results established that a protein of 215 kDa was induced during RANKL-stimulated osteoclastogenesis of RAW264.7 cell line infected with a retrovirus coding for a small hairpin RNA directed against firefly luciferase. For
RAW264.7 cell line infected with a retrovirus coding for a small hairpin RNA directed against Dock5, no protein of 215 kDa was detected during RANKL-stimulated osteoclastogenesis. Finally, the results confirmed that the protein DOCKS, such as its corresponding RNA, is induced during osteoclastogenesis, and that the obtained rabbit polyclonal anti-DOCK5 antibody is specific of the DOCK5 protein.
4) Dock5 mediates Rac activation in vivo
We therefore examined whether the DOCK5 protein, and more specifically its DHR2 domain, could activate small GTPases of the Rho-family -i.e., RACl/2 and cdc42-.
To this end a GFP protein fused to the DHR2 domain of DOCK5 (see Figure 3A) was generated.
In vivo GTP loading of Rac and cdc42 was analysed as previously described in COTE & VUORI (J Cell. ScL, vol.l 15, p: 4901-4913, 2002).
Briefly, 293 -T cells were transfected in six- wells plates with a vector coding for the GFP fusion protein comprising the DHR2 domain of DOCK5 (DHR2) or with a vector coding for GFP (GFP). 48 hours after transfection, cells were lysed in MLB buffer (25mM
HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgC12, 1 mM EDTA and 10% glycerol). The clarified lysates were incubated for 30 minutes with the GST-PAK-PBD fusion protein bound to Glutathione sepharose. The beads were washed extensively with MLB buffer and the bound GTP-loaded Rac and cdc-42 were detected by immunoblotting. Equal amount of input lysate were analysed by immunoblotting to verify the expression levels of Rac, cdc42, GFP-DHR2 and GFP proteins. GST-PAK-PBD was expressed and purified for these experiments as described previously in ABASSI & VUORI (EMBO J, vol.21, p:4571-4582, 2002).
The figure 3 B shows the expression levels of Rac, cdc42, GFP-DHR2 and GFP proteins in total cell lysates (total) and the protein detected after GTP -trapping.
The results show that the expression of the DHR2 domain in 293 -T cells induces the activation of endogeneous Rac but has no effect on cdc42 (figure 3B). Finally, the results established that the DHR2 domain of DOCK5 is able to activate the Rac GTPase, whereas it has no effect on cdc42.
5) ELMOl binds to the SH3 domain of DOCK5
293 -T cells were cotransfected as described previously with a vector coding for the ELMOl protein or deleted from the C-terminus (ΔT625) - (GUMIENNY et al, Cell, vol.107, p:27-41, 2001) and a vector coding GFP fusion proteins comprising the Full length DOCK5 protein (FL), the DHR2 domain, the DOCK5 protein sequence deleted from (i) the amino acids 1 to 559 of its N-terminus extremity (ΔNter), including the SH3 domain and half of the DHRl domain, or the DOCK5 protein sequence deleted from (ii) the amino acids 1 to 82 comprising the SH3 domain (ΔSH3) (see figure 3 E).
48 hours after transfection, cells were lysed in MLB buffer (25mM HEPES, pH 7.5,
150 mM NaCl, 1% NP-40, 10 mM MgC12, 1 mM EDTA and 10% glycerol). The clarified lysates were immunoprecipitated with anti-GFP antibody and the bound ELMOl protein was detected by immunoblotting. Equal amount of input lysate were analysed by immunoblotting to verify the expression levels of ELMOl protein.
The figure 3C and 3F show the expression levels of ELMOl protein in total cell lysates (total) and after immunopreciptation with anti-GFOP antibody (IP GFP), in cells cotransfeted with a vector coding for ELMOl protein and full length DOCK5 (FL), the DHR2 domain (DHR2), DOCK5 deleted from its SH3 domain (ΔSH3) or from its N-term domain (ΔNter).
The results show that deletion if Dock5 SH3 domain or coexpression of full lengh
ELMOl with full length Dock5 greatly increased its exchange activity on Rac thus establishing that the N-term domain of DOCK5, and more specifically its SH3 domain, is necessary for the binding of ELMOl to DOCK5 (Figure 3C). Figure 3F shows that Dock5
N-terminal domain binds Elmol C-terminus.
