+

WO2000031289A1 - Utilisation de trap activee pour rechercher des inhibiteurs specifiques de trap et technique aidant a l'identification d'un compose destine au traitement de maladies ou pathologies degeneratives provoquant une resorption osseuse accrue - Google Patents

Utilisation de trap activee pour rechercher des inhibiteurs specifiques de trap et technique aidant a l'identification d'un compose destine au traitement de maladies ou pathologies degeneratives provoquant une resorption osseuse accrue Download PDF

Info

Publication number
WO2000031289A1
WO2000031289A1 PCT/SE1999/002096 SE9902096W WO0031289A1 WO 2000031289 A1 WO2000031289 A1 WO 2000031289A1 SE 9902096 W SE9902096 W SE 9902096W WO 0031289 A1 WO0031289 A1 WO 0031289A1
Authority
WO
WIPO (PCT)
Prior art keywords
trap
activated
bone
screening
use according
Prior art date
Application number
PCT/SE1999/002096
Other languages
English (en)
Inventor
Göran Andersson
Barbro Ek-Rylander
Cornelia Oellig
Original Assignee
Pharmacia Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmacia Ab filed Critical Pharmacia Ab
Priority to EP99962612A priority Critical patent/EP1131463A1/fr
Priority to CA002351872A priority patent/CA2351872A1/fr
Priority to NZ511428A priority patent/NZ511428A/xx
Priority to AU19019/00A priority patent/AU759597B2/en
Priority to JP2000584097A priority patent/JP2002530117A/ja
Priority to IL14288099A priority patent/IL142880A0/xx
Publication of WO2000031289A1 publication Critical patent/WO2000031289A1/fr
Priority to NO20012453A priority patent/NO20012453L/no

