DE19737105A1 - New tissue-specific calpains, their production and use - Google Patents

Abstract

The invention relates to new tissue-specific calpaines and their production. The invention also relates to a method for screening for new calpaine inhibitors and their use.

Description

The invention relates to new tissue-specific calpains and their Manufacturing.

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.

So far there are three main isoforms of these ubiquitous calpains known, which is in vitro by their calcium-dependent activators distinguishability. Calpain I (= µCalpain) is through µ-molar calcium ion concentrations activated, while Calpain II (= mCalpain) only through millimolar concentrations of calcium ions is activated. Both calpains consist of two sub units, a large subunit with approx. 80 kDa and one small subunit of approx. 30 kDa. Both subunits of the active heterodimers have binding sites for calcium. The large subunit is made up of the following four protein domains (= I-IV) constructed: one protease domain (= domain II), one Calcium-binding domain (= domain IV) and two other domains (= Domain I and III), whose function is unclear. The little one 30 K subunit consists of a calcium binding sub unit (= IV ') and another subunit (= V), whose Function is unclear. In addition to these two types of calpains became a third intermediate in calcium activation Type (= µ / m 80K) found in the chicken (Wang K.K.W. et al., TiPS, Vol. 15, 1994: 412-419, Suzuki, K et al., Biol. Chem. Hoppe-Seyler, Vol. 376, 1995: 523-529).

In addition to these ubiquitous Calpainen were in the last Time two new tissue-specific expressed Calpaine identifi graces. nCL-1 (= p94) is one found in chickens, rats and humans coming, muscle-specific calpain, which is believed to be mono  could be active and consists only of the 80 kd subunit. In addition to nCL-1, there is a stomach-specific calpain that is divided into two Splicing variants nCL-2 and nCL-2 'can occur. nCL-2 'below differs from nCL-2 by the lack of calcium bin region (Sorimachi, H.S. et al., J. Biol. Chem. Vol. 268, No. 26, 1993: 19476-19482, Sorimachi, H: S: et al., FEBS Lett. 343, 1994: 1-5). A Calpain homologue was also found in Drosophila Protein (= CalpA) that interacts with actin and presumably one played an important role in embryonic development, found that has two different splicing variants (Mol. Cell. Biol. Vol. 15, No. 2, 1995: 824-834). The shorter one is also missing here Variant the calcium binding site.

It is believed that calpaine has various physiological effects Processes play an important role. A variety of cytos keletal, membrane-binding or regulatory proteins such as Protein kinase C, phospholipase C, spectrin, cytoskeleton proteins such as MAP2, muscle proteins, neurofilaments and neuropeptides, platelets Chenproteine, - "Epidermal Growth Factor" -, NMDA receptor and Pro stones involved in mitosis and other proteins are calpain substrates (Barrett M.J. et al., Life Sci. 48, 1991: 1659-69, Wang K.K. et al., Trends in Pharmacol. Sci., 15, 1994: 412-419). The normal physiological function of calpaine is still not clearly understood until today. You are with one Variety of physiological processes such as the Apoptosis, cell division and differentiation or at the Embryonate development involved.

With various pathophysiological processes and diseases increased calpain levels were measured, for example in: Ischemias 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 the central nervous system (e.g. trauma), Alzheimer's disease, HIV induced neuropathy, Parkinson's and Huntigton's disease etc. (see Wang K.K. above). One suspects a connection these diseases with an increased and persistent intracellu calcium levels. This makes calcium-dependent processes overactivated and are no longer subject to physiological Regulation. Accordingly, over-activation of calpaines also trigger pathophysiological processes.

Therefore, it has been postulated that inhibitors of calpain enzymes for the treatment of these diseases can be useful. Ver Various studies have confirmed this. So did Seung-Chyul Hong et al. (Stroke 1994, 25 (3), 663-669) and Bartus R.T. et al. (Neurological Res. 1995, 17, 249-258) a neuroprotective  Effect of calpain inhibitors in acute neurodegenerative Disorders as they appear after stroke. As well improved after experimental brain trauma calpaininhi Bitter recovery of memory deficits and neuromotor disorders (Saatman K.E. et al., Proc. Natl. Acad. Sci. USA, 93, 1996: 3428-3433). Gemstone C.L. et al. (Proc. Natl. Acad. Sci. USA, 92, 1995, 7662-7666) found one protective effect of calpain inhibitors on by hypoxia damaged kidneys. Yoshida K.I. et al. (Jap. Circ. LT. 59 (1), 1995, 40-48) could have beneficial effects of calpain inhibitors after cardiac damage caused by ischemia or Reperfusion were generated. Because calpain inhibitors release inhibit of β-AP4 protein has been a potential application as Therapeutic agent for Alzheimer's disease proposed (Higaki LT. et al., Neuron, 14, 1995: 651-659). The release of Interleukin-1α is also inhibited by calpain inhibitors (Watanabe N. et al., Cytokine, 6 (6), 1994: 597-601). Farther it has been found that calpain inhibitors exert cytotoxic effects Show tumor cells (Shiba E. et al., 20th Meeting Int. Ass. Breast Cancer Res., Sendai Jp, 1994, 25-28. Sept., Int. LT. Onco. 5 (Suppl.), 1994, 381). Also in the case of restenosis and Arthritis plays an important role in Calpain and Calpaininhibi gates 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 LT., 285, 1992: 857-862).

