WO1999010480A2

WO1999010480A2 - New tissue-specific calpaines, their production and their use

Info

Publication number: WO1999010480A2
Application number: PCT/EP1998/005275
Authority: WO
Inventors: Thomas Boehm; Neil T. Dear
Original assignee: Basf Aktiengesellschaft
Priority date: 1997-08-26
Filing date: 1998-08-19
Publication date: 1999-03-04
Also published as: CA2301815A1; WO1999010480A3; NO20000961L; ZA987660B; NO20000961D0; AU9263598A; KR20010023282A; BR9811365A; DE19737105A1; IL134361A0; CN1277634A; EP1009811A2; HUP0004027A2; JP2001513992A

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

New tissue-specific calpains, their production and use

description

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.

So far, three main isoforms of these ubiquitous calpains are known, which differ in vitro by their calcium-dependent activation. Calpain I (= μCalpain) is activated by μ-molar calcium ion concentrations, while 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. The large subunit is made up of the following four protein domains (= I - IV): a protease domain (= domain II), a calcium-binding domain (= domain IV) and two further domains (= domain I and III), whose function is unclear . The small 30 K subunit consists of a calcium-binding subunit (= IV) and another subunit (= V), the function of which is unclear. In addition to these two types of calpain, a third type of calcium activation (= μ / m 80K) was found in the chicken (Wang KKW, 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 calpaines, two new tissue-specific expressed calpains have recently been identified. nCL-1 (= p94) is a muscle-specific calpain found in chickens, rats and humans, which is believed to be a mono- could be active and consists only of the 80 kd subunit. In addition to 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. 26, 1993: 19476-19482, Sorimachi, H: S: et al., FEBS Lett. 343, 1994: 1-5). Also in Drosophila, a calpain-homologous protein (= CalpA), which interacts with actin and is believed to play an important role in embryonic development, was found to have two different splicing variants (Mol. Cell. Biol. Vol. 15, No. 2, 1995: 824-834). Here, too, the shorter variant lacks the calcium binding site.

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). However, 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.

Therefore, it has been postulated that inhibitors of calpain enzymes can be useful in the treatment of these diseases. Various studies have confirmed this. For example, Seung-Chyul Hong et al. (Stroke 1994, 25 (3), 663-669) and Bartus RT et al. (Neurological Res. 1995, 17, 249-258) a neuroprotective Effect of calpain inhibitors in acute neurodegenerative disorders, such as those occurring after stroke, shown. Likewise, after experimental brain trauma, calpain inhibitors improved the recovery of the memory deficits 5 that occurred and neuromotor disorders (Saatman KE et al., Proc. Natl. Acad. Sei. USA, 93, 1996: 3428-3433). Edelstein CL et al. (Proc. Natl. Acad. Sci. USA, 92, 1995, 7662-7666) found a protective effect of calpain inhibitors on kidneys damaged by hypoxia. Yoshida KI et al. (Jap. Circ. J. 59 (1),

10 1995, 40 - 48) were able to show favorable effects of calpain inhibitors after cardiac damage, which were produced by ischemia or reperfusion. Since calpain inhibitors inhibit the release of β-AP4 protein, a potential use as a therapeutic agent for Alzheimer's disease has been proposed (Higaki

15 J. et al., Neuron, 14, 1995: 651-659). The release of interleukin-lα is also inhibited by calpain inhibitors (Watanabe N. et al., Cytokine, 6 (6), 1994: 597-601). Furthermore, it has been found that calpain inhibitors show cytotoxic effects on tumor cells (Shiba E. et al., 20th Meeting Int. Ass.

20 Breast Cancer Res. , Sendai Jp, 1994, Sept. 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

25 J., 285, 1992: 857-862).

Further possible uses of calpain inhibitors can be found in Wang K.K (Trends in Pharmacol. Sci., 15, 1994: 412-419).

30 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. However, 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. In addition to low-molecular peptide calpain inhibitors, 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. Many 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.

To find selective inhibitors, it is necessary and desirable to provide further calpains for testing, which are expressed as tissue-specifically as possible, so that the inhibitors can be tested for their selectivity between the individual calpains.

In addition, other new calpains are sought-after proteins, since they are likely to be expressed differently in the various clinical pictures or diseases and play an important role in these diseases.

It was an object of the present invention to provide means for profiling and identifying calpain inhibitors which make it possible to identify calpain inhibitors which, on the one hand, have an inhibitory effect only on one calpain, and / or on the other hand have an inhibitory effect on several calpaines, and these as therapeutic To provide Target.

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:

(a) Leu-Gly-Asn-Lys-Ala, this sequence differing 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, the at position 81 of the sequences SEQ ID No. 1 and SEQ ID No. 3 lies, is changed;

(b) Ala-X-Ser-Cys-Leu-Ala, 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 compared to the corresponding sequence in human calpain I. No. 1 and SEQ ID No. 3 are changed; (c) Gly-Tyr-Thr- (His or Tyr) -Thr-X-Thr, the amino acids histidine, alanine and serine in positions 272, 273 and 275 against tyrosine, threonine and. against the corresponding sequence in human calpain I. 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 differing from the corresponding sequence in human calpain I in that the amino acid tryptophan in human calpain I, which has the position 298 in calpain I, against Leucine, which is located at 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 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. If a subsequent assay shows that these substances are not or only poorly permeable to cells, their cell permeability can be improved by derivatization. Human μ- and m-calpain protein sequences were used for a homology search in the EST database of the National Center for Biotechnology 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 one

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 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).