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)
    • 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
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates to the use of activated recombinant TRAP ( tartrate-resistant and purple acid phosphatases ) for screening for specific inhibitor of TRAP activity useful in the treatment of diseases or degenerative conditions resulting in increased bone resorption, such as tissue damages, bone metabolic disorders, osteoporosis.
  • activated recombinant TRAP tartrate-resistant and purple acid phosphatases
  • TRAP can be activated by proteolytic activation of TRAP e.g. by cleaving with a protease, papain-like enzyme.
  • TRAP is an enzyme expressed predominantly in bone resorbing cells (osteoclasts). Investigations in TRAP knockout mice show that the resorption process is disrupted so that, with increasing age, TRAP knockout mice become osteopetrotic. i.e. have an increased bone mineral content and more dense bone is formed. Osteoclasts prepared from these animals are functional and do resorb bone but to a lesser extent than wild type mouse osteoclasts.
  • Phosphatases are enzymes that remove organic phosphates from proteins.
  • the mammalian Purple Acid Phosphatases (PAPs) a group of enzymes to which Tartrate Resistant and purple Acid Phosphatase (TRAP) belongs, are characterized by a binuclear iron center at the active site.
  • these enzymes are also referred to as tartrate-resistant acid phosphatases (TRAPs) (EC 3.1.3.2) or type 5 acid phosphatases [4].
  • TRAPs are iron-containing, monomeric glycoproteins with molecular weights of around 35,000 Da [5].
  • the deduced amino acid sequences of human, rat and mouse TRAPs show a high degree of homology with the mammalian members of the PAP family, e.g uteroferrin (Uf) and bovine spleen PAP [6-9].
  • Uf uteroferrin
  • Mammalian PAPs contain a FeFe center, while a plant PAP from red kidney beans
  • KB PAP instead has a FeZn center [11].
  • the mammalian protein phosphatases calcineurin (type 2B) [12] and protein phosphatase type 1 (PP-1) [13] both contain a di- nuclear metal centre and also reveal a striking similarity to the plant PAP enzyme in the coordination environment of the active site, except for the absence of the tyrosine ligand.
  • PP-1 and calcineurin are serine/threonine protein phosphatases, suggesting that also PAPs may function as protein phosphatases. It has been shown that PAP enzymes exhibit a rather broad specificity as these enzymes can dephosphorylate both serine- and tyrosine-bound phosphate moieties in phosphoproteins [10, 14-19].
  • the binuclear iron center, low pH optimum ( ⁇ 5), high isoelectric point ( « 9) and insensitivity to inhibition by L(+) tartrate are features of TRAP that may be involved in the apparent substrate specificity at the low pH in the osteoclastic resorption area.
  • the TRAP enzyme is a cationic glycoprotein with a molecular mass of 35 kD and a monomeric 325 amino acid peptide structure.
  • the peptide sequence of rat bone TRAP displays 89-94% homology to TRAP enzyme of the human placenta, bovine spleen, and uteroferrin.
  • TRAP hydrolyzes aryl phosphates, nucleoside di- and triphosphates, pyrophosphate and phosphoproteins.
  • TRAP may mediate dephosphorylation of bone matrix proteins such as osteopontin and bone sialoprotein. Dephosphorylation of bone matrix proteins enables osteoclasts to migrate over the bone surface and TRAP is therefore likely to be involved in the attachment of osteoclasts to the bone surface.
  • PAP enzymes are highly expressed in certain cells of the monocyte- macrophage lineage, such as the bone-resorbing osteoclasts and certain activated macrophages in spleen, liver and lung [20-23], and TRAP has since long been used as a histochemical marker for these cells.
  • Orlando et al [29] managed to separate the monomeric and two-subunit variants of PAP from bovine spleen, and demonstrated a markedly higher specific enzyme activity associated with the two subunit form. Moreover, digestion of the single subunit form with the serine proteases trypsin or chymotrypsin generated the 23 kDa and 15 kDa disulfide-linked fragments characteristic of the two subunit form together with a significant enhancement of enzyme activity. Similar nicking and activation of the non- cleaved purified recombinant human and mouse PAPs were noted upon prolonged storage [17].
  • Inhibitors of TRAP are known, such as PGE2 [Quinn et al Calcif. Tissue Int (1997), 60 (1) 63-70], which has an influence on the formation of osteoclasts and thus reduce the amount of TRAP, hemin (ferric protoporphyrin) [Reddy et al, Blood (1996), 88 (6) 2288-2297], which regulates the TRAP on a gene level i.e. a lowering of the expression of TRAP and calcitonin which inhibits the realease of TRAP. Calcitonin, has an effect against osteoclasts and is used as medicament against osteoporosis.
  • the overall goal of this invention is to develop drugs for the treatment of osteoporosis.
  • Cysteine proteinases were chosen because enzymes belonging to this family appear to serve important roles in resorptive and degradative processes in cells of the monocyte-macrophage lineage [33-35].
  • TRAP represents a latent proenzyme with low enzymatic activity towards both tyrosine- and serine-containing phosphosubstrates.
  • cystein proteinase family plays an important role in degradative processes involving the TRAP enzymes by converting the TRAPs to enzymatically active and micro environmentally regulated species.
