CA2226919A1 - Neuroserpin - Google Patents

Abstract

There are described novel neuroserpins of the formulas I or II, including the separate coding and coded sequences of these compounds of the formulas I or II.
These compounds may be used as at least one active compound in a medicament.
The coded peptide sequences of these compounds may be used as targets for the development of pharmaceutical drugs.
The coding nucleotide sequences of these compounds may be used as components of vectors for gene therapeutical applications and for cell engineering.

Description

NEUROSERPIN
FIELD OF THE INVENTION
The present invention is directed to neuroserpins and to a medicament which contains these substances or has an influence on these substances.
DESCRIPTION OF THE PRIOR ART
The structural class of proteins denominated serpins is well known. So far, more than 60 members of the serpin family of proteins have been identified and characterized; see P.A. Patson and P.G.W. Gettins, Thrombosis and Haemostasis 72, pages 166-179 (1994).
SUMMARY OF THE INVENTION
It is an object of the present invention to provide novel neuroserpins, as well as 2o medicaments containing serpins. It is a further object of the present invention to provide novel applications of these substances andlor parts thereof.
The invention is characterized by the characteristics in the independent claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Preferred embodiments are defined in the dependent claims.
Neuroserpin is a newly discovered serine protease inhibitor, which is expressed predominantly in the brain; the expression in the brain takes place nearly exclusively in the neurons.

The newly found neuroserpins - neuroserpin of the human (compound of the formula I)) - neuroserpin of the mouse (compound of the formula II) differ structurally very much from the so far known serine protease inhibitors of the serpin family .
The serpin which is structurally most closely related to the new compounds, namely protease nexin-1 (PN-1 ), has only a 42 % amino acid sequence identity.
The neuroserpins of the human (compound of the formula I) and of the mouse (compound of the formula II) exhibit a very high structural similarity among each other.
i5 The identity of the amino acid sequences of the mature proteins of the compounds of the formulas I or II amounts to 88%.
Both the neuroserpin of the human (compound of the formula I) and the neuroserpin of the mouse (compound of the formula II) have a coding sequence of 1230 nucleotides. The coded peptide in both compounds has a length of 410 amino acids and contains a signal peptide of 16 amino acids. The mature protein in both compounds is composed of 394 amino acids. A particularly high degree of similarity is found in the segment forming the reactive site loop.
The reactive site loop between the amino acid positions 327 and 360 of the two compounds has the following sequence:
P~ P, EVNEEGSEAAAVSGMIAISR MAVLYPQVIVDHPF Partial sequence of the 3o compound of the formula I
EVNEEGSEAAAASGMIAISR MAVLYPQVIVDHPF Partial sequence of the compound of the formula II.

The capital letters represent standard single letter codes for amino acids.
The gap between R and M of the two sequences marks the location of the scissile bond of the reactive site loop. The amino acids denoted P1 and P1', which are flanking the putative scissile bond, are identical in both compounds (P1: Arg346; P1':
Met347).
The above mentioned segment including the P1/P1' site exhibits only one position out of 34 in which the amino acids of the human neuroserpin (compound of the formula I) and the murine neuroserpin (compound of the formula II) are not identical.
The non-identical amino acids are Va1338 and A1a338 and, thus, represent a conservative substitution (printed underscored).
The coded proteins of the compounds of the formulas 1 and II are potent inhibitors of the serine proteases tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and plasmin.
The protease inhibitory function of the inventive compounds is specific. No measurable inhibition of thrombin has been found.
Enzyme kinetic studies with tissue-type plasminogen activator revealed that 2o the inhibitory activity of the inventive compounds is approximately 100 times faster than that of protease nexin-I .
The inventive neuroserpins are unique when compared with the previously known serpins in that they are expressed almost exclusively in neurons.
The expression of the inventive compounds during neural development starts at the beginning of the time range in which restructuration processes of synapses are observed.
3o In the adult nervous system, expression of the inventive compounds is predominant in brain regions in which synapse plasticity occurs.
A particularly high expression of the inventive compounds is found in the cerebral cortex, the hippocampus) and the amygdala of the mouse.

In the deeper structures of the brain, in the brain stem, and in the spinal corcl of the adult mouse, a weaker expression of the inventive compounds is found.
In the adult peripheral nervous system) the inventive compounds are expressed in a subpopulation of the sensory ganglia neurons.
The gene expression pattern of the inventive compounds in the brain is extremely interesting, because these molecules are expressed in the adult nervous system predominantly in neurons of those regions that are thought to play an 1o important role in learning and in memory functions.
The gene expression pattern of the inventive compounds in the cerebral cortex is extremely interesting, because a reduction of the cellular differentiation in the cerebral cortex has been found to be associated with schizophrenia.
Another prominent characteristic of the inventive compounds consists therein that they are secreted by neurons.
This fact - together with the function as a protease inhibitor and the 2o expression pattern in the developing and adult brain - suggests that the inventive compounds play a role in the regulation of the extracellular proteolysis in brain areas which are involved in the processing and storage of learned behaviors, teamed emotions, or memory contents.
Together with the recently found evidence for a role of extracellular proteases, in particular tissue-type plasminogen activator, in neural plasticity (see Frey et al.) J. Neurosci. 16, pages 2057-2063, 1996; Huang et al., Proc. Natl.
Acad.
Sci. USA 93 pages 699-704, 1996), the expression pattern allows the assumption that the protease inhibitory activity of neuroserpin has a role in teaming and memory operations, for example operations which are involved in the processing and storage of learned behaviors, learned emotions, or memory contents.
The fact that neuroserpin is a potent inhibitor of tissue-type plasminogen activator (tPA) is particularly interesting) because tPA has been found to play a role in the pathogenesis of neuronal cell damage or neuronal cell death in the context of excitotoxin-induced epileptic seizures (see Tsirka et al., Nature 377. pages 340-344) 1995).
The gene expression pattern of the inventive compounds in the spinal cord 5 ~ and in the sensory ganglia is interesting, because these molecules are expressed in the adult nervous system in neurons of those brain regions that are thought to play a role in the processing of pain, as well as in the pathogenesis of pathological pain.
The inventive compounds were found in connection with a study aimed at to discovering proteins that are secreted from axons of neurons (see Stoeckli et al., Eur. J. Biochem. 180. pages 249-259, 1989). Using a combination of micromethods of protein purification, including isoelectric focusing, followed by hydrophobic interaction chromatography and preparative sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)) a novel protein, termed chicken neuroserpin thereafter, was isolated from the ocular vitreous fluid of the chicken embryo (see Osterwalder et al., EMBO J. 15, pages 2944, 1996). The purified chicken neuroserpin was successfully used to obtain partial amino acid sequence information by means of conventional solid-phase sequencing. Based on the amino acid sequence information, primer oligonucleotides were designed and synthesized, 2o which allowed the amplification of a complementary deoxyribonucleic acid (cDNA) fragment by conventional polymerase chain reaction (PCR). As a template for the PCR, first strand cDNA, generated by means of a reversed transcriptase from mRNA
from the retina of 14 days old chicken embryos, was used. The amplified cDNA
fragment was successfully used as a probe for the isolation of a full length cDNA
clone from a cDNA library. By conventional DNA sequencing of the cDNA of chicken neuroserpin the complete nucleotide sequence and the amino acid sequence deduced therefrom was obtained.
The cDNA of the compound of the formula I was cloned using conventional 3o PCR and library screening techniques based on its pronounced similarity with chicken neuroserpin. In a first step, a fragment of the cDNA was amplified by means of PCR using the DNA from cDNA libraries of human fetal brain and human fetal retina as template. The primer oligonucleotides used were designed and synthesized according to the known sequence of chicken neuroserpin. The cDNA fragment obtained by PCR was used as probe for the isolation of the complete cDNA of the compound of the formula I by the screening of two commercially available cDNA
libraries, one from human fetal brain (17n' to 18"' week of pregnancy) and one from human fetal retina. A full length cDNA clone was isolated from the human fetal retina cDNA library and the nucleic acid sequence was determined by conventional sequencing techniques. Clones from the human fetal brain cDNA library confirmed the sequence obtained.
The compound of the formula II was cloned using conventional PCR and library screening techniques based on its pronounced similarity with the chicken io neuroserpin. In a first step, total RNA from brains of ten days old mice was prepared.
Using the total RNA as template, first strand cDNA was synthesized using reverse transcriptase, and the resulting product was used as a template for PCR. The primer oligonucleotides used for the PCR were designed and synthesized according to the known sequences of chicken neuroserpin. The cDNA fragment obtained by PCR was used as probe for the isolation of the complete cDNA of the compound of the formula II by the screening a commercially available cDNA library from postnatal day mouse brain.
This procedure for the cloning can also be used for the isolation of the 2o homologous compounds of other species, such as rat, rabbit) guinea pig, cow, sheep, pig, primates) birds, zebra fish (Brachydanio rerio), Drosophila melanogaster, Caenorhabditis elegans etc.
The coding nucleotide sequences can be used for the production of proteins with the coded amino acid sequences of the compounds of the formulas I or II.
The coding sequences of the compounds of the formulas I or II can be used as starting compounds for the discovery and the isolation of alleles of the compounds of the formulas I or II. Both the polymerase chain reaction and the 3o nucleic acid hybridization can be used for this purpose.
The coding sequences of the compounds of the formulas I or II can be used as starting compounds for the discovery and the isolation of splice variants of the compounds of the formulas I or II. Both the polymerase chain reaction and the nucleic acid hybridization can be used for this purpose.