Other possible uses of calpain inhibitors are Wang K.K. (Trends in Pharmacol. Sci., 15, 1994: 412-419).

The most potent and selective calpain inhibitor is, of course occurring intracellular protein calpastatin. It both inhibits Calpain I and Calpain II, but not other cysteine or thiol proteases such as cathepsin B, L or papain. That from approx However, calpastatin consisting of 700 amino acids has the disadvantage that that it is for therapeutic options due to its size and the impassability of the cell membrane is out of the question. Next low molecular weight peptide calpain inhibitors have been identified Series of non-peptide inhibitors identified. disadvantage of these inhibitors is that they are unstable, rapidly metabolized be siert and are partially toxic. Many calpain inhibitors are also characterized by a lack of selectivity, d. H. they not only inhibit Calpain I and II but also others Cysteine proteases such as papain, chymotrypsin, or elastase Cathepsin B and L.  

There is therefore still a need for selective, high effective calpain inhibitors. For the screening for these selective, well-effective calpain inhibitors are highly specific Test systems required that allow selective inhibito identify. Usually the screening tests with the ubiquitous Calpainen Calpain I and Calpain II carried out.

It is necessary to find selective inhibitors and desirable further calpaine available for testing places that are expressed as tissue-specific as possible, so that the inhibitors on their selectivity between each Calpainen can be checked.

In addition, there are other new Calpaine proteins that are wanted they most likely with the different sick diseases or diseases are expressed differently and play an important role in these diseases.

The object of the present invention was to provide means for profiling tion and identification of calpain inhibitors places that make it possible to identify calpain inhibitors, on the one hand the inhibitory effect only against a calpain have, and / or on the other hand to several calpains have inhibitory effect and this as a therapeutic To provide Target.

The invention relates to a new tissue-specific calpaingen with the sequence SEQ ID NO. 1 or SEQ ID NC). 3 and the name CAPN6, its allelic variants, analogs or derivatives, which have a homology of 60 to 100% at the derived amino acid level, the calpaingenes, their all lian variants, analogs or derivatives containing the following sequences:

• (a) Leu-Gly-Asn-Lys-Ala,
  this sequence differs from the corresponding sequence in human calpain I in that the amino acid cysteine in human calpain I, which has position 115 in calpain I, against lysine, which is in position 81 of the sequences SEQ ID No. 1 and SEQ ID No. 3 lies, is changed;
• (b) Ala-X-Ser-Cys-Leu-Ala,
  where compared to the corresponding sequence in human calpain I, the amino acids alanine and threonine in positions 122 and 125 against serine and alanine in positions 88 and 91 in the sequences SEQ ID No. 1 and SEQ ID No. 3 are changed;
• (c) Gly-Tyr-Thr- (His or Tyr) -Thr-X-Thr,
  where, compared to the corresponding sequence in human calpain I, the amino acids histidine, alanine and serine in positions 272, 273 and 275 against tyrosine, youronin and threonine in positions 252, 253 and 255 in the sequences SEQ ID No. 1 and SEQ ID No. 3 are changed and in SEQ ID No. 3 additionally the tyrosine residue in position 274 in calpain I against histidine in position 254 in SEQ ID No. 3 is changed;
• (d) Arg-X-Arg-Asn-Pro-Leu-Gly
  this sequence differs from the corresponding sequence in human calpain I in that the amino acid tryptophan in human calpain I, which has position 298 in calpain I, against leucine, which is in position 286 of the sequences SEQ ID No. 1 and SEQ ID No. 3 lies, is changed and
  X denotes any natural amino acid in the sequences mentioned.

The invention also relates to a method for identification tion of calpain inhibitors, taking one calpain, its all mical variants or analogs coded according to a sequence Claim 1 isolated from tissues or cells and inhibition the cleavage of a substrate of the enzyme CAPN6 and in at least a further test inhibiting the cleavage of a substrate the enzyme calpain I and / or II is measured by test substances and selects the test substances that the enzyme CAPN6 and at least inhibit another of the Calpaine.

The invention further relates to a method for identi Fication of calpain inhibitors, characterized in that inhibition of cleavage of a substrate of the enzyme CAPN6 or the Calpaine I and / or II by test substances in cell systems and selects those test substances that pass through the cell membrane and the intracellular activity of the Inhibit enzyme CAPN6 and / or Calpaine I and / or II that the CAPN6 enzyme does not inhibit, but Calpain I and / or II enzymes or which inhibit the CAPN6 enzyme but not the calpain enzyme I and / or II. Also substances that have an activity in vitro show to the Calpainen without their cell mobility tested, are advantageously selected. Shows up in a subsequent assay that these substances do not or are only poorly cell-permeable, so their derivatization can Cell permeability can be improved.  