With 30% homology, 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). In a sequence comparison according to Lipman-Pearson (Ktupl 2, Gap Penalty 4, Gap Length Penalty 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., J. Biol. Chem. 264, 1989: 20106-20111) and Drosophila Calpain (= DMCLPN0CM_1) over partial sequences from 230-520 Amino acids were found to have slightly larger homologies from 32.8 to 39.5%, but overall they are lower than the homologies to tra-3 (Figure 1, Alignment, Clustal method with PAM25 residue weight tabl.). In addition to tra-3, CAPN6 has a pronounced Homology to the recently described CAPN5 -calpain on (44.2%

Homology file number P 19 718 248.8).

Sequence comparisons between the mouse and the human CAPN6 sequence in the most varied of databases gave homologies to CalpA, Tra-3 and human sequences with the designations C16980, T39424, AA16971, R93331 and G17331, the functions of which have not been specified. The sequence comparisons were carried out with the Genbank EST and databases at the National Center for Biotechnology Information (http: Hwww.ncbi .nlm.nih.yor) and the Wash-U database (Homo sapiens). A mouse EST sequence called AA050030 and called Calpain was also found in the databases. No further information could be found in the databases. The complete 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 calpains (so-called "EF-hand") 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.

Another special feature of the derived CAPN6 amino acid sequence is 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 and tyrosine in the mouse CAPN6 sequence. In addition, other amino acids exchanged were compared when comparing the CAPN6 sequences SEQ ID No. 1 and SEQ ID No. 3 with the human calpain I (= CAPNl, Aoki et al., FEBS Lett. 205, 1986: 313-317) in the region of the catalytic center. Thus, 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.

In addition to the replacement of the cysteine residue, the human CAPN6 sequence also 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. Probably 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.

Table 1: CAPN6 genomic sequences

* Amino acids of the active center (Arthur et al., FEBS Lett. 368, 1995: 397-400) ¹ low activity without leucine in m-calpain (Arthur et al., FEBS Lett. 368, 1995: 397-400)

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.

Starting from cDNA derived from (Ia) 17 mouse embryo mRNA, a modified RACE method (= rapid amplification of cDNA ends) according to Frohman et al. (Proc. Natl. Acad. Be. USA 85, 1988, 8998-9002) and Edwards et al. (Nucl. Acids

Res. 19, 1991, 5227-5232) using the above

Primers (Cal6 and Cal9) and sequences derived from EST AA050030, the entire sequence of the clone CAPN6 are cloned. The

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. The

The correctness of the sequence was verified by sequencing several cDNA

Clones confirmed with the sequence mentioned.

FIG. 2 shows the homologies between mouse CAPN5 (= nCL-3), human CAPN5 (= nCL-3), mouse CAPN6 (= nCL-4) and human CAPN6 (= nCL-4). Figure 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 the sake of clarity. These areas were marked with =. The conserved 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.

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. With the help of these phylogenetic analyzes, the vertebrate calpains could be divided into six different groups (Figure 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 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.

There it may play a role in disease processes of the 35 placenta such as gestosis (= preeclampsia), which occurs in 5 to 7% of all pregnancies. Preeclampsia (= pregnancy-induced high blood pressure) is one of the most common pregnancy-related complications, characterized by hypertension and proteinuria, often combined with excessive edema and possibly with seizures. 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. It is a multifactorial event 45 in which, for example, 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 in the level of vasopressinase and angiotensinase, which may be regulated by 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. TNFα and IFNγ induced apoptosis of the trophoblasts by regulating other calpains. CAPN6 appears to be involved in embryonic development.

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

The 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.

Ideally, 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.

The novel tissue-specific calpain CAPN6 according to the invention 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 test

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

To 88 μl cathepsin B (human liver cathepsin B 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 DMSO (final concentration: 100 μM up to 0.01 μM) added. This batch is preincubated for 60 minutes at room temperature (= 25 ° C.) and the reaction is then 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 ₅ o's are then determined from the maximum slopes.

Calpain I and II test

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

Incubated for 60 minutes at room temperature, with 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 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.

Because calpains are intracellular cysteine proteases,

Calpain inhibitors pass through the cell membrane to prevent the breakdown of intracellular proteins by calpain. 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.

Platelet test to determine the cellular activity of calpain inhibitors

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 μl at various concentrations of potential 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 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.

Tissue parts such as brain sections or cell cultures are also suitable for testing membrane permeability.

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 μ-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. In addition to the in vitro tests described and the cellular platelet test, all other 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. 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 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.

For the method according to the invention, 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. For example, 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.

