WO1999010480A2 - Nouvelles calpaines specifiques au tissu, leur production et leur utilisation - Google Patents

Nouvelles calpaines specifiques au tissu, leur production et leur utilisation Download PDF

Info

Publication number
WO1999010480A2
WO1999010480A2 PCT/EP1998/005275 EP9805275W WO9910480A2 WO 1999010480 A2 WO1999010480 A2 WO 1999010480A2 EP 9805275 W EP9805275 W EP 9805275W WO 9910480 A2 WO9910480 A2 WO 9910480A2
Authority
WO
WIPO (PCT)
Prior art keywords
calpain
leu
capn6
thr
gly
Prior art date
Application number
PCT/EP1998/005275
Other languages
German (de)
English (en)
Other versions
WO1999010480A3 (fr
Inventor
Thomas Boehm
Neil T. Dear
Original Assignee
Basf Aktiengesellschaft
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 Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Priority to BR9811365-8A priority Critical patent/BR9811365A/pt
Priority to EP98945265A priority patent/EP1009811A2/fr
Priority to CA002301815A priority patent/CA2301815A1/fr
Priority to JP2000507788A priority patent/JP2001513992A/ja
Priority to HU0004027A priority patent/HUP0004027A2/hu
Priority to KR1020007001916A priority patent/KR20010023282A/ko
Priority to IL13436198A priority patent/IL134361A0/xx
Priority to AU92635/98A priority patent/AU9263598A/en
Publication of WO1999010480A2 publication Critical patent/WO1999010480A2/fr
Publication of WO1999010480A3 publication Critical patent/WO1999010480A3/fr
Priority to NO20000961A priority patent/NO20000961L/no

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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)

