NL8902651A - Pharmaceutical compsn. with endo-proteolytic activity - comprises furin or its fragment, and is used to prevent obstruction of vital organs - Google Patents

Abstract

The compsn. contains an endoproteolytically active amt. of furin or furin-like enzyme, or a fragment or deriv. of furin or furin-like enzyme, together with pharmaceutically acceptable carriers, diluents or adjuvants. The enzyme is pref. obtd. from prokaryotic or eukaryotic cells which, via recombinant DNA or RNA engineering may express the enzyme as a fusion protein, when the enzyme has been spilt off from the fusion protein. Also claimed are: 1) A process for the in vitro cleavage of a protein, by using the enzyme in the presence of Ca(2+) ions; 2) A process for the mmicro (biological) prodn. of a protein by culturing cells expressing a proform of the protein; and 3) Mammalian cells containing recombinant-DNA-derived DNA encoding the enzyme.

Description

DESCRIPTION

Pharmaceutical preparation with endoproteolytic activity; method for endoproteolytic processing of proteins

The invention relates both to a pharmaceutical composition having an endoproteolytic activity and to a method of cleaving a protein in vitro by treating the protein with an endoproteolytically active enzyme.

The invention described herein results from a further investigation 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 genome upstream of the human fes / fps proto oncogene located fur gene. From said patent application and other publications of the same research group (Roebroek et al, Molec. Biol. Rep. 11, 1986, 117-125; Roebroek et al, EMBO J., 1986, 2197-2202; and Schalken et al, J. Clin Invest. & Q, 1987, 1545-1549), it appears that based on the limited DNA data available at that time, the function of the product of the fur gene could not be determined. However, it could be determined that furin is probably a membrane-associated protein that has a function in which certain recognition structures play a role. It has also been observed at the time that the fur gene is expressed as a 4.5 kb mRNA in liver, kidney, spleen, thymus and brain, although the expression in lung tissue is very low; in non-small cell lung carcinomas, on the other hand, a strongly increased expression appeared to occur, on the basis of which a usefulness of the lun gene as tumor marker has been suggested.

In the context of the research indicated above, the complete nucleotide sequence of a genomic DNA fragment of approximately 21 kbp, in which the fur gene is located, has now been determined (Van den Ouweland et al., Nucl. Acids Res. 12, 1989, 7101- 7102). On this basis, it was now possible to fully characterize the Xnc gene, both at the level of genomic organizational structure and at the level of the coding sequences. Furthermore, the amino acid sequence of the furin could also be derived from these coding nucleotide sequences.

A computer analysis of this amino acid sequence has now surprisingly revealed that furin is closely related to subtilisin-like proteases encoded in yeast by the KEX1 and KEX2 genes and apparently the higher eukaryotic form (the human form) of these endoproteases is. More specifically, the furin has been shown to exhibit a degree of homology to the catalytic domain of subtilisins such as thermitase from Thermoactinomyces vulgaris and subtilisin BPN 'from Bacillus amyloliquefaciens. and exhibits strikingly large homology with subtilisin-like proteases such as the expression product of the KEX1 gene from the yeast Kluyveromyces lactis and the expression product of the KEX2 gene from the yeast Saccharomyces cerevisiae. The 794 amino acids containing furin exhibits an overall homology of about 80.0% with amino acids 123-584 of the expression product of the KEX1 gene (i.e., 41.6% identical amino acids and 38%) in the region of amino acids 97 to 577. 3% conservative replacements) and an overall homology of approximately 78.9% with amino acids 134-597 of the expression product of the KEX2 gene (i.e., 39.4% identical amino acids and 39.5% conservative replacements). These amino acid regions of the yeast proteases include the subtilisin-like catalytic domains. The subtilisin-like domain of furin resides in an amino terminal furin fragment consisting of amino acids 114-383.

For the subtilisin-like proteases mentioned herein, reference is made to the following publications: Tanguy-Rougeau et al, FEBS Letters 234, 1988, 464-470; Mizuno et al., Biochem. Biophys. Res. Commun. 156, 19S8, 246-254; Meloun et al, FEBS Letters 1 & 3, 1985, 195-200; Markland et al., J. Biol. Chem. 242, 1967, 5198-5211; Mizuno et al., Biochem. Biophys. Res. Commun. 159, 1989, 305-311; Bathurst et al, Science 225, 1987, 348-350; Thomas et al., Science 241, 1988, 226-230; Fuller et al. PNAS USA M, 1989, 1434-1438; Julius et al, Cell 22-r 1984, 1075-1089; Bourbonnais et al, J. Biol. Chem. 263r 1988, 15342-15347; Cosman et al. Dev. Biol. Stand. M, 1988, 9-13; Schubert Wright et al, Nature 221, 1969, 235-242; Cunningham et al, Yeast, 1989, 25-33; Davidson et al, Nature 333, 1988, 93-96.

