CZ2000680A3 - Novel tissue-specific liquids, their preparation and use - Google Patents

Abstract

New tissue-specific calpains are prepared as described way. Also disclosed is a method for sorting new inhibitors calpaines and their uses.

Description

New tissue-specific calpains, their preparation and use

Technical field

The present invention relates to novel tissue-specific calpaines and their preparation.

The present invention further relates to methods for screening novel calpain inhibitors and their use.

BACKGROUND OF THE INVENTION

Calpains are intracellular non-lysosomal enzymes belonging to the family of cysteine proteases. They participate in the transmission of Ca ²⁺ dependent signals in eukaryotic cells, i.e., they control cellular functions in a manner that depends on the Ca ²⁺ concentration. Calpains are found ubiquitously in animal tissues and cells, for example in humans, chickens, rabbits or rats. Calpains have also been found in lower animals such as Drosophila melanogaster, Schistosoma or Caenorhabditis elegans. No calpains have been detected in yeast, fungi or bacteria.

Currently, three major isoforms of these ubiquitous calpaines are known and differ in their calcium-dependent in vitro activatability. Calpain I (= μ calpain) is activated by micromolar concentrations of calcium ions, whereas calpain II (= m calpain) is activated only by millimolar concentrations of calcium ions. Both calpains

- 2 • 00 · 0 0 0 0 · 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

00000 00 0 00 00 approximately the size of approximately the heterodimer bear subunit consists of two subunits, one large subunit of 80 kDa and one small subunit of 30 kDa. Both subunits of active calcium binding sites. Large of the following four protein regions (= I-IV): protease region (= region II), calcium-binding region (= region IV) and two additional regions (= regions)

I and III) whose function is unclear. The small 30 K subunit consists of a calcium-binding subunit (= IV) and another subunit (= V) whose function is unclear. In addition to these two types of calpain, a third type (= μ / m 80K) has been found in chickens, which is a transition between previous species in relation to calcium binding (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 everywhere found calpaines, two new tissue-specific expressed calpaines have recently been identified. nCL-1 (= p94) is a muscle-specific calpain found in chickens, rats and humans and which is believed to be active as a monomer and consists of only the 80 kd subunit. In addition to nCL-1, there is also gastric tissue-specific calpain, which can be found in two splice variants, nCL-2 and nCL-2 '. nCL-2 'differs from nCL-2 in the absence of a calcium binding region (Sorimachi, HS et al., J. Biol. Chem. Vol. 268, No. 26, 1993: 19476 - 19482, Sorimachi, HS et al., FEBS Lett., 343, 1994: 1-5). Calpain's homologous protein (= CalpA), which interacts with actin and is believed to play an important role in embryonic development and which has two different splice variants, has been found in Drosophila (Mol. Cell. Biol. Vol.

15, no. 2, 1995: 824-834). In this case, shorter variants also lack a calcium binding site.

Calpains are believed to play an important role in various physiological processes. A large number of cytoskeletal, membrane-bound or regulatory proteins such as protein kinase C, phospholipase C, spectrum, cytoskeletal proteins such as MAP2, muscle proteins, nerve fibers and neuropeptides, platelet proteins, epidermal growth factor, NMDA receptor and mitotic and other proteins are calpain substrates (Barrett MJ et al., Life Sci. 48, 1991: 1659-69; Wang KK et al., Trends in Pharmacol. Sci., 15, 1994: 412-419). However, the normal physiological function of calpaines is not yet well understood. They participate in a large number of physiological processes such as apoptosis, cell division and differentiation, or embryonic development.

Elevated calpain levels have been measured in various pathophysiological processes and disorders such as: ischemia of the heart (such as myocardial infarction), kidney or central nervous system (such as stroke), inflammation, muscular dystrophy, ocular cataracts (cataracts), central nervous system injuries ( trauma), Alzheimer's disease, HIV-induced neuropathy, Parkinson's disease and Huntington's disease and the like (see, for example, Wang KK, supra). It is believed that there is a link between these disorders and elevated and sustained levels of calcium in the cell. This results in calcium-dependent hyperactivated processes that are no longer physiologically controlled. Accordingly, calpain hyperactivation may also induce pathophysiological processes.

For this reason, it has been postulated that inhibitors of calpain enzymes may be useful for the treatment of the above disorders. This assumption has been confirmed by various studies. For example, Seung-Chyul Hong et al. (Stroke 1994, 25 (3), 663-669) and Bartus R.T. et al. (Neurological Res. 1995, 17, 249-258) that calpain inhibitors have neuroprotective acute neurodegenerative disorders to which stroke. Similarly, they may have demonstrated an effect upon after experimental improvement of brain injury recovery by calpain inhibitors of memory insufficiency and neuromotor disorders that occur (Saatman KE et al., Proc. Natl. Acad. Sci. USA, 93, 1996: 3428-3433 ). Edelstein C.L. et al. (Why.

Acad. Sci. USA, 92, 1995, 7662-7666) found that calpain inhibitors have a protective effect on the kidneys of hypoxia damage. Yoshida K.I. et al. (Jap. Circ. J. 59 (1), 1995, 40-48) have demonstrated that calpain inhibitors have beneficial effects following heart damage caused by ischemia or reperfusion. Since inhibitors inhibit the release of β-ΡΡ4 protein, it has been used in the treatment of Alzheimer's disease (Higaki J. et al., Neuron, 14, 1995: 651-659). Interleukin-α release is also inhibited by calpain inhibitors (Watanabe N. et al., Cytokine, 6 (6), 1994: 597 - 601). Calpain inhibitors have also been found to have cytotoxic effects on tumor cells (Shiba E. et al., 20th Meeting Int. Ass. Breast Cancer Res., Sendai Jp, 1994, 25-28 Sept., Int. J. Onco 5 (suppl.), 1994, 381). Calpaines also play an important role in restenosis and arthritis, and calpain inhibitors may have suggested a possible effect of calpain on pathology (March KL et al. Circ. Res. 72). , 1993: 413-423, Suzuki K. et al., Biochem J., 285,

1992: 857-862).

Another possible use of calpain inhibitors has been found in Wang K.K. (Trends in Pharmacol. Sci., 15, 1994: 412,419).

The most potent and selective calpain inhibitor is the naturally occurring intracellular protein calpastatin. This protein inhibits both calpain I and calpain II, but does not affect other cysteine and thiol proteases such as cathepsin B, L or papain. However, Calpastatin has the disadvantage that it is unsuitable for possible therapy due to its size, which is about 700 amino acids and the inability to cross the cell membrane. In addition to low molecular weight peptides that inhibit calpain, a number of non-peptide calpain inhibitors have been identified. The disadvantages of these inhibitors are their instability, rapid metabolism, and some of them are toxic. In addition, many calpain inhibitors have insufficient selectivity, which means that they not only inhibit calpain I and II, but also other cysteine proteases such as papain, chymotrypsin, elastase or cathepsin B and L.

Therefore, there remains a need for selective and highly potent calpain inhibitors. In order to identify selective inhibitors, highly specific assay systems are needed to screen for these selective and highly potent calpain inhibitors. Sorting and screening tests are usually performed using the ubiquitous occurrence of calpaines I and calpaines II.

In order to find selective inhibitors, it is necessary and desirable to find additional calpains that are expressed as tissue-specific as possible for testing so that the inhibitors can be tested for their selectivity between individual calpains.

In addition, other new calpains are sought because they are proteins that are very likely to be expressed differently in a variety of pathologies and disorders and play an important role in these disorders.

SUMMARY OF THE INVENTION

The present invention relates to methods and means for distinguishing and identifying calpain inhibitors which make it possible to identify calpain inhibitors which, on the one hand, have only one calpain inhibitory effect and / or, on the other hand, have multiple calpain inhibitory effect and administer them as a therapeutic target.