Definitions

  • the invention relates to new tissue-specific calpains and their production.
  • the invention further relates to methods for screening for new calpain inhibitors and their use.
  • Calpains belong to the intracellular, non-lysosomal enzymes from the group of cysteine proteases. They are involved in the Ca 2+ -dependent signal transduction in eukaryotic cells, ie they regulate cellular functions depending on the Ca 2+ concentration. Calpains occur ubiquitously in animal tissues or cells of, for example, humans, chickens, rabbits or in the rat. Calpains have also been found in lower animals such as Drosophila melanogaster, Schistosoma or Caenorhabditis elegans. No calpains have been found in yeasts, fungi or bacteria.
  • Calpain II mCalpain is only activated by millimolar concentrations of calcium ions.
  • Both Calpaine consist of two subunits, a large subunit with approx. 80 kDa and a small subunit of approx. 30 kDa. Both subunits of the active heterodimer have binding sites for calcium.
  • nCL-1 there is a stomach-specific calpain that can occur in two splicing variants, nCL-2 and nCL-2 '.
  • nCL-2 'differs from nCL-2 by the lack of the calcium-binding region Sorimachi, HS et al., J. Biol. Chem. Vol. 268, No.
  • CalpA calpain-homologous protein
  • Calpaine are believed to play important roles in various physiological processes.
  • a large number of cytoskeletal, membrane-binding or regulatory proteins such as protein kinase C, phospholipase C, spectrin, cytoskeleton proteins such as MAP2, muscle proteins, neurofilaments and neuropeptides, plate proteins, "epidermal growth factor", NMDA receptor and pro Teins involved in mitosis and other proteins are calpain substrates (Barrett MJ et al., Life Sei. 48, 1991: 1659-69, Wang KK et al., Trends in Pharmacol. Sei., 15, 1994: 412 - 419).
  • the normal physiological function of calpaine has not yet been clearly understood. They are involved in a variety of physiological processes such as apoptosis, cell division and differentiation or in embryonic development.
  • Increased calpain levels were measured in various pathophysiological processes and diseases, for example in: ischemia of the heart (e.g. heart attack), the kidney or the central nervous system (e.g. stroke), inflammation, muscle dystrophies, cataracts of the eyes (cataracts), injuries of the central nervous system (e.g. trauma), Alzheimer's disease, HIV-induced neuropathy, Parkinson's and Huntington's diseases, etc. (see Wang KK above). It is suspected that these diseases are related to an increased and persistent intracellular calcium level. As a result, calcium-dependent processes are overactivated and are no longer subject to physiological regulation. Accordingly, overactivation of calpains can also trigger pathophysiological processes.
  • Calpain also plays an important role in restenosis and arthritis, and calpain inhibitors can have a positive effect on the clinical picture (March K: L: et al. Circ. Res. 72, 1993: 413-423, Suzuki K. et al., Biochem
  • the most potent and selective calpain inhibitor is the naturally occurring intracellular protein calpastatin. It inhibits both calpain I and calpain II, but not other cysteine or thiol proteases such as cathepsin B, L or papain.
  • calpastatin which consists of approx. 700 amino acids, has the disadvantage that it is out of the question for therapeutic options due to the size and impassability of the cell membrane.
  • calpastatin which consists of approx. 700 amino acids, has the disadvantage that it is out of the question for therapeutic options due to the size and impassability of the cell membrane.
  • a number of non-peptide inhibitors have been identified. The disadvantage of these inhibitors is that they are unstable, are rapidly metabolized and some are toxic.
  • calpain inhibitors are also characterized by a lack of selectivity, ie they not only inhibit calpain I and II but also other cysteine proteases such as papain, chymotrypsin, elastase or cathepsin B and L. There is therefore still a need for selective, highly effective calpain inhibitors.
  • the screening for these selective, well-effective calpain inhibitors requires highly specific test systems that make it possible to identify selective inhibitors. The screening tests are usually carried out with the ubiquitous Calpainen Calpain I and Calpain II.
  • the invention relates to a new tissue-specific calpaingen with the sequence SEQ ID NO. 1 or SEQ ID NO. 3 and the designation CAPN6, its allelic variants, analogs or derivatives which have a homology of 60 to 100% at the derived amino acid level, the calpaingenes, their allelic variants, analogs or derivatives containing the following sequences:
  • X denotes any natural amino acid in the sequences mentioned.
  • the invention also relates to a method for identifying calpain inhibitors, in which a calpain, its allelic variants or analogs are isolated from tissues or cells and encoded by a sequence according to claim 1, and the inhibition of the cleavage of a substrate of the enzyme CAPN6 and in at least one further test measures the inhibition of the cleavage of a substrate of the enzymes Calpain I and / or II by test substances and selects the test substances which inhibit the enzyme CAPN6 and at least one other of the calpains.
  • the invention furthermore relates to a method for identifying calpain inhibitors, characterized in that the inhibition of the cleavage of a substrate of the enzyme CAPN6 or the calpains I and / or II is determined by test substances in cellular systems and such test substances are selected which select the Cross cell membrane and inhibit the intracellular activity of the enzyme CAPN6 and / or the Calpaine I and / or II, which do not inhibit the enzyme CAPN6, but which inhibit the enzyme Calpain I and / or II or which inhibit the enzyme CAPN6, but not the enzyme Calpain I and / or II. Substances which show an activity in vitro against the calpains without their cell mobility being tested are advantageously selected.
  • the amino acid sequence of the calpain CAPN6 can be found in the sequence SEQ ID NO: 2.
  • the amino acid sequence deduced taking into account an existing intron has a typical calpain signature, although an assignment to the known calpain subfamilies ⁇ Calpain, mCalpain, nCL-1 or nCL-2 is not possible due to the low homology.
  • the Calpain CAPN6 is a new, previously unknown Calpain.
  • the further investigations with this sequence from mice in the EST database also provided 4 human partial sequences (AA169715, C17331, C16980 and T39424) with a homology to the mouse CAPN6 sequence. These partial sequences could be assembled into a continuous sequence which codes for a 374 amino acid protein.
  • the protein sequence derived from the gene sequence SEQ ID N0: 1 shows the greatest homology to the known Caenorhabditis elegans gene tra-3 over the entire amino acid sequence.
  • the homology to the other known calpaines is between 20.9% (rats nCL-2) and 25.4% (mouse mCalpain).
  • Lipman-Pearson Ktupl 2, Gap Penalty 4, Gap Length Penalty 12
  • Both calpains have common properties that distinguish them from the other calpains.
  • CAPN5 and CAPN6 have, besides a shortened domain I, a modified C-terminal end which has no pronounced homology to domain IV of the other calpains.
  • the consensus sequence of the Ca 2 + _ binding site of the calpains lies in the domain IV.
  • This Ca 2+ binding site is missing in the case of CAPN5 and CAPN6, which means that no Ca 2+ may be bound to domain IV and the proteins are activated in another way. They are the only vertebrate calpains that lack the domain IV similar to calmodulin.
  • the amino acids alanine and threonine in positions 122 and 125 of the CAPNl have been changed to serine and alanine in positions 88 and 91 in the CAPN6.