Human µ- and m-calpain protein sequences were used for a homology search in the EST database of the National Center for Bio technology Information (http://www.ncbi.nlm.nih.gov) using the BLAST process program. A sequence with the name EST AA050030 was found which has a sequence typical for Calpaine. With the aid of this sequence, a mouse clone could be produced which codes for a gene whose gene product according to the invention was named CAPN6 (= nCL-4) as a new calpain. The nucleic acid sequence of the clone nCL-4 can be found in sequence SEQ ID NO: 1. The derived 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 µCalpain, 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. This sequence lacks both the typical start codon for methionine and the stop codon. This sequence is therefore probably only a partial sequence of the human orthologue for the cloned mouse sequence. The homology of both sequences over the 374 amino acids is 94.9% ( FIG. 1).

The protein sequence derived from the gene sequence SEQ ID NO: 1 shows with 30% homology the greatest homology to the known Caenorhabditis elegans gene tra-3 over the entire amino acid sequence. The homology to the other known acceptable calpaines is between 20.9% (rats nCL-2) and 25.4% (mouse mCalpain). In a sequence comparison according to Lipman-Pearson (Ktup1 2, Gap Penalty 4, Gap Length Pena: Lty 12) between CAPN6 and human CAPN1 (= CAN1_HUMAN, Aoki et al., FEBS Lett. 205, 1986: 313-317), human CAPN2 (= CAN2_HUMAN, Aoki et al., Biochemistry 27, 1988: 8122-8128), rat CAPN2 (= CAN2_RAT, Deluca et al., Biochim. Biophys. Acta 1216, 1993: 81-93), human CAPN3 (= CAN3_HUMAN , Richard et al., Cell 81, 27-40) rat CAPN3 (= CAN3_RAT, Sorimachi et al., LT. Biol. Chem. 264, 1989: 20106-20111) and Drosophila Calpain (= DMCLPNGCM_1) via partial sequences of 230 -520 amino acids, slightly larger homologies from 32.8 to 39.5% were found, but overall they are lower than the homologies to tra-3 ( Fig. 1, Alignment, Clustal method with PAM25 residue weight tabl.). In addition to tra3, CAPN6 has a pronounced homology to the recently described CAPN5 calpain (44.2% homology, file number P 19 718 248.8).

Sequence comparisons between the mouse and human CAPN6 sequence in Various databases gave homologies to CalpA, Tra-3 and human sequences with the designations C16980, T39424, AA16971, R93331 and G17331 about their functions no information were made. The sequence comparisons were made with the Genbank EST and databases at the National Center for Biotechnology Information (http: Hwww.ncbi.nlm.nih.yor) and the Wash-U database leads (Homo sapiens). A mouse was also found in the databases EST sequence labeled AA050030 and labeled as Calpain found. The databases could not provide any further information be removed. The full gene sequences of AA16971T, R93331, C17331 and AA050030 are unknown.

Both calpains (CAPN5 and CAPN6) have common properties that distinguish them from the other calpains. In comparison to 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 Calpaine (so-called "EF-hand") lies in the domain IV. This Ca ²⁺ binding site is absent 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 derived CAPN6 amino acid sequence has another Special feature an altered catalytic center. Only the Asn residue in position 284 is preserved. The cysteine residue in Position 81 and the histidine residue in position 252 were each exchanged for lysine or tyrosine in the mouse CAPN6 sequence. In addition, other amino acids exchanged in Ver equal to the CAPN6 sequences SEQ ID No. 1 and SEQ ID No. 3 with the human calpain I (= CAPN1, Aoki et al., FEBS Lett. 205, 1986: 313-317) in the area of the catalytic center. So are the amino acids alanine and threonine in positions 122 and 125 of CAPN1 against serine and alanine in positions 88 and 91 changed in CAPN6. The amino acids have also been changed Histidine, alanine, serine and tryptophan in positions 272, 273, 275 and 298 in CAPN1 to tyrosine, threonine, threonine and Leucine in positions 252, 253, 255 and 286 in CAPN6. The Assignment of the amino acids at the different positions of the Proteins CAPN1 and CAPN6 are done by sequence comparison and Mapping the conserved regions of the proteins to one another,  so that a maximum match between the proteins at the amino acid level. For example, the same Sequences in different proteins of a protein family very different places or positions can be localized.

The human CAPN6 sequence also shows the exchange of the Cysteine residue exchanges the tyrosine at position 254 against histidine (Table 1). A possible explanation could be for example, CAPN6 can be compared to the other Cal painen has changed substrate specificity or the active center Not critical for CAPN6 function is that it is an inhibitor other Calpaine or the CAPN6 is a pseudogene. This last option seems conserved due to the multitude of them ten amino acids are unlikely. Probably Cysteine residue in position 89 in the catalytic reaction function take the missing cysteine residue in position 81. Even if CAPN6 had no protease activity, which is very unlikely it could be involved in regulatory processes may be binding with other calpains competes for places on cofactors or substrates.

Table 1

CAPN6 genomic sequences

Tra-3 is at sexing Caenorhabditis elegans involved. In a cascade of several genes and their genes tra-3 decides whether Caenorhabditis Develop males or hermaphrodites (Kuwabara P.E. et al., TIG, Vol. 8, No. 5, 1992: 164-168). Tra-3 seems to be at the lock to be involved in matogenesis. Conservation of amino acids in the different domains between mouse CAPN6 and tra3 39.2%; 42.0%; 30.9% and 22% for domains I, II, III and T.