It has been found that this goal can be achieved based on a novel tissue-specific calpain gene having the sequence of SEQ ID NO. 1 or SEQ ID NO. 3 and the name of CAPN6, allelic variants, analogs or derivatives thereof having from 60 to 100% homology at the level of derived amino acid sequences, wherein the calpain genes, allelic variants, analogs or derivatives thereof comprise the following sequences:

• 99 99 99

9 9 9 · * »» ♦ · ♦

9 9 · · 9 9 9 9 9 * • · 9 9 9999999

99999 99 9 · · 9 »(a) Leu-Gly-Asn-Lys-Ala, wherein this sequence differs from the corresponding sequence in human calpain I by the amino acid cysteine in human calpain I located at position 115 in calpain I is replaced with lysine at position 81 of SEQ ID NO. 1 and SEQ ID NO. 3;

(b) Ala-X-Ser-Cys-Leu-Ala wherein, compared to the corresponding sequence in human calpain I, the amino acids alanine and threonine at positions 122 and 125 are replaced with serine and alanine at positions 88 and 91 of SEQ ID NO. . 1 and SEQ ID NO. 3;

(c) Gly-Tyr-Thr- (His or Tyr) -Thr-X-Thr wherein the amino acids histidine, alanine and serine at positions 272, 273 and 275 are replaced with tyrosine, threonine compared to the corresponding sequence in human calpain I and threonine at positions 252, 253, and 255 of SEQ ID NO. 1 and SEQ ID NO. 3 and SEQ ID NO. In addition, the tyrosine residue at position 274 in calpain I is replaced with histidine at position 254 of SEQ ID NO. 3;

(d) Arg-X-Arg-Asn-Pro-Leu-Gly, wherein this sequence differs from the corresponding sequence in human calpain I in that the tryptophan amino acid in human calpain I, which occupies position 298 in calpain 1, is replaced by leucine which is at position 286 of SEQ ID NO. 1 and SEQ ID NO. 3, a

X in said sequences is any natural amino acid.

The present invention also relates to a method of identifying a method of identification.

Calpain inhibitors, wherein calpain, its allelic variants or analogues encoded by the sequence of claim 1 is isolated from the tissue or cell and the inhibition of cleavage is measured. a CAPN6 enzyme substrate and, in at least one further assay, inhibiting the cleavage of a calpain I and / or II enzyme substrate by a test substance and selecting a test substance that inhibits the CAPN6 enzyme and at least one other calpain.

The present invention further relates to a method for identifying calpain inhibitors by determining inhibition of CAPN6 enzyme or calpain I and / or II substrate cleavage by a test substance in cellular systems and selecting test substances that cross the cell membrane and inhibit the intracellular activity of CAPN6 and / or calpaines I and / or II which do not inhibit CAPN6 but calpain I and / or II, or which inhibit CAPN6 but not calpain I and / or II. Substances which exhibit in vitro activity against calpaines without testing their ability to enter the cell are also preferably selected. If a subsequent test shows that these substances are incapable of, or very little able to enter the cell, their cell permeability can be improved by derivatization.

Sequences of human gamma calpain protein genes were used to determine homology in the EST database in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) using the BLAST program. A sequence, called EST AA050030, having a sequence typical of calpain has been found. Using this sequence, it was possible to prepare a mouse clone that encodes a gene whose new gene product has been designated CAPN6 as a new calpain (= nCL • fe fe · 9).

4). The nucleic acid sequence of clone nCL-4 is found as SEQ ID NO: 1. The deduced amino acid sequence of calpain CAPN6 is found as SEQ ID NO: 2.

The amino acid sequence that was derived considering the presence of an intron shows a typical calpain signature, although the association with known calpain subclasses represented by μ calpain, m calpain, nCL-1 or nCL-2 is not possible due to low homology. Kalpain CAPN6 is a new, previously unknown, calpain. Further exploration using this sequence in the mouse in the EST database yielded an additional 4 human partial sequences (AA169715, C17331, C16980 and T39424) that have homology to the mouse CAPN6 sequence. These partial sequences can be grouped into a contiguous sequence encoding a 374 amino acid protein. This sequence lacks both a typical methionine start codon and a stop codon. Therefore, this sequence is probably only part of the human ortholog sequence of the cloned murine sequence. The homology of the two 374 amino acid sequences is 94.9% (Fig. 1).

The protein sequence derived from the gene of SEQ ID NO: 1 shows 30% homology, which is the largest homology, with the known Caenorhabditis elegans tra-3 gene over the entire amino acid sequence. Homology with other known calpaines is from 20.9% (rat nCL-2) to 25.4% (mouse m calpain). When comparing sequences according to Lipman-Pearson method (Ktupl 2, Gap Penalty 4, Gap Length Penalty 12) between CAPN6 and human CAPNI (= 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:

T · · v v v v v v v v v v v v v v v v v v v v v v v v v

93), human CAPN3 (= CAN_3-HUMAN, Richard et al., Cell 81, 27-40), rat CAPN3 (= CAN3_RAT, Sorimachi et al., J. Biol. Chem. 264, 1989: 20106 - 20111) and Drosophily calpain (= DMCLPNOCM_1) over the partial sequences of 230-520 amino acids, slightly higher homologies from 32.8 to 39.5% were found, but these homologies are generally lower than homology to tra-3 (Fig. 1, Alignment, Clustal method with PAM25 residue weight tabl.). In addition to tra-3, CAPN6 exhibits marked homology to the recently described calpain CAPN5 (44.2% homology, set no. P 19 718 248.8).

Sequence comparison between murine and human CAPN6 sequences in a large number of databases revealed homology to CalpA, Tra3, and human sequences designated C16980, T39424, AA16971, R93331 and G17331 for which no information has been provided. Sequence comparison was performed using the EST gene bank and databases in the National Center for Biotechnology Information (http: //www.nebi.nim.nih.gov) and the Wash-U database (Homo sapiens). The murine EST sequence designated AA050030 and described as calpain was also found in the databases. No additional information was obtained from the databases. The complete sequences of the AA16971T, R93331, C17331 and AA050030 genes are unknown.

The two calpaines (CAPN5 and CAPN6) share common characteristics that distinguish them from other calpains. CAPN5 and CAPN6, unlike other calpains, have not only a truncated region I, but also a modified C-terminus that has no significant homology to the region IV of other calpaines. A common Ca ²⁺ -calpain binding region sequence (referred to as the EF hand) is located at the IV site. This Ca ²⁺ -binding site is • 4 4 4 · · · 4 4 4

4 · »44 # 4 4 4« • 4 »44 4« 444 «4« 4 »• 4» 4 4 4 4 ···

444 44 44 4 44 · * absent in the case of calpaines CAPN5 and CAPN6, which means that perhaps no Ca ^{2+ is} bound to the IV region and the proteins are activated differently. Thus, these are only vertebrate calpaines lacking the Calmodulin type IV region.

The deduced amino acid sequence of CAPN6 has another peculiarity in the shape of a modified catalytic center. Only the remainder of the Asn at position 284 is left. The cysteine residue at position 81 and the histidine residue at position 252 were replaced with lysine and tyrosine, respectively, in the mouse CAPN6 sequence. In addition, other replaced amino acids were identified in the catalytic center region as compared to the CAPN6 sequences of SEQ ID NO. 1 and SEQ ID NO. 3 with human calpain I (= CAPNI, Aoki et al., FEBS Lett. 205, 1986: 313-317). Thus, the amino acids alanine and threonine at positions 122 and 125 in the calpaines of CAPNI are changed to serine and alanine at positions 88 and 91 of the calpaines of CAPN6. Similarly, there are amino acid changes of histidine, alanine, lilac and tryptophan at positions 272, 273, 275, and 298 for the CAPNI calpains for tyrosine, threonine, threonine, and leucine at positions 252, 253, 255, and 286 for caplain CAPN6, respectively. The assignment of amino acids to different positions in the CAPN1 and CAPN6 proteins occurs by comparing the sequences and assigning the fixed regions of the proteins to one another for maximum protein matching at the amino acid level. Thus, it is possible, for example, for the same sequences to be located at very different sites or positions in different proteins in a set of proteins.

The human CAPN6 sequence has, in addition to replacing the cysteine residue, a tyrosine substitution at position 254 with histidine (Table 1). A possible explanation may be, for example, that CAPN6 has a substrate specificity that is different from the other.

4 4 * 9 • 4 · 44 * 4 ·

The calpaines or active center is not critical to the function of CAPN6, which is an inhibitor of other calpaines, or that CAPN6 is a pseudogen. This latter possibility seems unlikely due to the large number of amino acids stored. It is possible that the cysteine residue at position 89 can assume the function of the missing cysteine residue at position 81 in the catalyzed reaction. Even if CAPN6 had no protease activity, which is very unlikely, it could be involved in regulatory processes, possibly by competition with other calpains for binding sites on cofactors or substrates.

Table 1: CAPN6 genomic sequences of CAPN6

Amino acid no. 81 * 89 254 * 284 * 286 ¹ Mouse CAPN6 (129 ES DNA cells) TO C Y N L Human CAPN6 (HeLa DNA Cells) TO C H N L More calpains C S / C Y N W

* Active Center Amino Acids (Arthur et al., FEBS Lett. 368, 1995: 397-400) low leucine-free activity in m calpaines (Arthur et al., FEBS Lett. 368, 1995: 397-400)

Tra-3 is involved in sex determination in Caenorhabditis elegans. A cascade of several genes and their gene products, including tra-3, determines whether a male or hermaphrodite Caenorhabditis develops (Kuwabara P.E. et al., TIG, Vol. 8, No. 5, 1992: 164-168). Tra-3 appears to be involved in spermatogenesis. Conservation of amino acids in the different regions between mouse CAPN6 and tra-3 is 39.2%, 42.0%, 30.9%, respectively.

