polypeptides and polypeptide analogues

Details polypeptides and polypeptide analogues polypeptides and polypeptide analogues

1995-04-24

2001-06-12

Duffy, Patricia A. (Department: 1645)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S324000

Reexamination Certificate

active

06245739

ABSTRACT:

This invention relates to polypeptides which possess inhibitory activity against human elastase, processes for their preparation and pharmaceutical compositions containing them as well as to the genetic modification of hosts to express the said polypeptides, the hosts thereby obtained, the genetic material employed in the modification and vectors therefor.
Human leukocyte elastase (HLE) is a proteolytic enzyme which is present in the azurophil granules of human polymorphonuclear leukocytes. Under normal circumstances these granules fuse with phagosomes containing foreign material, such as bacteria, and the particles are metabolised by a combination of HLE and other lysomal enzymes. However, under certain pathological conditions the polymorphonuclear leukocytes become attached to host protein, such as elastin and collagen, and the contents of the granules, including HLE, may be released directly onto the tissue. The resulting degradative activity of HLE on the host tissue is believed to lead to tissue damage which may play a role in diseases such as emphysema (2), adult respiratory distress syndrome (1), psoriasis (3) and bullous dermatoses (4).
The potent proteolytic activity of HLE is known to be balanced to a certain extent by the antiproteolytic activity of inhibitors such as Alpha
1
-antitrypsin and Alpha
2
-macroglobulin. These inhibitors have been detected in serum as well as various tissues including epidermis (5,10). In addition, a low molecular weight inhibitor of human leukocyte proteases has been shown to be present in seminal fluid (11), cervical mucus (12), bronchial secretion (13) and parotid secretion (14) as well as in human serum (6). The inhibitor from all these sources was shown to be identical with antileukoprotease and its metabolites by immunoreactivity (6,15) or amino acid sequence analysis (16,17), and to also be a potent inhibitor of trypsin. Low molecular weight inhibitors have also been detected in bronchial mucus by Hochstrasser et al (22), and in psoriatic skin (21). The literature also reports other inhibitors of elastase (18,19) which also inhibit trypsin. In addition, elastase inhibitors have been detected in the submandibular glands of dogs, lions and cats (20).
According to one first feature of the present invention there is provided a polypeptide having all or part of the primary structure of formula I and fragments thereof, Ala-Gln-Glu-Pro-Val-Lys-Gly-Pro-Val-Ser-Thr-Lys-Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-Ile-Arg-Cys-Ala-Met-Leu-Asn-Pro-Pro-Asn-Arg-Cys-Leu-Lys-Asp-Thr-Asp-Cys-Pro-Gly-Ile-Lys-Lys-Cys-Cys-Glu-Gly-Ser-Cys-Gly-Met-Ala-Cys-Phe-Val-Pro-Gln. (Formula I) which polypeptide or fragments possess inhibitory activity against human leukocyte elastase, and compounds capable of being modified in vivo or in vitro to said polypeptide or fragments.
According to the present invention there is also provided a polypeptide which comprises all or part of the following primary structure and fragments thereof:
Lys-Gly-Pro-Val-Ser-Thr-Lys-Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-Ile-Arg-Cys-Ala-Met-Leu-Asn-Pro-Pro-Asn-
which polypeptide or fragments possess inhibitory activity against human leukocyte elastase, and compounds capable of being modified in vivo or in vitro to said polypeptide or fragments.
Conveniently, the polypeptide or fragments have the structure:
Lys-Gly-Pro-Val-Ser-Thr-Lys-Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-Ile-Arg-Cys-Ala-Met-Leu-Asn-Pro-Pro-Asn-X; wherein X comprises one or more amino acid residues and is preferably Arg-Cys-Leu-Lys-Asp-Thr-Asp-Cys-Pro-Gly-Ile-Lys-Lys-Cys-Cys-Glu-Gly-Ser-Cys-Gly-Met-Ala-Cys-Phe-Val-Pro-Gln.
The polypeptide may be preceded by one or more amino acid residues, for example Ala-Gln-Glu-Pro-Val-; or Y-Ala-Gln-Glu-Pro-Val-
wherein Y comprises one or more amino acids which may be, for example:
Asn-Gly-Gln-Asp-Pro-Val-Lys-Gly-Gln-Val-Ser-Val-Lys-Gly-Gln-Asp-Lys-Val-Lys-;
Gly/Ala-Gln/Val-Asp-Lys-Val-Lys-;