  • the amino acids histidine, alanine, serine and tryptophan in positions 272, 273, 275 and 298 in the CAPNl have also been changed to tyrosine, threonine, threonine and leucine in positions 252, 253, 255 and 286 in the CAPN6.
  • the assignment of the amino acids at the different positions of the proteins CAPN1 and CAPN6 is done by sequence comparison and assignment of the conserved regions of the proteins to one another, so that there is maximum agreement between the proteins at the amino acid level. For example, the same sequences can be localized in different proteins of a protein family at very different sites or positions.
  • the human CAPN6 sequence shows an exchange of the tyrosine in position 254 for histidine (Table 1).
  • a possible explanation could be, for example, that CAPN6 has a changed substrate specificity compared to the other calpains or that the active center is not critical for the CAPN6 function, that it is an inhibitor of other calpains or that CAPN6 is a pseudo gene. This last possibility seems unlikely due to the large number of conserved amino acids.
  • the cysteine residue in position 89 can take over the function of the missing cysteine residue in position 81 in the catalytic reaction. Even if CAPN6 had no protease activity, which is very unlikely, it could be involved in regulatory processes, possibly by competing with other calpains for binding sites on cofactors or substrates.
  • Tra-3 is involved in sex determination of Caenorhabditis elegans. In a cascade of several genes and their gene products, tra-3 also decides whether caenorhabditis males or hermaphrodites develop (Kuwabara P.E. et al., TIG, Vol. 8, No.5, 1992: 164-168). Tra-3 appears to be involved in spermatogenesis.
  • the conservation of the amino acids in the different domains between mouse CAPN6 and tra3 is 39.2%; 42.0%; 30.9% and 22% for domains I, II, III and T.
  • SEQ ID No. 1 encodes a protein with 641 amino acids and a molecular weight of 74.6 kDa.
  • the start codon methionine is preceded by a typical sequence for the translation start.
  • Figure 3 shows the phylogenetic family tree of the different Calpaine.
  • the phylogenetic analyzes for the compilation of this family tree were carried out using the nearest neighboring method (Saitou et al. Mol. Biol. Evol. 4, 1987, 406-425), with the gaps being excluded.
  • the vertebrate calpains could be divided into six different groups ( Figure 3, right side).
  • the CAPN5 and CAPN6 genes each form a separate group of calpains that are more similar to calpaine invertebrates than calpaine vertebrates.
  • the length of the horizontal lines is proportional to the phylogenetic distance of the different calpains.
  • the length of the vertical lines is irrelevant.
  • the sequences used to compile the phylogenetic family tree have the following SWISSPROT and EMBL numbers ("accession numbers"): human m (P17655), ⁇ (P07384), p94 (P20807); Rats m (Q07009), nCL-2 (D14480), p94 (P16259); Mouse p94 (X92523); Chickens m (D38026), ⁇ (D38027), ⁇ / m (P00789), p94 (D38028); Nematodes tra-3 (U12921); Drosophila Calp A (Q11002) and Dm (X78555), Schistosoma (P27730).
  • the human nCL-2 part sequence corresponds to the translation of EST clone AA026030 (Hellier et al., 1995, The Wash U-Merck EST project).
  • the deduced amino acid sequence of the EST clone AA026030 shows 5 a higher than usual homology after this analysis to the rat nCL-2 sequence. Since only a partial sequence was used for the analysis here, the phylogenetic family tree obtained for nCL-2 has a higher inaccuracy.
  • the nematode CPL1 sequence is the corrected version as used by Barnes and Hodjkin 10 (EMBOJ., 1996, 15: 4477-4484).
  • the first 7 exons as can be seen from the EMBL database (Accession No. L25598), were used, while the last 5 exons by connecting nucleotides 8028-8133, 15 8182-8239, 8729-8818, 8865-8983 and 9087 was preserved until the end.
  • the derived N-terminal sequence is unusual for calpains because of its length and its many glycine residues.
  • the new tissue-specific calpain CAPN6 according to the invention is only expressed in the tissue from the placenta (FIG. 4).
  • the human placenta is a rapidly growing and rapidly differentiating organ. It is also called "premalignant tissue” because it is a highly invasive tissue similar to that of malignant tumors. 25
  • Proteases play a central role in the development and differentiation of cells and thus also in the placenta.
  • the placenta is rich in proteases and protease inhibitors, which enable the development of the placenta 30 in a balanced equilibrium.
  • CAPN6 appears to play an important role here as a protease and / or may be involved in the regulation of other calpain cysteine proteases.
  • preeclampsia pregnancy-induced high blood pressure
  • preeclampsia pregnancy-induced high blood pressure
  • preeclampsia pregnancy-induced high blood pressure
  • preeclampsia during pregnancy is an important cause of maternal and child death, premature birth or, in mild cases, malnutrition or growth disorders in the embryo.
  • CAPN6 Another possible function of CAPN6 could be the regulation of the breakdown of somatostatin, glucagon and growth hormone in the placenta and thus influence the growth of the fetus.
  • the breakdown of maternal serum proteins could also be influenced by CAPN6, which also stimulate the growth of the fetus.
  • CAPN6 may take part in the complex process of the placenta mucosa during embryogenesis and thus controls the through e.g. TNF ⁇ and IFN ⁇ induced apoptosis of the trophoblasts by regulating other calpains. CAPN6 appears to be involved in embryonic development.
  • test substances to be tested for their inhibitory activity can be, for example, chemical substances, microbial or plant extracts. They are usually tested in addition to the test for their inhibitory activity towards CAPN6, Calpain I and / or II for their activity against cathepsin B or other thiol proteases.
  • good inhibitors should have little or no activity against cathepsin B, L, elastase, papain, chymotypsin or other cysteine proteases, but should have good activity against calpaines I and II.
  • novel tissue-specific calpain CAPN6 can be used to identify inhibitors with the method according to the invention which, for their inhibitory effect, can discriminate between the different calpaines calpain I, II, nCL-1, nCL-2 and / or nCL-4.
  • the various inhibitor tests were carried out as follows:
  • Cathepsin B inhibition was determined analogously to a method by S. Hasnain et al., J. Biol. Chem. 1993, 268, 235-240.
  • cathepsin B human liver cathepsin B from Calbiochem, diluted to 5 units in 500 ⁇ M buffer
  • an inhibitor solution prepared from the chemical substance to be tested
  • a microbial or vegetable extract and DMSO final concentration: 100 ⁇ M up to 0.01 ⁇ M
  • the activity of the calpain inhibitors was investigated in a colorimetric test with casein according to Hammarsten (Merck, Darmstadt) as the substrate. The test was performed in microtiter plates, according to the publication by Buroker-Kilgore and Wang in Anal. Biochem. 208, 1993, 387-392. Calpain I (0.04 U / test) from erythrocytes and Calpain II (0.2 U / test) from kidneys, both from pigs, from Calbiochem, were used as enzymes. The substances to be tested were with the enzyme for
  • the activity of calpain inhibitors can also be determined with the substrate Suc-Leu-Tyr-AMC. This fluorometric method is described in Zhaozhao Li et al, J. Med. Chem. 1993, 36, 3472-3480.
  • Calpain inhibitors pass through the cell membrane to prevent the breakdown of intracellular proteins by calpain.
  • Calpain mediated protein degradation in platelets has been described by ZhaozhaoLi et al. , J. Med. Chem., 36, 1993, 3472-3480. Human platelets were isolated from fresh sodium citrate blood from donors and adjusted to 10 7 cells / ml in buffer (5 mM Hepes, 140 mM NaCl and 1 mg / ml BSA, pH 7.3).
