EP0942995A1 - Neue calpaine, ihre herstellung und verwendung - Google Patents
Neue calpaine, ihre herstellung und verwendungInfo
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- EP0942995A1 EP0942995A1 EP97952002A EP97952002A EP0942995A1 EP 0942995 A1 EP0942995 A1 EP 0942995A1 EP 97952002 A EP97952002 A EP 97952002A EP 97952002 A EP97952002 A EP 97952002A EP 0942995 A1 EP0942995 A1 EP 0942995A1
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- 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)
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- 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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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)
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- 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
- G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
Definitions
- the invention relates to new calpains and their production.
- the invention further relates to methods for screening for new calpain inhibitors and their use.
- Calpains are intracellular, non-lysosomal
- Enzymes from the group of cysteine proteases 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 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 is divided into two Splicing variants nCL-2 and nC -2 'can occur.
- nC -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, platelet proteins, “epidermal growth factor”, NMDA receptor and proteins which are involved in mitosis, as well as other proteins, are calpain substrates (Barrett MJ et al., Life Sei. 48, 1991: 0 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.
- Increased calpain levels were measured in various pathophysiological processes and diseases 5, for example in: ischemia of the heart (e.g. heart attack), the kidney or central nervous system (e.g. stroke), inflammation, muscle dystrophies, cataracts of the eyes (cataracts), Central nervous system injuries (eg trauma), Alzheimer's disease, HIV-induced neuropathy, Parkinson's and Huntigton's disease, 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 inhibitors show cytotoxic effects on tumor cells (Shiba E. et al., 20th Meeting Int. Ass. 5 Breast Cancer Res., Sendai Jp, 1994, September 25-28, Int. J. Onco. 5 (Suppl.), 1994, 381). 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 0 J ., 285, 1992: 857-862).
- 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 approximately 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 approximately 700 amino acids
- calpastatin which consists of approximately 700 amino acids
- calpastatin which consists of approximately 700 amino acids
- calpastatin which consists of approximately 700 amino acids
- calpastatin which consists of approximately 700 amino acids
- calpastatin which consists of approximately 700 amino acids
- calpastatin which consists of approximately 700 amino acids
- calpastatin which consists of approximately 700 amino acids
- calpastatin which consists of approximately 700 amino acids
- calpastatin which
- the invention relates to a new calpain and its allelic variants, analogs or derivatives.
- the invention also relates to a method for the identification of calpain inhibitors, wherein the calpain nCL-3 coded by the sequence SEQ ID NO: 1 or SEQ ID NO: 6 is isolated from tissues or cells in which the enzyme nCL-3 is expressed and the inhibition of the cleavage of a substrate of the enzyme nCL-3 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 at least one of the calpains Show effect or select the test substances which do not inhibit the enzyme nCL-3, but which inhibit the enzymes Calpain I and / or II or which inhibit the enzyme nCL-3, but not the enzymes Calpain I and / or II or the nCL-3 and inhibit the enzymes Calpain I and / or II.
- 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 nCL-3 or of calpains I and / or II is determined by test substances in cellular systems and such test substances are selected which Pass through the cell membrane and inhibit the intracellular activity of the enzyme nCL-3 and / or Calpaine I and / or II.
- Calpain-specific primers were used in the so-called domain fingerprinting (Boehm T., Oncogene 8, 1993: 1385-1390) using genomic DNA Sequence signatures produced by means of PCR technology, which advantageously also contain intron sequences for better differentiation of the calpain sequences.
- This clone codes for a gene whose gene product was named nCL-3 as the new calpain.
- the derived amino acid sequence of calpain nCL-3 can be found in the sequence SEQ ID NO: 2.
- the amino acid sequence deduced taking into account an existing intron has one typical Calpain signature, whereby an assignment to the well-known Calpain subfamilies ⁇ Calpain, mCalpain, nCL-1 or nCL-2 is not possible due to the low homology (see Table 2).
- FIG. 3 shows the homology to known calpain subfamilies.
- the Calpain nCL-3 is a new, previously unknown Calpain.
- the intron shown in sequence SEQ ID NO: 5 was determined by comparison with the cDNA.
- nCL-3 has a shortened domain I and 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 is located in the domain IV.
- This Ca 2+ binding site is absent in the case of nCL-3, which means that possibly no Ca 2+ is bound to domain IV and the protein is activated in another way. It is the only vertebrate calpain that lacks domain IV similar to calmodulin.
