NEUROSERPIN

NEUROSERPINE

English 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.

Claims

30
CLAIMS
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

31
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.

32
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.

33
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".

34
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.

35
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.

36
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.

37
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

39
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

40
(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

41
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,

42
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.

Description

CA 02226919 1998-06-08
1
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.

CA 02226919 1998-06-08
2
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.

CA 02226919 1998-06-08
3
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.

CA 02226919 1998-06-08
4
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

CA 02226919 1998-06-08
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

CA 02226919 1998-06-08
6
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
20
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.

CA 02226919 1998-06-08
7
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.

CA 02226919 1998-06-08
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.

CA 02226919 1998-06-08
9
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

CA 02226919 1998-06-08
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.

CA 02226919 1998-06-08
11
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.

CA 02226919 1998-06-08
12
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
20
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.

CA 02226919 1998-06-08
13
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.

CA 02226919 1998-06-08
14
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.

CA 02226919 1998-06-08
Example 5:
Determination of the protease inhibitory function of the compounds of the
formulas I
and II
5
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%

CA 02226919 1998-06-08
16
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
4350-
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.

CA 02226919 1998-06-08
17
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

CA 02226919 1998-06-08
18
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

CA 02226919 1998-06-08
19
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.

CA 02226919 1998-06-08
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
Page 1
CA 02226919 1998-07-23

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
Page 2
CA 02226919 1998-07-23

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..
81
(xi) SEQUENCE DESCRIPTION: 1:
SEQ
ID
NO
GCGGAGCACA AGCATCCCGT CAGGGGTTGC
60
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
-15 -10
GTT CTG CAA AGTATG GCT ACA GCC TTC CCT GAG GAA GCCATT 159
GGG ACT
Val Leu Gln SerMet Ala Thr Ala Phe Pro Glu Glu AlaIle
Gly Thr
-5 1 5 10
GCT GAC TTG TCAGTG AAT ATG AAT CTT AGA GCC ACT GGTGAA 207
TAT CGT
Ala Asp Leu SerVal Asn Met Asn Leu Arg Ala Thr GlyGIu
Tyr Arg
15 20 25
GAT GAA AAT ATTCTC TTC TCT TTG ATT GCT CTT GCA ATGGGA 255
CCA AGT
Asp Glu Asn IleLeu Phe Ser Leu ale Ala Leu Ala MetGly
Pro Ser
30 35 40
ATG ATG GAA CTTGGG GCC CAA TCT CAG AAA GAA ATC CGCCAC 303
GGA ACC
Met Met Glu LeuGly Ala Gln Ser Gln Lys Glu Ile ArgHis
Gly Thr
45 50 55
TCA ATG GGA TATGAC AGC CTA AAT GAA GAA TTT TCT TTCTTG 351
AAA GGT
Ser Met Gly TyrAsp Ser Leu Asn Glu Glu Phe Ser PheLeu
Lys Gly
60 65 70
AAG GAG TTT TCAAAC ATG GTA GCT GAG AGC CAA TAT GTGATG 399
ACT AAA
Lys Glu Phe SerAsn Met Val Ala Glu Ser Gln Tyr ValMet
Thr Lys
Page 3
CA 02226919 1998-07-23