All 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.

In addition, all partial sequences which contain the catalytic center of the CAPN6 gene and / or further sequences of the CAPN6 gene and / or other calpain gene sequences and / or other sequences and which show enzymatic activity are to be understood as enzymatically active gene product of CAPN6.

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.

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 λ.

The term 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. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". In addition, 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. To increase the number of copies of the gene, 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. In addition to the described gene amplification, 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,

487ff), preferably 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) . For example, vectors which are derived from pBR or pUC or shuttle vectors are particularly suitable; pBluescript is very particularly suitable.

After isolation and sequencing, CAPN6 genes with nucleotide sequences are available which code for the amino acid sequence given in SEQ ID NO: 2 or its allele variants. Under 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.

Examples

Example 1: Cloning of the CAPN6 gene

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 consortάum (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).

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 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.

3. 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). 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.

4. Example: Cathepsin B test

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

To 88μL Cathepsin B (Cathepsin B from human liver

(Calbiochem), diluted to 5 units in 500 μM buffer), 2 μL of an inhibitor solution, prepared 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.

5. Example: Calpain test

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.

6. Example: Platelet test for determining the cellular activity of calpain inhibitors

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 μl

Pre-incubated concentrations of inhibitors (dissolved in DMSO).

Then calcium ionophore A 23187 (1 μM im

Test) and calcium (5 mM in the test) and a further incubation of 5 minutes at 37 ° C. After a centrifugation step, the

Platelets taken up in SDS-Page sample buffer, boiled for 5 minutes at 95 ° C. and the proteins separated in an 8% gel. The

Degradation of the two proteins actin binding protein (ABP) and talin was followed 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 LOG

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: BASF Aktiengesellschaft

(B) STREET: Carl Bosch Strasse

(C) LOCATION: Ludwigshafen

(D) FEDERAL STATE: Rhineland-Palatinate

(E) COUNTRY: Germany

(F) POSTCODE: D-67056

(ii) APPLICATION TITLE: New tissue-specific calpains, their manufacture and use

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPA)

(2) INFORMATION ABOUT SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2069 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Mus musculus

(vii) DIRECT ORIGIN:

(B) CLON: CAPN6

(ix) FEATURES:

(A) NAME / KEY: 5 'UTR

(B) LOCATION: 1..129

(ix) FEATURES:

(A) NAME / KEY: CDS

(B) LOCATION: 130..2055 (ix) FEATURES:

(A) NAME / KEY: 3 'UTR

(B) LOCATION: 2056..2069

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GGGGTTACCT GGCTAAGAGC AGCAGCAGCA GCAGCAGCAG CAGCAGCAGT AGCAGCAGCA 60

GCAGCAGCAG CAGCAGCAGC AGCAGCAGCA GCAGGGTTCC TGAGCTAACT CAGACCTAGT 120

TTGATAGCA ATG GGT CCT CCT CTG AAG CTC TTC AAA AAC CAG AAG TAC 168

Met Gly Pro Pro Leu Lys Leu Phe Lys Asn Gin Lys Tyr 1 5 10

CAA GAA CTG AAG CAG GAG TGC ATG AAG GAT GGC CGC CTT TTC TGT GAC 216 Gin Glu Leu Lys Gin Glu Cys Met Lys Asp Gly Arg Leu Phe Cys Asp 15 20 25

CCA ACC TTC CTA CCG GAG AAT GAT TCT CTG TTT TTC AAC CGG CTG CTT 264 Pro Thr Phe Leu Pro Glu Asn Asp Ser Leu Phe Phe Asn Arg Leu Leu 30 35 40 45

CCT GGG AAG GTG GTG TGG AAG CGT CCA CAG GAC ATT TCT GAT GAC CCC 312 Pro Gly Lys Val Val Trp Lys Arg Pro Gin Asp Ile Ser Asp Asp Pro

50 55 60

CAC CTG ATT GTG GGC AAC ATC AGC AAC CAC CAG CTG ATC CAG GGC AGA 360 His Leu Ile Val Gly Asn Ile Ser Asn His Gin Leu Ile Gin Gly Arg 65 70 75

TTG GGG AAC AAG GCA ATG ATC TCT GCA TTT TCC TGT TTG GCT GTT CAG 408 Leu Gly Asn Lys Ala Met Ile Ser Ala Phe Ser Cys Leu Ala Val Gin 80 85 90

GAG TCA CAC TGG ACA AAG GCA ATT CCC AAC CAC AAG GAT CAG GAA TGG 456 Glu Ser His Trp Thr Lys Ala Ile Pro Asn His Lys Asp Gin Glu Trp 95 100 105

GAT CCT CGA AAG CCA GAG AAA TAC GCT GGA ATC TTT CAC TTC CGC TTC 504 Asp Pro Arg Lys Pro Glu Lys Tyr Ala Gly Ile Phe His Phe Arg Phe 110 115 120 125

TGG CAT TTT GGA GAA TGG ACC GAG GTG GTG ATT GAT GAC TTG CTT CCC 552 Trp His Phe Gly Glu Trp Thr Glu Val Val Ile Asp Asp Leu Leu Pro 130 135 140