- CalpA is a tissue-specific expressed calpain homolog from Drosophila (Theopold V. et al., Mol. Cell. Biol., Vol. 15, No. 2, 1995: 824-834). It is expressed in some neurons in the central nervous system, in scattered cells of the midgut and in blood cells from Drosophila. CalpA found two different splicing variants. The shorter variant lacks the calcium binding site typical of Calpain.
- the homology at the amino acid level between CalpA and nCL-3 is 31.5% (see Table 2).
- 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 homology at the amino acid level between tra-3 and nCL-3 is 34.5% (see Table 2). NCL-3 may also be involved in gender determination.
- nCL-3 The greatest homology exists between nCL-3 and the human partial sequence ⁇ ST01106.
- the partial sequence EST01106 was obtained from a hippocampus library. None is known about the function (Nature 355, 6361, 1992: 632-634). The complete gene sequence and whether the sequence is a calpaingen is also unknown.
- the amino acid sequence (SEQ ID NO: 7) derived from the gene sequence SEQ ID NO: 6 shows 92.2% homology to the mouse nCL-3 sequence (see FIG. 4). This similarity corresponds to the amino acid level homology between human and mouse m-calpain (97%) and human and mouse p94 (93.5%) as our sequencing showed. EST01106 is therefore most likely the human ortholog of the mouse nCL-3 sequence.
- Figure 4 also shows the sequences of Caenorhabditis tra-3, Drosophila CalpA, mouse p94. Mouse m-calpain, human ⁇ -calpain and rats nCL-2. Amino acids that match between the different calpains and nCL-3 are indicated by dots.
- Dashes indicate gaps that have been introduced in order to achieve maximum match of the sequences.
- the C-terminal ends of the alternative splicing products of the nCL-3 and CalpA transcripts are indicated above and below the respective overall sequence, starting with the beginning of the different sequence. Stars indicate the remains conserved between all calpains.
- the two amino acid sequences corresponding to the oligonucleotides Cal6 and Cal9 were marked with boxes.
- the "splice sites" of the corresponding mouse nCL-3-DNA are marked by arrows.
- Figure 5 shows the phylogenetic family tree of the different Calpaine.
- the phylogenetic analyzes for the construction of this family tree were carried out using the closest neighboring method (Saitou et al. Mol. Biol. Evol. 4, 1987, 406-425) Exclusion of the gaps carried out.
- the vertebrate calpains could be divided into six different groups ( Figure 5, right side).
- the non-vertebrate calpains can be assigned to the nCL-3 group as the nearest neighbors or are in a separate group.
- the nCL-3 genes thus form a separate group of calpains, which are more similar to invertebrate calpains than to vertebrate calpains.
- the length of the horizontal lines is proportional to the phylogenetic distance of the different Calpaine.
- 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).
- nCL-3 gene structure is shown in Figure 6.
- the exon / intron splicing transitions within the coding sequence of the gene were determined by comparing the DNA sequences of genomic DNA and cDNA. 11 introns were found within the coding sequence.
- the position of the "splicing sites" is marked by arrows.
- the position of the genomic fragment first amplified with the primers Cal6 and Cal9 is marked by brackets.
- Figure 6b shows the location of the "splicing sites" of different Calpaine. Surprisingly, despite the relatively high homology, nCL-3 and tra3 do not have any common "splice sites". The agreement in the position of some "splice sites" between nCL-3 and the vertebrate calpaines indicates a common genesis. The question mark above the last preserved splice site in the chicken ⁇ / m-Calpaingen indicates that the published sequence around this interface is not in agreement with the original cDNA sequence.
- nCL-3 In addition to the nCL-3 gene shown in sequence SEQ ID NO: 1, a truncated form, which is shown in sequence SEQ ID NO: 3, was identified. The deduced amino acid sequence of the truncated nCL-3 gene can be found in the sequence SEQ ID NO: 4. This truncated nCL-3 gene, known as nCL-3 ', is believed to result from alternative splicing. Semi-quantitative RT-PCR analyzes with mRNA isolated from dE17 cells using primers that match the flanking Covering regions of the intron (see Figure 6) showed that the unsplit product accounted for approximately 0.5% of the n-CL3 mRNA.