22% for zones I, II, III and T.

«0 0 00 00 •»

0 0 0 0 0 * 00000 00000 99 99 0 · 9 9 9 9

0 · 0 0 0 ·

Starting from cDNA derived from (Ia) 17 mouse embryonic mRNA, it was possible using a modified RACE method (= rapid amplification of cDNA ends), Frohman et al. (Proc. Natl. Acad. Sci. USA 85, 1988, 8998-9002) and Edwards et al. (Nucl. Acids Res. 19, 1991, 5227-5322) and using the aforementioned primers (Cal6 and Cal9) and sequences derived from EST AA050030 to clone the complete sequence of the cloned CAPN6. SEQ ID NO. 1 encodes a protein of 641 amino acids and a molecular weight of 74.6 kDa. The methionine start codon is preceded by a typical translation start sequence. Sequence accuracy was confirmed by sequencing several cDNA clones having said sequence.

catalytic

Amino acid

Figure 2 shows homologies between mouse CAPN5 (= nCL-3), human CAPN5 (= nCL-3), mouse CAPN6 (= nCL-4), and human CAPN6 (= nCL-4). Giant. 2 also depicts the sequences of Caenorhabditis tra-3, human p94, murine m calpain, human μ calpain, and rat nCL-2. Amino acids that match between different calpains and CAPN6 are indicated by black rectangles. The bars show the gaps inserted in order to maximize sequence matching. For illustration, two sequences were omitted from the human p94 sequence. These areas were marked with =. The conserved amino acids in the centers were indicated by arrows, the sequences corresponding to CAL6 and CAL9 were underlined. The regions were labeled over the relevant sequence segments.

Figure 3 shows the phylogenetic pedigree of various calpaines. Phylogenetic analyzes for the construction of this 9 · * * * 9 9 9 9 9 9 9 9 9 · · · · · · · The pedigree was performed using the closest neighbor method (Saitou et al. Mol. Biol. Evol. 4, 1987, 406-425) omitting spaces. Using these phylogenetic analyzes, it was possible to divide vertebrate calpains into six different groups (Figure 3, right side). Non-vertebrate calpains can be designated as the closest neighbor of the CAPN5 - (= nCL-3-) and CAPN6 - (= nCL-4-) calpain groups and form their own group. Thus, the CAPN5 and CAPN6 genes each form their own group of calpaines, which are more similar to invertebrate calpain than vertebrate calpain. The length of the horizontal lines is proportional to the phylogenetic distance between the different calpains. The length of the vertical lines has no meaning. The sequences used to construct the phylogenetic family tree had the following designations SWISSPROT and EMBL (Accession Numbers): human m (P17655), μ (P07384), P94 (P20807); rat m (Q07009), nCL-2 (D14480), p94 (P16259); mouse p94 (X92523); chicken m (D38026), μ (D38027), μ / m (P00789), p94 (D38028); nematode tra-3 (U12921); Drosophila Calp A (Q11002) and Dm (X78555), Schistosoma (P27730). The human partial sequence of nCL-2 corresponds to the translation of the EST clone AA026030 (Hellier et al., 1995, The Wash U-Merck EST project).

Based on this analysis, the derived amino acid sequence of the EST clone AA026030 shows higher than usual homology to the rat nCL-2 sequence. Since only a partial sequence was used for this analysis, the inaccuracy of the phylogenetic pedigree obtained for nCL-2 is greater. The nematode CPL1 sequence is a corrected version used by Barnes and Hodjkin (EMBOJ., 1996, 15: 4477-4484).

99 99 9 9 9 9 9

9 9 9 9 9

9 9 9 9 9 9 9 9 9

9 999 * »999 99 99» · · 9 9 9999 • 9 9 99 9 9 9 9 9 9 «

The first 7 exons were used as obtained from the EMBL database (Accession No. L25598), while the last 5 exons were obtained by joining nucleotides 8028-8133, 8182-8239, 8729-8818, 8865-8983 and 9087 towards the end. The derived N-terminal sequence is unusual for calpains due to its length and the amount of their glycine residues.

The new tissue-specific calpain CAPN6 is expressed only in placental tissue (Figure 4). The human placenta is an organ that exhibits rapid growth and rapid differentiation. Therefore, it is also referred to as premalignant tissue as it is a highly invasive tissue similar to that of malignant tumors.

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, in a balanced equilibrium, allow placental development. CAPN6 appears to have an important role in this development as a protease and / or possibly involved in the regulation of other calpain cysteine proteases.

They may be involved in placental pathological processes such as gestosis (= preeclampsia), which occurs in 5 to 7% of all pregnancies. Preeclampsia (= high blood pressure induced by pregnancy) is one of the most common complications in pregnancy and is characterized by hypertension and proteinuria, often accompanied by excessive edema and, in some circumstances, convulsions. Preeclampsia during pregnancy is a serious cause of maternal and infant death, premature birth or, in milder cases, embryonic malnutrition or growth disorders. It is a multifactorial event involving, for example, genetic factors (recessive or

* 9 9 ft ftft ftftft ftftftft (* dominant genes), maternal immune tolerance, vasodilator / vasoconstrictor imbalance and probably CAPN6.

Women with pre-eclampsia have altered vasopressinase and angiotensinase levels, which may be affected by the regulatory way of calpain CAPN6.

Another possible function of CAPN6 may be to regulate the decomposition of somatostatin, glucagon and growth hormone in the placenta and thereby to control the growth of the fetus. The breakdown of maternal serum proteins, which also stimulates fetal growth, may also be affected by calpain CAPN6.

It is also possible that CAPN6 participates in complex phenomena in placental mucosa during embryogenesis and thus to control apoptosis induced by TNFα and IFNγ, for example trophoblasts, by regulating other calpaines. Thus, CAPN6 appears to be involved in embryo development.

In order to identify selective calpain inhibitors, it is necessary to have methods that are as specific as possible for identifying inhibitors. In this context, it is important that the selected inhibitors inhibit only the desired calpain or calpaines but not other cysteine proteases and thus intervene in physiological processes.

Test substances to be examined for their inhibitory activity may be, for example, chemicals, microbial or plant extracts. In addition to tests for their inhibitory activity for CAPN6, calpain I and / or calpain II, they can normally also be tested for their activity against cathepsin B.

ΦΦΦ »Φ φ φ φ Φ Φ Φ Φ φ

· Φ ΦΦΦ · · · · * ·

ΦΦΦΦ ΦΦ Φ ΦΦΦΦ

Dalším ΦΦ Φ · Φ ΦΦ ΦΦ or other thiol proteases. Ideally, good inhibitors should show little or no activity against cathepsin B, L, elastase, papain, chymotrypsin or other cysteine proteases, but show good activity against calpaines I and calpaines II.

Using the new tissue-specific calpaines of CAPN6, it is possible to use the method of the present invention to identify inhibitors that are capable of discriminating against their inhibitory action among different calpains from the group comprising calpain I, II, nCL-Ι, nCL-2 and / or nCL- 4.

Various inhibition tests have been performed for this purpose, which are listed below:

Cathepsin B Test

Inhibition of cathepsin B was determined by a method that is a variation of the procedure described by S. Hasnain et al., J. Biol. Chem. 268, 1993, 235-240.

μΐ of inhibitor solution prepared from the chemicals to be tested, microbial or plant extract and DMSO (final concentration: 100 μΜ to 0,01 μΜ) was added to 88 μΐ cathepsin B (human liver cathepsin B supplied by Calbiochem, diluted to 5 units in 500 μΜ buffer). This mixture is preincubated at ambient temperature (= 25 ° C) for 60 minutes and then the reaction is started by adding 10 μΐ 10 mM Z-Arg-Arg-pNA (in 10% DMSO buffer). The reaction is monitored with a microplate reader at 405 nm for 30 minutes. The IC50 values are determined from.

··· «··· ·« • «* * stoup stoup stoup stoup ání stoup stoup stoup stoup ání ání maxim maxim stoup stoup stoup stoup curves.