ACC ATC AAC GGA GAT CTG GTC TTC TCA TTC TCC ACC TCC ATG AAT GAG 600 Thr Ile Asn Gly Asp Leu Val Phe Ser Phe Ser Thr Ser Met Asn Glu 145 150 155

TTT TGG AAT GCT CTA CTG GAA AAA GCG TAT GCA AAG CTG CTG GGC TGT 648 Phe Trp Asn Ala Leu Leu Glu Lys Ala Tyr Ala Lys Leu Leu Gly Cys 160 165 170 TAT GAG GCT TTG GAT GGT CTG ACC ATC ACT GAT ATC ATC ATG GAC TTC 696

Tyr Glu Ala Leu Asp Gly Leu Thr Ile Thr Asp Ile Ile Met Asp Phe

175 180 185

ACT GGC ACA CTG GCT GAA ATC ATT GAC ATG CAG AAA GGA CGA TAC ACT 744 Thr Gly Thr Leu Ala Glu Ile Ile Asp Met Gin Lys Gly Arg Tyr Thr 190 195 200 205

GAT CTT GTT GAG GAG AAG TAC AAG CTG TTT GGA GAA CTG TAC AAA ACG 792 Asp Leu Val Glu Glu Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr 210 215 220

TTC ACC AAA GGA GGT CTA ATT TGC TGC TCC ATT GAG TCT CCC AGC CAG 840 Phe Thr Lys Gly Gly Leu Ile Cys Cys Ser Ile Glu Ser Pro Ser Gin 225 230 ^' 235

GAG GAA CAA GAA GTT GAA ACA GAC TGG GGA CTA CTG AAG GGT TAT ACC 888 Glu Glu Gin Glu Val Glu Thr Asp Trp Gly Leu Leu Lys Gly Tyr Thr 240 245 250

TAC ACC ATG ACT GAT ATT CGC AAG CTC CGT CTC GGA GAA AGA CTT GTG 936 Tyr Thr Met Thr Asp Ile Arg Lys Leu Arg Leu Gly Glu Arg Leu Val 255 260 265

GAA GTC TTC AGT ACT GAG AAG CTG TAT ATG GTT CGC CTA AGG AAC CCA 984 Glu Val Phe Ser Thr Glu Lys Leu Tyr Met Val Arg Leu Arg Asn Pro 270 275 280 285

TTG GGA AGA CAG GAA TGG AGT GGC CCC TGG AGT GAA ATT TCA GAG GAG 1032 Leu Gly Arg Gin Glu Trp Ser Gly Pro Trp Ser Glu Ile Ser Glu Glu 290 295 300

TGG CAG CAA CTG ACT GTA ACA GAT CGC AAG AAC CTA GGA CTT GTT ATG 1080 Trp Gin Gin Leu Thr Val Thr Asp Arg Lys Asn Leu Gly Leu Val Met 305 310 315

TCT GAT GAT GGA GAA TTT TGG ATG AGT CTG GAA GAT TTT TGC CAC AAC 1128 Ser Asp Asp Gly Glu Phe Trp Met Ser Leu Glu Asp Phe Cys His Asn 320 325 330

TTT CAC AAA CTG AAT GTC TGC CGC AAT GTG AAT AAT CCT GTT TTT GGC 1176 Phe His Lys Leu Asn Val Cys Arg Asn Val Asn Asn Pro Val Phe Gly 335 340 345

CGC AAG GAG CTG GAA TCA GTG GTG GGA TGT TGG ACT GTG GAT GAT GAC 1224 Arg Lys Glu Leu Glu Ser Val Val Gly Cys Trp Thr Val Asp Asp Asp 350 355 360 365

CCT CTG ATG AAC CGA TCA GGA GGT TGC TAT AAC AAC CGT GAT ACC TTC 1272 Pro Leu Met Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe 370 375 380 TTG CAG AAT CCT CAG TAC ATT TTC ACT GTG CCC GAG GAT GGC CAT AAA 1320

Leu Gin Asn Pro Gin Tyr Ile Phe Thr Val Pro Glu Asp Gly His Lys

385 390 395

GTC ATC ATG TCA CTG CAA CAG AAG GAC CTA CGC ACT TAC CGC CGA ATG 1368 Val Ile Met Ser Leu Gin Gin Lys Asp Leu Arg Thr Tyr Arg Arg Met 400 405 410

GGA AGA CCT GAT AAT TAC ATC ATT GGT TTT GAG CTC TTC AAG GTG GAG 1416 Gly Arg Pro Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Val Glu 415 420 425

ATG AAC CGA AGG TTC CGT CTT CAC CAT CTG TAT ATT CAG GAG CGT GCT 1464 Met Asn Arg Arg Phe Arg Leu His His Leu Tyr Ile Gin Glu Arg Ala 430 435 440 ^' 445