Starting from cDNA derived from (Ia) 17 mouse embryo mRNA Using a modified RACE method (= rapid ampli fication of cDNA ends) according to Frohman et al. (Proc. Natl. Acad. Sci. USA 85, 1988, 8998-9002) and Edwards et al. (Nucl. Acids  Res. 19, 1991, 5227-5232) using the above Primers (Ca16 and Ca19) as well as sequences derived from EST AA050030 deduce, the entire sequence of the clone CAPN6 can be cloned. The SEQ ID No. 1 encodes a protein with 641 amino acids and a molecular weight of 74.6 kDa. The start codon is methionine a typical sequence for the translation start upstream. The The correctness of the sequence was verified by sequencing several cDNA Clones confirmed with the sequence mentioned.

Fig. 2 shows the homologies between the mouse CAPN5 (= nCL-3), human CAPN5 (= nCL-3), mouse CAPN6 (= nCL-4) and human CAPN6 (= nCL-4). FIG. 2 also shows the sequences of Caenorhabditis tra-3, human p94, mouse m-calpain, human μ-calpain and rats nCL-2. Amino acids that match between the different calpains and CAPN6 are indicated by dark boxes. Dashes indicate gaps that have been introduced in order to achieve maximum match of the sequences. Two sequences were removed from the human p94 sequence for clarity. These areas were marked with =. The fourth amino acids of the catalytic center were marked with arrows. The amino acid sequences corresponding to CAL6 and CAL9 were underlined. The domain names were given above the relevant sequence segments.

Fig. 3 shows the phylogenetic pedigree of the different calpains. The phylogenetic analyzes for the establishment of this family tree were carried out using the next post-bar method (Saitou et al. Mol. Biol. Evol. 4, 1987, 406-425), with the gaps being excluded. With the help of these phylogenetic analyzes, the vertebrate calpains could be divided into six different groups ( Fig. 3, right side). The non-vertebrate calpains can be assigned to the CAPN5 - (= nCL-3-) and CAPN6 - (= nCL-4-) group as the next neighbors and are in a separate group. 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), µ (P07384), p94 (P20807); Rats m (Q07009), nCL-2 (D14480), p94 (P16259); Mouse p94 (X92523); Chickens m (D38026), µ (D38027), µ / m (P00789), p94 (D38028); Nematodes tra-3 (U12921); Drosophila Calp A (Q11002) and Dm (X78555), Schistosoma (P27730). The human nCL-2 partial sequence corresponds to the translation of the EST clone AA026030 (Hellier et al., 1995, The Wasch U-Merck EST project).

The deduced amino acid sequence of EST clone AA026030 has a higher than usual homology after this analysis to rats nCL-2 sequence. Since here is only a partial sequence for the analysis has been used, the phylogenetic obtained for nCL-2 Family tree a higher inaccuracy. The nematode CPL1 sequence is the corrected version like that of Barnes and Hodjkin (EMBOLT., 1996, 15: 4477-4484) was used.

The first 7 exons were like those from the EMBL database (Accession No. L25598) used during the last 5 exons by connecting nucleotides 8028-8133, 8182-8239, 8729-8818, 8865-8983 and 9087 received to the end has been. The derived N-terminal sequence is due to its Length and its many glycine residues unusual for calpaine.

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.

Proteases play a central role in development and Differentiation of cells and thus also in the placenta. The Placenta is rich in proteases and protease inhibitors that are found in a balanced balance the development of the placenta enable. CAPN6 appears to play an important role here as a protease to play and / or may be at the regulation of others Calpain cysteine proteases involved.

There it may play a role in disease processes Placenta such as gestosis (= preeclampsia) plays a role in occurs in 5 to 7% of all pregnancies. pre-eclampsia (= pregnancy-induced high blood pressure) is one of the most common pregnancy-related complications, charak terized by hypertension and proteinuria, often combined with eressive edema and possibly with seizures. Preeclampsia during pregnancy is an important one Cause of mother and child death, premature birth or in mild cases of malnutrition or growth disorders of the Emoryos. It is a multifactorial event genetic factors (recessive or dominant genes), maternal immune tolerance, vasodilator /  Vasoconstrictor imbalances and probably also CAPN6 is involved.

Women who have preeclampsia show a change vasopressinase and angiotensinase levels, which may is regulated by CAPN6.

Another possible function of CAPN6 could be regulation 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 caused by CAPN6 be influenced, which also stimulates the growth of the fetus lieren.

CAPN6 may be involved in complex placenta events mucous membrane during embryogenesis and thus controls the through z. B. TNFα and IFNγ induced apoptosis of the throphoblasts, regulating other calpains. CAPN6 seems to be at the To be involved in embryonic development.

For the identification of selective calpain inhibitors are the most specific methods for identifying the Inhibitors required. It is important that the selected Inhibitors only the desired calpaine or s inhibit, but not other cysteine proteases and thus in intervene physiological processes.

The test to be tested for its inhibitory activity Substances can, for example, chemical substances, micro organic or herbal extracts. They are usually in addition to testing for their inhibitory activity against CAPN6, Calpain I and / or II on their activity against cathepsin B or other thiol proteases tested.

Ideally, good inhibitors should have little or no Activity against cathepsin B, L, elastase, papain, chymo trypsin or other cysteine proteases, but a good one Have activity against calpains I and II.