As can be seen from the above publications, especially the expression product of the KEX2 gene 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 by this endopeptidase at the carboxyl site of pairs of basic amino acids containing arginine. The location of the enzyme is probably in a structure of the Golgi complex. The subtilisin-like domain and the Ca 2+ activation sequences lie in the amino terminal portion of the protein. In the yeast Saccharomyces cerevisiae, the endopeptidase is involved in the proteolytic processing of precursors (precursors) of killer toxin and mating pheromone alpha factor. The endopeptidase further appears to be capable of correct cleavage of the mouse neuroendocrine peptide precursor prepro-opiomelanocortin upon introduction into certain mutant mammalian cell lines with impaired proteolytic processing.

On the basis of the similarities found between the said known endopeptidases and the furin, it is postulated that the furin for the processing of proteins, more particularly the processing of precursor proteins from polypeptide hormones, growth factors, toxins, enzymes, or other types of biological relevant proteins can be used. In this context, an application in vitro can be envisaged, on the one hand, an application in vivo, i.e. in the context of a therapeutic treatment. For such applications, the human furin may be more suitable than the known known endopeptidases of non-human origin.

It is possible to obtain large amounts of the protein furin via recombinant DNA techniques. In prokaryotes, the fur gene can be expressed as a fusion protein with beta-galactosidase (püR vector system) or the anthranilate synthetase (pATH vector system). Another possibility is the synthesis of the fusion protein glutathione S-transferase furin (pGEX). The advantage of this approach is that the furin can be cleaved using thrombin. The furin can also be synthesized as such in prokaryotes by properly placing the cDNA behind a suitable promoter. The püR and pATH vector system are described in the aforementioned European patent application. pGEX is commercially available. Using the strong SV40 promoter, the fur cDNA can be expressed in suitable eukaryotic cells. In connection with glycosylation of the protein, this approach is preferred for certain purposes.

Purification of furin can be performed by standard biochemical techniques in the presence of protease inhibitors. Furin is active in a relatively acidic environment with a pH of 5.5, as it occurs in secretory granules, but the protein also retains its activity at pH 7.5. This makes it possible to use a 0.2 M sodium acetate buffer (pH 5.5) or Tris-HCl buffer (pH 7.0) in vitro. The activity of the enzyme furin depends on the presence of Ca2 + ions. A calcium concentration of 2-5 mM appears to be optimal for the in vitro enzyme activity. The presence of metal chelators such as EDTA will strongly inhibit the activity of furin. Furthermore, the presence must. of heavy metal ions such as Zn2 +, Hg2 + and Cu2 + are avoided. The substance o-phenanthroline binds heavy metals except Ca2 + and thus has no negative influence on the enzymatic activity of furin. Low concentrations of phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP) up to 5 mM do not inhibit. At higher concentrations of PMSF, the enzyme function is inhibited. An in vitro incubation for two hours at 37 ° C is sufficient for the processing of the protein to be cleaved.

Furine can be used for the endoproteolytic processing of various proteins. This makes it possible, among other things, to specifically cleave in vitro produced precursor proteins to produce biologically active preparations which may find their use as a medicine for the treatment of diseases in which the cleavage of the precursors does not take place or does not take place sufficiently. In general it can be stated that furin is useful in the processing of biologically relevant proteins.

The protein furin can also find use as a medicine, whereby patients who are deficient in an endoprotease can be treated by administration of furin, which still allows adequate processing of precursor proteins. This allows the cleavage products to perform their function and the possibly disturbingly high content of precursor proteins can be reduced.

Furin may also be useful for clearing depositions with substrate proteins in e.g. the bloodstream, so that obstructions of vital organs can be remedied by administering furin.

Furine is furthermore also applicable in commercial production of all kinds of biologically active substances (eg other enzymes) if processing is a production step therein.

The invention primarily relates to a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, diluents or excipients, as well as an endoproteolytically effective amount of furin or an endoproteolytic activity fragment or derivative of furin.

The proteolytic activity is also retained when the carboxy-terminal region containing the transmembrane domain is cleaved. According to the invention, instead of the complete furin, it is therefore also possible to use a furin fragment which still contains the part responsible for the proteolytic activity. A suitable fragment is, for example, the furin fragment, which consists of amino acids 114-383.

Furthermore, the activity of the furin or of an endoproteolytically active fragment of furin can be manipulated by mutation. Therefore, the invention also extends to derivatives of furin, which still have endoproteolytic activity.

. According to a preferred embodiment of such a pharmaceutical preparation, the furin or furin or endoproteolytic activity-containing fragment or derivative of furin used in the preparation is obtained from prokaryotic or eukaryotic cells, which are genetically manipulated with recombinant DNA or RNA. have acquired ability to express the furin, furin fragment, or furin derivative, whether or not in the form of a fusion protein, wherein in the event that the furin, furin fragment, or furin derivative is recognized by the cells as a fusion protein is produced, a work-up of the fusion protein has occurred in which the furin, furin fragment or furin derivative has been cleaved from the fusion protein.