  • aTRAP is a proteolytic modification of rTRAP.
  • the activity of aTRAP is about 10-20 times higher than for rTRAP.
  • the invention relates to the use of activated TRAP ( tartrate-resistant and purple acid phosphatases ) for screening for specific inhibitor of TRAP activity useful in the treatment of diseases or degenerative conditions resulting in increased bone resorption. such as tissue damages (e.g. inflammation, cancer), bone metabolic disorders, osteoporosis.
  • TRAP can be activated by proteolytic activation of TRAP e.g. by cleaving with a protease, papain-like enzyme.
  • the recombinant activated TRAP can be used as a screening tool to identify specific inhibitors of this enzyme and to develop drugs for the treatment of osteoporosis.
  • an enzyme expressed predominantly in bone, resorbing cells (osteoclasts) will modulate osteoclast activity.
  • An up-regulated bone turnover rate in combination with an imbalance between bone resorption and formation are key elements in postmenopausal osteoporosis, and using a TRAP inhibitor in patients with high bone turnover rate in postmenopausal osteoporosis is likely to shift the net effect of bone turnover to bone anabolism.
  • Recombinant rat TRAP has been a necessary tool for High Throughput Screening (HTS) and the results of such HTS show that it can be performed for the intended purpose.
  • HTS High Throughput Screening
  • Fig.l Protein composition and immunoblot analysis of recombinant and bone TRAP.
  • Fig.2. Fragmentation pattern after proteolytic digestion of recombinant TRAP.
  • Fig.3. pH-dependence for pNPP hydrolysis of intact and proteolytically cleaved TRAP.
  • Fig.4 a and b Differential sensitivity of intact and proteolytically cleaved TRAP to reducing agents.
  • Phosphothreonine (pT), phosphoserine (pS), phosphotyrosine (pY) and p- nitrophenylphosphate (pNPP) were purchased from Sigma.
  • the phosphopeptides RRA(pT)VA, END(pY)INASL and DADE(pY)LIPQQG came from Promega.
  • FRI(pS)HELDS (F9S) and EDEE(pS)EDEE were synthesized by Neosystem Laboratoire. France.
  • Osteopontin (OPN) was purified from milk according to procedure described under Methods. DEAE-Sephacel and Phenyl-Sepharose CL-4B were purchased from Pharmacia Biotech, Sweden.
  • protease inhibitors were purchased from: Papain-agarose (Pierce), Trypsin-agarose (Sigma), cathepsin B (Anawa. Switzerland), protease inhibitor cocktail Complete, Pefabloc, pepstatin, E-64 from Boehringer Mannheim. Germany.
  • Materials used for Western blot analysis were: immuno-PVDF membranes (Bio-Rad), colloidal gold (Bio-Rad), alkaline phosphatase- conjugated goat- anti rabbit IgG (Sigma), NBT/BCIP (nitrobluetetrazolium chloride/5-bromo-4-chloro-3- indolyl-phosphate p-toluidine salt; Bio-Rad).
  • Baculovirus-produced recombinant TRAP Baculovirus-produced recombinant TRAP (BaculoTRAP) was purified from the culture supernatant of recombinant Baculovirus-infected cells as described previously [10]. This preparation initially had a specific activity of 428 U/mg protein, which gradually dropped during prolonged storage at - 80°C. Bone TRAP was purified from the long bones of 40 3 week old Sprague Dawley rats. All operations were performed at 4°C. The dissected bones, free from soft tissue, were cut into small pieces and placed in homogenization solution with protease inhibitors (3 ml/g bone); 0.15 M KC1, 0.1 % Triton X-100.
  • Pefabloc (1 mg/ml), Pepstatin A (10 ⁇ g/ml). E-64 (10 ⁇ g/ml) and 5 mM EDTA.
  • a Polytron homogenizer (Brinkman Instruments Westbury, N.Y.) was used for homogenization during 10 seconds, with 1 minute intervals, until a homogenous suspension was achieved. The homogenate was cleared by centrifugation at 3.200 x g for 30 min. 5 % protamine sulfate was added dropwise to the supernatant during continuos stirring to a final concentration of 0.5 %, with further stirring for 30 min. The suspension was centrifuged for 30 min at 3,200 x g and the supernatant was adjusted to pH 6.5. The supernatant was loaded onto CM cellulose column and subsequent purification steps were performed as previously described [10].
  • P-nitrophenylphosphosphatase activity was assayed in 96-well plates using the p- nitrophenylphosphate (pNPP) as substrate in the incubation medium (150 ⁇ l) containing (final concentrations); 10 mM pNPP, 0.1 M sodium acetate pH 5.8, 0.15 M KC1, 0.1 % Triton X-100, 10 mM sodium tartrate, 1 mM ascorbic acid and 0.1 mM FeCl 3 .
  • the p- nitrophenol liberated after 1 hour of incubation at 37°C was converted into p- nitrophenylate by the addition of 100 ⁇ l of 0.3 M NaOH, and the absorbance was read at 405 nm.
  • 25 ⁇ g baculoTRAP or bone TRAP were digested with 100 ⁇ l (0.7 units) papain-agarose in 500 ⁇ l of incubation solution; 10 mM sodium acetate, pH 4.6, 0.1 % Triton X-100 and 2 mM DTT. Incubation was performed at room temperature for 24 h, with constant mixing of the gel. 25 ⁇ g baculoTRAP or bone TRAP were digested with 100 ⁇ l (5units) trypsin- agarose in 500 ⁇ l of incubation solution; 10 mM Tris pH 7.0 and 0.1 % Triton X-100. Incubation was performed at room temperature for lh with the suspension kept well mixed during the reaction period.
  • the proteolytic digestions above were stopped by centrifugation and the cleavage products of TRAP in the supernatant were further analyzed.
