Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1999-09-27
2002-04-30
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C514S002600, C514S012200, C530S324000, C530S330000
Reexamination Certificate
active
06379959
ABSTRACT:
FIELD OF THE INVENTION
The present invention, in general, refers to the use of carboxypeptidase protein inhibitors and of their natural variants or of forms redesigned by protein engineering as well as peptidomimetic molecules derived from the former as an anticancerous medicament, and more specifically, to control the invasion and the metastatic growth in tumors and to its use for therapeutic purposes.
1. BACKGROUND OF THE INVENTION
Protease inhibitors have been extensively related to processes of biomedical interest, such as the processing of hormone and neuropeptide precursors, inflammatory processes, tumoral processes, etc. (Avilés, F. X., editor of “Innovations in proteases and their inhibitors”, Walter de Gruyter, 1993; Trow, W. & Kennedy, A. R. eds. “Protease inhibitors as cancer chemopreventive agents”; Plenum Publishing Corp., 1993; Kennedy, A. R. (1994)
Cancer Res.,
54: 1995s-2005s). Among the protease inhibitors are the metalocarboxypeptidase inhibitors. One of them, the inhibitor from potato (from now on, referred to as “PCI”) has been extensively studied, from the structural and functional point of view, as an inhibitor by our research team (Molina, M. A. et al. (1992)
Gene,
116: 129-138; Oliva, B. et al. (1991)
Biochem. Biophys. Res. Comm.,
176: 616-621 and 627-632; Tapia et al. (1991)
J. Mol. Engineer.,
1: 249-266; Querol, E. et al. (1993) in “Innovations in Proteases and their Inhibitors” (F. X. Avilés, ed.) : 447-494. Walter de Gruyter, Berlin, 1993; Molina M. A. et al. (1994)
J. Biol. Chem.,
269: 22087-22094; Marino-Busjle, C. et al. (1994)
Applied Microb.
&
Biotech.,
41: 632-637; Oliva, B. et al. (1995)
J. Mol. Model.,
5:1-15).
A relevant structural feature of the PCI is that it is a small protein which contains a globular core stabilized by means of three disulfide bridges which constitute a cystine knot. We are referring to a topological pattern shared by other proteins, whether functionally related or not, which employ various cysteines concentrated in a central zone in order to create a stabilizing knot of disulfide bridges. This ensemble of proteins constitutes the so called group of knotins, because of the topological knot they form. We have to underline that, among the proteins which share this structural pattern with PCI, there are various cellular growth factors such as, for instance, the a and &bgr;-TGF, NGF, PDGF, EGF and those of the insulin family (Isaacs, N. (1995)
Curr. Opin. Struct. Biol.,
5:391-395; Lin, S et al. (1995)
Nature Struct. Biol.,
2, 835-837; Sun P. & Davies, D. (1995)
Ann. Rev. Biphys. Biolmol. Struct.,
24: 269-291). In the specific case of the PCI, the globular core contains 27 amino acid residues and is flanked by two tails of 7 residues (the N-terminal tail) and of 5 residues (the C-terminal tail). The disulfide bridges are between cysteines 8-24, 12-27 and 18-34. This is the most frequent natural form, the so called IIa form, although there are other isoforms (Hass, G. M. & Ryan, C. A. (1981)
Meth. Enzymol.,
80: 778-791).