  • Platelets (0.1 ml) were preincubated for 5 minutes in 1 ⁇ l at various concentrations of potential inhibitors (dissolved in DMSO). This was followed by the addition of calcium ionophore A 23187 (1 ⁇ M in the test) and calcium (5 mM in the test) and a further incubation of 5 minutes at 37 ° C. After a centrifugation step, the platelets were taken up in SDS-Page sample buffer, boiled at 95 ° C. for 5 minutes and the proteins were separated in an 8% gel. The breakdown of the two proteins actin binding protein (- ABP) and talin was monitored by quantitative densitometry. After the addition of calcium and ionophore, these proteins disappeared and new bands of less than 200 Kd molecular weight were formed. From this, the half maximum enzyme activity is determined with or as a control without an inhibitor.
  • - ABP actin binding protein
  • Tissue parts such as brain sections or cell cultures are also suitable for testing membrane permeability.
  • CAPN6 Inhibition of CAPN6 is carried out in cells which express this protein and which can be detected with a specific antibody. Are cells with z. B: Calcium and the corresponding ionophore stimulated, this leads to an activation of CAPN6. Takaomi Saido described in 1992 in J. Biochem. Vol. 11, 81-86 the autolytic transition of ⁇ -calpain after activation and the detection with antibodies. Corresponding antibodies are generated for the detection of CAPN6. Calpain inhibitors prevent autolytic transition and a corresponding quantification is possible with antibodies.
  • calpaint tests known to the person skilled in the art are suitable, such as the test for inhibition of glutamate-induced cell death on cortical neurons (Maulucci-Gedde MA et al., J. Neurosci. 7, 1987: 357-368 ), calcium-mediated cell death in NT2 cells (Squier MKT et al., J. Cell. Physiol., 159, 1994: 229-237, Patel T.
  • the calpain CAPN6 or its animal or human homologue is derived from tissues or cells in which the enzyme is usually or artificially expressed (for example by recombinant expression), such as advantageously the placenta or from cells or microorganisms, which contain at least one gene copy and / or a vector with at least one gene copy of the CAPN6 gene, its allelic variants or analogs, purified and used as a crude extract or as a pure enzyme.
  • the various calpain inhibitor tests are advantageously combined with the test for inhibition of CAPN6 enzyme activity by potential
  • Inhibitors performed. Inhibitors are selected so that they either only inhibit the enzyme CAPN6 and not the other calpains or, conversely, only the other calpains and not the enzyme CAPN6 or the enzyme CAPN6 and at least one other calpain. Inhibitors against CAPN6 can advantageously be used in the case of gestosis.
  • the various inhibitor tests are carried out in such a way that, in addition to the test for the inhibitory effect of the test substance against CAPN6, Calpain I and / or II as a control, the tests are carried out without the test substance. With this test arrangement, the inhibitory effects of the test substances can be easily recognized.
  • Another method according to the invention uses CAPN6 or its allelic variants, analogs or synthetic derivatives advantageously for protection against the enzymatic action of other calpains.
  • Another method according to the invention uses the enzyme CAPN6 for screening for new calpain inhibitors, these inhibitors advantageously advantageously generally all calpains or one can inhibit individual calpains such as calpain I, II, nCL-1, nCL-2 or CAPN6.
  • the various test substances can be tested individually or in parallel in test systems.
  • the test substances are advantageously screened for their inhibitory effect in parallel, automated test systems.
  • All substances are generally suitable for the inhibitor tests.
  • the substances come from classic chemical synthesis, from combinatorics, from microbial, animal or vegetable extracts.
  • Microbial extracts are, for example, fermentation broths, cell disruption of microorganisms or substances after biotransformation. Cell fractions are also suitable for the tests.
  • All prokaryotic or eukaryotic expression systems which are suitable for isolating an enzymatically active gene product are suitable for cloning the CAPN6 gene or its animal homologues or its human homologue, its allelic variants or analogs. Expression systems are preferred which enable expression of the CAPN6 gene sequences in bacterial, fungal or animal cells, very particularly preferably in insect cells.
  • An enzymatically active gene product is to be understood as CAPN6 proteins which, directly after isolation from the expression organism, for example from a prokaryotic or eukaryotic cell, or after renaturation, give an active protein which is capable of at least one known calpain substrate such as those mentioned above or to split itself through auto-catalysis.
  • calpain tests known to the person skilled in the art, such as in vitro tests such as the tests described above for calpain I and II, or cellular tests such as the platelet test, are suitable for determining the enzymatic activity. Tests which are based on a colorimetric assay (Buroker-Kilgore M. et al., Anal. Biochem. 208, 1993: 387-392) or on the basis of a fluorescence assay can be used as the detection possibility.
  • Host organisms include all prokaryotic or eukaryotic organisms that are suitable as host organisms, for example bacteria such as Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptococcus carnosus, yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, fungi such as Aspergillus niger, insect cells such as Spodoptera frugiperda, Trichoplusia cells or all other insect cells which are suitable for viral expression, or animal cells such as CV12, COS, CV1, COS To understand 3T3 or CHO or human cells.
  • bacteria such as Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptococcus carnosus
  • yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe
  • fungi such as Aspergillus niger
  • insect cells such
  • Expression systems are to be understood as the combination of the expression organisms mentioned above by way of example and the vectors which match the organisms, such as plasmids, viruses or phages such as the T7 RNA polymerase / promoter system or vectors with regulatory sequences for the phage ⁇ .
  • expression systems should preferably be understood to mean the combination of Escherichia coli and its plasmids and phages or the baculovirus system and the corresponding insect cells such as Spodoptera frugiperda.
  • a further 3 'and / or 5' terminal regulatory sequences are also suitable for the advantageous expression of the CAPN6 gene according to the invention.
  • These regulatory sequences are intended to enable targeted expression of the CAPN6 gene. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can preferably positively influence and thereby increase CAPN6 gene expression.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • Enhancers are understood to mean, for example, DNA sequences which bring about increased CAPN6 gene expression via an improved interaction between RNA polymerase and DNA.
  • One or more DNA sequences can be connected upstream and / or downstream of the CAPN6 gene with or without an upstream promoter or with or without a regulator gene, so that the gene is contained in a gene structure.
  • the gene expression of the CAPN6 gene can also be increased by increasing the CAPN6 gene copy number.
  • the CAPN6 gene is amplified, for example, in a CHO expression vector.
  • Vectors of the pED series - dicistronic vectors - which also contain the amplifiable marker gene dihydrofolate reductase are also suitable as vectors. Details can be found in the Current Protocols in Molecular Biology Vol 2, 1994.