Calpain I and Calpain II Test

The activity of calpain inhibitors was investigated in a colorimetric assay with Hammarsten casein (Merck, Darmstadt) as substrate. The assay was performed in microtiter plates as described by Buroker-Kilgore and Wang in Anal. Biochem. 208, 1993, 387-392. The enzymes used were calpain I (0.04 units / test) from erythrocytes and calpain II (0.2 units / test) from kidney, both of pigs, supplied by Calbiochem. The substances to be tested were incubated with the enzyme at ambient temperature for 60 minutes, the DMSO solvent concentration did not exceed 1%. After addition of the Bio-Rad color reagent, the optical density at 595 nm was measured using an SLT Easy Reader EAR 400. 50% enzyme activity is apparent from the optical densities determined at the maximum enzyme activity without inhibitors and enzyme activity without calcium addition.

The activity of calpain inhibitors can be further determined using the Suc-Leu-Tyr-AMC substrate. This fluorimetric method has been described by Zhaozhao Li et al., J. Med. Chem. 36, 1993, 34723480.

Since calpaines are intracellular cysteine proteases, it is necessary for calpain inhibitors to cross the cell membrane to prevent the breakdown of intracellular proteins by the calpain. Some known calpain inhibitors such as E64 and leupeptin cross the cell membrane only weakly and as a result show little effect on the cells, although • 9 • 9 9 Μ * 9

9 9 * * 99 99 9 9 • 99 9999 * 999

999 9 99 · 9 * 9 99 9

9 * 9 99 * 99 * 9 • • 9 * 9 * 9 9 99 9 * are good inhibitors of calpain. It is therefore advantageous to perform additional tests on the ability of potential calpain inhibitors to cross membranes, such as the human platelet assay.

Platelet assay to determine cellular activity of calpain inhibitors

Calpain-mediated protein degradation in platelets was performed as described by Zhaozhao Li et al., J. Med. Chem., 36, 1993 3472 - 3480 Human platelets were isolated from fresh sodium citrate blood of donors and the concentration was adjusted to 10 ⁷ cells / ml in buffer (5 mM HEPES, 140 mM NaCl and 1 mg / ml BSA, pH 7.3).

Plates (0.1 ml) were preincubated at 1 μΐ of various concentrations of potential inhibitors (dissolved in DMSO) for 5 minutes. Calcium iontophor A 23187 (1 μΜ in the assay) and calcium (5 mM in the assay) were then added and incubated at 37 ° C for 5 minutes. After the centrifugation step, the plates were removed in SDS-page sample buffer and boiled at 95 ° C for 5 minutes and proteins were fractionated in 8% gel. The decay of the two actin-binding protein (= ABP) and talin was followed by quantitative densitometry. Upon addition of calcium and ionophore, these proteins disappeared and a new band of molecular weight below 200 Kd appeared. From this, half the maximum activity of the enzyme with or without control was determined.

Other tissue sections, such as brain sections or cellular sections, are also suitable for testing the ability to cross membranes

- 20 • 4 · 4 · «4 · 4 · 4 4 · 4«

444 4 4444 4 4 44 4 • 4 4 4 4 4444

44 44 44 44 44 culture.

The CAPN6 inhibition assay is performed in cells that express this protein and allow it to be detected later using specific antibodies. If cells are stimulated, for example, with calcium and a suitable ionophore, this leads to the activation of calpaines CAPN6. Takaomi Saido is described in J. Biochem. 11, 1992, 81-86 autolytic transition of μ calpain after activation and detection using antibodies. Appropriate antibodies for detecting CAPN6 have been generated. Calpain inhibitors prevent autolytic transition and appropriate quantification is possible using antibodies.

In addition to the in vitro assays and cellular platelet assays described herein, other calpain assays known to those skilled in the art, such as the glutamate-induced cell death inhibition assay in cortical neurons, are suitable (MaulucciGedde MA et al., J. Neurosci. 7, 1987: 357 368), calcium-mediated cell death of NT2 cells (Skvier MKT et al., J. Cell. Physiol., 159, 1994: 229-237, Patel T. et al., Faseb Journal 590, 1996: 587-597) or analysis of 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 performing in vitro CAPN6 assays, calpain or an animal or human homolog thereof is purified from tissues or cells in which the enzyme is normally or artificially (for example based on recombinant expression) expressed, such as preferably the placenta, or from cells or microorganisms containing at least ·

4 ··

4 4 • · 3 ··· * * »

4 · 4 one gene copy and / or a vector comprising at least one copy of the CAPN6 gene, allelic variants or analogs thereof, and is used as a crude extract or as a pure enzyme.

In the methods of the present invention, various calpain inhibitors are preferably tested in combination with an assay for inhibiting CAPN6 enzyme activity by potential inhibitors. This allows the selection of inhibitors that inhibit only the CAPN6 enzyme and not other calpaines, or, conversely, only inhibit other calpaines and not the CAPN6 enzyme or CAPN6 enzyme and at least one other calpain. CAPN6 inhibitors can be advantageously used in the case of gestosis.

In addition, various inhibitor tests are performed in such a way that, in addition to the test substance inhibitory effect on calpain CAPN6, calpain I and / or calpain II, tests without test substance are performed as a control. This test arrangement facilitates the detection of inhibitory effects of test substances.

Another method of the present invention uses calpain CAPN6 or allelic variants, analogs or synthetic derivatives thereof preferably for protection against the enzymatic action of other calpaines.

Another method of the present invention employs the enzyme CAPN6 to screen for novel calpain inhibitors, which inhibitors are preferably capable of inhibiting all calpaines in general or individual calpains such as calpain I, calpain II, calpain nCL-1, calpain nCL-2 or calpain CAPN6. In addition, different test substances can be tested individually or in parallel in test systems. The substances to be tested are preferably sorted according to their inhibitory effect in parallel in the manner of their inhibiting effect in parallel by their parallel inhibition action. automated test systems.

In general, all substances are suitable for inhibition tests. Thus, these substances can be obtained, for example, by classical chemical synthesis, as a result of combinatorial chemistry procedures, or from microbial, animal or plant extracts. Microbial extracts are, for example, fermentation broths, disrupted cells of microorganisms or substances after biotransformation. Cell fractions are also suitable for assays.

For the cloning of the CAPN6 gene or its animal homologs or its human homologs, allelic variants or analogs thereof, all prokaryotic or eukaryotic expression systems are suitable for isolating an enzymatically active gene product. Preferred expression systems are those which allow the expression of CAPN6 gene sequences in bacterial cells, fungal cells or animal cells, and particularly preferably in insect cells. Enzymatically active gene product means CAPN6 proteins that, upon isolation from an expressing organism, for example, a prokaryotic or eukaryotic cell, or after renaturation, provide an active protein capable of cleaving at least one known calpain substrate such as those mentioned above, or itself autocatalysis.

Assays suitable for determining the enzymatic activity of calpaines are all assays known to those skilled in the art, such as in vitro assays such as those described above for calpain I and calpain II, or cellular assays such as platelet ftft. Ftft. Ftft. Ftftftft. Ftft. Ftftftft. Ftftftft ftft. ftftft ft ftftftft ftft · · * • ftft ftft · ftftftft • ftft ftft ftft ft ftft ftft tests. A possibility that can be used for detection is assays based on colorimetric testing (Buroker-Kilgore M. et al., Anal. Biochem. 208, 1993: 387-392) or fluorescence testing.

In addition, an enzymatically active CAPN6 gene product also means all partial sequences that contain the catalytic center of the CAPN6 gene and / or other CAPN6 gene sequences and / or other calpain gene sequences and / or other sequences that exhibit enzymatic activity.

By host organism is meant any prokaryotic or eukaryotic organism suitable as a host organism, for example bacteria such as Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptococcus carnosus, yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, fungi such as Aspergillus cells Spodoptera frugiperda, trichoplusia cells or other insect cells suitable for expression of viruses or animal cells such as CV1, COS, C127, 313 or CHO or human cells.

By expression system is meant a combination of an expression organism exemplified above and vectors suitable for the organism, such as plasmids, viruses or phages such as the T7 RNA polymerase / promoter system or vectors having phage λ regulatory sequences.

Expression systems preferably means a combination of Escherichia coli and its plasmids and phages, or a baculovirus system and corresponding insect cells such as Spodoptera at 0000000000

0 0 0 0 0000 0 0 0 _e <«00« 0 · * frugiperda.

In addition, additional 3 'and / or 5' terminal regulatory sequences are suitable for the preferred expression of the CAPN6 gene of the present invention.

These regulatory sequences aim to allow specific expression of the CAPN6 gene. This may mean, for example, depending on the host organism, that it is overexpressed only after the induction is expressed or overexpressed.

the gene is expressed or that it is immediately

In addition, regulatory sequences and factors may advantageously have a beneficial effect on CAPN6 gene expression and thereby increase it. Thus, regulatory elements can be enhanced, preferably at the transcription level, by using strong transcriptional signals such as promoters and / or enhancers. However, it is also possible, in addition, to enhance translation, for example, by improving mRNA stability.