The coding sequences of the compounds of the formulas I or II can be used as starting compounds for so-called "site-directed mutagenesis", in order to generate nucleotide sequences encoding the proteins that are defined by the compounds of the formulas I or II, or parts thereof, but whose nucleotide sequence is degenerated with respect to the compounds of the formulas I or II due to use of alternative codons.
The coding sequences of the compounds of the formulas I or II can be used 1o as starting compounds for the production of sequence variants exhibiting altered function by means of so-called site-directed mutagenesis.
The coding sequences can be used for the production of vectors for use in gene therapy and cell engineering.
The coding sequences can be used for the generation of transgenic animals overexpressing the coding and the coded sequences of the compounds of the formulas I or II.
2o The coding sequences can be used as probes for the isolation of the genes corresponding to the compounds of the formulas I or II. Both polymerase chain reaction and nucleic acid hybridization can be used for this purpose.
The coding sequences can be used for the diagnostics of disorders in the gene corresponding to the compound of the formula I.
The coding sequence can be used as a starting compound for gene technological modifications aimed at the production of medicaments or gene therapy vectors which exhibit changed properties as compared to the properties of the 3o compounds of the formulas I or II.
The coded sequences can be used as medicaments.
The coded sequences can be used as antigens for the production of antibodies.

g The coded sequences can be used as targets for drug development.
The coded sequences can be used for the spatial structure determination, for example by X-ray crystallography or nuclear magnetic resonance spectroscopy.
The coded sequence can be used for the computerized prediction of the protein structure.
See also Schrimpf et al., "Human Neuroserpin (P112):
cDNA Cloning and Chromosomal Localization to 3q26", Genomics, 40(1):55-52, 1997; Krueger et al, Developing and Adult Nervous System of the Mouse", The Journal of Neurc~sClence, 17(23:8984-8996, 1997; and Osterwalder et al, "The Axonally Secreted Serine Proteinase inhibitor, Neuroserpin, Inhibits Plasminogen Activators and Plasmin but Not Thrombin", Journal of Biological Chemistry, 273(4):2312-2321, 1998.
The following examples illustrate the present invention.

Example 1:
Cloning of the cDNA of the compound of the formula I (neuroserpin of the human) The cloning of the cDNA of the compound of the formula I was carried out basing on the nucleotide sequence of the chicken neuroserpin. As a first step, a fragment of the compound of the formula I was amplified using the polymerase chain reaction (PCR). As a matrix we used the DNA obtained from a cDNA library from the brain of a human fetus (17~' -18"' week of pregnancy) which is commercially available (oligo(dT)- and random-primed) human fetal brain cDNA library in the Lambda ZAP II
vector, cat. no. 936206, Stratagene). For the realization of the polymerase chain reaction two primers in the reading direction and two primers in the counter direction were designed and synthesized based on the nucleotide sequences of chicken >5 neuroserpin. To facilitate subsequent cloning, two of the synthetic PCR
primers contained restriction sites for BamHl at the 5' end.
In the reading direction (sense primers):
A: 5'-GCIATITA(CIT)TT(C/T)AA(AIG)GGIAA(T/C)TGGAA-3' 2o and B: 5'-GGGGGATCCGA(AIG)ACIGA(A/G)GTICA(A/G)ATICCIATGATG-3' In the counter direction (antisense primers):
C: 5'-CCCAT(A/G)AAIA(A/G)IACIGTICCNGT-3' and D: 5'-GGGGGATCCGG(AIG)TG(A/G)TCIACIATIAC(C/T)TGNGG-3' The PCR was Gamed out under standard conditions using the DNA
polymerase Amplitaq (Perkin Elmer) according to the recommendations of the producer. The PCR was carried out in two steps. In the first step, amplification was 3o carried out by means of the two outer primers (primer A and primer C). in the second step, amplification was carried out by means of the two inner primers (primer B and primer D). The amplified inner fragment had a length of 517 base pairs. It was cut with the appropriate restriction enzyme, and the resulting fragment of 436 base pairs was inserted into the Bluescript vector (Bluescript SK(-), Stratagene).
10° Lambda plaques of a human fetal retina cDNA library (Stratagene; cat. No. 937202) were screened under highly stringent conditions (Sambrook et al.) Molecular Cloning: A
laboratory manual, Cold Spring Harbor Laboratory Press, 1989), and several positive clones were found and isolated.
s From the positive Lambda Uni-ZAP XR phagemid clones the corresponding cDNA fragments were excised using the standard in vivo excision protocol recommended by the supplier (Stratagene). The resulting Bluescript plasmids were digested with EcoRl and Xhol in order to determine the length of the inserted segment. The longest inserts were subjected to nucleotide sequence determination.
1o The sequencing was carried out by means of the dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 77 pages 2163-2167, 1977), using the enzyme Sequenase 2.0 (USB). The computerized analysis of the sequences was carried out by means of the program package of the Genetics Computer Group (GCG, version 7, Unix, Silicon Graphics, Inc.).
is In this way the nucleotide sequence over the full length of the cDNA of 1577 base pairs was obtained.
With the described procedure for PCR cloning it is possible to find and to isolate also 2o variant forms of the compounds of the formula I, as for example their alleles or their splice variants. The described procedure for the screening of a cDNA library allows also the discovery and the isolation of compounds which hybridize under stringent conditions with the coding sequences of the compounds of the formula I.

Example 2:
cDNA cloning of the compound of the formula II ~(neuroserpin of the mouse}
The cloning of the cDNA of the component of the formula II was carried out based on the nucleotide sequences of chicken neuroserpin. As a first step, a fragment of the compound of the formula II was amplified by PCR. As a matrix, mRNA from the brain of 10 day old mice was used. Total RNA was isolated from the brains of 10 day old mice (ICR-ZUR) according to the method of Chomczynski and to Sacchi (Anal. Biochem. 1~2, pages 156-159,1987). The production of single stranded cDNA was carried out using oligo(dT) primer and a RNA-dependent DNA
polymerase (Superscript RNase H--Reverse Transcriptase; Gibco BRl-, Gaithersburg, MD) according to the instruction of the supplier. For the realization of the polymerase chain reaction two primers in the reading direction and two primers in the counter direction were designed and synthesized based on the nucleotide sequences of chicken neuroserpin. To facilitate subsequent cloning, two of the primers contained restriction sites for BamHl at their 5' ends.
The following primers were used:
In the reading direction (sense primers):
A: 5'-GCIATITA(Cll7TT(C/T}AA(A/G)GGIAA(T/C}TGGAA-3' and B: 5'-GGGGGATCCGA(A/G)ACIGA(A/G)GTICA(A/G)ATICCIATGATG-3' In the counter direction (antisense primers):
C: 5'-CCCAT(A/G)AAIA(A/G)IACIGTICCNGT-3' and D: 5'-GGGGGATCCGG(A/G)TG(A/a)TCIACIATIAC(CIT)TGNGG-3' 3o The polymerase chain reaction was carried out under standard conditions using the DNA polymerase AmpIiTaq (Perkin Elmer) according to the recommendations of the producer. The following PCR profile was employed:
93°C for 3 minutes, followed by 35 cycles of 93°C for 1 minute, 50°C for 2 minutes, and 70°C
for 2 minutes. Following the last cycle, the incubation was continued at 72°C for further 10 minutes.