  • Digestion of bacuio TRAP or bone TRAP with cathepsin B were performed at (final concentrations); 10 ng TRAP/ ⁇ l, 0.4 mU cathepsin B/ ⁇ l, 2 mM DTT, 50 mM sodium acetate and 1 mM EDTA, pH 5.5.
  • the incubations were performed at 37°C for 24 h and digestions were stopped with protease inhibitor cocktail Complete according to the instructions of the manufacturer.
  • Ostepontin OPN was purified from bovine milk essentially as published in [36]. Briefly, 1 liter raw milk was centrifuged for 15 min at 1.250 g and the non-fatty part was mixed with DEAE-Sephacel and rotated over-night at 4°C. Then the mix was first washed by centrifugation with 1.1 liter of 0.2 M NaCl in 10 mM phosphate buffer, pH 7.4 and then with 600 ml of 0,25 M NaCl in the same buffer. The mix was applied to a column and eluted with 0.3 M NaCl in 10 mM phosphate buffer pH 7.4.
  • the protein peak was pooled and adjusted to 4 M NaCl before applied to a Phenyl-Sepharose column (30 ml) (equilibrated with 4 M NaCl in 10 mM phosphate buffer pH 7.4). After wash with 4 M NaCl in 10 mM phosphate buffer pH 7.4 the protein was eluted with 2 M NaCl in the same buffer. The protein peak was pooled, adjusted to 5 M NaCl, and applied to a smaller (5 ml) Phenyl-Sepharose column equilibrated with 5 M NaCl in 10 mM phosphate buffer pH 7.4.
  • the protein was eluted with 2 M NaCl in 10 mM phosphate buffer pH 7.4.
  • the protein peak was pooled and the elution buffer was replaced with TBS (137 mM NaCl, 2 mM KCl, 25 mM Tris-HCl pH 7.4) by ultrafiltration with an Amicon cell equipped with a YM 10 filter.
  • SDS-polyacrylamide gel electrophoresis under reducing conditions was performed essentially according to the procedure described by Laemmli [38]. Proteins were blotted onto immuno-PVDF membranes. Colloidal gold was used for protein staining. Immunoblots were probed with polyclonal antiserum (diluted 1 : 100) raised in rabbits using rat recombinant TRAP as the immunogen [10] and the secondary antibody was alkaline phosphatase conjugated goat anti-rabbit IgG (diluted 1 : 500). Development was performed with NBT/BCIP. All operations were carried out according to the protocols of the manufacturers.
  • N-terminal amino acid sequence analysis was carried out by Edman degradation using a Hewlett Packard 1090 sequencer with adsorptive biphasic column technology. Approximately 20 ⁇ g of baculoTRAP digested with papain-agarose were loaded for sequence analysis.
  • Bone TRAP See Fig. 1, lanes 1 and 2
  • BaculoTRAP See Fig. , lanes 3 and 4
  • Fig. 1 denote the positions of molecular weight standards and to the right are the estimated molecular weight sizes of the major TRAP bands (in kDa).
  • Lanes 1 and 3 (1 ug of protein) were proteinstained and lanes 2 and 4 (0.5 ug of protein) were immunostained as described under Materials and Methods.
  • immunostained using a polyclonal antibody generated in rabbits using the purified recombinant rat TRAP as the immunogen some additional bands were visible.
  • the bands at 20 kDa and 16 kDa correspond to the disulphide-linked fragments contained in the two-subunit form [10]. Bands appearing on the proteinstained blots without a corresponding band on the immunostained neighbouring lane were considered as impurities. From densitometric analysis using the SigmaGel software, the purity of this preparation was estimated to around 90%.
  • the bone TRAP was purified from long bones of 3-week old rats using essentially the same procedure as for the recombinant TRAP.
  • this preparation (Fig 1, lanes 1 and 2), which had a specific activity of 1 , 165 U/mg protein, an apparent inverse proportion of monomeric and two-subunit forms compared to the recombinant enzyme preparation was noted (Fig 1, cf lanes 2 and 4). This preparation was considered approximately 40% pure using the densitometric analysis described above.
  • the TRAP enzyme from rat bone was mainly in the fragmented, two- subunit form and exhibited at least 5-6-fold higher catalytic activity compared to the mostly monomeric species with significantly lower specific activity contained in the recombinant TRAP preparation.
  • Example 2 Proteolytic cleavage in vitro of the monomeric recombinant TRAP BaculoTRAP was digested with papain or trypsin and compared with undigested BaculoTRAP and bone TRAP. 150 ng of TRAP was electrophoresed on a 12 % SDS- polyacrylamide gel under reducing conditions. The proteins were blotted onto a PNDF membrane and developed as described under Materials and Methods. Fig.2. shows the fragmentation pattern after proteolytic digestion of recombinant TRAP.
  • bovine spleen TRAP can be converted to the two-subunit form by limited proteolytic cleavage with either of the serine proteases trypsin or chymotrypsin with a significant increase in enzyme activity [29].
  • N-terminal sequence analysis was performed (data not shown).
  • two N-terminal sequences were detected; the predominant sequence starting with T-A-P-A-S-T, corresponding to amino acid residues 1-6 in the mature protein and a minor sequence V-A-R-T. corresponding to amino acids 161-164 in the deduced protein sequence [9].
  • the 2 N-terminal sequences detected were the A-P-A-S-T and R-T-Q-L-S-W, the latter corresponding to amino acids 163-168 [9].
  • Example 3 Effects of proteolytic cleavage in vitro on TRAP enzymatic parameters a) Cleavage of recombinant TRAP with the proteases trypsin and papain was associated with significant enhancement of enzymatic activity using pNPP as the substrate only with papain (See Table I).