GGG ACT TCC ACT TAT ATC GAC ACC CGT ACT GTG TTT CTG AGC AAG TAT 1512 Gly Thr Ser Thr Tyr Ile Asp Thr Arg Thr Val Phe Leu Ser Lys Tyr 450 455 460

CTG AAG AAG GGC AGC TAC GTG CTT GTT CCA ACC ATG TTC CAA CAT GGC 1560 Leu Lys Lys Gly Ser Tyr Val Leu Val Pro Thr Met Phe Gin His Gly 465 470 475

CGT ACC AGT GAA TTT CTG CTG AGG ATC TTC TCT GAA GTG CCC GTC CAG 1608 Arg Thr Ser Glu Phe Leu Leu Arg Ile Phe Ser Glu Val Pro Val Gin 480 485 490

CTC AGG GAA CTG ACC TTG GAC ATG CCC AAG ATG TCT TGC TGG AAC CTG 1656 Leu Arg Glu Leu Thr Leu Asp Met Pro Lys Met Ser Cys Trp Asn Leu 495 500 505

GCA CGT GGC TAC CCA AAG GTG GTT ACC CAG ATC ACT GTC CAC AGT GCT 1704 Ala Arg Gly Tyr Pro Lys Val Val Thr Gin Ile Thr Val His Ser Ala 510 515 520 525

GAG GGC CTG GAG AAG AAG TAT GCC AAT GAA ACT GTC AAT CCA TAT CTG 1752 Glu Gly Leu Glu Lys Lys Tyr Ala Asn Glu Thr Val Asn Pro Tyr Leu 530 535 540

ATC ATC AAA TGT GGA AAG GAG GAA GTC CGT TCC CCT GTC CAG AAG AAT 1800 Ile Ile Lys Cys Gly Lys Glu Glu Val Arg Ser Pro Val Gin Lys Asn 545 550 555

ACT GTG CAT GCC ATT TTT GAC ACG CAG GCC GTT TTC TAC AGA AGG ACC 1848 Thr Val His Ala Ile Phe Asp Thr Gin Ala Val Phe Tyr Arg Arg Thr 560 565 570

ACT GAC ATT CCT ATT ATC ATC CAG GTG TGG AAC AGC AGA AAA TTC TGT 1896 Thr Asp Ile Pro Ile Ile Ile Gin Val Trp Asn Ser Arg Lys Phe Cys 575 580 585 GAT CAG TTC CTG GGG CAG GTT ACT CTC GAT GCT GAC CCC AGC GAC TGC 1944

Asp Gin Phe Leu Gly Gin Val Thr Leu Asp Ala Asp Pro Ser Asp Cys

590 595 600 605

CGT GAT CTG AAA TCT CTG TAC CTG CGT AAG AAG GGT GGT CCT ACT GCC 1992 Arg Asp Leu Lys Ser Leu Tyr Leu Arg Lys Lys Gly Gly Pro Thr Ala 610 615 620

AAA GTC AAG CAA GGT CAC ATC AGC TTC AAA GTT ATC TCT AGC GAT GAT 2040 Lys Val Lys Gin Gly His Ile Ser Phe Lys Val Ile Ser Ser Asp Asp 625 630 635

CTC ACT GAG CTC TAAGTAGTCA TCATCAG 2069

Leu Thr Glu Leu 640

(2) INFORMATION ABOUT SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 641 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Gly Pro Pro Leu Lys Leu Phe Lys Asn Gin Lys Tyr Gin Glu Leu 1 5 10 15

Lys Gin Glu Cys Met Lys Asp Gly Arg Leu Phe Cys Asp Pro Thr Phe 20 25 30

Leu Pro Glu Asn Asp Ser Leu Phe Phe Asn Arg Leu Leu Pro Gly Lys 35 40 45

Val Val Trp Lys Arg Pro Gin Asp Ile Ser Asp Asp Pro His Leu Ile 50 55 60

Val Gly Asn Ile Ser Asn His Gin Leu Ile Gin Gly Arg Leu Gly Asn 65 70 75 80

Lys Ala Met Ile Ser Ala Phe Ser Cys Leu Ala Val Gin Glu Ser His

85 90 95

Trp Thr Lys Ala Ile Pro Asn His Lys Asp Gin Glu Trp Asp Pro Arg 100 105 110

Lys Pro Glu Lys Tyr Ala Gly Ile Phe His Phe Arg Phe Trp His Phe 115 120 125

Gly Glu Trp Thr Glu Val Val Ile Asp Asp Leu Leu Pro Thr Ile Asn 130 135 140 Gly Asp Leu Val Phe Ser Phe Ser Thr Ser Met Asn Glu Phe Trp Asn

145 150 155 160

Ala Leu Leu Glu Lys Ala Tyr Ala Lys Leu Leu Gly Cys Tyr Glu Ala 165 170 175

Leu Asp Gly Leu Thr Ile Thr Asp Ile Ile Met Asp Phe Thr Gly Thr 180 185 190

Leu Ala Glu Ile Ile Asp Met Gin Lys Gly Arg Tyr Thr Asp Leu Val 195 200 205

Glu Glu Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr Phe Thr Lys 210 215 220