The PCR was carried out in two steps. In the first step, amplification was carried out by means of the two outer primers (primer A and primer C). In the second step) amplification was carried out by means of the two inner primers (primer B and primer D). The amplified inner fragment had a length of 517 base pairs. It was cut with appropriate restriction enzymes, and the resulting fragment of 436 base pairs was inserted into the Bluescript vector (Bluescript SK(-), Stratagene). The amplified fragment was used for the screening of a Lambda cDNA library from the brain of days old mice (Uni ZAP XR) Cat. Nr. 937319, Stratagene). 2x108 Lambda plaques 1o were screened under highly stringent conditions (Sambrook et al., Molecular Cloning:
A laboratory manual, Cold Spring Harbor Laboratory Press, 1989), and 27 positive clones were found. Therefrom, 16 clones were isolated and analyzed.
From the positive Lambda Uni-ZAP XR phagemid clones the corresponding cDNA fragments were excised using the standard in vivo excision protocol recommended by the supplier (Stratagene). The resulting Bluescript plasmids were digested with EcoRl and Xhol in order to determine the length of the inserted segment. The longest inserts were subjected to nucleotide sequence determination.
The sequencing was carried out by means of the dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 77 pages 2163-2167, 1977), using the enzyme Sequenase 2.0 (USB). The computerized analysis of the sequences was carried out by means of the program package of the Genetics Computer Group (GCG, version 7, Unix, Silicon Graphics, Inc.).
In this way the nucleotide sequence over the full length of the cDNA of 2944 base pairs was obtained.
With the described procedure for PCR cloning it is possible to find and to isolate also variant forms of the compounds of the formula II, as for example their 3o alleles or their splice variants. The described procedure for the screening of a cDNA
library allows also the discovery and the isolation of compounds which hybridize under stringent conditions with the coding sequences of the compounds of the formula II.

Example 3:
Production of the coded protein of the compounds of the formulas I and II in a procaryotic expression stem The production of the coded protein of the compounds of the formulas I and II can be carried out in procaryotic and eucaryotic expression systems. In the following part, the production of the coded protein of the compound of the formula I
(human neuroserpin) in a procaryotic expression system is described.
The compound of the formula I was cytoplasmically expressed in E. coli with a stretch of six histidines fused to the carboxyterminus of the protein. A
fragment of the cDNA encoding amino acids 1 through 394 was amplified .in a PCR using the oligodeoxynucleotide primers 5'-AAT TTC TAG AGA AAG GAG ATA CAT ATG ACA
GGG GCC ACT TTC CCT-3' and 5'-GGG AAG CTT CTA GTG GTG ATG GTG GTG
GTG AAG TTC TTC GAA ATC ATG GTC C-3'. The cDNA fragment was cloned into the vector pAK400 (see Krebber et al., J. Immunol. Methods 201. pages 35-55, 1997) via the Xbal and Hindlll sites of the vector, allowing expression of the cDNA
from the lac operator/promoter located immediately upstream. For expression, a 2o colony of E. coli strain BL21 DE3 harboring the expression plasmid was precultured overnight at 37 °C in 100 ml LB medium containing 30 Ng/ml chloramphenicol. After inoculation of the same medium with the preculture, bacteria were grown at 25 °C
and induced with 1 mM Isopropyl-1-thio-b-D-galactosidase (IPTG) at an ODD of 0.5.
The bacteria were harvested by centrifugation 6 hrs after induction, resuspended in 25 Ni-NTA-binding buffer (1 M NaCI, 50 mM TrisCl pH8.0), and disrupted in a French press. The soluble protein extract was incubated over night at 4 °C
with 0.4 ml of Ni NTA resin (Qiagen, Chatsworth, CA). Following extensive washing with Ni-NTA
binding buffer, bound proteins were eluted with Ni-NTA-binding buffer containing 200 mM imidazole. The eluted protein was dialyzed against PBS and immediately frozen 30 at-80 °C.

Example 4:
Production of the coded protein of the compound of the formula II in a eucarxotic expression system.
The protein of the compound of the formula II was recombinantly expressed in human embryonic kidney cells (cell line 293) either in its unaltered form or, for single-step purification by metal chelate chromatography, fused carboxyterminally to a tag of six consecutive histidines. For heterologous expression of the unaltered form io of neuroserpin, a Spe I-Ssp I fragment from the lambda phage cDNA clone mmns 4.1, containing the full length open reading frame of mouse neuroserpin, 111 by of 5' untranslated region) and 100 by of 3' untranslated region, was cloned into the expression vector pcDNA3.1 (-)MycHisA (Invitrogen, Carlsbad, CA). The construct was electroporated and the cells were subsequently selected for neomycin resistance. Surviving cell clones were tested for expression of neuroserpin by a dot blot assay.
For heterologous expression of neuroserpin containing a carboxyterminal polyhistidine tag, the mouse neuroserpin cDNA was amplified in a polymerase chain 2o reaction using the oligonucleotides 5' -GC TCT AGA CAT ATG ACA GGG GCA ACG
TTC CCA-3' (5', sense) and 5'-GGG AAG CTT CTA GTG GTG ATG GTG GTG GTG
AAG TTC CTC AAA GTC ATG GC-3' (3') antisense, encoding an additional segment of six consecutive histidines). The entire amplification product was sequenced to exclude any polymerase chain reaction errors and a Sty I - Hind III fragment of the amplification product was used to replace a Sty I - Hind III fragment from the expression construct containing the unaltered form of mouse neuroserpin.
Transfection of the construct into 293 cells and subsequent selection of positive clones were done as described above.

Example 5:
Determination of the protease inhibitory function of the compounds of the formulas I
and II

a) Enzymes, inhibitors, and substrates Human urokinase-type plasminogen activator (100,000-300,000 ploughU/mg, Sigma U-8627)) porcine plasmin (3-5 U/mg, Sigma, P-8644) and human thrombin (50-100 NIHU/mg, Sigma, T-4648) were purchased from Sigma, St. Louis, MO.
io Human tissue-type plasminogen activator was initially from Sigma (400,000 IU/mg, Sigma T-7776} and at a later stage of the experiments from Genentech) South San Francisco, CA (ActivaseO, recombinant Allteplase, 580,000 IU/mg). The enzyme substrate S-2288 (H-D-Ile-Pro-Arg-para-nitroanilide), purchased from Chromogenix (Molndal, SV), was dissolved in water to a concentration of 25 mg/ml. Active enzyme 15 concentrations were determined by measuring the amidolytic activity of the proteinases in the presence of 1 mM S-2288, using values for substrate turnover of AA,~ = 0.275 min-'cm', 0.031 min-'cm', 0.030 min-'cm'', and 0.042 min''crri') for 4 nM
of thrombin, uPA, single-chain tPA, and plasmin) respectively, as indicated by the supplier of the substrate. Recombinant protein of the compounds of the formula I or II
2o was stored frozen, and thawed immediately before use. Protein concentrations were measured using amino acid analysis on an Aminoquant II equipped with the fluorescence detector 1046A (Hewlett-Packard, Palo Alto, CA). The concentrations of the compound of the formulas I and II were estimated by SDS-PAGE and silver staining. The concentration of the His-tagged from the comppounds of the formular I
or II was estimated using the Bradford protein assay (Bio-Rad, Glattbrugg, Switzerland) in combination with densitometric analysis of SDS-Polyacrylamid gels stained with Coomassie brilliant blue. Recombinant PN-1 (active concentration 1.2 mg/ml) was provided by Dr. D. Monard , Friedrich Miescher Institute, Basel, Switzerland and was stored frozen until use. Chemicals for reaction buffers were purchased from Sigma, if not indicated otherwise. For complex formation assays, a complexation buffer containing 67 mM Tris-HCI, pH 8.0, 133 mM NaCI, and 0.13%