Enhancers are, for example, DNA sequences that bring about increased expression of the CAPN6 gene through improved interaction between RNA polymerase and DNA.

One or more DNA sequences may be present upstream and / or downstream of the CAPN6 gene with or without a promoter upstream and with or without a regulatory gene, such that the gene is in the gene structure.

In addition, CAPN6 gene expression can be increased by increasing the number of copies of the CAPN6 gene. For example, amplification increases the number of copies of the CAPN6 gene

CHO expression vector.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • 0 ··

Suitable vectors are also those of the pED series - dicistronic vectors - which also contain an amplifiable marker gene of dihydrofolate reductase. Details can be found in Current Protocols in Molecular Biology, Vol. 2, 1994.

An increase in CAPN6 enzymatic activity over the parent enzyme can be achieved, for example, by modifying the CAPN6 gene or its animal homologs by classical mutagenesis such as UV irradiation or by chemical mutagens and / or specific mutagenesis such as site-directed mutagenesis, one or more deletions, insertions and / or substitutions. The enzymatic activity can be increased, for example, by modifying the catalytic center so that the substrate to be cleaved is more rapidly converted. Increased enzymatic activity can also be achieved, in addition to the gene amplification described above, also by eliminating factors that suppress enzyme biosynthesis and / or by synthesis of active CAPN6 proteins instead of inactive proteins. In this way, increased amounts of enzymes can be obtained for in vitro assays.

Preferably, CAPN6 or its animal homologs or human homologs may be cloned starting from genomic DNA or cDNA using, for example, PCR techniques (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, 239, 1988, 487 et al.) And CAPN6 can be preferably cloned using genomic DNA and particularly preferably using genomic DNA from a mouse cell or a human cell.

Suitable hosts for cloning are: · · · · · · · • · • · • · · · · · · · · · For example all strains of Escherichia coli, preferably Escherichia coli strain DH108. Suitable vectors for cloning are all vectors suitable for expression in Escherichia coli (see Molecular Cloning, Sambrook, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, second edition 1989, ISBN 087969-309-6). Particularly suitable examples are vectors derived from pBR or pUC, or shuttle vectors, and pBluescript is particularly suitable.

After isolation and sequencing, CAPN6 genes having nucleotide sequences that encode the amino acid sequence designated SEQ ID NO: 2 or allelic variants thereof can be obtained. By allelic variant is meant CAPN6 variants which have a homology of 60 to 100% at the amino acid level, preferably a homology of 70 to 100%, particularly preferably a homology of 80 to 100%. In particular, allelic variants include functional variants obtainable from the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 3 by deletion, insertion or substitution of nucleotides, but which retain CAPN6 activity and sequence (a) of 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 are present as in the CAPN6 genes, analogs or derivatives.

By CAPN6 analogues are meant, for example, its animal homologues, truncated sequences, single-stranded DNA or RNA coding and non-coding DNA sequences, especially non-coding RNA.

9 9 9 9

999 9999999 99 9

Examples of CAPN6 derivatives are those which can be enzymatically cleaved only with difficulty, if at all, such as the phosphonates or phosphothioates of the nucleic acids in which the phosphate group of the nucleic acid has been replaced by a phosphonate or thioate group.

A promoter that is present before said nucleotide sequence may also be modified by one or more nucleotide exchanges, insertions or insertions and / or deletions or deletions, but without impairing the functionality or activity of the promoter. In addition, promoter activity may be enhanced by modifying its sequence or may be completely replaced by more active promoters, even from heterologous organisms or promoters of synthetic origin.

The calpain inhibitors identified by the method of the present invention are useful in the preparation of medicaments for the treatment of disorders related to calpain dysfunction, for example disorders selected from the group of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerative or oncological diseases such as restenosis, arthritis central nervous system (e.g., stroke), inflammation, muscular dystrophy, cataracts (e.g., cataract), central nervous system injury (e.g., trauma), Alzheimer's disease, HIV-induced neuropathy, Parkinson's and Huntington's disease, preferably for the preparation of medicaments for treating disorders placenta such as gestosis or embryogenesis disorders. The CAPN6 gene sequences of the present invention are also preferably suitable for diagnosis of disorders or for gene therapy.

• · · · · · · ·

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Cloning of the CAPN6 gene

The mouse CAPN6 sequence (EMBL accession number Y12583) was cloned by the RACE method using tag 17 mouse embryos and averaged sequences derived from the EST sequence AA050030. A plasmid clone containing the corresponding EST sequences was obtained from the I.M.A.G.E consortium (Research Genetics Inc). The human CAPN6 homolog was obtained by searching for homology in the EST database using the mouse protein sequence and the tblastn algorithm. It was possible to combine the partial sequences that were found to give an incomplete sequence of human CAPN6 of 1083 nucleotides (SEQ ID NO: 3).

Example 2: Expression of the CAPN6 gene in various tissues

CAPN6 gene expression in various tissues was detected using a ³² P-labeled human cDNA fragment with human RNA major transfer, which is supplied by Klontech, which contains RNA from 50 different tissues. Hybridization and high stringency wash conditions were performed according to the manufacturer's instructions. The CAPN6 DNA fragment used in expression experiments was the 2.2 kb EcoRI / XhoI fragment containing EST AA050030. CAPN6 was only expressed in placental tissue (see Figure 4). As a control, transfer was performed with a human sample of everywhere present DNA for RNA determination.

Example 3: Determination of CAPN6 gene on chromosome

The human gene was localized using NIGMS human / rodent somatic cell hybrid mapping panel (Coriell Cell Repositories). The primer sequences that were used for PCR were as follows:

5'-gttgaaactgattggggtctg-3 'and' -ctgtcttcccaaggggtttctc-3 '.

PCR amplification was performed at an annealing temperature of 58 ° C and resulted in a 200 bp fragment. The resulting products were examined for consistency with the presence of human chromosomes and PCR products. The exact position of the gene in human chromosomes was found using the Stanford G3 RH Panel (Research Genetics) and transferring PCR results to a localization service at the Stanford Human Genome Center (http://www-shgc.stanford.edu). The human CAPN6 gene was found on the X chromosomes coupled to the DXS7356 marker.

Example 4: Cathepsin B Assay

Inhibition of cathepsin B was determined by a method similar to

described by S. Hasnain et al., J. Biol. Chem. 268, 1993, 235-240. 2 μΐ inhibitor solution prepared an inhibitor of a DMSO (final concentration: 100 μΜ to ž 0.01 μΜ) was added k 88

μΐ cathepsin B (human liver cathepsin B supplied by Calbiochem, diluted to 5 units in 500 μΜ buffer). This mixture is preincubated at ambient temperature (= 25 ° C) for 60 minutes and then the reaction is started by adding 10 μΐ of 10 mM Z-Arg-Arg-pNA (in 10% DMSO buffer). The reaction is monitored with a microplate reader at 405 nm for 30 minutes. IC ₅₀ values are determined from the maximum curve slope.

• 9 • 9 · 9 · 9 · 9 · 999 999 999 9 e e e e e e e e e e

JV Φ ·· «9« 9 9 · 99

Example 5: Calpain test

The activity of calpain inhibitors was investigated in a colorimetric assay with Hammarsten casein (Merck, Darmstadt) as substrate. The assay was performed in microtiter plates as described by Buroker-Kilgore and Wang in Anal. Biochem. 208, 1993, 387-392. The enzyme used was CAPN6, which was expressed in one of the systems described above and then purified. The substances were incubated with the enzyme at ambient temperature for 60 minutes, the DMSO solvent concentration did not exceed 1%. After addition of the Bio-Rad color reagents, the optical density at 595 nm was measured using an SLT Easy Reader EAR 400. 50% enzyme activity is apparent from the optical densities determined at the maximum enzyme activity without inhibitors and enzyme activity without calcium addition.

Example 6: Platelet assay to determine cellular activity of calpain inhibitors

Calpain-mediated protein degradation in platelets was performed as described by Zhaozhao Li et al., J. Med. Chem., 36, 1993 3472 - 3480 Human platelets were isolated from fresh sodium citrate blood of donors and the concentration was adjusted to 10 ⁷ cells / ml in buffer (5 mM Hepes, 140 mM NaCl and 1 mg / ml BSA, pH 7.3).