  • the TRAPs usually exhibit a pH-optimum for hydrolysis of phosphomonoesters in the range of 5.5-6.0 [39].
  • the recombinant TRAP as isolated exhibited a rather broad pH- optimum between 4.5-5.0, i.e by 1 pH unit lower than the rat bone TRAP (Fig 3).
  • cleaving the monomeric recombinant TRAP with papain as well as trypsin caused a shift in the optimal pH of pNPP hydrolysis to more basic pH-values. for trypsin 5.0-5.5 and for papain 5.5-6.0. This suggests that protonation reactions in amino acid residues involved in catalysis are affected, presumably by conformational changes induced by limited proteolytic cleavage.
  • the differential sensitivity of intact and proteolytically cleaved TRAP to reducing agents is shown inFig.4. Result: The di-iron containing TRAPs are redox-sensitive enzymes, due to a redox-active M2 site when present as the ferrous ion yields a catalytically active enzyme [3].
  • the TRAPs are present in an inactive diferric form, which can be rapidly activated by addition of reducing agents such as ascorbate. Following a maximal activation within 10 minutes, it was observed (Fig 4b) that prolonged pre-incubation with ascorbate (1 mM) in the presence of 0.1 mM FeCl, led to a time-dependent inactivation of the rat bone TRAP. This could be due to a conversion of the mixed-valent active enzyme to an inactive Fe(II)Fe(II) species [40]. However, although the intact recombinant TRAP did not show this tendency for inactivation under the same conditions (Fig 4a).
  • phosphotyrosine and two different phosphotyrosyl peptides were equally effective as pNPP as substrates with the Kcat/Km ratio between 10 4 - 10 3 .
  • the most effective of all substrates tested was the acidic phospho-seryl protein osteopontin from bovine milk.
  • the rat bone enzyme was more active, varying for different substrates between 4 to 19-fold higher compared to the recombinant TRAP.
  • Example 5 High Throughput Screen using recombinant TRAP
  • the assay measured the conversion of para-nitrophenyl Phosphate (pNPP) to paranitrophenol (pNP) by TRAP in the presence of test compounds.
  • pNPP para-nitrophenyl Phosphate
  • pNP paranitrophenol
  • 84073 compounds were tested.
  • 1012 compounds inhibited TRAP activity by 30% or better.
  • Retests of these compounds in duplicates showed 301 compounds remaining at 30% or better inhibition of TRAP activity.
  • 284 of the 301 compounds were evaluated in a confirmation assay rendering 217 reproducible, confirmed active TRAP inhibitors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physical Education & Sports Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Rheumatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Pain & Pain Management (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne l'utilisation de phosphatases acides résistant au tartrate (TRAP) servant à la recherche d'inhibiteurs spécifiques de l'activité TRAP qui conviennent pour le traitement de maladies ou de pathologies dégénératives ayant comme conséquences une résorption osseuse accrue, telles que des lésions tissulaires, des troubles du métabolisme osseux ou l'ostéoporose. Les phosphatases acides résistant au tartrate peuvent être activées par action protéolytique, par clivage à l'aide d'une protéase de type papaïne.
PCT/SE1999/002096 1998-11-19 1999-11-16 Utilisation de trap activee pour rechercher des inhibiteurs specifiques de trap et technique aidant a l'identification d'un compose destine au traitement de maladies ou pathologies degeneratives provoquant une resorption osseuse accrue WO2000031289A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP99962612A EP1131463A1 (fr) 1998-11-19 1999-11-16 Utilisation de trap activee pour rechercher des inhibiteurs specifiques de trap et technique aidant a l'identification d'un compose destine au traitement de maladies ou pathologies degeneratives provoquant une resorption osseuse accrue
CA002351872A CA2351872A1 (fr) 1998-11-19 1999-11-16 Utilisation de trap activee pour rechercher des inhibiteurs specifiques de trap et technique aidant a l'identification d'un compose destine au traitement de maladies ou pathologies degeneratives provoquant une resorption osseuse accrue
NZ511428A NZ511428A (en) 1998-11-19 1999-11-16 Use of activated trap for screening for specific inhibitor of trap and method for aiding in the identification of a compound for use in the treatment of diseases or degenerative conditions resulting in increased bone resorption
AU19019/00A AU759597B2 (en) 1998-11-19 1999-11-16 Use of activated trap for screening for specific inhibitor of trap and method for aiding in the identification of compound for use in the treatment of diseases or degenerative conditions resulting in increased bone resorption
JP2000584097A JP2002530117A (ja) 1998-11-19 1999-11-16 Ｔｒａｐの特異的インヒビターのスクリーニング用の活性化ｔｒａｐの使用および骨吸収の増加を引き起こす疾患または変性状態の治療用の化合物同定の補助的方法
IL14288099A IL142880A0 (en) 1998-11-19 1999-11-16 Use of activated trap for screening for specific inhibitor of trap and method for aiding in the identification of a compound for use in the treatment of diseases or degenerative conditions resulting in increasing bone resorption
NO20012453A NO20012453L (no) 1998-11-19 2001-05-18 Anvendelse av aktivert TRAP ved utvelgelse av spesifikke inhibitorer for TRAP, samt anvendelse derav ved utvelgelse avmedikamenter mot öket benresorpsjon