PEG 8000 was used. Inhibition buffer contained 10 mM phosphate buffer, pH 7.2, 140 mM NaCI, 4 mM KCI, 0.1 % PEG 8000; and 0.2 mg/ml bovine serum albumin (BSA; from Serva, Heidelberg, D). Coating solution contained 1 % BSA, 0.5% w/v PEG 8000, and 0.01 % v/v Triton X-100.
b) Complex formation assays In an Eppendorf reaction tube, proteinases and inhibitors were mixed in 30 NI
of complexation buffer and incubated for 30 min at 37 °C. The reaction was stopped by adding an equal volume of two-fold concentrated sample buffer for SDS-PAGE
(containing 6% SDS, 10% f3-mercaptoethanol, 30% glycerol, 31.3 mM Tris-HCI, pH
6.8) and by immediately boiling the sample for 5 min. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Laemmli (see Laemmli, Nature 227. pages 680-685, 1970). Electrotransfer of the proteins onto nitrocellulose (Schleicher & Schuell, Dassel, D) was carried out according to Towbin et al. (see Towbin et al.) Proc. Natl. Acad. Sci. USA 76 4354, 1979) at 30 V for 16 h or at 100 V for 1-2 h at 4 °C.
Immunodetection of neuroserpin was performed using the polyclonal antisera R35 or R61, and the BM
Chemiluminescence Western blotting kit (Boehringer, Mannheim) D) according to the supplier's recommendations, or goat anti rabbit IgG conjugated to peroxidase (Bio-2o Science products, Emmenbrucke, CH) at a dilution of 1I1,000.

d) Amidolytic assays Enzyme inhibition was determined by mixing enzyme and inhibitor in a 96 well plate in 98 NI of inhibition buffer. The final concentrations of enzymes were as follows: uPA, 9.1 nM; tPA, 7.9 nM; thrombin, 18.8 nM; plasmin, 12.7 nM. The concentrations of the inhibitors were 150 nM. The compound of the formula I or II
was incubated with the respective proteases for 10 min at room temperature.
After preincubation, the amidolytic reactions were started simultaneously by adding 2 NI of substrate solution (25 mg/ml S-2288) to each well. Residual amidolytic activity was determined by measuring the hydrolysis over time (velocity) using an ELISA
reader io (Dynatech, Denkendorf) D).
e) Determination of kinetic parameters The kinetics of the interaction between the proteins of the compounds of the formulas I or II and tPA, uPA) or plasmin, was determined by the progress curve is method (see Morrison and Walsh, Adv. Enzymol. Relat. Areas Mol. Biol. 61 pages 201-301, 1988). Reactions were started by adding a constant) catalytic amount of enzyme (tPA) 1.6 nM; uPA, 3.6 nM; plasmin, 1.4 nM) to the inhibition buffer containing a fixed substrate concentration (1.08 mM S-2288) and variable inhibitor concentrations (ranging from 4.6 nM to 46.8 nM), preincubated at 37~1 °C. Tight-2o binding conditions were avoided by using sufficiently high substrate and inhibitor concentrations. Because the interaction between serpins and serine proteinases is assumed to follow slow-binding kinetics, product formation was described in the following equation [P] = vst + (vs - vs ) (1- e-k~' ) / k'+d 25 where v, and uZ represent the velocities at steady-state and at zero-time, respectively, k' represents the apparent first-order rate constant for approach to the steady-state and d is a displacement factor compensating for small uncertainties in absorbance at the start of the reaction. For each of several inhibitor concentrations, vs, vZ, k' and d were determined by fitting the above equation to the data sampled from progress curves. The association and dissociation constants were determined from the following relationship:
k' k~ + 1 + [S ] / Km [I ]
where Km of S-2288 was 3 x 10~ M, 2 x 10~ M, 1 x 10-' M) and 9 x 10-3 M for thrombin, uPA, single-chain tPA, and plasmin, respectively, as indicated by the supplier. An absorption coefficient e,,~ = 10'500 M-'cm' for the released para-nitroaniline was used to determine the product concentrations.
In this way it was found that neuroserpins are potent inhibitors of tissue-type to plasminogen activator, urokinase-type plasminogen activator, and, to a lesser extent, plasmin. In contrast, no significant inhibition of thrombin was found. The kinetic measurements determined that the association of tPA with neuroserpin occurs approximately 100 times faster than the association of tPA with protease nexin-1.
Example 6:
Determination of the aene a ~ression pattern of the compound of the formula I
I
2o The gene expression pattern of the compound of the formula II was determined by visualization of the mRNA in frozen sections.
In situ hybridization was carried out essentially as described previously (Schaeren-Wiemers and Gerfin-Moser, Histochemistry 100. pages 431-440, 1993).
Briefly, tissues were quickly removed from ICR-ZUR mice killed by asphyxiation with C02 and immediately frozen in a bed of pulverized dry ice. To obtain mouse embryos of determined gestational age, the onset of pregnancy was determined by the appearance of a vaginal plug and counted as embryonic day 0 (EO).
Sacrificed embryos were checked for correct gestational age employing the criteria established 3o by Theiler (see Theiler, The house mouse: Atlas of Embryonic Development, Springer, New York, 1989}. For postnatal mice, the day of birth was taken as P0.
Cryosections were cut at 12 - 20 Nm and thaw mounted on poly-L-lysine coated slides, fixed in PBS containing 4% paraformaldehyde and acetylated with acetic anhydride. Following prehybridization in hybridization buffer containing 5x SSC, 50%
formamide, 5x Denhardt's solution, 250 Ng/ml total yeast RNA, and 500 Ng/ml DNA
from herring sperm, hybridization was carried out at 55°C overnight using approximately 0.25 Ng/ml digoxigenin-labeled riboprobes diluted in hybridization buffer. Sections were then subjected to low (2x SSC) and high (0.1 x SSC / 50%
formamide at 55°C) stringency washes. Hybridized riboprobe was detected employing an AP-coupled anti-digoxigenin antibody (Boehringer) and the AP
substrates nitrotetrazolium blue and X-phosphate (Boehringer). As a control for the specificity of the labeling, in each hybridization experiment sections adjacent to those 1o hybridized with the antisense neuroserpin cRNA were incubated with an equal concentration of a sense riboprobe transcribed from the same template. Control sections showed no staining except for strong labeling in the intestinal mucosa of embryos older than E15) which was probably due to endogenous intestinal AP. In addition, we have carried out in situ hybridization with four different antisense i5 riboprobes from non-overlapping regions of the mouse neuroserpin cDNA (nt 1 -365, nt 464 - 788, nt 788 - 1211, and nt 2037 - 2395). The staining obtained with these riboprobes was qualitatively identical to that obtained with the antisense riboprobe transcribed from the full length cDNA.
2o In this way it was shown that the onset of the gene expression of the compound of the formula II in the developing mouse nervous system occurs relatively late and coincides with synapse reorganization processes. In the central nervous system of the adult mouse, the compound of the formula II is expressed predominantly in areas which are involved on neural plasticity, such as the cerebral 25 cortex) the hippocampus, and the amygdala. Lower expression levels were observed in various neuronal subpopulations of the deeper brain structures) as well as in the brain stem and the spinal cord.