- nCL-3 is expressed in many tissues with different intensities (FIG. 1). In the mRNA analyzes examined, nCL-3 is clearly expressed in the skin, the kidneys, the heart, the lungs, the thymus and in the liver.
- nCL-3 gene is also expressed to different extents in humans. Low expression was detected in all tissues examined. Strong expression was found in the colon, testicles, kidney, liver and trachea.
- mice orthologist nCL-3 gene was localized between 44 and 53 cM on mouse chromosome 7.
- 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 nCL-3, calpain I and / or II for their activity against cathepsin B or other thiol proteases.
- 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 calpain nCL-3 according to the invention, inhibitors can be identified with the methods according to the invention, which can discriminate their inhibitory effect between the different calpaines Calpain I, II, nCL-1, nCL-2 and / or nCL-3.
- 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 DSMO final concentration: 100 ⁇ M up to 0.01 ⁇ M
- the activity of the calpain inhibitors was examined 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 incubated with the enzyme for 60 minutes at room temperature, a concentration of 1% of the solvent DMSO not being exceeded.
- 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 for the maximum activity of the enzyme without inhibitors and the activity of the enzyme without the addition of calcium.
- 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 must cross the cell membrane to prevent calpain from breaking down intracellular proteins.
- Some known calpain inhibitors, such as E 64 and leupeptin, only poorly cross the cell membranes and accordingly show, although they are good calpain inhibitors, only poor activity on cells. It is therefore advantageous to carry out an additional test for the permeability of potential calpain inhibitors such as the human platelet test.
- ABP actin binding protein
- Tissue parts such as brain sections or cell cultures are also suitable for testing membrane permeability.
- nCL-3 Inhibition of nCL-3 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 nCL-3. Takaomi Saido described in 1992 in J. Bio- chem. Vol. 11, 81-86 the autolytic transition of ⁇ -calpain after activation and the detection with antibodies. Appropriate antibodies are generated for the detection of nCL-3. Calpain inhibitors prevent the autolytic transition and a corresponding quantification is possible with antibodies.
- the calpain nCL-3 or its animal or human homologue is made from tissues or cells in which the enzyme is expressed, such as the kidney, the thymus, the liver, the lung, or from cells or microorganisms which contain at least one gene copy and / or a vector with at least one gene copy of the nCL-3 gene, purified and used as a crude extract or as a pure enzyme.
- the various calpain inhibitor tests are advantageously carried out in combination with the test for inhibition of the nCL-3 enzyme activity by potential inhibitors.
- Inhibitors are selected so that they either only inhibit the enzyme nCL-3 and not the other calpains or, conversely, only the other calpains and not the enzyme nCL-3 or the enzyme nCL-3 and at least one other calpain.
- 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 nCL-3, 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 easily be recognized.
- Another method according to the invention uses the enzyme nCL-3 for the screening for new calpain inhibitors, these inhibitors advantageously being able to generally inhibit all calpains or individual calpains such as calpain I, II, nCL-1, nCl-2 or nCL-3.
- the different test substances can be used individually or tested 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 nCL-3 gene or its animal homologs or its human homologues. Expression systems are preferred which enable expression of the nCL-3 gene sequences in bacterial, fungal or animal cells, very particularly preferably in insect cells.
- An enzymatically active gene product is to be understood as nCL-3 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 yourself through autocatalysis.
- 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.
- nCL-3 Contain the center of the nCL-3 gene and / or further sequences of the nCL-3 gene and / or other calpain gene sequences and / or other sequences and show enzymatic activity, to be understood by the enzymatic active gene product of nCL-3.
- prokaryotic or eukaryotic organisms which are suitable as host organisms are, for example, bacteria such as Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptococcus carnosus, yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, fungi, Trypsoptera as such as Aspergillusiperia nod Cells or any other insect cells that are for viral expression is suitable, or to understand animal cells such as CV1, COS, C127, 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, Trypsoptera as such as Aspergillusiperia nod Cells or any other insect cells that are for viral expression is
- 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 is preferably to be understood as the combination of Escherichia coli and its plasmids and phages or the baculovirus system and the corresponding insect cells such as Spodoptera frugiperda.
- Additional 3 'and / or 5' terminal regulatory sequences are also suitable for the advantageous expression of the nCL-3 gene according to the invention.
- regulatory sequences are intended to enable the targeted expression of the nCL-3 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 have a positive influence on nCL-3 gene expression and thereby increase it.