Gly Gly Leu Ile Cys Cys Ser Ile Glu Ser Pro Ser Gin Glu Glu Gin 225 230 235 240

Glu Val Glu Thr Asp Trp Gly Leu Leu Lys Gly Tyr Thr Tyr Thr Met 245 250 255

Thr Asp Ile Arg Lys Leu Arg Leu Gly Glu Arg Leu Val Glu Val Phe 260 265 270

Ser Thr Glu Lys Leu Tyr Met Val Arg Leu Arg Asn Pro Leu Gly Arg 275 280 285

Gin Glu Trp Ser Gly Pro Trp Ser Glu Ile Ser Glu Glu Trp Gin Gin 290 295 300

Leu Thr Val Thr Asp Arg Lys Asn Leu Gly Leu Val Met Ser Asp Asp 305 310 315 320

Gly Glu Phe Trp Met Ser Leu Glu Asp Phe Cys His Asn Phe His Lys 325 330 335

Leu Asn Val Cys Arg Asn Val Asn Asn Pro Val Phe Gly Arg Lys Glu 340 345 350

Leu Glu Ser Val Val Gly Cys Trp Thr Val Asp Asp Asp Pro Leu Met 355 360 365

Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe Leu Gin Asn 370 375 380

Pro Gin Tyr Ile Phe Thr Val Pro Glu Asp Gly His Lys Val Ile Met 385 390 395 400

Ser Leu Gin Gin Lys Asp Leu Arg Thr Tyr Arg Arg Met Gly Arg Pro 405 410 415

Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Val Glu Met Asn Arg 420 425 430 Arg Phe Arg Leu His His Leu Tyr Ile Gin Glu Arg Ala Gly Thr Ser

435 440 445

Thr Tyr Ile Asp Thr Arg Thr Val Phe Leu Ser Lys Tyr Leu Lys Lys 450 455 460

Gly Ser Tyr Val Leu Val Pro Thr Met Phe Gin His Gly Arg Thr Ser 465 470 475 480

Glu Phe Leu Leu Arg Ile Phe Ser Glu Val Pro Val Gin Leu Arg Glu 485 490 495

Leu Thr Leu Asp Met Pro Lys Met Ser Cys Trp Asn Leu Ala Arg Gly 500 505 510

Tyr Pro Lys Val Val Thr Gin Ile Thr Val His Ser Ala Glu Gly Leu 515 520 525

Glu Lys Lys Tyr Ala Asn Glu Thr Val Asn Pro Tyr Leu Ile Ile Lys 530 535 540

Cys Gly Lys Glu Glu Val Arg Ser Pro Val Gin Lys Asn Thr Val His 545 550 555 560

Ala Ile Phe Asp Thr Gin Ala Val Phe Tyr Arg Arg Thr Thr Asp Ile 565 570 575

Pro Ile Ile Ile Gin Val Trp Asn Ser Arg Lys Phe Cys Asp Gin Phe 580 585 590

Leu Gly Gin Val Thr Leu Asp Ala Asp Pro Ser Asp Cys Arg Asp Leu 595 600 605

Lys Ser Leu Tyr Leu Arg Lys Lys Gly Gly Pro Thr Ala Lys Val Lys 610 615 620

Gin Gly His Ile Ser Phe Lys Val Ile Ser Ser Asp Asp Leu Thr Glu 625 630 635 640

Leu

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1125 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Homo sapiens

(vii) DIRECT ORIGIN:

(B) CLON: CAPN6

(ix) FEATURES:

(A) NAME / KEY: CDS

(B) LOCATION: 2..1125

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

G CTT GTT GAG GAG AAG TAC AAG CTA TTC GGA GAA CTG -TAC AAA ACA 46

Leu Val Glu Glu Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr 1 5 10 15

TTT ACC AAA GGT GGT CTG ATC TGC TGT TCC ATT GAG TCT CCC AAT CAG 94 Phe Thr Lys Gly Gly Leu Ile Cys Cys Ser Ile Glu Ser Pro Asn Gin

20 25 30

GAG GAG CAA GAA GTT GAA ACT GAT TGG GGT CTG CTG AAG GGC CAT ACC 142 Glu Glu Gin Glu Val Glu Thr Asp Trp Gly Leu Leu Lys Gly His Thr 35 40 45

TAT ACC ATG ACT GAT ATT CGC AAA ATT CGT CTT GGA GAG AGA CTT GTG 190 Tyr Thr Met Thr Asp Ile Arg Lys Ile Arg Leu Gly Glu Arg Leu Val 50 55 60

GAA GTC TTC AGT GCT GAG AAG CTG TAT ATG GTT CGC CTG AGA AAC CCC 238 Glu Val Phe Ser Ala Glu Lys Leu Tyr Met Val Arg Leu Arg Asn Pro 65 70 75

TTG GGA AGA CAG GAA TGG AGT GGC CCC TGG AGT GAA ATT TCT GAA GAG 286 Leu Gly Arg Gin Glu Trp Ser Gly Pro Trp Ser Glu Ile Ser Glu Glu 80 85 90 95