Example 7:
Determination of the chromosomal localization of the gene corresponding to the com~~ound of the formula I.
s The chromosomal localization of the gene corresponding to the compound of the formula I was determined by fluorescence in situ hybridization (FISH).
Metaphase chromosome spreads of a healthy donor were prepared from peripheral blood lymphocytes by standard cytogenetic procedures. FISH was performed essentially as io described (see Wiegant et al., Genomics 10. pages 345-349, 1991 ). The purified 1.6 kb insert of the human neuroserpin full length cDNA clone and a genomic clone (with an approximate insert size of 15 kb) were labeled either with biotin-14-dUTP
(Bio Nick Labeling System, Gibco BRL, Life Technologies) Gaithersburg, MD) or with digoxigenin-11-dUTP (Boehringer Mannheim). Biotin-labeled cDNA was detected by 15 successive application of avidin-fluorescein-isothiocyanate (FITC) and biotinylated anti-avidin (Vector Labs., Burlingame, CA). Digoxigenin-labeled genomic sequence was detected with an anti-digoxigenin Fab fragment conjugated to rhodamine (Boehringer Mannheim). Slides mounted in antifadant solution (Vectashield, Vector Labs.) were counterstained either with PI/DAPI or DAPI (propidium iodide/4',6-2o diamidino-2-phenylindole), respectively. A Zeiss Axioplan epifluorescence microscope was used for conventional fluorescence microscopy. Images were recorded by Photometrics CCD ICAF camera (Tuscon, AZ) controlled with Smart Capture imaging software (Vysis, Inc., Framingham, MA).
In this way it was found that the gene corresonding to the compound of the formula I in the human is located in the region q26 of chromosome 3.
In the following part statements concerning the compounds of the formulas 1 or II are given:

2226919.seq SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT:
SONDERGGER, Peter (ii) TITLE
OF
INVENTION:
NEUROSERPIN

(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Institute of Biochemistry, University of Zurich, (B) STREET: Wintherthurerstrasse 190, (C) CITY: Zurich (E) COUNTRY: SWITZERLAND

(F) ZIP: CH-8057 (v) COMPUTER READABLE FORM:

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

(D) SOFTWARE: TXT ASCII

(vi) CURRENTAPPLICATION DATA:

(A) APPLICATION NUMBER: 2,226,919 (B) FILING DATE: 1998/02/13 (C) CLASSIFICATION: 06C12N-00015/15 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT
INFORMATION:

(A) NAME:

(B) REGISTRATION NUMBER:

(C) REFERENCE/DOCKET NUMBER:

(ix) TELECOMMUNICATION
INFORMATION:

(A) TELEPHONE:

(B) TELEFAX:

(2) INFORMATION FOR SEQ ID NO: 1: Neuroserpin of the Human (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1577 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to m-RNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapiens (D) DEVELOPMENT STAGE: fetal (F) TISSUE TYPE: retina 2226919.seq (vii) IMMEDIATE SOURCE:
(A) LIBRARY: oligo(dT)-primed human fetal retina cDNA

library in the lambda Uni-ZAP XR vector catalog Nr. 937202, Stratagene, La Jolla, CA, USA

(vii) IMMEDIATE
SOURCE:

(A) LIBRARY: oligo(dT)-and random-primed human fetal brain (17-18 weeks of gestation) cDNA library in the lambda Uni-ZAP XR vector catalog Nr. 936206, Stratagene, La Jolla, CA, USA

(B) CLONE:
IVb (viii}POSITION GENOME:

(A) CHROMOSOME/SEGMENT:
Chromosome 3 / Segment q26 (ix) FEATURE:

(A) NAME/KEY:signal peptide (B) LOCATION:82.. 129 (ix} FEATURE:

(A) NAME/KEY:mature peptide (B) LOCATION:130.. 13l1 (ix) FEATURE:

(A) NAME/KEY:coding sequence (B} LOCATION:82.. I311 (ix) FEATURE:

(A) NAME/KEY:reactive site loop (B) LOCATION:l108.. 1209 a mino acid P1: 1165.. 1l67 a mino acid P1': 1168.. 1170 (ix) FEATURE:
(A) NAME/KEY: inhibiting segment (B) LOCATION: 1108.. 1209 amino acid Pl: l165.. 1167 amino acid P1': 1l68.. 1170 2226919.seq (ix) FEATURE

(A) NAME/KEY: polyAsignal (B) LOCATION: 1535..1540 (ix) FEATURE

(A) NAME/KEY: polyAsegment (B) LOCATION: 1560..1577 (ix) FEATURE

(A) NAME/KEY: 3'UTR

(B) LOCATION: 1312..1559 (ix) FEATURE

(A) NAME/KEY: 5'UTR

(B) LOCATION: 1..

(xi) SEQUENCE DESCRIPTION: 1:
SEQ
ID
NO

GCGGAGCACA AGCATCCCGT CAGGGGTTGC

GTCCGCCGAG AGGTGTGTGG
CACAAGCTCC

GAGGCTTGAA TTC GGA CTC TTC TCT TTGCTG 11l ACTGTTACAA CTT
T
ATG
GCT

Met Ala Phe Gly Leu Phe Ser LeuLeu Leu GGG ACT

Val Leu Gln SerMet Ala Thr Ala Phe Pro Glu Glu AlaIle Gly Thr TAT CGT

Ala Asp Leu SerVal Asn Met Asn Leu Arg Ala Thr GlyGIu Tyr Arg CCA AGT

Asp Glu Asn IleLeu Phe Ser Leu ale Ala Leu Ala MetGly Pro Ser GGA ACC

Met Met Glu LeuGly Ala Gln Ser Gln Lys Glu Ile ArgHis Gly Thr AAA GGT

Ser Met Gly TyrAsp Ser Leu Asn Glu Glu Phe Ser PheLeu Lys Gly ACT AAA

Lys Glu Phe SerAsn Met Val Ala Glu Ser Gln Tyr ValMet Thr Lys 2226919.seq Lys Ile Ala Asn Ser Leu Phe Val Gln Asn Gly Phe His Val Asn Glu Glu Phe Leu Gln Met Met Lys Lys Tyr Phe Asn Ala Ala Val Asn His Val Asp Phe Ser Gln Asn Val Ala Val Ala Asn Tyr Ile Asn Lys Trp Val Glu Asn Asn Thr Asn Asn Leu Val Lys Asp Leu Val Ser Pro Arg Asp Phe Asp Ala Ala Thr Tyr Leu Ala Leu Ile Asn Ala Val Tyr Phe Lys Gly Asn Trp Lys Ser Gln Phe Arg Pro Glu Asn Thr Arg Thr Phe Ser Phe Thr Lys Asp Asp Glu Ser Glu Val Gln Ile Pro Met Met Tyr Gln Gln Gly Glu Phe Tyr Tyr Gly Glu Phe Ser Asp Gly Ser Asn Glu Ala Gly Gly Ile Tyr Gln Val Leu Glu Ile Pro Tyr Glu Gly Asp Glu Ile Ser Met Met Leu Val Leu Ser Arg Gln Glu Val Pro Leu Ala Thr Leu Glu Pro Leu Val Lys Ala Gln Leu Val Glu Glu Trp Ala Asn Ser Val Lys Lys Gln Lys Val Glu Val Tyr Leu Pro Arg Phe Thr Val Glu 2226919.seq CAG GAA ATT GAT TTA AAA GAT GTT TTG AAG GCT CTT GGA ATA ACT GAA l023 Gln Glu Ile Asp Leu Lys Asp Val Leu Lys Ala Leu Gly Ile Thr Glu Ile Phe Ile Lys Asp Ala Asn Leu Thr Gly Leu Ser Asp Asn Lys Glu Ile Phe Leu Ser Lys Ala Ile His Lys Ser Phe Leu Glu Val Asn Glu GAA GGC TCA GAA GCT GCT GCT GTC TCA GGA ATG ATT GCA ATT AGT AGG 1l67 Glu Gly Ser Glu Ala Ala Ala Val Ser Gly Met Ile Ala Ile Ser Arg Met Ala Val Leu Tyr Pro Gln Val Ile Val Asp His Pro Phe Phe Phe CTT ATC AGA AAC AGG AGA ACT GGT ACA ATT CTA TTC ATG GGA CGA GTC l263 Leu Ile Arg Asn Arg Arg Thr Gly Thr Ile Leu Phe Met Gly Arg Val ATG CAT CCT GAA ACA ATG AAC ACA AGT GGA CAT GAT TTC GAA GAA CTT 13l1 Met His Pro Glu Thr Met Asn Thr Ser Gly His Asp Phe Glu Glu Leu GTATATATTT AGGATTTGTG TTTTACAGTA TATCTTAAGA TAATATTTAA AATAGTTCCA l431 GATAA.AAACA ATATATGTAA ATTATAAGTA ACTTGTCAAG GAATGTTATC AGTATTAAGC 1491 AACAT GT GAA AAAAA.P. 15 7 7 (2) INFORMATION FOR SEQ ID N0: 2: Neuroserpin of the Mouse (Mus musculus) (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2944 base pairs (B) TYPE: nucleid acid 2226919.seq (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULE TYPE: c-DNA to m-RNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus (D) DEVELOPMENT STAGE: postnatal day 20 (F) TISSUE TYPE: brain (vii) IMMEDIATE SOURCE:
(A) LIBRARY: oligo(dT)-primed mouse brain cDNA
library in the lambda Uni-ZAP-XR vector, from Balb c mice, postnatal day 20, catalog Nr. 937319, Stratagene, La Jolla, CA, USA
(C) CLONE: mmns 4.1 (ix) FEATURE:
(A) NAME/KEY: signal peptide (B) LOCATION: 98.. 145 (ix) FEATURE:
(A) NAME/KEY: mature peptide (B) LOCATION: Z46.. 1327 (ix) FEATURE:
(A) NAME/KEY: coding sequence (B) LOCATION: 98.. 1327 (ix) FEATURE:
(A) NAME/KEY: reactive site loop (B) LOCATION: 1124.. 1225 amino acid P1: 1181.. 1183 amino acid P1': 1184.. 1185 (ix) FEATURE:
(A) NAME/KEY: inhibiting segment (B) LOCATION: 1124.. 1225 amino acid P1: 1181.. 1183 amino acid P1': l184.. 1186 (ix) FEATURE:

2226919.seq (A) NAME/KEY: polyAsignal (B) LOCATION: l551..1556 2910 and 2905..

(ix) FEATURE:

(A) NAME/KEY: polyAsegment (B) LOCATION: 2930..2944 (ix) FEATURE:

(A) NAME/KEY: 3'UTR

(B) LOCATION: 1328..2929 (ix) FEATURE:

(A) NAME/KEY: 5'UTR

(B) LOCATION: 1..

(xi) SEQUENCE DESCRIPTION: 2:
SEQ ID NO

CGGCACGAGA TCTCCAGCAT CCCGAGCGGG

TCCGGAGCAG
TCTCAGCCTG
CCCAGCATCC

GTGAAGGAGA
CTTGAAACCA
TCCCATC
ATG
ACT
TAC
CTT
GAA
CTG

Met Thr Tyr Leu Glu Leu AGT GTG

Leu AlaLeu LeuAla Leu Gln Val Thr Gly Ala ThrPhe Pro Ser Val TCA AAC

Asp GluThr IleThr Glu Trp Val Met Tyr Asn HisLeu Arg Ser Asn ATT TTC

Gly ThrGly GluAsp Glu Asn Leu Ser Pro Leu SerIle Ala Ile Phe CTT GCT

Leu AlaMet GlyMet Met Glu Gly Gln Gly Ser ThrArg Lys Leu Ala TAT GGT

Glu IleArg HisSer Met Gly Glu Leu Lys Gly GlyGlu Glu Tyr Gly TCT ATG

Phe SerPhe LeuArg Asp Phe Asn Ala Ser Ala GluGlu Asn Ser Met 2226919.seq CAA TAT GTG ATG AAA CTT GCC AAT TCG CTC TTT GTA CAA AAT GGA TTT 45l Gln Tyr Val Met Lys Leu Ala Asn Ser Leu Phe Val Gln Asn Gly Phe His Val Asn Glu Glu Phe Leu Gln Met Leu Lys Met Tyr Phe Asn Ala 105 l10 115 Glu Val Asn His Val Asp Phe Ser Gln Asn Val Ala Val Ala Asn Ser Ile Asn Lys Trp Val Glu Asn Tyr Thr Asn Ser Leu Leu Lys Asp Leu l35 140 145 150 Val Ser Pro Glu Asp Phe Asp Gly Val Thr Asn Leu Ala Leu Ile Asn 155 l60 165 Ala Val Tyr Phe Lys Gly Asn Trp Lys Ser Gln Phe Arg Pro Glu Asn l70 175 l80 Thr Arg Thr Phe Ser Phe Thr Lys Asp Asp Glu Ser Glu Val Gln Ile l85 190 195 Pro Met Met Tyr Gln Gln Gly Glu Phe Tyr Tyr Gly Glu Phe Ser Asp Gly Ser Asn Glu Ala Gly Gly Ile Tyr Gln Val Leu Glu Ile Pro Tyr Glu Gly Asp Glu Ile Ser Met Met Leu Ala Leu Ser Arg Gln Glu Val Pro Leu Ala Thr Leu Glu Pro Leu Leu Lys Ala Gln Leu Ile Glu Glu Trp Ala Asn Ser Val Lys Lys Gln Lys Val Glu Val Tyr Leu Pro Arg 2226919.seq Phe Thr Val Glu Gln Glu Ile Asp Leu Lys Asp Ile Leu Lys Ala Leu Gly Val Thr Glu Ile Phe Ile Lys Asp Ala Asn Leu Thr Ala Met Ser Asp Lys Lys Glu Leu Phe Leu Ser Lys Ala Val His Lys Ser Cys Ile Glu Val Asn Glu Glu Gly Ser Glu Ala Ala Ala Ala Ser Gly Met Ile Ala Ile Ser Arg Met Ala Val Leu Tyr Pro Gln Val Ile Val Asp His Pro Phe Leu Tyr Leu Ile Arg Asn Arg Lys Ser Gly Ile Ile Leu Phe Met Gly Arg Val Met Asn Pro Glu Thr Met Asn Thr Ser Gly His Asp Phe Glu Glu Leu ' 2226919.seq CATCAAGTGG TACATTGGTG TGCCAGGAAA ATAGATGTAA TTACTTTATT AA.A.A.A.AGTTC 2207 TACAACACTT GAAATGCAGT CGTTAAA.A.AT ATGGAGACAT TTATAGGCAA TACCCATGAA 2627

Claims (44)

1. Neuroserpins of the formulas I and II
I: Neuroserpin of the human II: Neuroserpin of the mouse Including the separate, coding and coded sequences of these compounds of the formulas I or II, including the separate partial sequences of the coding and coded sequences of these compounds of the formulas I or II, including the coding or coded sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the coding or coded sequences or partial sequences of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coding or coded sequences, or parts thereof, of the compounds of formulas I or II, whose biological activity is equal or similar to that of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequence of the formulas I or II, including the sequences hybridizing to the coding sequences, or parts thereof, under stringent conditions, including the translation products of the sequences hybridizing to the coding sequences of the compounds of the formulas I or II, or to parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of formulas I or II, or parts thereof, but, as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.

2. Medicament, characterized in that it contains as at least one active compound either the coded sequence or the coding sequence of the compound of the formula I or of the formula II, or the separate partial sequences of the coded and coding sequences of these compounds of the formulas I or II, including the coding or coded sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the coding or coded sequences or partial sequences of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coding or coded sequences, or parts thereof, of the compounds of formulas I or II, whose biological activity is equal or similar to that of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequence of the formulas I or II, including the sequences hybridizing to the coding sequences, or parts thereof, under stringent conditions, including the translation products of the sequences hybridizing to the coding sequences of the compounds of the formulas I or II, or to parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of formulas I or II, or parts thereof, but, as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.

3. Medicament, characterized in that it contains as at least one active compound a substance which changes the function of the coded sequence of the compounds of formulas I or II, for example, in that it reduces or increases the protease inhibitory activity of the coded protein, or a part thereof, or in that it shortens or prolongs the time of presence of the coded protein at its place of action in the body.

4. Medicament, characterized in that it contains as at least one active compound a substance which changes the expression of the coding or coded sequences of the compounds of formulas I or II, for example in that it enhances or inhibits the transcription of the mRNA or in that it enhances or inhibits the translation of the coded sequences of the compounds of formulas I or II.

