Peptides with antiproliferative properties

Details Peptides with antiproliferative properties Peptides with antiproliferative properties

1999-06-07

2003-12-09

Helms, Larry R (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S303000, C514S002600, C514S003100

Reexamination Certificate

active

06660830

ABSTRACT:

The present invention pertains to peptides having antiproliferative properties, nucleic acids (DNA/RNA) coding for said peptides, peptide nucleic acids that are structurally homologous to said nucleic acids and the use of said peptides, said nucleic acids, said peptide nucleic acids and/or pharmaceutical compositions thereof in biotechnology, in molecular biology, bioinformatics, as well as the diagnosis and therapy of hyperproliferative diseases, particularly cancer and atherosclerosis.
In oncology there are a variety of different treatment regimens primarily based on surgical procedures, chemotherapy or radiation therapy. However, these methods have not provided the desired results in cancer therapy. According to current statistics in the United States of America every third person living there develops cancer and every fifth person is going to die therefrom. This number is likely to apply to all industrialized nations all over the world and has not significantly changed for more than a decade. Moreover, it is to be noted that the efficacy of treating cancer has not substantially improved during the past twenty years since the five-year survival rate has essentially stayed the same for most cancer types (R. N. Proctor “The Sciences”, March/April 1995, pp. 20-24).
Therefore, the objective of the present invention is to provide a means to inhibit the growth of a variety of different types of cancer eventually resulting in a regression of the tumors.
This objective is achieved by a peptide by a nucleic acid (DNA/RNA), by a peptide nucleic acid, by a pharmaceutical composition and by the use of said peptides, by the use of the DNA/RNA, by the use of the peptide nucleic acid and by the use of a pharmaceutical composition the present invention. Preferred embodiments are illustrated in the dependent claims.
The present inventor has found a promising approach in the treatment of cancer which is to utilize specific peptides that block substances causing cancer, particularly oncogenic proteins. The peptides of the present invention are characterized by a short sequence length which renders them economical in terms of their synthesis. Moreover, they are able to efficiently penetrate cells and, as a result, are capable of neutralizing intracellular proteins that promote the development of cancer.
It has previously been found that in a variety of cancer types tumor suppressor genes are deleted or mutated which in turn results in an insufficient provision of the corresponding gene products. This finally leads to the development of cancer (A. G. Knudson, Proc. Natl. Acad. Sci., USA (1993), vol.90, pp 10914-10921; A. J. Levine, Sci. Am. “Science & Medicine”, Jan./Feb. 1995, pp.28-37).
Defects in tumor suppressor proteins, such as RB1, also seem to be important for the pathogenesis of other hyperproliferative diseases, e.g. atherosclerosis. The present inventor has found that it is sufficient to introduce parts of a tumor suppressor protein into cells in order to block growth factors that accelerate the uncontrolled growth of cells. It is not possible to introduce the entire tumor suppressor protein since such a protein is too long and is in most cases is subject to proteolytic degradation, thus preventing the protein from exerting its effect. On the other hand, if only parts of tumor suppressor proteins are utilized, said parts are too rapidly degraded as well, i.e. they are not sufficiently stable.