- the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". But there is also a reinforcement of the
- Translation possible, for example by improving the stability of the mRNA.
- Enhancers are understood to mean, for example, DNA sequences which bring about increased nCL-3 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 nCL-3 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 nCL-3 gene can also be increased by increasing the nCL-3 gene copy number.
- the nCL-3 gene is amplified, for example, in a CHO expression vector.
- Vectors of the pED series - dicistronic vectors - are also suitable as vectors contain the amplifiable marker gene dihydrofolate reductase. Details can be found in the Current Protocols in Molecular Biology Vol 2, 1994.
- nCL-3 enzyme activity can be achieved, for example, compared to the starting enzyme by changing the nCL-3 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, Achieve deletion (s), 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 which repress enzyme synthesis and / or by synthesizing active instead of inactive nCL-3 proteins. In this way, increased amounts of enzyme can be made available for the in vitro tests.
- nCL-3 or its animal homologues can advantageously be derived from genomic DNA or cDNA using, for example, the PCR technique (see Molecular Cloning, Sambrok, 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, 487ff), preferably nCL-3 can be selected using genomic DNA and particularly preferably using genomic DNA Clone 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.
- nCL-3 genes with nucleotide sequences are available which code for the amino acid sequence given in SEQ ID NO: 2 or its allele variants. Allele variants are understood to mean nCL-3 variants which have 60 to 100% homology at the amino acid level, preferably 70 to 100%, very particularly preferably 80 to 100%. Allelic variants include, in particular, functional variants which are obtained by deleting, inserting or substituting nucleotides from the SEQ ID NO: 1 or SEQ ID NO: 6 sequence are available, but the nCL-3 activity is retained.
- Analogs of nCL-3 mean, for example, its animal homologs, shortened sequences such as nCL-3 '(see SEQ ID NO: 3), single-stranded DNA or RNA of the coding and non-coding DNA sequence, particularly antisense RNA.
- nCL-3 Derivatives of nCL-3 are, for example, those derivatives which cannot be cleaved enzymatically or are difficult to cleave like 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 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, including organisms of other species.
- the cal pain inhibitors identified by the methods according to the invention are suitable for the manufacture of medicaments for the treatment of diseases selected from the group of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerative or oncological diseases such as restenosis, arthritis, ischemia of the heart or the like 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 Huntigton's disease.
- diseases selected from the group of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerative or oncological diseases such as restenosis, arthritis, ischemia of the heart or the like 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 Huntigton's disease.
- nCL-3 gene sequences according to the invention are also advantageously suitable for diagnosing diseases, for example for diagnosing muscular dystrophy or for gene therapy. Examples
- Genomic DNA from mouse cells ES E14 was used to clone the nCL-3 gene with the sequence SEQ ID NO: 1.
- nCL-3 gene was cloned into the vector pBluescript (SK +) with the enzyme EcoRV (see Holton et al., Nucleic Acids Research, Vol. 19, No. 5, 1156ff.).
- the pBluescript vector with the cloned-in nCL-3 gene, the Escherichia coli strain DH10B according to Maniatis et al.
- the human sequence of the nCL-3 gene with SEQ ID NO: 6 can also be cloned in this way, it being possible to start from 0.1 ng cDNA or 0.5 ⁇ g genomic DNA.
- the cloning batch may also advantageously contain 0.1% Triton X-100.
- Example 2 Expression of the nCL-3 gene in different mouse tissues
- mRNA was isolated from dE12 embryos and from mouse, kidney, heart, lung, brain, thymus and small intestine tissue.
- the tissues were dispersed in liquid nitrogen and in 10 ml of a solution of 4 M guanidinium isothiocyanate, 25 mM Na citrate, 0.5% sarcosyl and 72 ul 2-mercaptoethanol resuspended. Then 1 ml of Na acetate (pH 4.0), 10 ml of water-saturated phenol and 2 ml of chloroform were added with mixing. The samples were centrifuged for 20 minutes (5000 xg, 4 ° C). The precipitate was discarded.
- RNA was precipitated from the supernatant with a volume of isopropanol at ⁇ 20 ° C. (precipitation for at least one hour) and centrifuged again for 30 minutes (19000 ⁇ g, 4 ° C.).