  • CAPN6 enzyme activity can be increased compared to the parent enzyme, for example, by altering the CAPN6 gene or its animal homologues through classic mutagenesis such as UV radiation or treatment with chemical mutants and / or through targeted mutagenesis such as site directed mutagenesis, deletion ( en), insertion (s) and / or substitution (s).
  • An increase in enzyme activity can be achieved, for example, by changing the catalytic center so that the substrate to be cleaved is converted more quickly.
  • increased enzyme activity can also be achieved by eliminating factors that repress enzyme synthesis and / or by synthesizing active instead of inactive CAPN6 proteins. In this way, increased amounts of enzyme can be made available for the in vitro tests.
  • CAPN6 or its animal homologues or its human homologue can advantageously be derived from genomic DNA or cDNA using, for example, the PCR technique (see Molecular Cloning, Sambrook, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, Chapter 14, 1-35, ISBN 0-87969-309-6 and Saiki et al., Science, 1988, vol. 239,
  • CAPN6 can be cloned using genomic DNA and particularly preferably using genomic DNA from mouse cells or human cells.
  • Suitable host organisms for cloning are, for example, all Escherichia coli strains, preferably the Escherichia coli strain DH10B.
  • Suitable vectors for cloning are all vectors which are suitable for expression in Escherichia coli (see Molecular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, ISBN 0-87969-309-6) .
  • vectors which are derived from pBR or pUC or shuttle vectors are particularly suitable; pBluescript is very particularly suitable.
  • CAPN6 genes with nucleotide sequences are available which code for the amino acid sequence given in SEQ ID NO: 2 or its allele variants.
  • AI- All variants are to be understood as CAPN6 variants which have 60 to 100% homology at the amino acid level, preferably 70 to 100%, very particularly preferably 80 to 100%.
  • Allelic variants comprise, in particular, functional variants which can be obtained by deleting, inserting or substituting nucleotides from the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, but the CAPN6 activity is retained and the sequences (a ) Leu-Gly-Asn-Lys-Ala, (b) Ala-X-Ser-Cys-Leu-Ala, (c) Gly-Tyr-Thr- (His or Tyr) -Thr-X-Thr and (d) Arg-X-Arg-Asn-Pro-Leu-Gly as well as in the CAPN6 genes, analogs or derivatives are present.
  • Analogs of CAPN6 include, for example, its animal homologues, shortened sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence, particularly antisense RNA.
  • Derivatives of CAPN6 are, for example, those derivatives which are not or only difficult to cleave enzymatically, such as the nucleic acid phosphonates or phosphothioates, in which the phosphate group of the nucleic acids has been replaced by a phosphonate or thioate group.
  • the promoter which is connected upstream of the specified nucleotide sequence can also be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoter being impaired. Furthermore, the effectiveness of the promoter can also be increased by changing its sequence, or it can be completely replaced by more effective promoters of organisms of other species or of synthetic origin.
  • the calpain inhibitors identified by the method according to the invention are suitable for the manufacture of medicaments for the treatment of diseases in the case of calpain malfunctions, for example in diseases selected from the group of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerative or oncological diseases such as restenosis, arthritis, ischemia Hearts, the kidney or the central nervous system (e.g. stroke), inflammation, muscular dystrophies, cataracts of the eyes (cataracts), injuries to the central nervous system (e.g. trauma), Alzheimer's disease, HIV-induced neuropathy, Parkinson's and Huntington's disease preferred for production of medicines for the treatment of diseases of the placenta such as gestosis or embryogenesis.
  • the CAPN6 gene sequences according to the invention are advantageously also suitable for diagnosing diseases or for gene therapy.
  • the mouse CAPN6 sequence (EMBL accession number Y12583) was cloned by the RACE method using day 17 mouse embryos and primer sequences derived from the sequence EST AA050030. A plasmid clone containing the corresponding EST sequences was obtained from the I.M.A.G.E consort ⁇ um (Research Genetics Inc.). The human CAPN6 homolog was obtained through a homology search in the EST database using the mouse protein sequence and the tblastn algorithm. The partial sequences found could be combined to form an incomplete sequence of the human CAPN6 with a length of 1083 nucleotides (SEQ ID NO: 3).
  • CAPN6 The expression of the CAPN6 gene in the different tissues was determined with the aid of a 32 P-labeled human cDNA fragment with a human RNA master blot from Clontech, which contains RNA from 50 different tissues. The hybridization and the highly stringent washing conditions were carried out according to the manufacturer's instructions. A 2.2 kb EcoRI / XhoI fragment which contains the EST AA050030 was used as the CAPN6 cDNA fragment for the expression experiments. CAPN6 was only expressed in placenta tissue (see FIG. 4). As a control, the blot was carried out with a human ubiquitin DNA sample in order to determine the RNA loading.
  • the gene was localized in humans using the NIGMS human / rodent somatic cell hybrid "mapping panel" (Coriell Cell Repositories).
  • the following primers were used as primer sequences for the PCR reaction: 5 '-gttgaaactgattggggtctg-3' and 5 '-ctgtcttcccaaggggtttctc-3'.
  • the PCR amplification was carried out at an "annealing" temperature of 58 ° C. and resulted in a 200 bp fragment. The results were examined for correspondence between the presence of human chromosomes and the PCR product.
  • the exact localization of the gene in the human chromosome was carried out with the aid of the "Stanford G3 RH Panel" (Research Genetics) and transmission of the PCR results to the Stanford Human Genome Center localization service (http://www-shgc.stanford.edu).
  • the human CAPN6 gene was found on the X chromosome coupled to the DXS7356 marker.
  • Cathepsin B inhibition was determined analogously to a method by S. Hasnain et al., J. Biol. Chem. 1993, 268, 235-40.
  • the activity of the calpain inhibitors was examined in a colorimetric test with casein according to Hammarsten (Merck, Darmstadt) as substrate. The test was performed in the microtiter plate, according to the publication by Buroker-Kilgore and Wang in Anal. Biochemistry 208, 387-392 (1993).
  • CAPN6 which was expressed in one of the systems described above and subsequently purified, was used as the enzyme. The substances were incubated with the enzyme for 60 minutes at room temperature, a concentration of 1% of the solvent DMSO not being exceeded. After adding the Bio-Rad color reagent, the optical density was measured at 595 nm in the Easy Reader EAR 400 from SLT. The 50% activity of the enzyme results from the optical densities, which were determined at the maximum activity of the enzyme without inhibitors and the activity of the enzyme without the addition of calcium.
  • Calpain-mediated protein degradation in platelets was performed as described by ZhaozhaoLi et al., J. Med. Chem., 1993, 36, 3472-3480. Human platelets were isolated from fresh sodium citrate blood from donors and adjusted to 10 7 cells / ml in buffer (5 mM Hepes, 140 mM NaCl and 1 mg / ml BSA, pH 7.3). Platelets (0.1ml) are mixed for 5 minutes with 1 ⁇ l
  • ABSP actin binding protein
  • the half maximum enzyme activity is determined from this.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • ORGANISM Mus musculus
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTISENSE NO