Through the new tissue-specific Calpain CAPN6 according to the invention can identify inhibitors with the method according to the invention be adorned for their inhibitory effect between the different Calpainen Calpain I, II, nCL-1 nCL-2 and / or nCL-4 can discriminate.  

The various inhibitor tests were carried out as follows guided:

Cathepsin B test

Cathepsin B inhibition was analogous to a method by S. Hasnain et al., LT. Biol. Chem. 1993, 268, 235-240.

To 88 µl cathepsin B (cathepsin B from human liver from Calbiochem, diluted to 5 units in 500 µM buffer), 2 µl of an inhibitor solution, prepared from the chemical substance to be tested, a microbial or vegetable extract and DSMO (final concentration: 100 µM to 0.01 µM) added. This mixture is incubated for 60 minutes at room temperature (= 25 ° C.) and then the reaction is started by adding 10 μl of 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 IC ₅₀ 's are then determined from the maximum gradients.

Calpain I and II test

The activity of the calpain inhibitors was determined in a colori metric test with casein according to Hammarsten (Merck, Darmstadt) examined as a substrate. The test was in microtiter plates, according to the publication by Buroker-Kilgore and Wang in anal. Biochem. 208, 1993, 387-392. As enzymes Calpain I (0.04 U / test) was made from erythrocytes and Calpain II (0.2 U / test) from kidneys, both from pigs, from Calbiochem, used. The substances to be tested were with the enzyme for Incubated for 60 minutes at room temperature, one concentration was not exceeded by 1% of the solvent DMSO. After The addition of the Bio-Rad color reagent was used to measure the optical Density at 595 nm in the Easy Reader EAR 400 from SLT. The 50% activity of the enzyme results from the optical Densities at the maximum activity of the enzyme without inhibi gates and the activity of the enzyme without the addition of calcium were determined.

The activity of calpain inhibitors can also be with the substrate Suc-Leu-Tyr-AMC can be determined. This fluorometric method is with Zhaozhao Li et al, LT. Med. Chem. 1993, 36, 3472-3480 described.

Because calpains are intracellular cysteine proteases, Calpain inhibitors cross the cell membrane to breakdown to prevent intracellular proteins by calpain. Some  known calpain inhibitors, such as E 64 and leupeptin, overcome the cell membranes poorly and show dementia speaking, although they are only good calpain inhibitors poor effect on cells. It is therefore advantageous additional test for the membrane passage of potential Calpain inhibitors such as the human platelet test.

Platelet test to determine the cellular activity of Calpain inhibitors

Calpain mediated protein degradation in platelets has been described by ZhaozhaoLi et al., LT. 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) are mixed for 5 minutes in 1 µl Concentrations of potential inhibitors (dissolved in DMSO) incubated. Calcium ionophore A 23187 was then added (1 µM in the test) and calcium (5 mM in the test) and another Incubate for 5 minutes at 37 ° C. After a centrifugation the plates were opened in SDS-Page sample buffer taken, cooked at 95 ° C for 5 minutes and the proteins in one 8% gel separated. The breakdown of the two actin proteins binding protein (= ABP) and valley in was determined by quantitative Densitometry tracked. After the addition of calcium and ionophore these proteins disappeared and new bands of less than 200 Kd molecular weight. This becomes the half maximum Enzyme activity determined with or as a control without inhibitor.

Are also suitable for testing membrane passage Tissue parts such as brain sections or cell cultures.

Inhibition of CAPN6 test is performed in cells that express this protein and combine it with a specific Antibodies can be detected. Are cells with z. B: calcium and stimulates the corresponding ionophore, this leads to a Activation of CAPN6. Takaomi Saido described in 1992 in LT. Organic chem. Vol. 11, 81-86 the autolytic transition of µ-calpain after activation and detection with antibodies. Appropriate Antibodies are generated for the detection of CAPN6. Calpain Inhibitors prevent autolytic transition and ent Talking quantification is possible with antibodies.  

In addition to the described in vitro tests such as the cellular one All other known to those skilled in the art are suitable for platelet tests Calpain tests such as the glutamate inhibition test induced Cell death on cortical neurons (Maulucci-Gedde M.A. et al., LT. Neurosci. 7, 1987: 357-368), calcium-mediated cell death in NT2 cells (Squier M.K.T. et al., LT. Cell. Physiol., 159, 1994: 229-237, Patel T. et al., Faseb Journal 590, 1996: 587-597) or analysis in tissue samples for degradation products of proteins such as Spectrin, MAP2 or Tau (Ami Arai et al., Brain Research, 1991, 555, 276-280, James Brorson et al., Stroke, 1995, 26, 1259-1267).

For the in vitro tests of CAPN6, the Calpain CAPN6 or his animal or his human homologue from tissues or Cells in which the enzyme is usually or artificial (e.g. by recombinant expression) is expressed as more advantageous as the placenta or from cells or microorganisms that at least one gene copy and / or a vector with at least a gene copy of the CAPN6 gene, its allelic variants or Contain analogs, purified and as raw extract or as pure enzyme used.

The various Calpain inhibitor tests advantageously in combination 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 others Calpaine or just flipped the other Calpaine and not that CAPN6 enzyme or CAPN6 enzyme and at least one other Calpain. Inhibitors against CAPN6 can advantageously in Case of gestose can be used.

