Pharmaceutical composition having an endoproteolytic activity

Details Pharmaceutical composition having an endoproteolytic activity Pharmaceutical composition having an endoproteolytic activity

1997-10-22

2001-08-14

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C424S094630, C424S094640

Reexamination Certificate

active

06274365

ABSTRACT:

The invention relates to both a pharmaceutical composition having an endoproteolytic activity and a process for the (micro)biological production of a protein and for the in vitro cleavage of a protein, in particular a precursor protein by processing the protein with an endoproteolytically active enzyme.
The invention described herein is the result of a further study into the possible physiological significance of furin, a human protein described in European patent application EP-A-0 246 709, which is the expression product of the fur gene located in the genome upstream of the human fes/fps proto-oncogene. The patent application referred to and other publications by the same research group (Roebroek et al., Molec.Biol.Rep. 11, 1986, 117-125; Roebroek et al., EMBO J. 6, 1986, 2197-2202; and Schalken et al., J.Clin. Invest. 80, 1987, 1545-1549) show that on the basis of the limited DNA data then available, it was impossible to determine the function of the product of the fur gene. What could be determined was that the furin is probably a membrane-associated protein which has a function in which certain recognition structures play a role. It was also observed at the time that the fur gene is expressed as a 4.5 kb mRNA in liver, kidney, spleen, thymus and brain, whereas the expression in lung tissue is very slight; in non-small-cell lung carcinomas, on the other hand, a highly increased expression was found to occur, on the ground of which the fur gene was suggested to have a utility as a tumor marker.
Within the framework of the above research, the complete nucleotide sequence of a genomic DNA fragment of about 31 kbp containing the fur gene has meanwhile been determined (Van den Ouweland et al., Nucl.Acids Res. 17, 1989, 7101-7102), while the nucleotide sequence of the corresponding fur cDNA has also been determined (Van den Ouweland et al., Nucl. Acids Res. 18, 1990, 664). On the basis thereof it is now possible for the fur gene to be completely characterized, at both the level of genomic organization structure and the level of the encoding sequences. From these encoding nucleotides sequences, the amino acid sequence of the furin can also be derived.
A computer analysis of this amino acid sequence has now surprisingly revealed that furin is highly similar to subtilisin-like proteases as encoded in yeast by the KEX1 gene of
Kluyveromyces lactis
and the KEX2 gene of
Saccharomyces cerevisiae,
and that furin is evidently the higher-eukaryotic form (found in Man and in animals, such as monkey, cat, rat, mouse, chicken and Drosophila) of this endoproteases. More specifically it has been found that the furin exhibits a certain degree of homology with the catalytic domain of the hitherto described bacterial subtilisins (about 20 enzymes), such as thermitase of
Thermoactinomyces vulgaris
and subtilisin BPN′ of
Bacillus amyloliquefaciens,
and exhibits a striking high homology with subtilisin-like proteases, such as the expression product of the KEX1 gene of the yeast
Kluyveromyces lactis
and the expression product of the KEX2 gene of the yeast
Saccharomyces cerevisiae.
The furin, which contains 794 amino acids, exhibits in the domain of the amino acids 97 to 577 an overall homology of about 80.0% with the amino acids 123-584 of the expression product of said KEX1 gene (i.e. 41.6% identical amino acids and 38.3% conservative substitutes) and an overall homology of about 78.9% with the amino acids 134-597 of the expression product of said KEX2 gene (i.e., 39.4% identical amino acids and 39.5% conservative substitutes). These amino acid regions of the yeast proteases comprise the subtilisin-like catalytic domains. The subtilisin-like domain of furin is situated in an amino-terminal furin fragment comprising the amino acids 108-464.
With regard to the subtilisin-like proteases, reference is made to the following publications: Tanguy-Rougeau et al., FEBS Letters 234, 1988, 464-470; Mizuno et al., Biochem. Biophyx. Res. Commun. 156, 1988, 246-254; Meloun et al. FEBS Letters 183, 1985, 195-200; Marklan et al., J.Biol.Chem. 242, 1967, 5198-5211; Mizuno et al., Biochem. Biophys. Res. Commun. 159, 1989, 305-311; Bathurst et al., Science 235, 1987, 348-350; Thomas et al., Science 241, 1988, 226-230; Foster et al., Biochemistry 29, 1990 347-354; Fuller et al., PNAS USA 86, 1989, 1434-1438; Julius et al., Cell 37, 1984, 1075-1089; Bourbonnais et al., J.Biol. Chem. 263, 1988, 15342-15347; Cosman et al., Dev.Biol. Stand. 69, 1988, 9-13; Schubert Wright et al., Nature 221, 1969, 235-242; Cunningham et al., Yeast 5, 1989, 25-33; Davidson et al., Nature 333, 1988, 93-96.
As shown by the above publications, it is especially the expression product of the KEX2 gene of the yeast species
Saccharomyces cerevisiae
which has been well studied and characterized. It is a membrane-associated, calcium ion dependent endopeptidase with an enzyme specificity for paired basic amino acid residues; substrate proteins are cleaved at the carboxyl site of pairs of basic amino acids containing arginine by this enzyme, which is to be defined here as a “restriction” endopeptidase (by analogy to the nomenclature in restriction endonucleases in which a given nucleotide sequence is determinative of the cleavage of the DNA). The location of the enzyme is probably in a structure of the Golgi complex. The subtilisin-like domain and the Ca
2+
activation sequences are in the aminoterminal part of the protein. In the yeast
Saccharomyces cerevisiae,
the endopeptidase is involved in the proteolytic processing of precursors of killer toxin and pairing pheromone alpha factor, i.e., of pro-killer toxin and pro-alpha factor. Furthermore, the endopeptidase is found to be capable of correctly cleaving the mouse neuroendocrine-peptide precursor prepro-opiomelanocortin after introduction into certain mutant mammalian cell lines with disturbed proteolytic processing, and to be capable of processing proalbumin to mature albumin and to be capable of processing the precursor of the plasma C protein.
On the ground of the established similarities between the above known endopeptidase and the furin, it is postulated that the furin is a restriction endopeptidase which can be used for the processing of proteins, more specifically the processing of precursor proteins of polypeptide hormones, growth factors, toxins, enzymes, or other types of biologically relevant proteins. In this connection, in vitro applications are conceivable on the one hand, and in vivo applications on the other, including an application within the framework of a therapeutic treatment. For such applications, the human furin may be more suitable than the above known endopeptidases of non-human origin, or more generally an animal “furin” may be more suitable than an endopeptidase from lower organisms. The same applies to analogues or relatives of furin not yet isolated, referred to herein as furin-like enzymes, belonging to a larger family of restriction-endoproteolytic enzymes of which furin is the first-found representative. The various members of this family will exhibit a high structural resemblance, although the sequence homology may be quite low, possibly as low as below 50% homology. Within this family, it will be possible to distinguish several enzyme classes, such as a group of furin-like enzymes involved in the processing of constitutively secreted proteins and a group of furin-like enzymes involved in the processing of proteins whose secretion is regulated (secretion through secretory granula). It is possible that each of these furin-like enzymes is characteristically expressed in a limited number of cell types in which the enzyme is active as a processing enzyme. A limited degree of overlap between the cell and tissue distribution of these enzymes is also conceivable and could very well be responsible for the known phenomenon of cell type-dependent differential processing of precursors.
The pituitary proteins PC1 and PC2, described recently by Seidah et al., DNA and Cell Biol. 9, 1990, 415-424, constitute examples of such furin-like enzymes.
Throug

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