6) The SH3 domain of DOCK5 inhibits Rac activation in vivo
In vivo GTP loading of Rac was determined as previously in the presence of different domains of the DOCK5 protein and, eventually, the simultaneous presence of the ELMOl protein.
The figure 3D shows the expression levels of Rac in total cell lysates (total) and the
RAC-GTP protein detected after GTP trapping in the cells transfected with a vector coding for the GFP protein (GFP), for the DHR2 domain of DOCK5 (DHR2), for the DOCK5 protein deleted from its SH3 domain (ΔSH3), for the DOCK protein (FL), eventually cotransfected with a vector coding for the ELMOl protein (FL+Elmol).
The results show as previously that the expression of the DHR2 domain is able to activate the Rac GTPase and that the SH3 domain inhibits this activation (Figure 3D). In fact, the deletion of the SH3 domain results in the activation of the Rac GTPase by the deleted DOCK5 protein. Finally, the binding of ELMOl to the SH3 domain results in the activation of the Rac GTPase.
7) DOCK5 is a major activator of Rac in osteoclats.
RAW264.7 cell lines stimulated with RANKL were infected as described previously with a retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5).
Furthermore, the levels of active Rac in TCL from Dock5+ + and Dock5" " osteoclasts were determined. Dock5v" mice were obtained by gene trap (Laurin et al. 2008) to generate Dock5 deficient osteoclasts. The in vivo GTP loading of Rac was determined as disclosed previously.
The figure 4 shows the average of three independent experiments with active Rac levels set to 1 in control shLuc and Dock5+/+ osteoclasts. Error bars : SD.
The figure 4A show the expression levels of Rac in total cell lysates (total Rac) and the RAC-GTP protein detected after GTP trapping in the cells infected with a retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5).
The figure 4B shows that Dock5"Λ osteoclasts have reduced active Rac levels compared to the control level of Dock5+/+ osteoclasts.
The results established that the inhibition of DOCK5 expression results in a decrease of the levels of active RAC (i.e., 40%) in osteoclasts expressing Dock5 shRNAs and osteoclasts derived from Dock5 KO BMMs as compared to controls. Thus, DOCK5 is an essential exchange factor of RAC in osteoclasts.
8) DOCK5 is necessary for mineralised matrix resorption
RAW264.7 cell lines were infected as described previously with a retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5), and then osteoclastogenesis was stimulated with RANKL. The obtained cells were then cultured on calcium phosphate substrates to induce the formation of the actin ring. After 48 hours, cells were fixed and stained for actin using rhodamine-labeled Phalloidin to reveal the sealing zone (figure 5).
The figure 6 shows the polymerisation of actin in RAW264.7 cell lines stimulated with RANKL which have been infected with a retrovirus coding for either small hairpin RNA directed against firefly luciferase (shLuc) or dock5 (shDock5) and the mineralised matrix resorption in the presence of said osteoclasts.
The results show that in the osteoclasts, the DOCK5 protein is associated with the podosome and with the sealing zone (data not shown). The osteoclasts wherein DOCK5 expression was inhibited show a default of contraction and of sealing zone formation. The measure of mineralised matrix resorption surface by VON KOSSA staining shows a strong decrease of the resorption by osteoclasts wherein D0CK5 expression was decreased.
9) Confirmation by osteoclasts from DockS^'mice
BMMs (bone marrow macrophages) isolated from Dock5+ + and Dock5~Λ mice were differentiated into osteoclasts in the presence of 100 ng/ml RANKL and 10 ng/ml M-CSF. TCL (total cell extracts) were prepared at days 0, 3 and 4 and subjected to western blot with antibodies against Dock5 and β-gal and against tubulin for normalization.
Osteoclasts derived from Dock5v~ BMMs express Dock5 truncated after aminoacid 1115, between DHRl and DHR2 domains, and fused to a β-geo cassette (Figure 7A). Furthermore, the differentiated osteoclasts were fixed and stained with TRAP and
Hoeschst at day 5 to determine the number of MNCs (multinucleated cells). Figure 7B (average and SD from four independent experiments **: significant difference, p<0.01, Mann & Whitney test) shows that the efficiency of TRAP positive MNCs formation was reduced in Dock5~Λ BMMs as compared to Dock5+/+ and osteoclasts were smaller. Furthermore, in order to show that osteoclasts differentiated from Dock5~A BMMs can't assemble a sealing zone, they were seeded on calcium-phosphate substrate to induce the formation of the actin ring. After 48 hours, cells were fixed and stained for actin using rhodamine-labeled Phalloidin (green) to reveal the sealing zone and with Hoeschst dye to stain nuclei (blue) (data not shown). It was observed that on calcium-phosphate substrates, sealing zone assembly and resorption was defective in DockS" " osteoclasts.