2226919.seq
75 80 85 90
AAA ATT GCC AAT TCC TTG TTT GTG CAA AAT GGA TTT CAT GTC AAT GAG 447
Lys Ile Ala Asn Ser Leu Phe Val Gln Asn Gly Phe His Val Asn Glu
95 100 105
GAG TTT TTG CAA ATG ATG AAA AAA TAT TTT AAT GCA GCA GTA AAT CAT 495
Glu Phe Leu Gln Met Met Lys Lys Tyr Phe Asn Ala Ala Val Asn His
110 115 120
GTG GAC TTC AGT CAA AAT GTA GCC GTG GCC AAC TAC ATC AAT AAG TGG 543
Val Asp Phe Ser Gln Asn Val Ala Val Ala Asn Tyr Ile Asn Lys Trp
125 130 135
GTG GAG AAT AAC ACA AAC AAT CTG GTG AAA GAT TTG GTA TCC CCA AGG 591
Val Glu Asn Asn Thr Asn Asn Leu Val Lys Asp Leu Val Ser Pro Arg
140 145 150
GAT TTT GAT GCT GCC ACT TAT CTG GCC CTC ATT AAT GCT GTC TAT TTC 639
Asp Phe Asp Ala Ala Thr Tyr Leu Ala Leu Ile Asn Ala Val Tyr Phe
155 160 165 170
AAG GGG AAC TGG AAG TCG CAG TTT AGG CCT GAA AAT ACT AGA ACC TTT 687
Lys Gly Asn Trp Lys Ser Gln Phe Arg Pro Glu Asn Thr Arg Thr Phe
175 180 185
TCT TTC ACT AAA GAT GAT GAA AGT GAA GTC CAA ATT CCA ATG ATG TAT 735
Ser Phe Thr Lys Asp Asp Glu Ser Glu Val Gln Ile Pro Met Met Tyr
190 195 200
CAG CAA GGA GAA TTT TAT TAT GGG GAA TTT AGT GAT GGC TCC AAT GAA 783
Gln Gln Gly Glu Phe Tyr Tyr Gly Glu Phe Ser Asp Gly Ser Asn Glu
205 210 2I5
GCT GGT GGT ATC TAC CAA GTC CTA GAA ATA CCA TAT GAA GGA GAT GAA 831
Ala Gly Gly Ile Tyr Gln Val Leu Glu Ile Pro Tyr Glu Gly Asp Glu
220 225 230
ATA AGC ATG ATG CTG GTG CTG TCC AGA CAG GAA GTT CCT CTT GCT ACT 879
Ile Ser Met Met Leu Val Leu Ser Arg Gln Glu Val Pro Leu Ala Thr
235 240 245 250
CTG GAG CCA TTA GTC AAA GCA CAG CTG GTT GAA GAA TGG GCA AAC TCT 927
Leu Glu Pro Leu Val Lys Ala Gln Leu Val Glu Glu Trp Ala Asn Ser
255 260 265
GTG AAG AAG CAA AAA GTA GAA GTA TAC CTG CCC AGG TTC ACA GTG GAA 975
Val Lys Lys Gln Lys Val Glu Val Tyr Leu Pro Arg Phe Thr Val Glu
270 275 280
Page 4
CA 02226919 1998-07-23

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
285 290 295
ATT TTC ATC AAA GAT GCA AAT TTG ACA GGC CTC TCT GAT AAT AAG GAG 1071
Ile Phe Ile Lys Asp Ala Asn Leu Thr Gly Leu Ser Asp Asn Lys Glu
300 305 310
ATT TTT CTT TCC AAA GCA ATT CAC AAG TCC TTC CTA GAG GTT AAT GAA 1119
Ile Phe Leu Ser Lys Ala Ile His Lys Ser Phe Leu Glu Val Asn Glu
315 320 325 330
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
335 340 345
ATG GCT GTG CTG TAT CCT CAA GTT ATT GTC GAC CAT CCA TTT TTC TTT 1215
Met Ala Val Leu Tyr Pro Gln Val Ile Val Asp His Pro Phe Phe Phe
350 355 360
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
365 370 375
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
380 385 390 394
TAAGTTACTT TATTTGAATA ACAAGGAAAA CAGTAACTAA GCACATTATG TTTGCAACTG 1371
GTATATATTT AGGATTTGTG TTTTACAGTA TATCTTAAGA TAATATTTAA AATAGTTCCA l431
GATAA.AAACA ATATATGTAA ATTATAAGTA ACTTGTCAAG GAATGTTATC AGTATTAAGC 1491
TAATGGTCCT GTTATGTCAT TGTGTTTGTG TGCTGTTGTT TAAAATAAAA GTACCTATTG 1551
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
Page 5
CA 02226919 1998-07-23

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:
Page 6
CA 02226919 1998-07-23

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..
97
(xi) SEQUENCE DESCRIPTION: 2:
SEQ ID NO
CGGCACGAGA TCTCCAGCAT CCCGAGCGGG
60
TCCGGAGCAG
TCTCAGCCTG
CCCAGCATCC
GATTGCAGGT 115
GTGAAGGAGA
CTTGAAACCA
TCCCATC
ATG
ACT
TAC
CTT
GAA
CTG
Met
Thr
Tyr
Leu
Glu
Leu
-15
CTT GCTTTG CTGGCC TTG CAA GTG ACA GGG GCA ACGTTC CCA 163
AGT GTG
Leu AlaLeu LeuAla Leu Gln Val Thr Gly Ala ThrPhe Pro
Ser Val
-10 -5 1 5
GAT GAAACC ATAACT GAG TGG GTG ATG TAT AAC CACCTT CGA 211
TCA AAC
Asp GluThr IleThr Glu Trp Val Met Tyr Asn HisLeu Arg
Ser Asn
10 15 20
GGC ACCGGG GAAGAT GAA AAC CTC TCT CCA CTA AGCATT GCC 259
ATT TTC
Gly ThrGly GluAsp Glu Asn Leu Ser Pro Leu SerIle Ala
Ile Phe
25 30 35
CTT GCGATG GGAATG ATG GAG GGG CAA GGA TCT ACTAGG AAA 307
CTT GCT
Leu AlaMet GlyMet Met Glu Gly Gln Gly Ser ThrArg Lys
Leu Ala
40 45 50
GAA ATCCGC CATTCA ATG GGA GAG CTG AAA GGT GGTGAA GAA 355
TAT GGT
Glu IleArg HisSer Met Gly Glu Leu Lys Gly GlyGlu Glu
Tyr Gly
55 60 65 70
TTT TCTTTC CTGAGG GAT TTT AAT GCC TCT GCC GAAGAA AAC 403
TCT ATG
Phe SerPhe LeuArg Asp Phe Asn Ala Ser Ala GluGlu Asn
Ser Met
75 80 85
Page 7
CA 02226919 1998-07-23