5. Medicament according to claim 2, 3, or 4, characterized in that it prevents or reduces the growth, the expansion, the infiltration and the metastasis of primary and metastatic tumors, as for example brain tumors or tumors of the retina.

6. Medicament according to claim 2, 3, or 4, characterized in that it contributes to the minimization of the tissue destruction in stroke, including brain infarction and ischemia, intracerebral hemorrhage, and subarrachnoid hemorrhage, as for example by exerting a protecting effect on the cells of the so-called penumbra zone surrounding the necrotic tissue.

7. Medicament according to claim 2, 3, or 4, characterized in that it contributes to the minimization of the tissue destruction in traumatic brain injury, as for example by exerting a protective effect on the cells of the so-called zone surrounding the necrotic tissue.

8. Medicament according to claim 2, 3, or 4, characterized in that it prevents, ameliorates or cures the negative effects caused by neurodegenerative diseases.

9. Medicament according to claim 2, 3, or 4, characterized in that it prevents, ameliorates or cures the negative effects caused by neuroinflammatory diseases, as for example multiple sclerosis.

10. Medicament according to claim 2, 3, or 4, characterized in that it reduces or prevents negative effects on brain tissue caused by epileptic seizures.

11. Medicament according to claim 2, 3, or 4, characterized in that it contributes to the rescue of endangered neurons, as for example neurons endangered by hypoxia and ischemia, excitotoxicity, neuroinflammatory diseases and processes, epileptic seizures, and cancerous neoformations.

12. Medicament according to claim 2, 3, or 4, characterized in that it contributes to axonal regeneration and/or restoration of synaptic integrity and functions.

13. Medicament according to claim 2, 3, or 4, characterized in that it prevents, ameliorates, or cures retinal disorders, as for example retinal degeneration and retinal neoangiogenesis.

14. Medicament according to claim 2, 3, or 4, characterized in that it prevents the apoptosis of cells of the nervous system.

15. Medicament according to claim 14, characterized in that the apoptosis is an apoptosis in connection with damages of the nervous tissue, for example infarct of the brain and ischemic stroke, or hemorrhage of the brain, or trauma of the brain.

16. Medicament according to claim 14, characterized in that the apoptosis is an apoptosis in connection with damages of the nervous tissue, which occur due to lack of oxygen or glucose or due to intoxication.

17. Medicament according to claim 14, characterized in that the apoptosis is an apoptosis in connection with epileptic seizures.

18. Medicament according to claim 14, characterized in that the apoptosis is an apoptosis in connection with neurodegenerative diseases and inherited genetic deficiencies of the nervous system.

19. Medicament according to claim 2, 3, or 4, characterized in that it influences the regeneration of injured, damaged, underdeveloped, or maldeveloped brain tissue and/or nervous tissue.

20. Medicament according to claim 2, 3, or 4, characterized in that it enhances the reorganization of the brain or nervous areas that have remained intact after brain and/or nerve injuries or after the destruction or damage of brain areas.

21. Medicament according to claim 2, 3, or 4, characterized in that it prevents, ameliorates, or cures pathological pain syndromes.

22. Medicament according to claim 2, 3, or 4, characterized in that it contributes to the improvement of the brain performance in healthy persons, as well as in persons with reduced brain performance.

23. Medicament according to claim 2, 3, or 4, characterized in that it ameliorates the learning and memory functions in healthy persons, as well as in persons with reduced learning and memory functions.

24. Medicament according to claim 2, 3, or 4, characterized in that it ameliorates or cures disorders in the field of disorders of the psychic wellness, or the psychosomatic state of health, as for example nervosity or ~inner unrest".

25. Medicament according to claim 2, 3, or 4, characterized in that it prevents, ameliorates or cures disorders in the field of the emotional functions, as for example states of anxiety.

26. Medicament according to claim 2, 3, or 4, characterized in that it prevents, ameliorates or cures psychiatric disorders.

27. Medicament according to claim 26, characterized in that the psychiatric disorder is a disorder in the field of schizophrenia and schizophrenia-like disorders, including chronic schizophrenia, chronic schizo-affective disorders, unspecific disorders, including acute and chronic schizophrenia of various symptomatologies, as for example severe, non-remitting ~Kraepelinic" schizophrenia, or as for example the DSM-III-R-prototype of the schizophrenia-like disorders, including episodic schizophrenic disorders, including delusionic schizophrenia-like disorders, including schizophrenia-like personality disorders, as for example schizophrenia-like personality disorders with mild symptomatics, including schizotypic personality disorders, including the latent forms of schizophrenic or schizophrenia-like disorders, including non-organic psychotic disorders.

28. Medicament according to claim 26, characterized in that the psychiatric disorder is a disorder in the field of the endogenic depressions or in the field of manic or manic-depressive disorders.

29. Medicament according to claim 2, 3, or 4, characterized in that it prevents, ameliorates or cures disorders of the brain due to at least one protease.

30. Medicament according to claim 29, characterized in that the protease is tissue-type plasminogen activator, abbreviated as tPA, urokinase-type plasminogen activator, abbreviated as uPA, or plasmin.

31. Medicament according to claim 29, characterized in that it reduces, prevents, or cures side effects of therapeutically administered tissue-type plasminogen activator, or urokinase-type plasminogen activator, or another type of plasminogen activator, or plasmin.

32. Use for the production of recombinant proteins of the coding nucleotide sequences of the compounds of the formulas I or II, including the separate partial sequences of the coding sequences of the compounds of the formulas I or II, including the coding nucleotide sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the coding sequences or partial sequences thereof of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coding sequences, or parts thereof, of the compounds of formulas I or II, whose translation products have a biological activity equal or similar to that of the translation products of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequence of the formulas I or II, including the sequences hybridizing to the coding sequences of the compounds of the formulas I or II, or parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of the formulas I or II, or parts thereof, but, as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.

33. Use as targets for the development of pharmaceutical drugs, for example for the inhibition or the enhancement of the protease inhibitory activity of the coded proteins of the formulas I or II, of proteins with the coded amino acid sequences of the compounds of the formulas I or II, including the proteins with the separate partial sequences of the coded amino acid sequences of the compounds of the formulas I
or II, including the proteins with the coded sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the proteins with the coded amino acid sequences or partial sequences thereof of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coded sequences, or parts thereof, of the compounds of formulas I or II, whose biological activity is equal or similar to the coded sequences of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequences of the formulas I or II, including the proteins with the coded amino acid sequences, or partial sequences thereof, of the nucleotide sequences hybridizing to the coding sequences of the compounds of the formulas I
or II, or parts thereof, under stringent conditions.

34. Use as targets for the development of pharmaceutical drugs, for example for the enhancement or the inhibition of the protease inhibitory activity of the coded proteins of the formulas I or II, of the species-homologous proteins, or parts thereof, of the compounds of the formulas I or II, as for example the species-homologous proteins of the rat, the rabbit, the cow, the sheep, the pig, the primates, the birds, the zebra fish, the fruit fly (Drosophila melanogaster), etc., including the partial sequences thereof, including the splice variants of the species-homologous proteins, including the alleles of the species-homologous proteins, including the translation products of the sequences hybridizing under stringent conditions to the corresponding species-homologous compounds of the formulas I or II, or their splice variants, or their alleles, of the coding sequences or partial sequences of the compounds of formulas I or II.

35. Use for the spatial structure determination, for example the spatial structure determination by means of crystallography or nuclear resonance spectroscopy, of the proteins with the coded amino acid sequences of the compounds of the formulas I or II, including the proteins with the separate partial sequences of the coded amino acid sequences of the compounds of the formulas I
or II, including the proteins with the coded sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the proteins with the coded amino acid sequences, or partial sequences thereof, of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coded sequences, or parts thereof, of the compounds of the formulas I or II, whose biological activity is equal or similar to that of the coded sequences of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequences of the formulas I or II, including the translation products with the sequences hybridizing to the coding sequences of the compounds of the formulas I or II, or parts thereof, under stringent conditions, including the species-homologous proteins of the compounds of the formulas I or II, for example the species-homologous proteins of the rat, the rabbit, the cow, the sheep, the pig, the primates, the birds, the zebra fish, the fruit fly (Drosophila melanogaster), etc., including the partial sequences thereof, as for example the separate catalytic domains.