Finally, to demonstrate that Dock5~Λ osteoclasts can't form resorption pit, derived from Dock5"Λ BMMs were differentiated on bone sliced for 5 days, fixed and observed by scanning electron microscopy. The results show that when seeded on bone slices, Dock5" ~ osteoclasts did not form resorption pits.
Moreover, in order to show that Dock5~ " osteoclasts are defective for bone resorption, the levels of collagen degradation peptide (CTx) were determined in the medium of Dock5+/+ and Dock5"Λ osteoclasts after 5 days of differentiation and bone slices were stained. Figure 7C shows average and SD of three osteoclast-seeded wells from one experiment, representative of three independent experiments. The measurement of collagen telopeptide (CTx) confirmed that the resorbing activity of Dock5" " osteoclasts was defective (Figure 7C).
10) Suppression of Dock5 impairs RAC activation in osteoclasts. The levels of osteoclastic markers in wild type and Dock5 deficient osteoclasts derived from BMM of Dock5+/+ or Dock5"Λ animals or from control and Dock5 shRNA expressing RAW264.7 cells. Total RNA was prepared from Dock5+/+ and Dock5"A BMMs grown for 5 days in the presence of M-CSF only (black bars) or in the presence of RANKL and M-CSF to obtain osteoclasts (white bars). The levels of indicated gene mRNAs relative to Gapdh mRNA were determined by RT-PCR.
The results of figure 8A show that the expression of osteoclast differentiation markers is normal in osteoclasts differentiated from DockS"7" BMMs. This indicated osteoclast maturation was not affected and suggested Dock5 deficiency did not impair the capacity of osteoclasts to respond to M-CSF and RANKL in vitro.
Moreover, the ability of Dock5~Λ preosteoclasts to respond to M-CSF and RANKL was not the result of a compensatory increase in Dockl or Dock2 expression as their mRNA levels were identical as in Dock5+/+ (Figure 8B).
Preosteoclasts prepared from Dock5+/+ and Dock5"Λ BMMs were stimulated with M- CSF or RANKL for the inducated amount of time. The levels of ERK, ρ38 and Akt phosphorylation in TCL were determined by western blot.
The results show that M-CSF-driven phosphorylation ERK and p38MAP kinase (Figure 8C) and RANKL-driven phosphorylation of Akt (Figure 8D) were unaffected in Dock57" preosteoclasts as compared to controls.
Finally, these results established that DOCK5 is a new therapeutic target for limiting bone loss in menopause, osteoporosis, osteopenia due to bone metastases, periarticular erosions in rheumatoid arthritis, primary hyperparathyroidism, hypercalcemia of malignancy, Paget's disease of bone, periodontal disease, immobilization induced osteopenia, or in glucocorticoid treatment. Because of the specific osteoclasts DOCK5 expression, the targeting of DOCK5 may limit side effects such as the ones observed with drugs for treating bone loss.
11) Identification of DOCK5 inhibitor
In order to identify DOCK5 inhibitors, which inhibitors can be useful for treating bone loss associated disease, we use the Yeast Exchange Assay (YEA) as disclosed in DE TOLEDO et al. (FEBS, vol.480, p:287-292, 200) and International Patent application PCT WO 2005/064007.
Briefly, we transform a yeast strain TAT7 (Mata, trpl, his3, Ieu2, ura3, ade2, LYS::
(LexAop)4-HIS3, URA3:: (LexAop)8-lacZ) provided by J. CAMONIS) with vectors expressing the DHR2 domain of DOCK5 fused to a myc-tag (SEQ ID NO: ...), the wild type Rac GTPase fused to LexA and its effector PAK fused to the transactivation domain ofGAL4.
In the obtained transformed yeast, the expression of the DHR2 domain of DOCKS induces the activation of Rac, which activated Rac interacts with its effector PAK resulting in the expression of reporter genes β-Gal and His3 (see Figure 6).