- the precipitate was resuspended in 300 ⁇ l and again precipitated in the cold with Na acetate / ethanol and washed with 70% ethanol, centrifuged (19000 ⁇ g, 4 ° C.) and dissolved in 300 ⁇ l water.
- the mRNA concentration at 250 nm was then determined in a photometer and in an agarose gel with a 5 ⁇ l mRNA sample against a reference.
- the cDNA was produced according to a protocol from GIBCO as follows:
- 2 ⁇ l oligo (dT) and 12 ⁇ l DEPC dH 0 were added to 5 ⁇ g mRNA (isolated according to the method described above). This mixture was incubated at 70 ° C. for 10 minutes and then placed on ice. 2 ⁇ l 10 ⁇ buffer, 2 ⁇ l 25 mM MgCl 2 , 1 ⁇ l 10 mM dNTPs, 2 ⁇ l 0.1 M DTT were added to the sample in the order given. After 5 minutes of incubation at 23 ° C., 1 ⁇ l of SuperScript Reverse Transcriptase was added and the whole incubation continued for 10 minutes at 25 ° C., 50 minutes at 42 ° C. and 15 minutes at 70 ° C.
- RNAse H and 79 ⁇ l dH0 were added and the reaction was carried out for 20 min at 37 ° C and 15 min at 70 ° C. 1 ul of this cDNA was used for the RT-PCR reaction.
- PCR reaction for the detection of the expression of nCL-3 was carried out as follows:
- Human hippocampus marathon-ready cDNA (Clontech) was used for the human sequence of the 5 'and 3' end.
- cDNA from day 12 mouse embryos was used as described in Example 2.
- the human 3 'end could not be isolated with the kit reverse primer.
- the cloning was carried out with the aid of a forward primer complementary to the human EST sequence and a reverse primer which corresponded to the last 6 amino acids of the mouse nCL-3 sequence (5 '-teagacage-cgtgagagagg-3').
- Cosmid clones with the mouse nCl-3 gene were isolated from a cosmid library, which was produced from genomic ES mouse cell DNA by cloning in the Vector pSuperCos (Stratagene). The library was divided into 348 “pools” of 1000 clones. Positive “pools” were identified using PCR analysis using fertilization of nCL-3-specific primers identified. These "pools” were then plated out and screened. Positive clones were identified by colony hybridization with 32 P-labeled mouse nCL-3 cDNA fragments.
- the expression of the human nCL-3 was examined by hybridizing a human RNA master blot (Clontech) with a 32 P-labeled human nCL-3 fragment (nucleotide 1-928 coding for amino acids 1-295). The hybridization and the stringent washing conditions were carried out according to the manufacturer's instructions.
- the gene was localized in the mouse by PCR analysis of genomic DNA isolated from mouse somatic hamster cell hybrids as described by Williamson et al. (Mamm. Genome 6, 1995, 429-432) using a set of DNAs as described by Schupp et al. (Immunogenet. 45, 1997, 180-187). 5'-tgca-cagcctacagcataag-3 'and 5' -tcagacagccgtgagagagg-3 'were used as primer sequences. With the help of these primers an approximately 2.7 kb mouse fragment and no hamster DNA could be amplified. The PCR reactions were carried out using the "Expand Long Template PCR System (Boehringer Mannheim) according to the manufacturer's instructions at an" annealing "temperature of 58 ° C.
- the gene was localized in humans using the NIGMS human / rodent somatic cell hybrid "mapping panels" (Coriell Cell Repositories).
- the following primers were used as primer sequences for the PCR reaction: 5 '-acttcatcttc-tggcttcttgacttc-3' and 5 '-gctgcatcaaccacaaggacac-3'.
- the PCR amplification was carried out at an "annealing" temperature of 58 ° C. and resulted in a 600 bp fragment. The results were examined for correspondence between the presence of human chromosomes and the PCR product.
- Cathepsin B inhibition was determined analogously to a method by S.Hasnain et al., J. Biol. Chem. 1993, 268, 235-40.
- an inhibitor solution prepared from inhibitor and DMSO (final concentrations: 100 ⁇ M to 0.01 ⁇ M), are added to 88 ⁇ L cathepsin B (cathepsin B from human liver (Calbiochem), diluted to 5 units in 500 ⁇ M buffer). This mixture is preincubated for 60 minutes at room temperature (25 ° C.) and the reaction is then started by adding 10 ⁇ L 10 mM Z-Arg-Arg-pNA (in buffer with 10% DMSO). The reaction is monitored for 30 minutes at 405 nM in a microtiter plate reader. The IC50 is then determined from the maximum gradients.
