Peptide that kills growing but not stationary cells

Details Peptide that kills growing but not stationary cells Peptide that kills growing but not stationary cells

2000-11-06

2004-02-24

Mosher, Mary E. (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

8 to 10 amino acid residues in defined sequence

C530S325000, C530S300000, C530S501000, C530S333000, C530S333000, C514S016700, C514S013800

Reexamination Certificate

active

06696546

ABSTRACT:

BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced in parentheses by number. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The in-depth study of the molecular mechanisms behind cancer has enabled the design of small molecules that specifically target cancer cells and kill them. This will hopefully reduce host toxicity, but still be lethal to cancer cells. One avenue of intervention for these molecules is manipulating the cell cycle.
Cyclin-dependent kinases (cdk) are significant players in regulating the entry into, and progression of, the eukaryotic cell cycle (reviewed in 1, 2). Notably, selective killing of transformed cells by cyclin/cdk 2 antagonists has already been shown (30).
An important cdk substrate involved in gene expression is poly adenylate polymerase (PAP). This enzyme catalyzes the polyadenylation of the 3′ poly(A) tail found in almost all eukaryotic messenger ribonucleic acids (mRNA). The polyadenylation reaction affects, and is affected by, steps in mRNA synthesis, and so can constitute a significant point of regulation, utilized by the cell, to control gene expression (reviewed in 3). A growing body of evidence suggests this to be the case in early development (4), in cell differentiation (5, 6) and in M-phase (mitotic phase) of the cell cycle. Regulation of polyadenylation via control of PAP activity, including the effect of phosphorylation of PAP on its activity in vitro and in vivo assays has been shown (7, 8, 9).
All known vertebrate PAPs contain a C-terminal serine- and threonine-rich domain with multiple cyclin-dependent kinase sites. These sites are phosphorylated in vitro and in vivo by cyclin B/p34cdc2 (10). Cyclin B/p34cdc2 is a member of the cdk family, with all members being heterodimers containing a kinase subunit (the cdk) and a regulatory subunit (the cyclin). Cyclin binding to the catalytic subunit imparts upon the cdk much of its substrate specificity (11, 12, 13, 14, 15). Some cdk substrates contain a sequence, the cyclin recognition motif (CRM), which is thought responsible for the interaction of cdk substrates with the cyclin. This sequence, the CRM, is now known to be shared by inhibitors (p21, p27 and p57) and substrates (e.g., E2F-1, p130, p107, and pRB) alike (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26).
The present application discloses that PAP binds directly to both G1- and G2-type cyclins, and that cyclin binding is mediated by a stretch of amino acids with similarity to the consensus CRM. Furthermore, the present application discloses an 8-mer peptide, spanning PAP's CRM, which regulates phosphorylation of CRM containing proteins such as the retinoblastoma gene product (pRB) by cdks. Notably, low concentrations of the 8-mer peptide inhibit cdk-mediated pRB phosphorylation.
The present application discloses that this 8-mer peptide, spanning PAP's CRM, is lethal to growing cells. If the cell is dividing, the peptide kills the cell. However, if the cell is stationary, the peptide actively protects the cell from death. CRM peptides previously reported to kill cells (30) are much more limited in effect and only kill some cancer cells, leaving other immortalized cells untouched. In contrast, the membrane permeable PAP CRM peptide sequence disclosed in this application (SEQ ID NO: 3) kills all dividing cells tested thus far, including cells the prior art cannot.
More importantly, the peptide disclosed here is much more effective in killing cells with an inactive pRB than it is in killing normal growing cells (up to 50 times more). The peptide is thus more efficient at killing the dividing cells of the type found in neoplasias (up to 90% of neoplasias are thought to have an inactive pRB) than other dividing cells in the body, such as those in the gut. Potentially, the peptide will specifically target cancer cells and kill them whilst showing reduced host toxicity as compared to other treatments.
SUMMARY OF THE INVENTION
The present invention provides a peptide comprising the amino acid sequence shown in SEQ ID NO: 1, wherein the peptide is a purified peptide or a synthetic peptide. The present application also provides a peptide comprising the amino acid sequence shown in SEQ ID NO: 3, wherein the peptide is a purified peptide or a synthetic peptide.
The invention provides a method of killing a dividing cell, which comprises applying to the cell an amount of any of the peptides described herein effective to kill the cell.
The invention provides a method of treating a tumor in a subject, which comprises administering to the subject an amount of any of the peptides described herein effective to treat the tumor.
The invention provides a method of treating an abnormality in a subject, which comprises administering to the subject an amount of any of the peptides described herein effective to alleviate the abnormality, wherein the abnormality is alleviated by killing dividing cells.
The invention provides a pharmaceutical composition comprising any of the peptides described herein and a pharmaceutically acceptable carrier.
The invention provides the use of any of the peptides described herein for the preparation of a pharmaceutical composition for treating an abnormality, wherein the abnormality is alleviated by killing dividing cells.
The invention provides a method of protecting a non-dividing cell from cell death which comprises applying to the cell an amount of any of the peptides described herein effective to protect the cell.


REFERENCES:
patent: WO 93/11227 (1993-06-01), None
Bolton, et al., 2000, Eur J Neurosci, 12(8):2847-55.*
Bond, G.L., Prives, C., and Manley, J.L. Poly(A) polymerase phosphorylation is dependent on novel interactions with cyclins. Mol. Cell. Biol. 20(14) : 5310-5320, Jul. 2000.
Chen, Y.-N. P., Sharma, S.K., Ramsey, T.M., Jiang, L., Martin, M.S., Baker, K., Adams, P.D., Bair, K.W, and Kaelin, W.G. Jr. Selective killing of transformed cells by cyclin/cyclin-dependen kinase 2 antagonists. Proc. Natl. Acad. Sci. 96: 4325-4329, Apr. 1999.

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