In order to modify yeast cell membrane permeability, a mutation in the Erg6 gene has been introduced as disclosed in BLANGY et al (Biol. Cell., vol.98(9), ρ:511-22, 2006). This mutation of the Erg6 gene increases the entry of the screened compounds in the yeast cells, and thus enables to limit the concentration of the screened compounds.
For screening DOCK5 inhibitors, which can be useful for treating bone loss diseases, the transformed yeast is contacted with several chemical or peptidic molecules, and the chemical or peptidic molecules inhibiting the expression of reporter genes β-Gal and His3 are selected for further testing in the bone loss model disclosed in 8 and then in bone loss diseases models.
The yeast strain TAT7 was used to identify DOCK5 inhibitors. The strain was seeded, in a 96-well culture plate in a selective medium devoid of histidine or in a non selective medium where histidine is added. 2560 compounds were screened to select the ones which inhibit the growth of the strains in a selective medium without having effect on the growth in a non selective medium. DMSO was used as a control. The compounds were tested at a concentration of 200μM in presence of 1% DMSO. The growth of the yeasts was measured by optical density at 600 nm at t= 2 hours, 15 hours, 20 hours and 24 hours after seeding. The inhibiting compounds were defined as follows:
-At time n, the growth derivative Cr (medium) = (OD600Tn-OD600T2)/Tn-T2 in test medium (-HIS) and in toxic medium (+HIS).
- At each time and for each plate, the Cr (-HIS) and Cr (+HIS) medium control was calculated on the control.
- the ratio R(compound)= Cr (-HIS) and Cr(+HIS) and R was calculated for each plate
- the inhibition rate was determined by dividing by the control ratio I(compound)= R(compound)/R(control) *
-the selected compounds are those showing a ratio I(compound) < 0.9 at each time.
Results are shown in table 2.
55 compounds were thus selected as inhibiting the activation of RACl/2 by Dock5.
12) Toxicity test on osteoclast precursors.
The selected compounds were then tested for their toxicity on osteoclast precursors. Since these cells do not express Dock5, a Dock5 inhibitor should not affect their growth. RAW264.7 cells used as osteoclasts inhibitors were allowed to grow for 72 hours with 10 to lOOμM of compound. The growth of the cells was compared to control cells which were grown with 0.5% DMSO.
The results are presented in table 2. The optimal concentration was the determined for the compounds which were not toxic (the concentration which does not affect the growth of the cells).
13) Toxicity test on differentiated osteoclasts. The compounds were tested for their toxicity on differentiated osteoclasts at the concentration determined above. RAW264.7 cells differentiated in osteoclasts were allowed to grow for 72 hours in presence of the tested compounds. The tartrate-resistant acid phosphatase (TRAP) was then revealed in osteoclasts by coloration (SUDA et al, 1997). This osteoclasts specific labeling permits to visualize the cell morphology. The cell morphology was then compared to control cells which were allowed to grow in presence of 0.5% DMSO. The compounds were then classified in 3 categories:
compounds which induce the death of all the osteoclasts after 72 hours (-)
compounds which induce morphological anomalies and/or death of part of the osteoclasts (+/-)
Compounds which do not induce visible modifications of the osteoclasts.
The results are shown in table 2.
14) Resorption inhibition test
The identified compounds were used at the same concentration as defined above on osteoclats seeded on minerOalised matrix resorption surface of calcium phosphate (Osteologic Biocoat Clontech Reference 354609) during 72 hours. Then the mineralised matrix was coloured with silver nitrate in order to show the resorbed areas. The compounds were classified in 3 categories:
Compounds that totally prevent resorption in 72 hours (-)
- Compounds that induce an attenuated resorption compared to the control (+/-)
Compounds that do not visibly modify the osteoclats resorption activity compared to the control. (+)
The compounds of the resorption categories (+/-) and (-) represent new inhibitors of the bone resorption. They were used at a concentration of 10 to 100 μM.
To confirm the results, the compounds were then tested in vivo in a mouse which presents osteoporose. Table 1 : compound identified by the screening method of the present invention
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Table 2
Figure imgf000058_0002
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001

Claims

1. A method for identifying a compound which inhibits the activation of RAC GTPase by DOCK5 protein comprising the steps of:
- coexpressing at least the DHR2 domain of DOCK5 and the RAC proteins in a cell, wherein said at least the DHR2 domain of DOCK5 protein induces the conversion of inactive RAC, which inactive RAC is bound to GDP, to active RAC, which active RAC is bound to GTP.
contacting or not said cell with said compound,
determining the conversion of inactive RAC to active RAC in the presence or absence of said compound, and
selecting the compound inhibiting the conversion of inactive RAC to active RAC.