- the activity of the calpain inhibitors was examined in a colorimetric test with casein according to Hammarsten (Merck, Darmstadt) as a 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). 29/30, 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 is derived from optical densities that were determined without the enzyme inhibitors and the activity of the En ⁇ zyms without the addition of calcium at the maximum activity.
- Example Platelet test for determining the cellular activity of calpain inhibitors
- Platelets (0.1 ml) are preincubated for 5 minutes with 1 ⁇ l of various concentrations of 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 for 5 minutes at 95 ° C. and the proteins were separated in an 8% gel.
- ABSP actin binding protein
- ORGANISM Mus musculus
- MOLECULE TYPE cDNA
- HYPOTHETICAL NO
- ANTISENSE NO
- ORGANISM Mus musculus
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTISENSE NO
- ORGANISM Mus musculus
- MOLECULE TYPE cDNA
- HYPOTHETICAL NO
- ANTISENSE NO
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Application Number | Priority Date | Filing Date | Title |
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DE19650142 | 1996-12-04 | ||
DE1996150142 DE19650142A1 (de) | 1996-12-04 | 1996-12-04 | Neue Calpaine, ihre Herstellung und Verwendung |
DE19718248 | 1997-04-30 | ||
DE1997118248 DE19718248A1 (de) | 1997-04-30 | 1997-04-30 | Neue Calpaine, ihre Herstellung und Verwendung |
PCT/EP1997/006644 WO1998024916A1 (de) | 1996-12-04 | 1997-11-28 | Neue calpaine, ihre herstellung und verwendung |
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EP97952002A Withdrawn EP0942995A1 (de) | 1996-12-04 | 1997-11-28 | Neue calpaine, ihre herstellung und verwendung |
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US (1) | US6569665B1 (de) |
EP (1) | EP0942995A1 (de) |
JP (1) | JP2001506846A (de) |
KR (1) | KR20000057365A (de) |
CN (1) | CN1245536A (de) |
AU (1) | AU737504B2 (de) |
BR (1) | BR9713849A (de) |
CA (1) | CA2274304A1 (de) |
CZ (1) | CZ9902005A3 (de) |
HU (1) | HUP0000498A3 (de) |
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AUPQ656500A0 (en) * | 2000-03-28 | 2000-04-20 | Autogen Pty Ltd | A method of treatment and agents for same |
WO2005082860A1 (en) * | 2004-02-27 | 2005-09-09 | National Research Council Of Canada | Tetracyclines and their use as calpain inhibitors |
CA2631071A1 (en) * | 2008-05-09 | 2009-11-09 | Tong-Jun Lin | Inhibition of calpain reduces allergic inflammation |
CN104458709A (zh) * | 2013-09-12 | 2015-03-25 | 中国药科大学 | 一种筛选钙激活中性蛋白酶-1抑制剂高通量筛选方法 |
CN105334329B (zh) * | 2015-12-10 | 2017-07-28 | 中国农业科学院农产品加工研究所 | 钙蛋白酶磷酸化水平的测定方法 |
WO2018071216A1 (en) * | 2016-10-11 | 2018-04-19 | University Of Iowa Research Foundation | Methods and compositions for treating genetic eye diseases |
WO2021072196A1 (en) * | 2019-10-11 | 2021-04-15 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions and methods for measuring and inhibiting calpain-5 activity |
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EP0717110A1 (de) * | 1994-11-22 | 1996-06-19 | Association Francaise Contre Les Myopathies | LGMD-Gen |
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HUP0000498A2 (hu) | 2000-06-28 |
WO1998024916A1 (de) | 1998-06-11 |
HUP0000498A3 (en) | 2003-08-28 |
BR9713849A (pt) | 2000-02-29 |
CA2274304A1 (en) | 1998-06-11 |
KR20000057365A (ko) | 2000-09-15 |
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NO992690D0 (no) | 1999-06-03 |
NO992690L (no) | 1999-08-03 |
JP2001506846A (ja) | 2001-05-29 |
AU5557698A (en) | 1998-06-29 |
AU737504B2 (en) | 2001-08-23 |
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CZ9902005A3 (cs) | 1999-09-15 |
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