The various inhibitor tests are carried out this in addition to the test on the inhibitory effect of the test substance compared to CAPN6, Calpain I and / or II as controls the tests is carried out without the test substance. Through this test arrangement The inhibitory effects of the test can easily be determined recognize substances.

Another method according to the invention uses CAPN6 or its allelic variants, analogs or synthetic derivatives advantageously for protection against the enzymatic action other calpaine.

Another method according to the invention uses the enzyme CAPN6 for screening for new calpain inhibitors, these In general, inhibitors advantageously all or one calpaine  inhibit individual calpains such as calpain I, II, nCL-1, nCl-2 or CAPN6 can. The different test substances can be used individually or tested in parallel in test systems. Advantageously are the test substances in parallel, automated test systems screened for their inhibitory effects.

All substances are generally suitable for the inhibitor tests. For example, the substances come from the classic chemical synthesis, from combinatorics, from microbial, animal or vegetable extracts. Under microbial Extracts are, for example, fermentation broths, cell opening conclusions of microorganisms or substances according to Biotrans to understand formation. Cell fractions are also for the tests suitable.

For cloning the CAPN6 gene or its animal homo logen or its human homologue, its allelic variants or analogues are all prokaryonic or eukaryotic expression systems used to isolate an enzymatic active gene product are suitable. Expressions are preferred systems that express the CAPN6 gene sequences in bacteria len, fungal or animal cells are particularly preferred in insect cells. Under enzymatically active gene pro CAPN6 proteins are to be understood directly after isolation from the expression organism such as from a pro karyotic or eukaryotic cell or after renaturation result in an active protein that is capable of at least one well-known calpain substrate such as those mentioned above or via car catalysis to split itself.

All are for the determination of the enzymatic activity Calpain tests known to those skilled in the art, such as in vitro tests such as those above described tests for Calpain I and II or cellular tests such as the platelet test is suitable. It can be used as a detection tests are used that are based on a colorimetric Assays (Buroker-Kilgore M. et al., Anal. Biochem. 208, 1993: 387-392) or based on a fluorescence assay.

In addition, all partial sequences that are the catalytic Center of the CAPN6 gene and / or further sequences of the CAPN6 gene and / or other calpain sequences and / or other sequences contain and show enzymatic activity, under enzymatic to understand the active gene product of CAPN6.

Among host organisms are all prokaryotic or eukaryon organisms that are suitable as host organisms for example bacteria such as Escherichia coli, Bacillus subtilis,  Streptomyces lividans, Streptococcus carnosus, yeasts like Saccharomyces cerevisiae, Schizosaccharomyces pombe, mushrooms like Aspergillus niger, insect cells such as Spodoptera frugiperda, Trichoplusia cells or any other insect cells that are responsible for viral expression is suitable, or animal cells such as Understand CV1, COS, C127, 3T3 or CHO or human cells.

The expression systems are the combination of the above expression organisms mentioned by way of example and those related to Vectors suitable for organisms such as plasmids, viruses or phages like the T7 RNA polymerase / promoter system or vectors with to understand regulatory sequences for the phage λ.

Combinations are preferred under the term expression systems tion from Escherichia coli and its plasmids and phages or the baculovirus system and the corresponding insect cells such as Understand Spodoptera frugiperda.

For the advantageous expression of the CAPN6 gene according to the invention are further 3 'and / or 5, regulatory terminals Sequences suitable.

These regulatory sequences are designed to target expression of the CAPN6 gene. This can vary depending on, for example Host organism mean that the gene only after induction ex is primed or overexpressed, or that it expresses immediately and / or is overexpressed.

The regulatory sequences or factors can be preferred have a positive effect on CAPN6 gene expression and thereby increase. This can reinforce the regulatory elements advantageously at the transcription level by strong transcription signals such as promoters and / or enhancers be used. But there is also a reinforcement of the Translation possible by, for example, the stability of the mRNA is improved.

“Enhancers” are understood to mean, for example, DNA sequences which have an improved interaction between RNA polymerase and DNA cause increased CAPN6 gene expression.

The CAPN6 gene with or without an upstream promoter or with or without a regulator gene, one or more DNA sequences can be and / or downstream, so that the gene is in a gene structure is included.  

The gene expression of the CAPN6 gene can also be by increasing the CAPN6 gene copy number. To increase the For example, the CAPN6 gene is copied in a CHO Expression vector amplified. Also suitable as vectors 5 vectors from the pED series - dicistronic vectors - that too contain the amplifiable marker gene dihydrofolate reductase. Details can be found in the Current Protocols in Molecular Biology Vol 2, 1994 are taken.

For example, an increase in CAPN6 enzyme activity can be seen compared to the parent enzyme by altering the CAPN6 gene or of its animal homologues through classic mutagenesis such as UV Irradiation 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 implemented more quickly. Also one can increased enzyme activity in addition to the described gene amplification by eliminating factors that affect enzyme synthesis repress and / or by synthesis more active than inactive CAPN6 proteins can be achieved. This way you can increase Enzyme quantities for the in vitro tests are made available.