TGG CAG CAA CTG ACT GCA TCA GAT CGC AAG AAC CTG GGG CTT GTT ATG 334 Trp Gin Gin Leu Thr Ala Ser Asp Arg Lys Asn Leu Gly Leu Val Met 100 105 110

TCT GAT GAT GGA GAG TTT TGG ATG AGC TTG GAG GAC TTT TGC CGC AAC 382 Ser Asp Asp Gly Glu Phe Trp Met Ser Leu Glu Asp Phe Cys Arg Asn 115 120 125

TTT CAC AAA CTG AAT GTC TGC CGC AAT GTG AAC AAC CCT ATT TTT GGC 430 Phe His Lys Leu Asn Val Cys Arg Asn Val Asn Asn Pro Ile Phe Gly 130 135 140

CGA AAG GAG CTG GAA TCG GTG TTG GGA TGC TGG ACT GTG GAT GAT GAT 478 Arg Lys Glu Leu Glu Ser Val Leu Gly Cys Trp Thr Val Asp Asp Asp 145 150 155 CCC CTG ATG AAC CGC TCA GGA GGC TGC TAT AAC AAC CGT GAT ACC TTC 526 Pro Leu Met Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe 160 165 170 175

CTG CAG AAT CCC CAG TAC ATC TTC ACT GTG CCT GAG GAT GGG CAC AAG 574 Leu Gin Asn Pro Gin Tyr Ile Phe Thr Val Pro Glu Asp Gly His Lys 180 185 190

GTC ATT ATG TCA CTG CAG CAG AAG GAC CTG CGC ACT TAC CGC CGA ATG 622 Val Ile Met Ser Leu Gin Gin Lys Asp Leu Arg Thr Tyr Arg Arg Met 195 200 205

GGA AGA CCT GAC AAT TAC ATC ATT GGC TTT GAG CTC TTC AAG GTG GAG 670 Gly Arg Pro Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Val Glu 210 215 220

ATG AAC CGC AAA TTC CGC CTC CAC CAC CTC TAC ATC CAG GAG CGT GCT 718 Met Asn Arg Lys Phe Arg Leu His His Leu Tyr Ile Gin Glu Arg Ala 225 230 235

GGG ACT TCC ACC TAT ATT GAC ACC CGC ACA GTG TTT CTG AGC AAG TAC 766 Gly Thr Ser Thr Tyr Ile Asp Thr Arg Thr Val Phe Leu Ser Lys Tyr 240 245 250 255

CTG AAG AAG GGC AAC TAT GTG CTT GTC CCA ACC ATG TTC CAG CAT GGT 814 Leu Lys Lys Gly Asn Tyr Val Leu Val Pro Thr Met Phe Gin His Gly 260 265 270

CGC ACC AGC GAG TTT CTC CTG AGA ATC TTC TCT GAA GTG CCT GTC CAG 862 Arg Thr Ser Glu Phe Leu Leu Arg Ile Phe Ser Glu Val Pro Val Gin 275 280 285

CTC AGG GAA CTG ACT CTG GAC ATG CCC AAA ATG TCC TGC TGG AAC CTG 910 Leu Arg Glu Leu Thr Leu Asp Met Pro Lys Met Ser Cys Trp Asn Leu 290 295 300

GCT CGT GGC TAC CCG AAA GTA GTT ACT CAG ATC ACT GTT CAC AGT GCT 958 Ala Arg Gly Tyr Pro Lys Val Val Thr Gin Ile Thr Val His Ser Ala 305 310 315

GAG GAC CTG GAG AGG AGG TAT GCC AAT GGA ACT GTA AAC CCA TAT TTG 1006 Glu Asp Leu Glu Arg Arg Tyr Ala Asn Gly Thr Val Asn Pro Tyr Leu 320 325 330 335

GTC ATC AAA TGT GGA AAG GAG GAA GTC CGT TCT CCT GTC CAG AAA AAT 1054 Val Ile Lys Cys Gly Lys Glu Glu Val Arg Ser Pro Val Gin Lys Asn 340 345 350

ACA GTT CAT GCC ATT TTT GAC ACC CAT GCC ATT TTC TAC AGA AGG ACC 1102 Thr Val His Ala Ile Phe Asp Thr His Ala Ile Phe Tyr Arg Arg Thr 355 360 365 ACG GAC ATT CCT ATT ATA GTA CA 1125

Thr Asp Ile Pro Ile Ile Val 370

(2) INFORMATION ABOUT SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 374 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Leu Val Glu Glu Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr Phe 1 5 10 15