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9803959-7 1998-11-19
SE9803959A SE9803959D0 (sv) 1998-11-19 1998-11-19 Inhibitors

Publications (1)

Publication Number Publication Date
WO2000031289A1 true WO2000031289A1 (fr) 2000-06-02

Family

ID=20413342

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1999/002096 WO2000031289A1 (fr) 1998-11-19 1999-11-16 Utilisation de trap activee pour rechercher des inhibiteurs specifiques de trap et technique aidant a l'identification d'un compose destine au traitement de maladies ou pathologies degeneratives provoquant une resorption osseuse accrue

Country Status (9)

Country Link
EP (1) EP1131463A1 (fr)
JP (1) JP2002530117A (fr)
AU (1) AU759597B2 (fr)
CA (1) CA2351872A1 (fr)
IL (1) IL142880A0 (fr)
NO (1) NO20012453L (fr)
NZ (1) NZ511428A (fr)
SE (1) SE9803959D0 (fr)
WO (1) WO2000031289A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040684A3 (fr) * 2000-11-14 2002-11-07 Bayer Ag Regulation de phosphatase acide pourpre d'origine humaine
WO2002055710A3 (fr) * 2001-01-11 2003-04-24 Bayer Aktiengesellschaft Regulation de l'acide phosphatase pourpre humain

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ALLEN S.H. ET AL: "Purification and Characterization of Human Bone Tartrate-Resistant Acid Phosphatase", JOURNAL OF BONE AND MINERAL RESEARCH, vol. 4, no. 1, 1989, pages 47 - 55, XP002922390 *
CHEMICAL ABSTRACTS, 1 January 1900, Columbus, Ohio, US; abstract no. 121:50499, ZHENG M H, ET AL: "Tartrate Resistant Acid Phosphatase Activity in Rat Cultured Osteoclasts is Inhibited by a Carboxyl Terminal Peptide (Osteostatin) from Parathyroid Hormone- Related Protein" XP002949831 *
DATABASE DIALOG MEDLINE 1 January 1900 (1900-01-01), BERGHUIS H M, ET AL: "Effects of TGF-Beta 2 on Mineral Resorption in Cultured Embryonic Mouse Long Bones; 45Ca Release and Osteoclast Differentiation and Migraation", XP002949835, Database accession no. 08175308 *
DATABASE DIALOG MEDLINE 1 January 1900 (1900-01-01), HARA K, ET AL: "The Inhibitory Effect of Vitamin K2 (Menatetrenone) on Bone Resorption may be Related to its Side Chain", XP002949836, Database accession no. 08517952 *
DATABASE DIALOG MEDLINE 1 January 1900 (1900-01-01), QUINN J M, ET AL: "Inhibitory and Stimulatory Effects of Prostaglandins on Osteoclast Differentiation", XP002949834, Database accession no. 09282609 *
DATABASE DIALOG MEDLINE 1 January 1900 (1900-01-01), WEIR E C, ET AL: "Colony Stimulating Factor-1 Plays a Role in Osteoclast Formation and Function in Bone Resorption Induced by Parathyroid Hormone-Related Protein", XP002949833, Database accession no. 09106185 *
DATABASE DIALOG MEDLINE 1 January 1900 (1900-01-01), ZAIDI M, ET AL: "Inhibition of Osteoclastic Acid Phosphatase Abolishes Bone Resorption", XP002949832, Database accession no. 06564242 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002040684A3 (fr) * 2000-11-14 2002-11-07 Bayer Ag Regulation de phosphatase acide pourpre d'origine humaine
WO2002055710A3 (fr) * 2001-01-11 2003-04-24 Bayer Aktiengesellschaft Regulation de l'acide phosphatase pourpre humain