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
90 95 100
CAT GTC AAT GAG GAA TTC TTG CAA ATG CTG AAA ATG TAC TTT AAT GCA 499
His Val Asn Glu Glu Phe Leu Gln Met Leu Lys Met Tyr Phe Asn Ala
105 l10 115
GAA GTC AAC CAT GTG GAC TTC AGT CAA AAT GTG GCT GTG GCT AAC TCC 547
Glu Val Asn His Val Asp Phe Ser Gln Asn Val Ala Val Ala Asn Ser
120 125 130
ATC AAT AAA TGG GTG GAG AAT TAT ACA AAC AGT CTG TTG AAA GAT CTG 595
Ile Asn Lys Trp Val Glu Asn Tyr Thr Asn Ser Leu Leu Lys Asp Leu
l35 140 145 150
GTG TCT CCG GAG GAC TTT GAT GGT GTC ACT AAT TTG GCC CTC ATC AAT 643
Val Ser Pro Glu Asp Phe Asp Gly Val Thr Asn Leu Ala Leu Ile Asn
155 l60 165
GCT GTA TAT TTC AAA GGA AAC TGG AAG TCT CAG TTT AGA CCT GAA AAT 691
Ala Val Tyr Phe Lys Gly Asn Trp Lys Ser Gln Phe Arg Pro Glu Asn
l70 175 l80
ACC AGA ACT TTC TCC TTC ACG AAA GAT GAT GAA AGT GAA GTG CAG ATT 739
Thr Arg Thr Phe Ser Phe Thr Lys Asp Asp Glu Ser Glu Val Gln Ile
l85 190 195
CCA ATG ATG TAT CAA CAA GGA GAA TTT TAT TAT GGT GAA TTT AGT GAT 787
Pro Met Met Tyr Gln Gln Gly Glu Phe Tyr Tyr Gly Glu Phe Ser Asp
200 205 210
GGA TCC AAT GAG GCT GGT GGT ATC TAC CAA GTC CTT GAG ATA CCC TAT 835
Gly Ser Asn Glu Ala Gly Gly Ile Tyr Gln Val Leu Glu Ile Pro Tyr
2I5 220 225 230
GAG GGA GAT GAG ATC AGC ATG ATG CTG GCA CTG TCC AGA CAG GAA GTC 883
Glu Gly Asp Glu Ile Ser Met Met Leu Ala Leu Ser Arg Gln Glu Val
235 240 245
CCA CTG GCC ACA CTG GAG CCT CTG CTC AAA GCA CAG CTG ATC GAA GAA 931
Pro Leu Ala Thr Leu Glu Pro Leu Leu Lys Ala Gln Leu Ile Glu Glu
250 255 260
TGG GCA AAC TCT GTG AAG AAA CAA AAG GTG GAA GTG TAC TTG CCC AGG 979
Trp Ala Asn Ser Val Lys Lys Gln Lys Val Glu Val Tyr Leu Pro Arg
265 270 275
TTC ACT GTG GAA CAG GAA ATT GAT TTA AAA GAC ATC TTG AAA GCC CTT 1027
Page 8
CA 02226919 1998-07-23