The peptides according to the present invention are therefore synthetic fusion poly-peptides consisting of the components (A) and (B), wherein these components are connected to each other as AB or BA. The first component (A) comprises an effective part of a tumor suppressor protein—in such a form as first found by the inventor—or is hydropathically homologous thereto. The second component (B) comprises a sequence that stabilizes the first component (A). According to the invention the peptide of the present invention binds to a growth factor or a growth factor receptor, preferably to the sequence LXCXE (SEQ. ID. NO: 1) therein, more preferably to the LXCXE (SEQ. ID. NO: 1) sequence in insulin, i.e. to LVCGE (SEQ. ID. NO: 2) (Radulescu et al., Biochemical and Biophysical Research Communications 1995, vol. 206, pp. 97-102). Since the growth factors IGF-1 and IGF-2 comprise the sequence FVCGD (SEQ. ID. NO: 3), which sequence is hydropathically homologous to the segment LVCGE (SEQ. ID. No: 2) in insulin (R. Radulescu & C. Wendtner, J. Mol. Recognition 1992, vol. 5, pp. 133-137) the peptide of the invention binds to IGF-1 and IGF-2 as well.
The term “hydropathically homologous” is used herein according to the teaching of Kyte and Doolittle (J. Kyte & R. F. Doolittle, J. Mol. Biol. 1982, vol. 157, pp. 105-132) and J. E. Blalock and Smith, E. M. (Biochemical and Biophysical Research Communications, Vol. 121, no. 1, pp. 203-207 (1984)).
The component (A) is further characterized by the fact that it is hydropathically complementary to a fragment of a growth factor or of a growth factor receptor according to the complementary peptide theory (J. E. Blalock, Trends in Biotechnology, vol. 8, pp. 140-144, June 1990) or according to the three-dimensional configuration, respectively. Examples for growth factors are: insulin, IGF-1, IGF-2, EGF, FGF, angiogenin, NGF and PDGF.
According to the present invention the effective regions for component (A) of the peptide according to the present invention may be derived from the following tumor suppressor gene products: RB1, P107, P130, WT1, TP53, NF1, NF2, VHL, APC, NB1, MLM, MEN1, BCNS, RCC, BRCA1, BRCA2, DCC, MTS1=p16, MTS 2, p21, p27 and others. In a preferred embodiment, the component (AN comprises a section of RB1, in particular amino acids 649-654 of RB1, i.e. LFYKKV (SEQ. ID. No: 4) or is hydropathically homologous thereto.
The component (B) of the fusion peptide has the property of stabilizing said component (A) as a cofactor so that the latter is not proteolytically degraded in the cell. This may be achieved by the peptides of the present invention rapidly arriving at the cell nucleus where they are less prone to proteolytic degradation (R. Fahraeus et al., Current Biology 1996, vol.6, no. 1, pp. 84-91) or by using branched components (B) such as polylysine branches or polylysine cores or by using D-amino acids. In a preferred embodiment, the component (B) is a polylysine core (R. Radulescu et al., Biochemical and Biophysical Research Communications, 1995, vol. 206, pp. 97-102; and G. Fassina, EPO 0 481 930 A2) or a nuclear localization sequence (NLS), particularly a SV40 NLS or Penetratin or a bipartite NLS or the RNP A1 NLS (M9 region). For the construction of peptides which are preferably directed to the cell nucleus general reference is made to Sheldon et al., Proc. Natl. Acad. Sci. USA, vol. 92, pp. 2056-2060 (1995); Dingwall et al., Trends in Biochemical Sciences 16, pp. 478-481 (1991), M. S. Moore, Current Biology, vol. 6, no. 2, pp. 137-140 (1996); D. A. Jans, FASEB Journal, 1994, vol 8, pp. 841-847; J. Moroianu & J. F. Riordan PNAS, 1994, vol. 91, pp. 1677-1681 and D.Derossi et al., Journal of Biological Chemistry, 1994, vol. 269, pp. 10444-10450.
In a preferred embodiment, the component (A) has the following sequence or is homologous thereto in at least two positions:
NH
2
-LFYKKV-COOH (P1) (SEQ. ID. No: 4)
From the above the following preferred peptides according to the present invention may be derived:
NH
2
-[LFYKKVGGG]
4
-[KRG]
2
-KG-COOH (P2) (SEQ. ID. No: 6)
This peptide is a combination of the sequence P1 and the polylysine core [GGG]
4
-[KRG]
2
KG (SEQ. ID. No: 7)
NH
2
-[LFYKKVGGG]
4
-[K]
2
-KG-COOH (P2i) (SEQ. ID. No: 8)
This peptide is a combination of the sequence P1 and the polylysine core [GGG]
4
-[K]
2
-KG (SEQ. ID. No: 9).
NH
2
-[dLdFdYdKdKdV-GGG]
4
-[1KdRG]
2
-1KG-COOH (P3) (SEQ. ID. No: 6)
This peptide is a combination of the sequence P4 and the polylysin core [GGG]
4
-[1KdRG]
2
-1KG (SEQ. ID. No: 7).
NH
2
-&

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