2. The method of claim 1, wherein the selected compound is useful for treating disease - associated bone loss.
3. The method of claim 2, wherein said disease associated with bone loss is selected in the group comprising osteoporosis, osteopenia due to bone metastases, periarticular erosions in rheumatoid arthritis, primary hyperparathyroidism, hypercalcemia of malignancy, Paget's disease of bone, periodontal disease, immobilization induced osteopenia, and glucocorticoid treatment.
4. The method of any one of claims 1 to 3, wherein said method further comprises the step of testing the inhibition of bone resorption by the selected compound.
5. The method of any one of claims 1 to 4, wherein said DOCK5 protein refers to a polypeptide comprising the DHR2 domain of the protein DOCK5 corresponding to the amino acid 1132 to 1661 of the DOCK5 protein from Mus musculus SEQ ID NO:1 and derivatives thereof.
6. The method of any one of claims 1 to 5, wherein said DOCK5 protein corresponds to SEQ ID NO:4 corresponding to Homo sapiens D0CK5 protein.
7. The method of any one of claims 1 to 6, wherein the RAC protein corresponds to SEQ ID NO:2 and derivatives thereof.
8. The method of any one of claims 1 to 7, wherein said method further comprises the expression of any protein capable to interact with the active RAC protein and not with the inactive RAC protein.
9. The method of claim 8, wherein said cell further comprises a reporter gene under the control of a promoter sequence, and said active RAC and protein interacting with are each fused either with a transactivation domain or with a DNA binding domain specific of said promoter sequence, wherein the interaction of active RAC with the interacting protein results in the induction of expression of the reporter gene.
10. The method of any of claims 8 and 9, wherein the protein interacting with active RAC protein is chosen in the group comprising PAKl protein which corresponds to the SEQ ID NO:3 and derivatives thereof.
11. A method for the selection of compounds, which permit to decrease the level of expression of a DOCKS gene in diseases associated with bone loss comprising the step of: a) contacting a test compound with an host cell expressing a reporter nucleic acid comprising a nucleic acid sequence coding for a reporter placed under the control of a promoter, which promoter comprises all or part of the promoter sequence of DOCK5 gene or a derivative thereof, and
b) measuring the level of expression of the reporter.
12. A method for identifying a compound which inhibits the activation of RACl/2 GTPase by inhibiting the binding of ELMOl to the SH3 domain of DOCK5 comprising the steps of:
a) contacting a test compound with the ELMOl protein or a derivative thereof; b) determining the possible binding of said test compound to the ELMOl protein or the derivative thereof; and optionally c) selecting the compound inhibiting the conversion of inactive RAC 1/2 to active RAC 1/2.
13. A method for identifying a compound which inhibits the activation of RAC 1/2
GTP ase by inhibiting the binding of ELMOl to the SH3 domain of DOCK5 comprising the steps of:
a) contacting a test compound with the ELMOl protein or the derivative thereof and a polypeptide comprising at least the SH3 domain of DOCK5 or the derivative thereof; b) measuring the binding between said ELMOl protein and said polypeptide in the presence or in the absence of said compound; and optionally c) selecting the compound inhibiting the conversion of inactive RAC 1/2 to active RACl/2.