2226919.seq
Phe Thr Val Glu Gln Glu Ile Asp Leu Lys Asp Ile Leu Lys Ala Leu
280 285 290
GGG GTC ACT GAA ATT TTC ATC AAA GAT GCA AAT TTG ACT GCC ATG TCA 1075
Gly Val Thr Glu Ile Phe Ile Lys Asp Ala Asn Leu Thr Ala Met Ser
295 300 305 310
GAT AAG AAA GAG CTG TTC CTC TCC AAA GCT GTT CAC AAG TCC TGC ATT 1123
Asp Lys Lys Glu Leu Phe Leu Ser Lys Ala Val His Lys Ser Cys Ile
315 320 325
GAG GTT AAT GAA GAA GGG TCA GAA GCC GCT GCA GCC TCC GGA ATG ATT I171
Glu Val Asn Glu Glu Gly Ser Glu Ala Ala Ala Ala Ser Gly Met Ile
330 335 340
GCG ATT AGT AGG ATG GCT GTG CTG TAC CCT CAG GTT ATT GTC GAC CAT 1219
Ala Ile Ser Arg Met Ala Val Leu Tyr Pro Gln Val Ile Val Asp His
345 350 355
CCA TTT CTC TAT CTT ATC AGG AAC AGG AAA TCT GGC ATA ATC TTA TTC 1267
Pro Phe Leu Tyr Leu Ile Arg Asn Arg Lys Ser Gly Ile Ile Leu Phe
360 365 370
ATG GGA CGA GTC ATG AAC CCT GAA ACA ATG AAT ACA AGT GGC CAT GAC 1315
Met Gly Arg Val Met Asn Pro Glu Thr Met Asn Thr Ser Gly His Asp
375 380 385 390
TTT GAG GAA CTT TAAATGACGA CGTTTGAGTA CAAAGAAAGC AGGAACAAAG 1367
Phe Glu Glu Leu
394
CACATTATGT TTGCAAGTGG TATATATTTA GGATTTCTGT TTTATAGTGT TACTTAGGGA 1427
AATATTTAAA TAGTTCTGGA TAGTAGTAAT CCATGTGACC TATAAGTTAG CCTGTCAAAA 1487
GCTGTTATCA GTATAAAGAG TATGGTCCCA TTGTGTCATT GTGTCTGGTG TGCTGCTGTT 1547
TAAAATAAAA GTACATATTG AAACTGTGAA CCACTTTTTT TCATTTTGAA AGTAGTTGTA 1607
GTCTATACAA TACTATGTCT GAGATTTGAA ACCTATGCTG TTTCTTTAGG AATTGTAGTA 1667
AAATGATCCT ACAAGGCAAA ATGTAGAAAC TGTTGTTTCT GAGTTTCTTC ATAATCATGC 1727
AGAATCAAAC ACCAAAGTAA GCAACATACA TATATATATA TAATAAGCAA TACTGTGAAG 1787
GGGAGGCCAA AAGGCAGAGA AATTGAGATT GTTATTTAGT GTGGCATTCC ATGACAAAAG 1847
ATTTAGGAGG AAATGTGGGA TATGTAAGAC CCATAGATGT ATATTTTGTA TATCTGTAGT 1907
ATTATACTTT TAATTTATTA AAGTATAACT CTTTTATTTA TTTTTAAAAG TTTCCTGTGA 1967
Page 9
CA 02226919 1998-07-23

' 2226919.seq
ACCAATATGC CACATGACTC TACTAGCAAG TTCAGATATC TCATTAGCTA TTCTGGATGA 2027
CATCAAGAGG CCTCATGGAG GGAATCCCGT GTACCATTTA CGTTTTAGTG ATTTTTTGTG 2087
ATGTTCACAC AAAGATGAAA TCACATTGTT GCACACTCTC TAGACTATAT CCAAGAAAGG 2147
CATCAAGTGG TACATTGGTG TGCCAGGAAA ATAGATGTAA TTACTTTATT AA.A.A.A.AGTTC 2207
CTGGTATTGT GCATCATATG GAATCAGTGC TGCTTAAACT TAGTACGTCC TGCTGACACC 2267
TGGTCACTTA TTACAAATAT AGGTTCTTAT CCAGGATGTC TAAGGTAGAG TGGGAACCAC 2327
AGCTTTCTAT CATTACTGAC ATCCAAATGA TGCCGCAGAT ATCTGACCAT AGCCTTTGCT 2387
GAGAGTCCCT TGGGTTGCAA TGTCGTACTT GAAGTCAGCC TCACATTTTC ACAGACTGAG 2447
ATTGGAGAGA TGAGGGTGCA GGGAGGAGAT AATCTACACT AGTGATACGA TGCCTTTGTC 2507
AAGCACTGGT GTGATCTCGA AGTATTCTAG TACACACTCT AGATAAATTC TTCTGTACAT 2567
TACAACACTT GAAATGCAGT CGTTAAA.A.AT ATGGAGACAT TTATAGGCAA TACCCATGAA 2627
AGAATTTATG ACTATCCGAG GACACAGTAC TTAACAATGA ATCTTTTACA GCTTATATTT 2687
TCAGAGGACT TGTAGTTTAT TCATAAATCT TCATGTTATT GTACAATAGT GCTCTTGTTT 2747
TCATTTATAA TTTATGAAGC TGAGATGCTG GTGTTAATTC AGTGTTCACA TTCTCTGCTA 2807
AGAACAGTCT TTATCTCTGT ATCCTTCTTG TTAATATGAC ATCTATAGCT ATATCTATAT 2867
GTTCATTAGT TAAACAAATG TATGGCCTGT AAGGAAGAAT AAACATTATT ATGCAATCAT 2927
GTAAAAAAAA AAAAAAA 2944
Page 10
CA 02226919 1998-07-23