Plates (0.1 ml) were preincubated at 1 μΐ of various concentrations of potential inhibitors (dissolved in DMSO) for 5 minutes. Calcium ionophore A 23187 (1 μΜ in the assay) and calcium (5 mM in the assay) were then added and incubated at 37 ° C for a centrifugation step in sample buffer and cooked while the proteins were fractionated with actin-binding protein (= quantitative densitometry). ionophore these proteins have higher molecular weight lower r half maximum

31 - • · · • · · · • · · • »9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9999999 · · «» • · • ·· ·· for 5 minutes. After design platičky excluded in SDS-page temperature 95 ° C Mon time 5 minutes and τ 8% gel. Disintegration two proteins ABP) a talinu was tracked

because after adding calcium and zely, a new belt of up to 200 Kd appeared. From this, enzyme activity was determined.

• · »» »» »

LIST OF SEQUENCES (1)

(i) (A) (B) (C) (D) (E) (F)

APPLICANT:

NAME: BASF Aktiengesellschaft STREET: Carl Bosch Strasse CITY: Ludwigshafen COUNTRY: Rheinland-Pfalz COUNTRY: Germany

POST CODE: D-67056 (ii) APPLICATION NAME:

Novel tissue-specific calpains, their preparation and use (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER-PROCESSABLE FORM:

(A) MEDIA TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE:

PatentIn Release # 1.0, Verse * 1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2069 base pairs (B) TYPE: Nucleic acid (C) FIBER NUMBER: single · 4 444 4444 «· 4444 4 4 4 * · 4 4 4444444 44 4» 4 44 4 4 4 4 · • 4 44 4 44 44 (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iii) TEMPLATE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus (vii) IMMEDIATE SOURCE:

(B) CLONE: CAPN6 (ix) FEATURES:

(A) NAME / KEY: 5'UTR (B) POSITION: 1 ... 129 (ix) FEATURES:

(A) NAME / KEY: CDS (B) POSITION: 130 ... 2055 (ix) FEATURES:

(A) NAME / KEY: 3'UTR (B) POSITION: 2056 ... 2069 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ft · · ♦ · ft ·· · ftft * · · · · · ft «· ft ft ftftftft · <· ft ·

GGGGTTACCT GGCTAAGAGC GCAGCAGCA GCAGCAGCAG CAGCAGCAGT AGCAGCAGCA 60

GCAGCAGCAG CAGCAGCAGC AGCAGCAGCA GCAGGGTTCC TGAGCTAACT CAGACCTAGT 120

TTGATA.GCA ATG GGT CCT CCT CTC AAG CTC TTC AAA

Met Gly Pro Pro Leu Lys Phu Lys Asn Gin Lys Tyr

10

CAA GAA CTG AAG CAG GAG TGC Cys 20 May ATG AAG GAT GGC CGC Arg 25 CTT TTC TGT GAC Asp 216 Gin Glu 15 Dec Leu Lys Gin Glu Met Lys Asp Gly Leu Phe Cys CCA ACC TTC CTA CCG GAG AAT GAT TCT CTG TTT TTC AAC CGG CTG CTT 264 For Thr Phe Leu For 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 For Gly Lys Wall Wall Trp Lys Arg For Gin Asp Ile Ser Asp Asp For 50 55 60 CAC CTG ATT GTG GGC AAC ATC AGC AAC CAC CAG CTG ATC CAG GGC AGA 360 His Leu Ile Wall 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 Wall 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 For Asn His Lys Asp Gin Glu Trp 95 100 ALIGN! 105 GAT CCT CGA AAG CCA GAG AAA TRAY GCT GGA ATC TTT CAC TTC CGC TTC 504 Asp For Arg Lys For 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 Wall Wall Ile Asp Asp Leu Leu For 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 Wall Phe Ser Phe Ser Thr Ser Met Asn Glu

145 150 155 • 99 9 9 9 9 9 9 9

9 9 9

9 · · ·

TTT Phe TGG AAT GCT CTA CTG GAA AAA GCG TAT GCA AAG CTG 648 Trp Asn 160 Ala Leu Leu Glu Lys 165 Ala Tyr Ala Lys Gly Cys 170 MELT 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 TRAY 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 TRAY AAG CTG TTT GGA GAA CTG TRAY AAA ACG 792 Asp Leu Wall 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 For Ser Gin 225 230 235 GAG GAA CAA GAA GTT GAA ACA GAC TGG GGA CTA CTG AAG GGT MELT ACC 888 Glu Glu Gin Glu Wall Glu Thr Asp Trp Gly Leu Leu Lys Gly Tyr Thr 240 245 250 TRAY 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 Wall 255 260 265 GAA GTC TTC AGT ACT GAG AAG CTG MELT ATG GTT CGC CTA AGG AAC CCA 984 Glu Wall Phe Ser Thr Glu Lys Leu Tyr Met Wall Arg Leu Arg Asn For 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 For 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 Wall Thr Asp Arg Lys Asn Leu Gly Leu Wall 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 «• • • * * * * * 325 325 325 325 325 325

9 • 9

TTT CAC AAA CTG AAT GTC 1176 Phe His Lys Leu Asn Val 335 Cys 340 Arg Asn Val Asn Asn For Val 345 Phe Gly CGC AAG GAG CTG GAA . TCA . GTG GTG GGA . TGT TGG ACT GTG GAT 'GAT GAC 1224 Arg Lys Glu Leu Glu Ser Wall Wall Gly Cys Trp Thr Wall Asp Asp Asp 350 355 360 365 CCT CTG ATG AAC CGA TCA GGA GGT TGC MELT AAC AAC CGT GAT ACC TTC 1272 For Leu Met Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe 370 375 380 TTG CAG AAT CCT CAG TRAY ATT TTC ACT GTG CCC GAG GAT GGC CAT AAA 1320 Leu Gin Asn For Gin Tyr Ile Phe Thr Wall For Glu Asp Gly His Lys 385 390 395 GTC ATC ATG TCA CTG CAA CAG AAG GAC CTA CGC ACT TRAY CGC CGA ATG 1368 Wall Ile Met Ser Leu Gin Gin Lys Asp Leu Arg Thr Tyr Arg Arg Met 400 405 410 GGA AGA CCT GAT AAT TRAY ATC ATT GGT TTT GAG CTC TTC AAG GTG GAG 1416 Gly Arg For Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys Wall Glu 415 420 425 ATG AAC CGA AGG TTC CGT CTT CAC CAT CTG MELT 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 MELT ATC GAC ACC CGT ACT GTG TTT CTG AGC AAG MELT 1512 Gly Thr Ser Thr Tyr Ile Asp Thr Arg Thr Wall Phe Leu Ser Lys Tyr 450 455 460 CTG AAG AAG GGC AGC TRAY GTG CTT GTT CCA ACC ATG TTC CAA CAT GGC 1560 Leu Lys Lys Gly Ser Tyr Wall Leu Wall For 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 Wall For Wall 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 For Lys Met Ser Cys Trp Asn Leu 495 500 505

9 9 9

9

9999 9 9999

9

GCA CGT GGC TAC CCA AAG ATC ACT GTC CAC His AGT Ser GCT Ala 525 1704 Ala 510 Arg Gly Tyr Pro Lys 515 Val Thr Gin Ile 520 Thr Wall GAG GGC CTG GAG AAG AAG TAT GCC AAT GAA ACT GTC AAT CCA MELT CTG 1752 Glu Gly Leu Glu Lys Lys Tyr Ala Asn Glu Thr Wall Asn For 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 For Wall Gin Lys Asn 545 550 555 ACT GTG CAT GCC ATT TTT GAC ACG CAG GTT TTC TRAY AGA AGG ACC 1848 Thr Val His Ala Ile Phe Asp Thr Gin Ala Wall 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 For 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 For 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 Wall Ile Ser Ser Asp Asp 625 630 635 CTC ACT GAG CTC TAAGTAGTCA TCATCAG 2069

Leu Thr Glu Leu 640 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 641 amino acids (B) TYPE: amino acid

• 9 • 9

4 ·

«

9

4

(D) TOPOLOGY: linear (ii) MOLECULA TYPE: protein (xi) SEQUENCE DESCRIPTION SEQ ID NO: 2 Met Gly Pro Pro Leu Lys Leu Phe Lys Asn Gin Lys Tyr Gin Leu 1 5 10 15 Lys Gin Glu Cys Met Lys Asp Gly Arg Leu Phe 20 May 25 30 Leu Pro Glu Asn Asp Ser Leu Phe Phe Asn Arg Leu Leu For Gly Lys 35 40 45 Val Val Trp Lys Arg Pro Gln Asp Ile Ser Asp Asp For His Leu Ile 50 55 60 Val Gly Asn Ile Ser Asn His Gin Leu Gle Gly Arg Leu Gly Asn 65 70 75 80 Lys Ala Met Ile Ser Ala Phe Ser Cys Leu Ala Glu Glu Ser His 85 90 95 Trp Thr Lys Ala Ile Pro Asn His Lys Asp Gin Glu Asp Asp Arg 100 ALIGN! 105 110 Lys Pro Glu Lys Tyr Ala Gly Ile Phe His Phe Arg Phe 115 120 125 Gly Glu Trp Thr Glu Val Val Ile Asp Asp Leu Leu For Thr Ile Asn 130 135 140 Gly Asp Leu Val Phe Ser Thr Ser Met Asn 145 150 155 160 Ala Leu Leu Glu Lys Ala Tyr Ala Lys Leu Gly Cys Tyr Glu Ala 165 170 175 Leu Asp Gly Leu Thr Ile Thr Asp Ile Ile Met Phe Thr Gly Thr 180 185 190