CAPN6 or its animal homologues or its human homologue can advantageously be based on genomic DNA or cDNA using, for example, the PCR technique (see mole kular 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) cloning, CAPN6 can preferably be performed using genomi DNA and particularly preferably using genomi cloning 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. All vectors are for cloning suitable for expression in Escherichia coli are (see Molecular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, ISBN 0-87969-309-6). Vectors, for example, are particularly suitable, derived from pBR or pUC or shuttle vectors, whole pBluescript is particularly suitable.

After isolation and sequencing there are CAPN6 genes with nucleotides sequences available for those specified in SEQ ID NO: 2 Encode amino acid sequence or its allele variations. Under  Allele variants are to be understood as CAPN6 variants, the 60 to 100 % Homology at the amino acid level, preferably 70 to 100%, completely particularly preferably have 80 to 100%. Include allelic variants in particular functional variants, which are deleted, inserted tion or substitution of nucleotides from the in SEQ ID NO: 1 or SEQ ID NO: 3 sequence available, wherein 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.

Examples of CAPN6 analogs are its animal ones Homologs, truncated sequences, single-stranded DNA or RNA coding and non-coding DNA sequence particularly antisense To understand RNA.

Derivatives of CAPN6 are, for example, those derivatives that are enzy are not cleavable or difficult to cleave like nucleic acid phosphonates or phosphothioates, in which the phosphate group of Nucleic acids replaced by a phosphonate or thioate group has been.

Also the promoter that precedes the specified nucleotide sequence is switched on by one or more nucleotide exchanges, be changed by insertion (s) and / or deletion (s) without but that the functionality or effectiveness of the promoter is impaired. Furthermore, the promoter can also by Change in its sequence increases its effectiveness or completely through more effective promoters of other organisms or synthetic origin.

The Cal pain inhibitors are suitable for the manufacture of medicines Treatment of diseases in calpain malfunctions, for example wise in diseases selected from the group of cardiovascu laral, immunological, inflammatory, allergic, neurological human, neurodegenerative, or oncological diseases such as for example restenosis, arthritis, ischemia of the heart, the Kidney or central nervous system (e.g. stroke), inflammation exercises, muscular dystrophies, cataracts of the eyes (cataracts), Central nervous system injuries (e.g. trauma), Alzheimer's Disease, HIV-induced neuropathy, Parkinson's and Huntig's tonian disease preferred for the manufacture of medicines for Treatment of diseases of the placenta such as B. Gestose or the Embryogenesis.  

The CAPN6 gene sequences according to the invention are advantageous also used to diagnose diseases or genes therapy.

Examples example 1 Cloning of the CAPN6 gene

The mouse CAPN6 sequence (EMBL accession number Y12583) was created with the RACE method using day 17 mouse embryos and Primer sequences derived from the LAST AA050030 sequence were cloned. A plasmid clone that contains the corresponding EST Sequences was included by the I.M.A.G.E consortium (Research Genetics Inc.). The human CAPN6 homolog was over a homology search in the EST Davenbank using the Mouse protein sequence and the tblastn algorithm obtained. The ge partial sequences found could lead to an incomplete sequence of human CAPN6 with a length of 1083 nucleotides be added (SEQ ID NO: 3).

Example 2 Expression of the CAPN6 gene in different tissues

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 in accordance with 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 with a human ubiquitin DNA sample was used to determine the RNA loading.

3rd example Localization of the CAPN6 gene on the chromosome

The gene was localized in humans using the NIGMS Human / rodent somatic cell hybrid "mapping panel" (Coriell Cell Repositories). As primer sequences for the PCR reaction uses the following primers: 5'-gttgaaactgattggggtctg-3 'and 5, -ctgtcttcccaaggggtttctc-3 '. The PCR amplification was carried out with an annealing temperature of 58 ° C performed and resulted in a 200 bp fragment. The results were checked for agreement between the presence of humans chromosomes and the PCR product. The exact The gene was localized in the human chromosome with Help from the "Stanford G3 RH Panel" (Research Genetics) and About  Communication of the PCR results to the localization service of the Stanford Human Genome Center (http://www.-shgc.stanford.edu). The The human CAPN6 gene was linked to the X chromosome DXS7356 marker found.

4th example Cathepsin B test

Cathepsin B inhibition was analogous to a method by S. Hasnain et al., LT. Biol. Chem. 1993, 268, 235-40.

To 88 µL cathepsin B (cathepsin B from human liver (Calbiochem), diluted to 5 units in 500 µM buffer) are 2 µL of an inhibitor solution made from inhibitor and DMSO (final concentrations: 100 µM to 0.01 µM). This mixture is preincubated for 60 minutes at room temperature (25 ° C.) and the reaction is then started by adding 10 μL 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 IC _{50 is then} determined from the maximum gradients.

5th example Calpain test

The activity of the calpain inhibitors was measured in a colorimetri test with casein according to Hammarsten (Merck, Darmstadt) as Substrate examined. The test was in the microtiter plate, according to the publication by Buroker-Kilgore and Wang in Anal. Biochemistry 208, 387-392 (1993). As an enzyme was CAPN6, which in one of the systems described above was expressed and then purified. The sub were punched with the enzyme for 60 minutes at room temperature incubated with a concentration of 1% of the solvent DMSO was not exceeded. After adding the Bio-Rad color The optical density was measured at 595 nm in reagent the Easy Reader EAR 400 from SLT. The 50% activity of the Enzyme results from the optical densities that the maxi paint activity of the enzyme without inhibitors and the activity of Enzyme were determined without the addition of calcium.