A great number of studies have described the expression of proteases and peptidases in different types of tumoral cells (Chen, W. T. (1992)
Curr. Opin. Cell. Biol.,
4: 802-809; Birkedal-Hansen, H. (1995)
Curr. Opin. Cell. Biol.,
7: 728-735). Some of these proteases are related to the processing of peptidic hormones, growth factors, etc. For instance, the existence of proteases responsible for the mobilization of the &bgr;FGF (basic fibroblast growth factor) of the extracellular matrix, a factor which has been involved in tumoral angiogenesis (Liotta, L. A. et al. (1991)
Cell,
327-336. Folkman, J. (1995)
New England J. Med.,
333: 1757-1763). At the same time, the TGF-&bgr;1 factor is also mobilized. It shows opposite effects, thus regulating the angiogenesis process (Mignati, P. & Rifkin, D. B., (1993)
Physiolog. Rev.,
73: 161-195). The process which has been more intensively studied is the role played by the proteases in invasiveness and metastasis. A feature of the invasive processes, either those which take place in the primary tumor or those related to the establishment of metastasis, is the degradation of the extracellular matrix as a result of which the tumoral cells can penetrate the adjacent tissues (the main components of the extracellular matrix are proteins and proteoglycans) (Hynes, R. O. (1994)
Bioessays,
16:663-669). In fact, there is a positive relationship between the level of expression of proteases and the aggressivity and malignancy of the tumor due to the greater invasiveness of the tissues of the tumoral cell. These proteases are not specific to the tumoral cells but are also produced by normal cells during the normal recomposition processes of the tissue (wound healing, morphogenesis, etc). The difference is that the tumoral cells links this proteolysis with the motility resulting in an invasion in times and sites which are inappropriate.
Due to the important role which is played by proteases in the tumoral processes and in practically all their stages (concretely, in transformation, invasiveness, adherence and metastasis), various studies have been published in which the possible role of the protease inhibitors as antitumoral agents is analysed (Troll, W. & Kennedy, A. R., editors “Protease inhibitors as cancer chemopreventive agents”, 1993). Protease inhibitors would employ their antitumoral properties at various levels among which we may mention:
1. Blocking the transformation of normal cells in tumoral cells, a process in which proteases seem to be involved (Billings, P. C., et al. (1989)
Carcinogenesis,
10: 687-691).
2. Blocking the proteolytic cascade involved in the process of invasiveness and metastasis (Kohn, E. C. & Liotta, L. A. (1995)
Cancer Res.,
55: 1856-1862).
3. Altering the processing of growth factors required for the development of the tumor.
Among the relevant studies in relation to the present invention are the following: Billings, P. C. et al. (1989)
Carcinogenesis,
10: 687-691, where it is described that the addition of PCI in low concentrations (5 &mgr;g/mL) inhibits in vitro the tumoral transformation induced by irradiation in embryonic cells of mice. However, up to now, any effect of the PCI in the growth and survival of already transformed cells or in tumoral cells, such as it is described in the present invention, cannot be found in scientific literature. On the other hand, Billings, P. C., et al. (1991)
Carcinogenesis,
12: 653-657, explain that the chemotrypsin inhibitor 1 of the potato suppresses the transformation provoked by the irradiation in CH3/10T1/2 cells, in vitro. Billings, P. C. et al. (1991)
Eur. J. Cancer,
27: 903-908, describe another inhibitor derived from soya, the so called Bowman-Birk (from now on, referred to as “BBI”) which, in mice, reduces cancer of the colon induced by dimethylhydrazine and in (1992)
Proc. Natl. Acad. Sci. USA,
889: 3120-3124, characterizes the enzyme target of the BBI as a gelatinase. It has also been described that BBI suppresses carcinogenesis in the intestines (Kennedy, A. R. et al. (1996)
Cancer Res.,
56: 679-682).
Another relevant aspect for the present invention is the role that growth factors play in the tumoral process. The normal cells are dependent on various growth factors to complete their cellular cycle or to get out of phase G
0
(quiescence). In tumoral cells, the system of signal transduction of any of these factors is often found to be altered. For instance, the tumoral cell may present an autocrine loop, or an altered receptor which would be active even in the absence of the growth factor. However, tumoral cells always need some growth factor (IGF-I type) for their proliferation and for the development of the tumor so their absence may provoke apoptosis (Aaronson, S. A. (1991)
Science,
254: 1146-1153; Thompson, C. B. (1995)
Science,
267, 1456-1462). The migration of the tumoral cell may be regulated by autocrine factors (produced and/or secreted by the same cell), paracrine (growth factors such as those of the PDGF, FGF, EGF and IGF families, whos
Aviles Puigvert Francesc X.
Blanco Aparicio Carmen
De Llorens Duran Rafael
Fernandez Salas Esther
Frazier Marsha L.
Slobodyansky Elizabeth
Steinberg & Raskin, P.C.
Universitat Autonoma de Barcelona
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