Also Published As

Publication number Publication date
NZ511428A (en) 2003-06-30
NO20012453L (no) 2001-07-19
JP2002530117A (ja) 2002-09-17
NO20012453D0 (no) 2001-05-18
AU1901900A (en) 2000-06-13
IL142880A0 (en) 2002-03-10
CA2351872A1 (fr) 2000-06-02
EP1131463A1 (fr) 2001-09-12
AU759597B2 (en) 2003-04-17
SE9803959D0 (sv) 1998-11-19

Similar Documents

Publication Publication Date Title
LJUSBERG et al. Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases
Österlund et al. Domain-structure analysis of recombinant rat hormone-sensitive lipase
Mitić et al. Human tartrate-resistant acid phosphatase becomes an effective ATPase upon proteolytic activation
Peeples et al. Albumin, a new biomarker of organophosphorus toxicant exposure, identified by mass spectrometry
Lübke et al. Proteomics of the lysosome
Ljusberg et al. Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts
Gaidukov et al. The 192R/Q polymorphs of serum paraoxonase PON1 differ in HDL binding, lipolactonase stimulation, and cholesterol efflux
Ferrington et al. Catalytic site-specific inhibition of the 20S proteasome by 4-hydroxynonenal
Maejima et al. Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation
CHU et al. Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides
Kaartinen et al. Transglutaminase-catalyzed cross-linking of osteopontin is inhibited by osteocalcin
Alves et al. A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity
US6329137B1 (en) Intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses
Liniger et al. Cleavage of trypanosome surface glycoproteins by alkaline trypsin-like enzyme (s) in the midgut of Glossina morsitans
AU759597B2 (en) Use of activated trap for screening for specific inhibitor of trap and method for aiding in the identification of compound for use in the treatment of diseases or degenerative conditions resulting in increased bone resorption
US6451548B1 (en) Methods for screening for specific inhibitors of trap and identifying compounds for treatment of diseases or conditions resulting in increased bone resorption using activated TRAP
Jang et al. Caspase-7 mediated cleavage of proteasome subunits during apoptosis
US20030219849A1 (en) Fluorescent phospholipase assay, phospholipase A2 inhibitor and stimulator, and the use thereof
Chattopadhyay et al. Calmodulin binds to the cytoplasmic domain of angiotensin-converting enzyme and regulates its phosphorylation and cleavage secretion
EP1699460B1 (fr) Utilisation d'inhibiteurs enzymatiques de h-prune pour la prevention et le traitement des metastases de tumeurs sur-exprimant h-prune
Xing et al. Low molecular weight protein tyrosine phosphatase (LMW-PTP) and its possible physiological functions of redox signaling in the eye lens
JP4585342B2 (ja) 不全角化を抑制する物質のスクリーニング方法、同方法によりスクリーニングされた物質及び不全角化を抑制する方法
Zenger et al. Long bone osteoclasts display an augmented osteoclast phenotype compared to calvarial osteoclasts
Wang et al. Expression and proteolytic processing of mammalian purple acid phosphatase in CHO-K1 cells
US7348437B2 (en) Proteomic analysis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA IL JP NO NZ US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 142880

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 511428

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 19019/00

Country of ref document: AU

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 584097

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref document number: 2351872

Country of ref document: CA

Ref country code: CA

Ref document number: 2351872

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1999962612

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1999962612

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 19019/00

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1999962612

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载