However, another possibility is to use as a source of the furin cells which are naturally capable of producing furin, e.g. a suitable tumor cell line.

A particularly preferred embodiment of the invention relates to a pharmaceutical preparation containing furin itself.

An alternative particularly preferred embodiment of the invention relates to a pharmaceutical composition containing an amino-terminal fragment of furin, comprising at least amino acids 114-383 of furin.

The invention further relates to a method of cleaving a protein in vitro by treating the protein with an endo-proteolytically active enzyme, wherein the protein of the present invention is treated with furin or an endoproteolytically active in the presence of Ca 2+ ions fragment, derivative or fusion protein of furin as an endoproteolytically active enzyme.

The treatment will usually be carried out at physiologically occurring pH and temperature values, i.e. at a pH in the range of 4-9 and at a temperature of about 37 ° C.

It is preferred here that the treatment be carried out at a pH of 5-7.5, preferably 5.5-7.0.

It is also preferred according to the invention that the treatment is carried out at a temperature of 20-50 ° C, preferably 30-40 ° C.

It is further preferred according to the invention that the treatment is carried out at a calcium concentration of 1-10 mM, preferably 2-5 mM.

According to a particularly preferred embodiment of the invention, the treatment is carried out in the presence of o-phenanthroline or an equivalent agent for binding ions of heavy metals other than calcium.

A further explanation of the invention is given with reference to the accompanying figures.

Figure 1 shows the amino acid sequence (in the one-letter code) of the 794 amino acids furin.

Figure 2 schematically shows the similarities between the expression products encoded by the genes furf KEX1 and KEX2. The regions in the expression products of the genes KEX1 and KEX2f located between the triangles are the regions in which there is a striking homology with the furin. The shaded areas represent the subtilisin-like domains of the three proteins. Vertical bars indicate the relative positions of cysteine residues in these domains. The arrows labeled D, H or S indicate the relative positions of the three essential amino acids in the subtilisin-like catalytic domain, where D = Asp, H = His, and S = Ser. The arrows shown above the bars point to potential sites of N-linked glycosylation.

Figure 3 compares the amino acid sequence of amino acids 114-383 in the amino terminal region of furin with that of the catalytic domains of the subtilisins thermitase, subtilisin BPN ', and subtilisin Carlsberg. Also included in this comparison are the subtilisin-like domains of the expression products of the genes KEX1 (residues 123-388) and KEX2 (residues 134-400). All sequences are aligned with furin. The numbers listed above the sequences correspond to the amino acid sequence of furin, with amino acids 114 to 383 numbered from 1 to 270. Furine homologous amino acids are boxed. The asterisks (*) indicate the essential amino acids of the active site known for subtilisin.

Claims (10)

A pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, diluents or excipients, as well as an endoproteolytically effective amount of furin or an endoproteolytic activity fragment or derivative of furin. Pharmaceutical preparation according to claim 1, containing furin or an endoproteolytic activity fragment or derivative of furin, obtained from prokaryotic or eukaryotic cells, which by genetic manipulation with recombinant DNA or RNA have obtained the ability to produce the furin, furin fragment or furin derivative, whether or not in the form of a fusion protein, wherein in the case where the furin, furin fragment or furin derivative is produced by the cells as a fusion protein, a work-up of the fusion protein has occurred in which the furin, furin fragment or furin derivative has been cleaved from the fusion protein. Pharmaceutical preparation according to claim 1 or 2, containing furin itself. A pharmaceutical composition according to claim 1 or 2, containing an amino terminal fragment of furin, comprising at least amino acids 114-383 of furin. A method for cleaving a protein in vitro by treating the protein with an endoproteolytically active enzyme, wherein the protein is treated in the presence of Ca 2+ ions with fufine or an endoproteolytically active fragment, derivative or fusion protein of furin as an endoproteolytic active enzyme. The method of claim 5, wherein the treatment is carried out at a pH of 5-7.5, preferably 5.5-7.0. A method according to claim 5 or 6, wherein the treatment is carried out at a calcium concentration of 1-10 mM, preferably 2-5 mM. The method of any one of claims 5-7, wherein the treatment is carried out in the presence of o-phenanthroline or an equivalent agent for binding ions of heavy metals other than calcium. A method according to any of claims 5-8, wherein the treatment is carried out at a temperature of 20-50 ° C, preferably 30-40 ° C. The method of any one of claims 5-9, wherein the treatment is applied to a polypeptide hormone precursor protein, a growth factor, a toxin, an enzyme, or another type of biologically relevant protein.