Asp-Lys-Val-Lys-; or
Gly/Val-Lys-
Most preferably the polypeptide has the structure of formula I
Conveniently, the polypeptide or fragments of the present invention possess inhibitory activity which is specific for serine proteases, in that they possess inhibitory activity against proteases such as human leukocyte elastase and porcine pancreatic elastase; but do not possess any significant inhibitory activity against trypsin. Preferred polypeptides or fragments of the invention do not possess any significant inhibitory activity against human cathepsin G, Alpha-chymotrypsin and plasmin.
Polypeptides, or fragments thereof, of the present invention may have an isoelectronic point at about pH 9.7 (as determined by isoelectronic focussing).
Although they may be prepared by genetic engineering techniques the preferred polypeptides of the present invention may also be obtained from psoriatic scales of human skin.
According to the present invention there is also provided a polypeptide which possesses inhibitory activity against human leukocyte elastase, said polypeptide being obtainable from psoriatic scales of human skin and possessing one or more of the following characteristics:
a) a molecular weight of about 9k (as determined by SDS-PAGE);
b) an isoelectronic point at about pH 9.7 (as determined by isoelectronic focusing);
c) inhibitory activity against porcine pancreatic elastase in addition to human leukocyte elastase;
d) no significant activity against trypsin, human cathepsin G,
Alpha-chymotrypsin and plasmin;
or a fragment thereof possessing inhibitory activity against human leukocyte elastase.
Polypeptides obtainable from human skin include those obtainable directly by means of isolation and purification procedures and also fragments of such polypeptides.
Preferred polypeptides of the present invention contain one of the following amino acid sequences:
a)-Ala-Gln-Glu-Pro-Val-Lys-Gly-Pro-
and most preferably a N-terminal sequence comprising the above sequence preceded by X,
where X represents an amino acid sequence which may contain 19 amino acids, which sequence is conveniently
Asn-Gly-Gln-Asp-Pro-Val-Lys-Gly-Gln-Val-Ser-Val-Lys-Gly-Gln-Asp-Lys-Val-Lys-
b)-Ala-Gln-Glu-Pro-Val-Lys-Gly-Pro-Val-Ser-Thr-Lys-Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-
and most preferably a N-terminal sequence comprising the above sequence preceded by X,
where X represents an amino-acid sequence which may contain six amino acids, which sequence is conveniently
Gly/Ala-Gln/Val-Asp-Lys-Val-Lys-
c)-Ala-Gln-Glu-Pro-Val-Lys-Gly-Pro-Val-Ser-Thr-Lys-Pro-Gly-Ser-Cys-
and most preferably a N-terminal sequence which comprises the above sequence preceded by X,
where X represents an amino acid sequence which may contain four amino acids, which sequence is conveniently
Asp-Lys-Val-Lys-
d) Ala-Gln-Glu-Pro-Val-Lys-Gly-Pro-Val-Ser-Thr-Lys-Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-Ile-Arg-Cys-Ala-Met-Leu-Asn-Pro-Pro-Asn-Arg-Cys-Leu-Lys-Asp-Thr-
and most preferably a N-terminal sequence which comprises the above sequence preceded by X, where X represents an amino acid sequence which may contain two amino acid residues which are conveniently
Gly/Val-Lys
e) Lys-Gly-Pro-Val-Ser-Thr-Lys-Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-Ile-Arg-Cys-Ala-Met-Leu-Asn-Pro-Pro-Asn-
and most preferably a polypeptide having an N-terminal sequence which comprises the above sequence.
Particularly preferred polypeptides are as defined in the first aspect of the present invention, especially the polypeptide of formula I.
The polypeptide of formula I also possesses inhibitory activity against protinase 3.
The polypeptides and fragments thereof of the present invention are obtainable in a biologically pure and homogenous form. The present invention also provides a polypeptide as herein defined unaccompanied by associated native glycosylation.
In addition to polypeptides as hereinbefore defined, the present invention also embraces other products which possess inhibitory activity against human leukocyte elastase, such as polypeptide analogues. Therefore, according to the present invention there is also provided polypeptide analogues of the polypeptides as hereinbefore defined which analogues possess inhibitory activi

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