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Neurology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Neurosurgery (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Rheumatology (AREA)
  • Cardiology (AREA)
  • Psychiatry (AREA)
  • Pain & Pain Management (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pulmonology (AREA)
  • Hospice & Palliative Care (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Analytical Chemistry (AREA)

Abstract

L'invention concerne de nouvelles calpaines spécifiques au tissu, leur production et leur utilisation. L'invention concerne également un procédé de tri de nouveaux inhibiteurs de calpaines et leur utilisation.
PCT/EP1998/005275 1997-08-26 1998-08-19 Nouvelles calpaines specifiques au tissu, leur production et leur utilisation WO1999010480A2 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
BR9811365-8A BR9811365A (pt) 1997-08-26 1998-08-19 Gene de calpaìna capn6, construção de gene, sequência de aminoácidos, processos para identificar inibidores de calpaìna e para preparar a enzima capn6 e suas variantes ou análogos alélicos, e, usos de um inibidor de calpaìna, de uma sequência de aminoácidos e de genes, do mrna anti-sentido e de um gene de calpaìna e suas variantes ou análogos alélicos
EP98945265A EP1009811A2 (fr) 1997-08-26 1998-08-19 Nouvelles calpaines specifiques au tissu, leur production et leur utilisation
CA002301815A CA2301815A1 (fr) 1997-08-26 1998-08-19 Nouvelles calpaines specifiques au tissu, leur production et leur utilisation
JP2000507788A JP2001513992A (ja) 1997-08-26 1998-08-19 新規の組織特異的カルパイン、その製造およびその使用
HU0004027A HUP0004027A2 (en) 1997-08-26 1998-08-19 New tissue-specific calpaines, their production and their use
KR1020007001916A KR20010023282A (ko) 1997-08-26 1998-08-19 신규 조직 특이적 칼파인, 그의 제조 방법 및 용도
IL13436198A IL134361A0 (en) 1997-08-26 1998-08-19 New tissue-specific calpaines, their production and their use
AU92635/98A AU9263598A (en) 1997-08-26 1998-08-19 New tissue-specific calpaines, their production and their use
NO20000961A NO20000961L (no) 1997-08-26 2000-02-25 Nye vevs-spesifikke calpainer, samt fremgangsmåte for fremstilling og anvendelse derav