36. Use for the prediction of the protein structure by means of computerized protein structure prediction methods, of the coded amino acid sequences of the compounds of the formulas I or II, including the separate partial sequences of the coded amino acid sequences of the compounds of the formulas I or II, including the coded sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the coded amino acid sequences, or parts thereof, of the corresponding alleles of the compounds of the formulas I
or II, including all sequence variants of the coded sequences, or parts thereof, of the compounds of the formulas I or II, whose biological activity is equal or similar to that of the coded sequences of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequences of the formulas I or II, including the amino acid sequences of the translation products of the sequences hybridizing to the coding sequences of the compounds of the formulas I or II, or parts thereof, under stringent conditions, including sequences of the species-homologous compounds of the compounds of the formulas I or II, for example the sequences of the species-homologous compounds of the rat, the rabbit, the cow, the sheep, the pig, the primates, the birds, the zebra fish, the fruit fly (Drosophila melanogaster), etc., including the partial sequences of the species-homologous compounds, as for example the sequences of the catalytic domains of the species-homologous compounds.

37. Use as targets for the development of pharmaceutical drugs , for example for the inhibition or the enhancement of the protease inhibitory activity of the coded proteins of the formulas I or II, of the spatial structure of the coded amino acid sequences of the compounds of the formulas I or II, including the spatial structures of the separate partial sequences of the compounds of the formulas I or II, including the spatial structure of the coded sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the spatial structure of the coded sequences or partial sequences of the corresponding alleles of the compounds of the formulas I or II, including the spatial structure of all sequence variants of the coded sequences, or parts thereof, of the compounds of formulas I or II, whose biological activity is equal or similar to the coded sequences of the compounds of the formulas I or II, for example sequence variants of the

38 compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequences of the formulas I or II, including the spatial structures of the translation products of the sequences hybridizing to the coding sequences of the compounds of the formulas I or II, or parts thereof, under stringent conditions, including the spatial structures of the species-homologous compounds of the compounds of the formulas I or II, as for example the spatial structures of the species homologous compounds, or parts thereof, of the rat, the rabbit, the cow, the sheep, the pig, the primates, the birds, the zebra fish, the fruit fly (Drosophila melanogaster), etc..
38. Use in gene therapeutical applications in humans and in animals, as for example as parts of gene therapy vectors or as for example as parts of artificial chromosomes, of the coding nucleotide sequences of the compounds of the formulas I or II, including the separate partial sequences of the coding sequences of these compounds of the formulas I or II, including the coding sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I
or II, including the coding sequences or partial sequences of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coding sequences, or parts thereof, of the compounds of the formulas I or II, whose translation products exhibit a biological activity which is equal or similar to the that of the translation products of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequences of the compounds of the formulas I or II, including the sequences hybridizing to the coding sequences, or parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of the formulas I or II, or parts thereof, but as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.

39. Use for so-called cell engineering applications for the production of gene technologically mutated cells, which produce the coded sequences, or parts thereof, of the compounds of the formulas I or II, for example for cell-therapeutical applications as a medicament according to claim 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 of the coding nucleotide sequences of the compounds of the formulas I or II, including the separate partial sequences of the coding sequences of these compounds of the formulas I or II, including the coding sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the coding sequences or partial sequences of the corresponding alleles of the compounds of the formulas I or 11, including all sequence variants of the coding sequences, or parts thereof, of the compounds of the formulas I or II, whose translation products exhibit a biological activity which is equal or similar to that of the translation products of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequence of the compounds of the formulas I
or II, including the sequences hybridizing to the coding sequences, or parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of formulas I or II, or parts thereof, but as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.

40. Use as antigens for the production of antibodies, as for example antibodies that inhibit or promote the protease inhibitory function or antibodies that can be used for immunohistochemical studies, of the coded amino acid sequences of the compounds of the formulas I or II, including the separate partial sequences of the coded amino acid sequences of the compounds of the formulas I or II, including the coded sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the coded sequences or partial sequences of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coded sequences, or parts thereof, of the compounds of the formulas I or II, whose biological activity is equal or similar to that of the coded sequences of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequence of the compounds of the formulas I or II, including the translation products or parts thereof, of the sequences hybridizing to the coding sequences of the compounds of the formulas I or II, or parts thereof, under stringent conditions, including the coded sequences of the species-homologous compounds of the compounds of the formulas I or II, as for example the coded sequences of the species-homologous compounds of the rat, the rabbit, the cow, the sheep, the pig, the primates, the birds, the zebra fish, the fruit fly (Drosophila melanogaster), etc., including the separate partial sequences of the coded sequences of the species-homologous compounds of the compounds of the formulas I or II, as for example the coded amino acid sequence of the catalytic domain, or one or more of the other domains or segments.

41. Use for the production of transgenic animals, as for example transgenic mice, of the coding nucleotide sequences of the compounds of the formulas I or II, including the separate partial sequences of the coding sequences of these compounds of the formulas I or II, including the coding sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I
or II, including the coding sequences, or partial sequences, of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coding sequences, or parts thereof, of the compounds of the formulas I or II, whose translation products exhibit a biological activity which is equal or similar to that of the translation products of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequences of the compounds of the formulas I
or II, including the sequences hybridizing to the coding sequences, or parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of the formulas I or II, or parts thereof, but as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.

42. Use for the inactivation or the mutation of the corresponding gene by means of gene targeting techniques, as for example the elimination of the gene in the mouse through homologous recombination or the replacement of the gene by a mutated form thereof, of the coding nucleotide sequences of the compounds of the formulas I or II, including the separate partial sequences of the coding sequences of these compounds of the formulas I or II, including the coding sequences, or partial sequences, of the corresponding splice variants of the compounds of the formulas I
or II, including the coding sequences, or partial sequences, of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coding sequences, or parts thereof, of the compounds of the formulas I or II, whose translation products exhibit a biological activity which is equal or similar to that of the translation products of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequence of the compounds of the formulas I
or II, including the sequences hybridizing to the coding sequences, or parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of the formulas 1 or II, or parts thereof, but as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.

43. Use for the diagnostics of disorders in the gene corresponding to the compound of the formula I, of the coding nucleotide sequences of the compounds of the formulas I or II, including the separate partial sequences of the coding sequences of these compounds of the formulas I or II, including the coding sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the coding sequences, or partial sequences, of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coding sequences, or parts thereof, of the compounds of the formulas I or II, whose translation products exhibit a biological activity which is equal or similar to that of the translation products of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequences of the compounds of the formulas I or II, including the sequences hybridizing to the coding sequences, or parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of the formulas I or II, or parts thereof, but as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.

44. Use as a starting sequence for gene technological modifications aimed at the production of medicaments or gene therapy vectors which exhibit changed properties as compared with the corresponding medicaments or gene therapy vectors containing the coding nucleotide sequence of the compounds of formulas I or II, for example changed protease inhibitory activity, changed protease inhibitory specificity, or changed pharmacokinetic characteristics, of the coding nucleotide sequences of the compounds of the formulas I or II, including the separate partial sequences of the coding sequences of these compounds of the formulas I or II, including the coding sequences or partial sequences of the corresponding splice variants of the compounds of the formulas I or II, including the coding sequences, or partial sequences, of the corresponding alleles of the compounds of the formulas I or II, including all sequence variants of the coding sequences, or parts thereof, of the compounds of the formulas I or II, whose translation products exhibit a biological activity which is equal or similar to that of the translation products of the compounds of the formulas I or II, for example sequence variants of the compounds of the formulas I or II, which differ in the not conserved amino acid sequence positions of the sequences of the compounds of the formulas I or II, including the sequences hybridizing to the coding sequences, or parts thereof, under stringent conditions, including the nucleotide sequences coding the proteins coded by the compounds of the formulas I or II, or parts thereof, but as a result of the use of different alternative codons, are degenerated with regard to the nucleotide sequences defined by the compounds of the formulas I or II.