Thr Lys Gly Gly Leu Ile Cys Cys Ser Ile Glu Ser Pro Asn Gin Glu 20 25 30

Glu Gin Glu Val Glu Thr Asp Trp Gly Leu Leu Lys Gly His Thr Tyr 35 40 45

Thr Met Thr Asp Ile Arg Lys Ile Arg Leu Gly Glu Arg Leu Val Glu 50 55 60

Val Phe Ser Ala Glu Lys Leu Tyr Met Val Arg Leu Arg Asn Pro Leu 65 70 75 80

Gly Arg Gin Glu Trp Ser Gly Pro Trp Ser Glu Ile Ser Glu Glu Trp

85 90 95

Gin Gin Leu Thr Ala Ser Asp Arg Lys Asn Leu Gly Leu Val Met Ser 100 105 110

Asp Asp Gly Glu Phe Trp Met Ser Leu Glu Asp Phe Cys Arg Asn Phe 115 120 125

His Lys Leu Asn Val Cys Arg Asn Val Asn Asn Pro Ile Phe Gly Arg 130 135 140

Lys Glu Leu Glu Ser Val Leu Gly Cys Trp Thr Val Asp Asp Asp Pro 145 150 155 160

Leu Met Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe Leu 165 170 175

Gin Asn Pro Gin Tyr Ile Phe Thr Val Pro Glu Asp Gly His Lys Val 180 185 190

Ile Met Ser Leu Gin Gin Lys Asp Leu Arg Thr Tyr Arg Arg Met Gly 195 200 205 Arg Pro Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Val Glu Met

210 215 220

Asn Arg Lys Phe Arg Leu His His Leu Tyr Ile Gin Glu Arg Ala Gly 225 230 235 240

Thr Ser Thr Tyr Ile Asp Thr Arg Thr Val Phe Leu Ser Lys Tyr Leu 245 250 255

Lys Lys Gly Asn Tyr Val Leu Val Pro Thr Met Phe Gin His Gly Arg 260 265 270

Thr Ser Glu Phe Leu Leu Arg Ile Phe Ser Glu Val Pro Val Gin Leu 275 280 285

Arg Glu Leu Thr Leu Asp Met Pro Lys Met Ser Cys Trp Asn Leu Ala 290 295 300

Arg Gly Tyr Pro Lys Val Val Thr Gin Ile Thr Val His Ser Ala Glu 305 310 315 320

Asp Leu Glu Arg Arg Tyr Ala Asn Gly Thr Val Asn Pro Tyr Leu Val 325 330 335

Ile Lys Cys Gly Lys Glu Glu Val Arg Ser Pro Val Gin Lys Asn Thr 340 345 350

Val His Ala Ile Phe Asp Thr His Ala Ile Phe Tyr Arg Arg Thr Thr 355 360 365

Asp Ile Pro Ile Ile Val 370

Claims

claims

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 calpain genes, their allelic variants, analogs or derivatives containing the following sequences:

(b) Ala-X-Ser-Cys-Leu-Ala, 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, the amino acids histidine, alanine and serine in positions 272, 273 and 275 against tyrosine, threonine and. compared to the corresponding sequence in human calpain I. 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;

X denotes any natural amino acid in the sequences mentioned.

Sign.

2. gene construct containing a CAPN6 calpaingen, its allelic variants or analogs according to claim 1, which is functionally linked to one or more regulatory signals to increase gene expression.

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 for identifying calpain inhibitors, wherein a calpain, its allelic variants or analogs are encoded by a sequence according to claim 1 from tissues or cells and the inhibition of cleavage of a substrate of the enzyme CAPN6 and in at least one further test the inhibition of the cleavage measures 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.

6. The method according to claim 5, characterized in that one selects the test substances that do not inhibit the enzyme CAPN6, but the enzymes Calpain I and / or II.

7. The method according to claim 5, characterized in that one selects the test substances which inhibit the enzyme CAPN6, but not the enzymes Calpain I and / or II.

8. The method according to claims 5 to 7, characterized in that one selects the test substances that pass through the cell membrane in cellular systems.

9. A process for the preparation of the enzyme CAPN6 and its allergic variants or analogs, characterized in that at least one copy of the gene sequences for CAPN6, its allelic variants or analogs according to claim 1 is cloned into a vector and in a host organism corresponding to the vector Gene expressed for the enzyme CAPN6, its allelic variants or analogs and then isolated the enzyme from the host organism.

10. The method according to claim 9, characterized in that a vector is used which enables the expression of the genes, the allelic variants or analogs in prokaryotic or eukaryotic cells.

11. The method according to claim 9, characterized in that the host organism is bacteria, fungal or animal

Cells used.

5 12. The method according to claim 9, characterized in that insect cells are used as the vector baculoviruses and as the host organism.

13. Use of a calpain inhibitor identifiable according to claims 5 to 8 for the manufacture of medicaments for

Treatment of diseases with a malfunction of calpain.

14. Use of a calpain inhibitor according to claim 13 for the manufacture of medicaments for the treatment of diseases selected from the group of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerative or oncological diseases.

20. 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 the production of antibodies.

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17. Use of a gene sequence, its allelic variants or analogs according to claim 1 for the production of antisense mRNA.

18. Use of the antisense mRNA according to claim 17 for the manufacture of medicaments for the treatment of diseases in which a calpain malfunction is present.

19. Use of a calpaing gene and its allelic variants or analogs according to claim 1 for the diagnosis of diseases or in gene therapy.

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