* 4 4 4 4 4

4 4 4 4

4 · 4 ·

4 4 4 4

444 44 44 ·

4444

44 • 4 4 · · ·

4 4 4 «

4 4 4

Leu Glu Ile Ile Asp Met Gin Lys Wall 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 For Ser Gin Glu Glu Gin 225 230 235 240 Glu Wall 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 cly Glu Arg Leu Wall Glu Wall Phe 260 265 270 Ser Thr Glu Lys Leu Tyr Met Wall Arg Leu Arg Asn For Leu Gly Arg 275 280 285 Gin Glu Trp Ser Gly For Trp Ser Glu Ile Ser Glu Glu Trp Gin Gin 290 295 300 Leu Thr Wall Thr Asp Arg Lys Asn Leu Gly Leu Wall 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 Wall Cys Arg Asn Wall Asn Asn For Wall Phe Gly Arg Lys Glu 340 345 350 Leu Glu Ser Wall Wall Gly Cys Trp Thr Wall Asp Asp Asp For Leu Met 355 360 365 Asn Arg Ser Gly Gly Cys Tyr Asn Asn Arg Asp Thr Phe Leu Gin Asn 370 375 380 For Gin Tyr Ile Phe Thr Wall For Glu Asp Gly His Lys Wall Ile Met 385 390 395 400 Ser Leu Gin Gin Lys Asp Leu Arg Thr Tyr Arg Arg Met Gly Arg For

405 410 415

Asp Asn Ir Ile Ile Gly Phe Glu Leu Phe Lys Val Glu Met Asn Arg 420 425 430 # * ♦ ·· *

Arg Phe Arg Leu His His Leu Tyr 435,440

Thr Tyr Ile Thr Arg Thr Val 450 455

Gly Ser Tyr Val Leu Val Pro Thr 465,470

Glu Phe Leu Arg Ile Phe Ser 485

Leu Thr Leu Asp Met For Lys Met 500

Tyr Pro Lys Val Val Thr Gin Ile 515,520

Glu Lys Lys Tyr Ala Asn Glu Thr 530,535

Cys Gly Lys Glu Val Arg Ser 545 550

Ala Ile Phe Thr Gin Ala Val 565 ti

Pro Ile Ile Gle Val Trp Asn 580

Leu Gly Gin Val Thr Leu Asp Ala 595 600

Lys Ser Leu Lys Lys 610 615

Gin Gly His Ile Ser Phe Lys Val 625 630

Ile Gin Glu Arg Ala 445 Gly Thr Ser Phe Leu Ser Lys 460 Tyr Leu Lys Lys Met Phe Gin 475 His Gly Arg Thr Ser 480 Glu Wall 490 For Wall Gin Leu Arg 495 Glu Ser 505 Cys Trp Asn Leu Ala 510 Arg Gly Thr Wall His Ser Ala 525 Glu Gly Leu Wall Asn For Tyr 540 Leu Ile Ile Lys For Wall Gin 555 Lys Asn Thr Wall His 560 Phe Tyr 570 Arg Arg Thr Thr Asp 575 Ile Ser 585 Arg Lys Phe Cys Asp 590 Gin Phe Asp For Ser Asp Cys 605 Arg Asp Leu Gly Gly For Thr 620 Ala Lys Wall Lys Ile Ser Ser 635 Asp Asp Leu Thr Glu 640

Leu

·

4

4

4 • 4 *

44 · 4 »4 44 · 9

4 4 · ·· · 9 4 4 • * * 4 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1125 base pairs (B) TYPE: nucleic acid (C) FIBER NUMBER: single

(D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iii) HYPOTHETICAL: No (iii) TEMPLATE: No (vi) ORIGINAL SOURCE

(A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE:

(B) CLONE: CAPN6 (ix) FEATURES:

(A) NAME / KEY: CDS (B) POSITION: 2 ... 1125 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

G CTT GTT GAG GAG AAG TRAY AAG CTA TTC GGA GAA CTG TRAY AAA ACA 46 Leu Wall Glu Glu Lys Tyr Lys Leu Phe Gly Glu Leu Tyr Lys Thr 1 5 10 15 Dec

TTT ACC AAA GGT GGT GTC GTC GTC GTC GTC GTC GTC GTC GTC GTC GTC GTC GTC GTC

25 30

42 • · »» »» • ·· » • »♦ ·· • · ♦ · · • · · · • «« • · GAG GAG CAA GAA GTT GAA ACT GAT TGG GGT CTG CTG AAG GGC CAT ACC 142 Glu Glu Gin Glu 35 Wall Glu Thr Asp Trp 40 Gly Leu Leu Lys Gly 45 His Thr MELT ACC ATG ACT GAT ATT CGC AAA ATT CGT CTT GGA GAG AGA CTT GTG 190 Tyr Thr Met 50 Thr Asp Ile Arg Lys 55 Ile Arg Leu Gly Glu 60 Arg Leu Wall GAA GTC TTC AGT GCT GAG AAG CTG MELT ATG GTT CGC CTG AGA AAC CCC 238 Glu Wall 55 Phe Ser Ala Glu Lys 70 Leu Tyr Met Wall Arg 75 Leu Arg Asn For TTG GGA AGA CAG GAA TGG AGT GGC CCC TGG AGT GAA ATT TCT GAA GAG 286 Leu 80 Gly Arg Gin Glu Trp 85 Ser Gly For Trp Ser 90 Glu Ile Ser Glu Glu 95 TGG CAG CAA CTG ACT GCA TCA GAT CGC AAG AAC CTG GGG CTT GTT ATG 334 Trp Gin Gin Leu Thr 100 ALIGN! Ala Ser Asp Arg Lys 105 Asn Leu Gly Leu Wall 110 Met TCT GAT GAT GGA GAG TTT TGG ATG AGC TTG GAG GAC TTT TGC CGC AAC 382 Ser Asp Asp Gly 115 Glu Phe Trp Met Ser 120 Leu Glu Asp Phe Cys 125 Arg Asn TTT CAC AAA CTG AAT GTC TGC CGC AAT GTG AAC AAC CCT ATT TTT GGC 430 Phe His Lys 130 Leu Asn Wall Cys Arg 135 Asn Wall Asn Asn For 140 Ile Phe Gly CGA AAG GAG CTG GAA TCG GTG TTG GGA TGC TGG ACT GTG GAT GAT GAT 478 Arg Lys 145 Glu Leu Glu Ser Wall 150 Leu Gly Cys Trp Thr 155 Wall Asp Asp Asp CCC CTG ATG AAC CGC TCA GGA GGC TGC MELT AAC AAC CGT GAT ACC TTC 526 For 160 Leu Met Asn Arg Ser 165 Gly Gly Cys Tyr Asn 170 Asn Arg Asp Thr Phe 175 CTG CAG AAT CCC CAG TRAY ATC TTC ACT GTG CCT GAG GAT GGG CAC AAG 574 Leu Gin Asn For Gin 180 Tyr Ile Phe Thr Wall 185 For Glu Asp Gly His 190 Lys GTC ATT ATG TCA CTG CAG CAG AAG GAC CTG CGC ACT TRAY CGC CGA ATG 622 Wall Ile Met Ser 195 Leu Gin Gin Lys Asp 200 Leu Arg Thr Tyr Arg 205 Arg Met

♦ fe

- 43 ·· · · fe · · · · fefe • fefe • fefe fefe fefe · • fefe • fefefe • fe fefefe fe fefefe • fe · fe feefe feefefe · fe fefe