14. A compound chosen in the group consisting in:
- 4-[5-(4-bromophenyl)-3-(4-nitrophenyl)-4,5-dihydro-lH-pyrazol-l-yl]-4- oxobutanoic acid;
- 2,2,2-trichloro-N-(l,l-dioxido-2,3-dihydro-3-thienyl)-N-(4- methylphenyl)acetamide;
- 3-(3-chlorophenyl)-7-methyl-4-methylene-3,4-dihydro-2(l H)-quinazolinone;
- 3-[4-(3-bromobenzylidene)-3-methyl-5-oxo-4,5-dihydro-lH-pyrazol-l- yljbenzoic acid;
- N-2, 1 ,3-benzothiadiazol-4-yl-5-bromo-2-furamide;
- 1 -acetyl-4-(2-chloro-4-nitrophenyl)-2-methylpiperazine;
- 3-(3-methoxybenzylidene)-5-(4-methylphenyl)-2(3H)-furanone;
- 3-[5-(3,4-dichlorophenyl)-2-furyl]acrylic acid; - (2-chloro-4-{[5-(2-chlorophenyl)-6-(ethoxycarbonyl)-7-methyl-3-oxo-5H-
[1,3 ]thiazolo[3 ,2-a]pyrimidin-2(3 H)-ylidene]methyl } -6-methoxyphenoxy)acetic acid;
- 4- { [4-(diphenylmethyl)- 1 -piperazinyl] sulfonyl } -2, 1 ,3 -benzothiadiazole;
- 4-[4-phenyl-5-(2-thienyl)- 1 H-imidazol-2-yl]- 1 ,2-benzenediol;
- N-(3,4-dimethoxyphenyl)-4-[methyl(phenylsulfonyl)amino]benzamide;
- l-[(2-hydroxyphenyl)carbonothioyl]-3-phenyl-5-(trifluoromethyl)-4,5-dihydro- lH-pyrazol-5-ol;
- 2-methoxyethyl 4-[(4-tert-butylbenzoyl)amino]benzoate;
- N-(2,3-dichlorophenyl)-3-(5-methyl-2-furyl)acrylamide;
- N-(4-fluorophenyl)-3 -[3 -(trifluoromethyl)phenyl] acrylamide;
- 3-(2-furylmethyl)-2-(2-hydroxyphenyl)-2,3-dihydro-4(lH)-quinazolinone;
- N-(4-ethoxyphenyl)-2-{[5-(4-methoxyphenyl)-l,3,4-oxadiazol-2- yl]thio } acetamide;
- 5-(4-nitrobenzylidene)-2-thioxo-3-[3-(trifluoromethyl)phenyl]-l,3-thiazolidin-4- one;
- (3,5-dichlorophenyl)[(phenylsulfonyl)carbonyl]amine;
- N-(2-bromophenyl)-3 -(5-methyl-2-furyl)acrylamide;
- 2-(2-chlorophenoxy)-N-[2-chloro-5-(trifluoromethyl)phenyl]acetamide;
- N-[4-(4-acetyl-l-piperazinyl)phenyl]propanamide;
- 8-[(dimethylamino)methyl]-9-hydroxy-2-methyl-4H-pyrido[ 1 ,2-a]pyrimidin-4- one; - 4-tert-butyl-N-[l-{[(2-methoxyphenyl)amino]carbonyl}-2-(2- thienyl)vinyl]benzamide;
- 2-chloro-N-(3-chloro-4-methoxyphenyl)benzamide;
- 2,6-di-tert-butyl-4-(2,3 -dihydro- 1 H-perimidin-2-yl)phenol;
- 3-benzyl-2-(2,6-dichlorophenyl)-2,3-dihydro-4(lH)-quinazolinone;
- 1 -(3,4-dichlorobenzyl)- 1 H-indole-3-carbaldehyde;
- N-[5-(l-adamantyl)-l,3,4-thiadiazol-2-yl]-N'-phenylurea;
- N-(3,4-dichlorophenyl)-N'- {5-[(4-methylphenoxy)methyl]-l ,3,4-thiadiazol-2- yl}urea;
- N-(2,3-dihydro-l,4-benzodioxin-6-yl)-2-(l-naphthyloxy)acetamide;
- N-[4-(4-acetyl- 1 -piperazinyl)phenyl]-4-ethoxy-3-nitrobenzamide;
- N-(2-chlorophenyl)-3 -(4-fluorophenyl)acrylamide;
- l-[(dimethyl-lambda~4~-sulfanylidene)amino]-2-methoxy-4-nitrobenzene;
- 5-benzylidene-l-(2-chlorophenyl)-2,4,6(lH,3H,5H)-pyrimidinetrione;
- 4-ethyl-5,6-dimethyl-2-phenylpyrimidine;
- 2-(3 -chlorobenzylidene)- 1 H-indene- 1 ,3 (2H)-dione;