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19737105A DE19737105A1 (de) 1997-08-26 1997-08-26 Neue gewebsspezifische Calpaine, ihre Herstellung und Verwendung
DE19737105.1 1997-08-26

Publications (2)

Publication Number Publication Date
WO1999010480A2 true WO1999010480A2 (fr) 1999-03-04
WO1999010480A3 WO1999010480A3 (fr) 1999-05-27

Family

ID=7840195

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/005275 WO1999010480A2 (fr) 1997-08-26 1998-08-19 Nouvelles calpaines specifiques au tissu, leur production et leur utilisation

Country Status (13)

Country Link
EP (1) EP1009811A2 (fr)
JP (1) JP2001513992A (fr)
KR (1) KR20010023282A (fr)
CN (1) CN1277634A (fr)
AU (1) AU9263598A (fr)
BR (1) BR9811365A (fr)
CA (1) CA2301815A1 (fr)
DE (1) DE19737105A1 (fr)
HU (1) HUP0004027A2 (fr)
IL (1) IL134361A0 (fr)
NO (1) NO20000961L (fr)
WO (1) WO1999010480A2 (fr)
ZA (1) ZA987660B (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072320A1 (fr) * 2000-03-28 2001-10-04 Autogen Research Pty Ltd Methode de traitement de troubles metaboliques et agents utiles pour ladite methode
WO2002081684A2 (fr) * 2001-04-04 2002-10-17 Universite De Montreal Nucleotides de calpaine antisens et utilisations correspondantes

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19928021A1 (de) * 1999-06-18 2000-12-21 Basf Ag Neue Calpaine und deren Verwendung
AU7476900A (en) * 1999-09-09 2001-04-10 Millennium Pharmaceuticals, Inc. 26176, a novel calpain protease and uses thereof
WO2001073073A2 (fr) * 2000-03-28 2001-10-04 Bayer Aktiengesellschaft Regulation d'enzymes apparentees a des proteases neutres humaines
JP4593404B2 (ja) * 2005-08-29 2010-12-08 シスメックス株式会社 液体試料吸引監視方法及び装置、並びに液体試料分析装置

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002634A1 (fr) * 1994-07-15 1996-02-01 Cephalon, Inc. Calpaine active exprimee par un baculovirus
EP0799892A2 (fr) * 1996-04-05 1997-10-08 Takeda Chemical Industries, Ltd. Calpain, sa préparation et son utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002634A1 (fr) * 1994-07-15 1996-02-01 Cephalon, Inc. Calpaine active exprimee par un baculovirus
EP0799892A2 (fr) * 1996-04-05 1997-10-08 Takeda Chemical Industries, Ltd. Calpain, sa préparation et son utilisation

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
EMBL Datenbank Emhum1, Eingang Af029232 Zugriffsnummer Af029232, 20.November 1997 Crotty P.L.: XP002097048 *
EMBL Datenbank Emhum1, Eingang Hscanpx Zugriffsnummer AJ000388, 22. Juli 1997 Belsito et al.: 'A novel X-linked calpain-like protease'; unpublished XP002097047 *
EMBL Datenbank Emrod, Eingang Af067793 Zugriffsnummer Af067793, 11. Juni 1998 Shimizu T.: 'Cellular genes modulated by transactivator Tax of HTLV1'; unpublished XP002097049 *
EMBL Datenbank Emrod, Eingang Mmcalpain Zugriffsnummer Y12582, 1. July 1997 Matena et al. "Genomic organization of mouse Capn5 and Capn6 genes confirms that they are a distinct calpain subfamily" XP002097045 -& MATENA ET AL.: "Genomic organization of mouse Capn5 and Capn6 genes confirms that they are a distinct calpain subfamily" GENOMICS, Bd. 48, 1998, Seiten 117-120, XP002097044 *
EMBL Datenbank Trrod, Eingang O35646 Zugriffsnummer O35646, 1. Januari 1998 Matena et al. "Calpain-like protease" XP002097046 & MATENA ET AL.: "Genomic organization of mouse Capn5 and Capn6 genes confirms that they are a distinct calpain subfamily" GENOMICS, Bd. 48, 1998, Seiten 117-120, *
SUZUKI K ET AL: "CALPAIN: NOVEL FAMILY MEMBERS, ACTIVATION, AND PHSIOLOGICAL FUNCTION" BIOLOGICAL CHEMISTRY HOPPE-SEYLER, Bd. 376, September 1995, Seiten 523-529, XP002061847 *
WANG K K W ET AL: "CALPAIN INHIBITION: AN OVERVIEW OF ITS THERAPEUTIC POTENTIAL" TRENDS IN PHARMACOLOGICAL SCIENCES, Bd. 15, 1. November 1994, Seiten 412-417, XP000568894 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072320A1 (fr) * 2000-03-28 2001-10-04 Autogen Research Pty Ltd Methode de traitement de troubles metaboliques et agents utiles pour ladite methode
WO2002081684A2 (fr) * 2001-04-04 2002-10-17 Universite De Montreal Nucleotides de calpaine antisens et utilisations correspondantes
WO2002081684A3 (fr) * 2001-04-04 2003-07-17 Univ Montreal Nucleotides de calpaine antisens et utilisations correspondantes