6th example Platelet test to determine cellular Calpain inhibitor activity

Calpain-mediated protein degradation in platelets has been described by ZhaozhaoLi et al., LT. 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 (0.1 ml) are pre-incubated for 5 minutes with 1 µl of various concentrations of inhibitors (dissolved in DMSO). This was followed by the addition of calcium ionophore A 23187 (1 μM 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. The breakdown of the two proteins actin binding protein (ABP) and valley in was monitored by quantitative densitometry, since after the addition of calcium and ionophore these proteins disappeared and a new band in the range of 200 Kd molecular weight was formed. The half maximum enzyme activity is determined from this.

SEQUENCE PROTOCOL

Claims (19)

1. CAPN6-Calpaingen with the sequence SEQ ID NO. 1 or SEQ ID NO. 3, 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:

• (a) Leu-Gly-Asn-Lys-Ala,
  this sequence differs from the corresponding sequence in human calpain I in that the amino acid cysteine in human calpain I, which has position 115 in calpain I, against lysine, which is in position 81 of the sequences SEQ ID No. 1 and SEQ ID No. 3 lies, is changed;
• (b) Ala-X-Ser-Cys-Leu-Ala,
  where compared to the corresponding sequence in human calpain I, the amino acids alanine and threonine in positions 122 and 125 against serine and alanine in positions 88 and 91 in the sequences SEQ ID No. 1 and SEQ ID No. 3 are changed;
• (c) Gly-Tyr-Thr- (His or Tyr) -Thr-X-Thr,
  where, compared to the corresponding sequence in human calpain I, the amino acids histidine, alanine and serine in positions 272, 273 and 275 against tyrosine, threonine and threonine in positions 252, 253 and 255 in the sequences SEQ ID No. I and SEQ ID No. 3 are changed and in SEQ ID No. 3 additionally the tyrosine residue in position 274 in calpain I against histidine in position 254 in SEQ ID No. 3 is changed;
• (d) Arg-X-Arg-Asn-Pro-Leu-Gly
  this sequence differs from the corresponding sequence in human calpain I in that the amino acid tryptophan in human calpain I, which has position 298 in calpain I, against leucine, which is in position 286 of the sequences SEQ ID No. 1 and SEQ ID No. 3 lies, is changed and
  X denotes any natural amino acid in the sequences mentioned.

2. Gene construct containing a CAPN6 calpaingen, its alleli rule variants or analogs according to claim 1, the functio nell with one or more regulation signals to increase gene expression is functionally linked. 3. Amino acid sequences encoded by CAPN6 genes, allelic Variants or analogs according to claim 1. 4. amino acid sequences according to claim 3, characterized in that it is enzymatically active proteins. 5. A method of identifying calpain inhibitors, wherein a Calpain, its allelic variants or analogues encoded by a sequence according to claim 1 from tissues or cells isolated and inhibiting the cleavage of a Substrate of the enzyme CAPN6 and in at least one other Test the inhibition of the cleavage of a substrate of the enzymes Calpain I and / or II measures through test substances and the test selects substances that contain the enzyme CAPN6 and at least one inhibit another of the Calpaine. 6. The method according to claim 5, characterized in that one selects the test substances that the CAPN6 enzyme does not inhibit, however, the enzymes Calpain I and / or II. 7. The method according to claim 5, characterized in that one selects the test substances that inhibit the enzyme CAPN6, but not the enzymes Calpain I and / or II. 8. The method according to claims 5 to 7, characterized shows that one selects the test substances, which in cellular systems cross the cell membrane. 9. Process for the preparation of the enzyme CAPN6 and all of it lischen variants or analogs, characterized in that to have at least one copy of the gene sequences for CAPN6, his allelic variants or analogs according to claim 1 in one Vector cloned and in a host corresponding to the vector organism the gene for the enzyme CAPN6, its allelic Expressed variants or analogues and then the enzyme isolated from the host organism. 10. The method according to claim 9, characterized in that one a vector used in prokaryotic or eukaryon cells express the genes, the allelic expression Variants or analogs possible.   11. The method according to claim 9, characterized in that as host organism bacteria, fungal or animal Cells used. 12. The method according to claim 9, characterized in that one as vector baculoviruses and as host organism insect cells used. 13. Use of a calpain inhibitor identifiable according to claims 5 to 8 for the manufacture of medicaments Treatment of diseases in which a calpain malfunction is present. 14. Use of a calpain inhibitor according to claim 13 for the manufacture Provision of medicines for the treatment of diseases selected from the group of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerati ven or oncological diseases. 15. Use of an amino acid sequence according to claim 3 in test systems. 16. Use of an amino acid sequence according to claim 3 for Her position of antibodies. 17. Use of a gene sequence, its allelic variants or analogs according to claim 1 for the preparation of antisense mRNA. 18. Use of the antisense mRNA according to claim 17 Provision of medicines for the treatment of diseases who have a calpain malfunction. 19. Use of calpaing and its allelic Variants or analogs according to claim 1 for the diagnosis of Diseases or in gene therapy.