- 5-{5-[(3-methyl-5-oxo-l-phenyl-l,5-dihydro-4H-pyrazol-4-ylidene)methyl]-2- furyl } - 1 H-isoindole- 1 ,3 (2H)-dione;
- N-(2,5-dimethylphenyl)-3-(4-methoxyphenyl)acrylamide;
- 2-({2-[(4-nitrophenyl)amino]ethyl}amino)ethanol;
- N-(3 -methoxyphenyl)-4-propoxybenzamide;
- 2-(4-hydroxyphenyl)-3 -phenyl-2,3 -dihydro-4( 1 H)-quinazolinone; - 4-methyl- 1 -(2-nitrobenzoyl)piperidine;
- 2-hydroxy-N'-[(2-methylphenyl)sulfonyl]benzohydrazide;
- 4-(l,3-benzothiazol-2-yl)butanoic acid;
- 4-(3-methylbenzylidene)-l-phenyl-3,5-pyrazolidinedione;
- 4-(2,4-dichlorophenoxy)-N-(2-ethoxyphenyl)butanamide;
- N-(2-methoxyphenyl)-N'-(phenylsulfonyl)benzenecarboximidamide;
- N-[2-(2-chloro-5-iodophenyl)- 1 ,3-benzoxazol-5-yl]-2-methylpropanamide;
- 5-(4-butoxyphenyl)-3-cyclohexyl-l ,2,4-oxadiazole;
- N-(3,4-dichlorophenyl)-N'-4H-l,2,4-triazol-4-yl urea;
- 6-chloro-4-phenyl-3-[3-(3,4,5-trimethoxyphenyl)acryloyl]-2(lH)-quinolinone;
- 6-bromo-4-phenyl-3-[3-(3,4,5-trimethoxyphenyl)acryloyl]-2(lH)-quinolinone; and
- N-( 1 H- 1 ,2,3 -benzotriazol- 1 -ylmethyl)-4-nitro- 1 ,2,5-oxadiazol-3 -amine for treating and/or preventing bone loss diseases in a subject in need thereof.
15. A pharmaceutical composition comprising at least one compound chosen in the group consisting in - 4-[5-(4-bromophenyl)-3-(4-nitrophenyl)-4,5-dihydro-lH- pyrazol-l-yl]-4-oxobutanoic acid;
- 2,2,2-trichloro-N-(l , 1 -dioxido-2,3-dihydro-3-thienyl)-N-(4- methylphenyl)acetamide;
- 3 -(3 -chlorophenyl)-7-methyl-4-methylene-3 ,4-dihydro-2( 1 H)-quinazolinone;
- 3-[4-(3-bromobenzylidene)-3-methyl-5-oxo-4,5-dihydro-lH-pyrazol-l- yl]benzoic acid;
- N-2, 1 ,3-benzothiadiazol-4-yl-5-bromo-2-furamide; - 1 -acetyl -4-(2-chloro-4-nitroρhenyl)-2-methylpiperazine;
- 3-(3-methoxybenzylidene)-5-(4-methylphenyl)-2(3H)-fiiranone;
- 3-[5-(3,4-dichlorophenyl)-2-furyl]acrylic acid;
- (2-chloro-4-{[5-(2-chlorophenyl)-6-(ethoxycarbonyl)-7-methyl-3-oxo-5H- [l,3]thiazolo[3,2-a]pyrimidin-2(3H)-ylidene]methyl}-6-methoxyphenoxy)acetic acid;
- 4- {[4-(diphenylmethyl)-l -piperazinyl]sulfonyl} -2, 1 ,3-benzothiadiazole;
- 4-[4-phenyl-5-(2-thienyl)-lH-imidazol-2-yl]-l,2-benzenediol;
- N-(3,4-dimethoxyphenyl)-4-[methyl(phenylsulfonyl)amino]benzamide;
- 1 -[(2-hydroxyphenyl)carbonothioyl]-3-phenyl-5-(trifluoromethyl)-4,5-dihydro- lH-pyrazol-5-ol;
- 2-methoxyethyl 4-[(4-tert-butylbenzoyl)amino]benzoate;
- N-(2,3-dichlorophenyl)-3-(5-methyl-2-furyl)acrylamide;
- N-(4-fluorophenyl)-3 -[3 -(trifluoromethyl)phenyl] acrylamide;
- 3-(2-furylmethyl)-2-(2-hydroxyphenyl)-2,3-dihydro-4(lH)-quinazolinone;
- N-(4-ethoxyphenyl)-2-{[5-(4-methoxyphenyl)-l,3,4-oxadiazol-2- yl]thio}acetamide;
- 5-(4-nitrobenzylidene)-2-thioxo-3-[3-(trifluoromethyl)phenyl]- 1 ,3-thiazolidin-4- one;