Also Published As

Publication number Publication date
EP1009811A2 (fr) 2000-06-21
KR20010023282A (ko) 2001-03-26
DE19737105A1 (de) 1999-03-04
ZA987660B (en) 2000-02-25
IL134361A0 (en) 2001-04-30
NO20000961D0 (no) 2000-02-25
JP2001513992A (ja) 2001-09-11
NO20000961L (no) 2000-04-14
BR9811365A (pt) 2000-08-22
AU9263598A (en) 1999-03-16
WO1999010480A3 (fr) 1999-05-27
CN1277634A (zh) 2000-12-20
HUP0004027A2 (en) 2001-03-28
CA2301815A1 (fr) 1999-03-04

Similar Documents

Publication Publication Date Title
DE69533135T2 (de) In baculovirus exprimiertes aktives calpain
DE69723074T2 (de) Mch4 und mch5, apoptotische proteasen, dafür kodierende nukleinsäure und verfahren zur verwendung
EP1161524B1 (fr) CELLULES COEXPRIMANT UNE PROTEINE PRECURSEUR AMYLOIDE ET UNE $g(a)-SECRETASE, ET LEURS UTILISATIONS DANS DES METHODES D'ESSAI ET A DES FINS DE DIAGNOSTIC
WO1999010480A2 (fr) Nouvelles calpaines specifiques au tissu, leur production et leur utilisation
EP0942995A1 (fr) Nouvelles calpaines, leur preparation et leur utilisation
DE69935327T2 (de) Endonuclease für dns-schaden mit breiter spezifität
DE60034880T2 (de) Ceramidasegen
DE10242016A1 (de) Verfahren zur Identifizierung BHS-spezifischer Proteine und Fragmente davon
EP0616642B1 (fr) Nouvelles proteines inhibitrices de thrombine provenant de la sangsue des campagnes
DE19718248A1 (de) Neue Calpaine, ihre Herstellung und Verwendung
EP0728209B1 (fr) Enzyme de conversion de l'endotheline
DE60217711T2 (de) Ptp10d nukleinsäuren und peptide in der regulation von energie-homeostase
DE19650142A1 (de) Neue Calpaine, ihre Herstellung und Verwendung
EP0904384A2 (fr) Proteine apparentee a la tyrosine-phosphatase
DE69921505T2 (de) Serinprotease-inhibitor
EP0840789B1 (fr) Proteine apparentee a la transcetolase
MXPA00001719A (es) Novedosas calpainas especificas de tejido, su preparacion y uso
DE19545126A1 (de) ATP- und Nukleinsäure-bindendes Protein mit Helikase-Eigenschaften
EP1098985A1 (fr) Genes de la famille des proteines de la boite dead, leurs produits d'expression et leur utilisation
DE3841873A1 (de) Neue serinprotease-inhibitor-proteine, diese enthaltende arzneimittel, dna-sequenzen, die fuer diese proteine codieren und verfahren zur herstellung dieser proteine, arzneimittel und dna-sequenzen
DE4403665A1 (de) Endothelinkonversionsenzym (ECE)
DE19905128A1 (de) Humane antibiotische Proteine
WO1999058647A2 (fr) Nuclease humaine desadenylante, sa preparation et son utilisation
WO2000008143A2 (fr) Gene isole sur le bras court du chromosome humain 17
CZ2000680A3 (cs) Nové tkáňově-specifické kalpainy, jejich příprava a použití

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 134361

Country of ref document: IL

Ref document number: 98810562.4

Country of ref document: CN

AK Designated states

Kind code of ref document: A2

Designated state(s): AL AU BG BR BY CA CN CZ GE HU ID IL JP KR KZ LT LV MK MX NO NZ PL RO RU SG SI SK TR UA US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AL AU BG BR BY CA CN CZ GE HU ID IL JP KR KZ LT LV MK MX NO NZ PL RO RU SG SI SK TR UA US

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

WWE Wipo information: entry into national phase

Ref document number: 1998945265

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 09485866

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: PA/a/2000/001719

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 92635/98

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2301815

Country of ref document: CA

Ref document number: 2301815

Country of ref document: CA

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: PV2000-680

Country of ref document: CZ

WWE Wipo information: entry into national phase

Ref document number: 1020007001916

Country of ref document: KR

WWP Wipo information: published in national office

Ref document number: PV2000-680

Country of ref document: CZ

WWP Wipo information: published in national office

Ref document number: 1998945265

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1020007001916

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 1020007001916

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 1998945265

Country of ref document: EP

WWR Wipo information: refused in national office

Ref document number: PV2000-680

Country of ref document: CZ