GGA Gly AGA CCT GAC AAT TAC ATC ATT GGC TTT GAG 670 Arg For Asp Asn 210 Tyr Ile Ile Gly Phe Glu Leu Phe 220 Lys wall Glu ATG AAC CGC AAA TTC CGC CTC CAC CAC CTC TRAY 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 MELT ATT GAC ACC CGC ACA GTG TTT CTG AGC AAG TRAY 766 Gly Thr Ser Thr Tyr Ile Asp Thr Arg Thr Wall Phe Leu Ser Lys Tyr 240 245 250 255 CTG AAG AAG GGC AAC MELT GTG CTT GTC CCA ACC ATG TTC CAG CAT GGT 814 Leu Lys Lys Gly Asn Tyr Wall Leu Wall For 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 Wall For Wall 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 For Lys Met Ser Cys Trp Asn Leu 290 295 300 GCT CGT GGC TRAY CCG AAA GTA GTT ACT CAG ATC ACT GTT CAC AGT GCT 958 Ala Arg Gly Tyr For Lys Wall Wall Thr Gin Ile Thr Wall His Ser Ala 305 310 315 GAG GAC CTG GAG AGG AGG MELT GCC AAT GGA ACT GTA AAC CCA MELT TTG 1006 Glu Asp Leu Glu Arg Arg Tyr Ala Asn Gly Thr Wall Asn For Tyr Leu 320 325 330 335 GTC ATC AAA TGT GGA AAG GAG GAA GTC CGT TCT CCT GTC CAG AAA AAT 1054 Wall Ile Lys Cys Gly Lys Glu Glu Wall Arg Ser For Wall Gin Lys Asn 340 345 350 ACA GTT CAT GCC ATT TTT GAC ACC CAT GCC ATT TTC TRAY AGA AGG ACC 1102 Thr Wall 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 For Ile Ile Wall

370 ·· (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 374 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: protein

(Xi) DESCRIPTION SEQUENCE: SEQ ID NO: 4: Leu Val 1 Glu Glu Lys 5 Tyr Lys Leu Phe Gly Glu Leu 9 Tyr Lys Thr Phe Thr Lys Gly Gly Leu Ile Cys Ser Ile Glu 25 For Asn Gin Glu 30 Glu Gin Glu 35 Val Glu Thr Asp Trp 40 Gly Leu Leu Lys Gly His Thr Tyr Thr Met 50 Thr Asp Ile Arg Lys Ile 55 Arg Leu Gly Glu 60 Arg Leu Val Glu Val Phe 65 Ser Ala Glu Lys Leu Tyr 70 Met Val Arg Leu Arg Asn Pro Leu Gly Arg Gin Glu Trp Ser Gly Pro Trp Ser Glu Ile 90 Ser Glu Glu Trp Gin Gin Leu Thr Ala 100 ALIGN! Ser Asp Arg Lys Asn Leu Gly Leu Val Met 110 Asp Asp Gly 115 Glu Phe Trp Met Ser 120 Leu Glu Asp Phe Cys Arg Asn Phe 126 His Lys Leu Asn Val Cys Arg Asn 135 Val Asn Asn Pro 140 Ile Phe Gly Arg

* 9 · 9 9 9

9 9 9 • 99 9 99 • 9 9 * 4

9 9 9

9 9 · 4

Lys 145 Glu Leu Glu Ser Wall 150 Gly Cys Trp Thr Val Asp Asp Asp Pro 155 160 Leu Met Asn Arg Gly Gly Cys Tyr Asn Asn Arg. Asp Thr Phe Leu 165 170 175 Gin Asn Pro Gin Tyr Ile Phe Thr For Glu Asp Gly His Lys Wall 180 185 190 Ile Met Ser Leu Gin Gin Lys Asp Leu Arg Thr Tyr Arg Arg Met Gly 195 200 205 Arg For Asp Asn Tyr Ile Ile Gly Phe Glu Leu Phe Lys 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 Wall Phe Leu Leu 245 250 255 Lys Lys Gly Asn Tyr Wall 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 For Lys Wall Gin Thr Ile Thr Val His Ser Ala Glu 305 310 315 320 Asp Arg Tyr Ala Asn Gly Thr Val Asn For Tyr Leu Wall 325 330 335 Ile Lys Cys Glu Glu Val Arg Ser For Val Gin Lys Asn Thr 340 345 350 Wall His Ala Ile Phe Asp Thr His Ala Ile Phr Tyr Arg Arg Thr Thr

355 360 365

Asp Ile For Ile Ile Val

Claims (19)

PATENT CLAIMS A CAPN6 calpain gene having the sequence of SEQ ID NO. 1 or SEQ ID NO. 3, allelic variants, analogs or derivatives thereof having homology of from 60 to 100% at the derived amino acid level, wherein the calpain genes, allelic variants, analogs or derivatives thereof comprising the following sequences: (a) Leu-Gly-Asn-Lys-Ala, wherein this sequence differs from the corresponding sequence in human calpain I in that the amino acid cysteine in human calpain I, located at position 115 in calpain I, is replaced by lysine which is located at position 81 of SEQ ID NO. 1 and SEQ ID NO. 3; (b) Ala-X-Ser-Cys-Leu-Ala wherein, compared to the corresponding sequence in human calpain I, the amino acids alanine and threonine at positions 122 and 125 are replaced by serine and alanine at positions 88 and 91 of SEQ ID NO. 1 and SEQ ID NO. A gene construct comprising the CAPN6 calpain gene, allelic variants or analogs thereof of claim 1 and which is operably linked to one or more regulatory signals to increase gene expression. The amino acid sequence encoded by the CAPN6 gene, allelic variant or analogue of claim 1. 3; (c) Gly-Tyr-Thr- (His or Tyr) -Thr-X-Thr, wherein compared to the corresponding sequence in human calpain I, the amino acids histidine, alanine and serine at positions 272, 273, and 275 are replaced by tyrosine, threonine, and threonine at positions 252, 253 and 255 of SEQ ID NO. 1 and SEQ ID NO. 3 and in SEQ ID NO. 3, the tyrosine residue at position 274 in calpain I is additionally replaced by histidine at position 254 of SEQ ID NO. 3; 4 4 4 • 4 44 4 4 4 4 9 · • 4 • 9 9 The amino acid sequence of claim 3, which is a sequence of an enzymatically active protein. Method for identifying calpain inhibitors, characterized in that calpain, allelic variants or analogues encoded by the sequence of claim 1 are isolated from tissues or cells, the inhibition of CAPN6 enzyme substrate cleavage by the test substances is measured, and the inhibition is measured in at least one further assay. cleavage of the substrate of the enzymes calpain I and / or calpain II by the test substances, and test substances that inhibit the CAPN6 enzyme and at least one other calpain are selected. Method according to claim 5, characterized in that * 9 • 99 is selected 99 · 9 9 99999 Method according to claim 5, characterized in that test substances are selected which inhibit the enzyme CAPN6 but do not inhibit the enzymes calpain I and / or calpain II. Method according to any one of claims 5 to 7, characterized in that test substances which cross the cell membrane of the cell systems are selected. 9 ♦ 4 · • 4444 9. A method of preparing a CAPN6 enzyme and allelic variants or analogs thereof, comprising cloning at least one copy of the CAPN6 gene sequences, allelic variants or analogs thereof of claim 1 into a vector, and expressing the CAPN6 enzyme, allelic variants or analogs thereof in a vector. a host organism suitable for the vector and subsequent isolation of the enzyme from the host organism. 9 9 « 99 9 test substances which do not inhibit the CAPN6 enzyme but inhibit the enzymes calpain I and / or calpain II. (D) Arg-X-Arg-Asn-Pro-Leu-Gly, wherein this sequence differs from the corresponding sequence in human calpaines I by the amino acid tryptophan in human calpaines I, located at position 298 in calpaines I is replaced by the leucine found at position 286 of SEQ ID NO. 1 and SEQ ID NO. 3, a X in said sequences is any natural amino acid. 9 9 9999 8 9 9 9 9 9 9 9 9 9 9 Method according to claim 9, characterized in that a vector is used which allows the expression of genes, allelic variants or analogs in prokaryotic or eukaryotic cells. The method according to claim 9, characterized in that bacterial cells, fungal cells or animal cells are used as the host organism. 12. The method of claim 9, wherein the vector is a baculovirus and the host organism is an insect cell. 13. Use kalpainového inhibitor, who can be identified in accordance with any of the claims 5 to 8 for drug production for the treatment of diseases in which occurs ke calpain disfunction. Use of a calpain inhibitor according to claim 13 for the manufacture of a medicament for the treatment of diseases selected from the group consisting of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerative or oncological diseases. Use of the amino acid sequence of claim 3 in assay systems. Use of the amino acid sequence of claim 3 for the preparation of antibodies. Use of the gene sequence, allelic variants or analogs thereof according to claim 1 for the preparation of a templar mRNA. Use of the templar mRNA of claim 17 for the manufacture of a medicament for the treatment of diseases in which calpain dysfunction occurs. Use of the calpain gene and allelic variants or analogs thereof according to claim 1 for the diagnosis of a disease or in gene therapy.