- (3,5-dichlorophenyl)[(phenylsulfonyl)carbonyl]amine;
- N-(2-bromophenyl)-3 -(5-methyl-2-furyl)acrylamide;
- 2-(2-chlorophenoxy)-N-[2-chloro-5-(trifluoromethyl)phenyl]acetamide; - N-[4-(4-acetyl-l -piperazinyl)phenyl]propanamide;
- 8-[(dimethylamino)methyl]-9-hydroxy-2-methyl-4H-pyrido[ 1 ,2-a]pyrimidin-4- one;
- 4-tert-butyl-N-[ 1 - { [(2-methoxyphenyl)amino]carbonyl} -2-(2- thienyl)vinyl]benzamide;
- 2-chloro-N-(3 -chloro-4-methoxyphenyl)benzamide;
- 2,6-di-tert-butyl-4-(2,3-dihydro-lH-perimidin-2-yl)phenol;
- 3-benzyl-2-(2,6-dichlorophenyl)-2,3-dihydro-4(lH)-quinazolinone;
- 1 -(3,4-dichlorobenzyl)- 1 H-indole-3-carbaldehyde;
- N-[5-(l-adamantyl)-l,3,4-thiadiazol-2-yl]-N'-phenylurea;
- N-(3,4-dichlorophenyl)-N'-{5-[(4-methylphenoxy)methyl]-l,3,4-thiadiazol-2- yljurea;
- N-(2,3-dihydro-l,4-benzodioxin-6-yl)-2-(l-naphthyloxy)acetamide;
- N-[4-(4-acetyl- 1 -piperazinyl)phenyl]-4-ethoxy-3-nitrobenzamide;
- N-(2-chlorophenyl)-3-(4-fluorophenyl)acrylamide;
- l-[(dimethyl-lambda~4 — sulfanylidene)amino]-2-methoxy-4-nitrobenzene;
- 5-benzylidene-l-(2-chlorophenyl)-2,4,6(lH,3H,5H)-pyrimidinetrione;
- 4-ethyl-5,6-dimethyl-2-phenylpyrimidine;
- 2-(3-chlorobenzylidene)-lH-indene-l,3(2H)-dione;
- 5-{5-[(3-methyl-5-oxo-l-phenyl-l,5-dihydro-4H-pyrazol-4-ylidene)methyl]-2- furyl} - 1 H-isoindole- 1 ,3 (2H)-dione;
- N-(2,5-dimethylphenyl)-3-(4-methoxyphenyl)acrylamide; - 2-( {2-[(4-nitrophenyl)amino]ethyl}amino)ethanol;
- N-(3 -methoxyphenyl)-4-propoxybenzamide;
- 2-(4-hydroxyphenyl)-3-phenyl-2,3-dihydro-4(lH)-quinazolinone;
- 4-methyl- 1 -(2-nitrobenzoyl)piperidine;
- 2-hydroxy-N'-[(2-methylphenyl)sulfonyl]benzohydrazide;
- 4-(l,3-benzothiazol-2-yl)butanoic acid;
- 4-(3-methylbenzylidene)-l-phenyl-3,5-pyrazolidinedione;
- 4-(2,4-dichlorophenoxy)-N-(2-ethoxyphenyl)butanamide;
- N-(2-methoxyphenyl)-N'-(phenylsulfonyl)benzenecarboximidamide;
- N-[2-(2-chloro-5-iodophenyl)- 1 ,3-benzoxazol-5-yl]-2-methylpropanamide;
- 5-(4-butoxyphenyl)-3-cyclohexyl-l,2,4-oxadiazole;
- N-(3,4-dichlorophenyl)-N'-4H-l,2,4-triazol-4-yl urea;
- 6-chloro-4-phenyl-3-[3-(3,4,5-trimethoxyphenyl)acryloyl]-2(lH)-quinolinone;
- 6-bromo-4-phenyl-3-[3-(3,4,5-trimethoxyphenyl)acryloyl]-2(lH)-quinolinone; and
- N-(I H-1 ,2,3-benzotriazol-l -ylmethyl)-4-nitro- 1 ,2,5-oxadiazol-3-amine and, optionally, a pharmaceutically acceptable support for treating and/or preventing bone loss diseases in a subject in need thereof.
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JP6016788B2 (en) * 2011-05-16 2016-10-26 国立大学法人九州大学 Low molecular weight compound controlling Rac activation by DOCK-A subfamily molecule and use thereof
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