Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-04-07
2003-05-27
McKane, Joseph K. (Department: 1627)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S014800, C514S015800, C514S016700, C530S326000, C530S327000, C530S328000
Reexamination Certificate
active
06569833
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to substances having the property of binding to cyclin dependent kinase (cdk), and in particular to substances having this property derived from analysis of fragments of p16 protein. The present invention also relates to pharmaceutical compositions comprising these substances and their use in methods of medical treatment, especially in the treatment of hyperproliferative disorders. The invention also relates to methods and uses of these substances in identifying compounds having related activities.
BACKGROUND TO THE INVENTION
Phosphorylation of the Rb gene product (pRb) by members of the cyclin dependent kinase (cdk) family is an important step in the cells commitment to undergo mitosis. This step is regulated in the later part of the G1 phase of the cell cycle at what is known as the restriction (R) point (6). The cdks are key regulatory factors through which both positively and negatively acting cell signal transduction factors merge. Mitogenic stimulation induces an active complex between the D-cyclin, and cdk4 or cdk6 that is capable of phosphorylating pRb in late G1. These kinases are also the targets for cell growth inhibitory signals arising from contact inhibition, growth factor starvation or TGF-&bgr;. The inhibitory signals can block kinase activity by inducing the production or activity of different members of the two rapidly enlarging families of INK4 and p21/KIP cdk-inhibitors that either interfere directly with the kinases or with the cyclin-kinase complexes (7). The family of INK proteins that have been identified consists of p15, p16, p18 and p19 (20, 22, 23).
However, unlike p21
cipl/WAF1
, which is indirectly linked to tumour suppression activity through p53 transcriptional stimulation (8), the INK4p16 gene is itself deleted or mutated in a large number of human tumours (9-15). Germ line mutations in INK4p16 have been associated with an increased risk of developing melanoma (9,10). The 156 amino acid product of the INK4p16 gene is known as CDKN2 or p16INK4a (referred to in this application as “p16”).
SUMMARY OF THE INVENTION
We set out to identify and study the region of p16 that interacts with cyclin dependent kinases such as cdk4 and cdk6, and to investigate applications of these properties, in particular the possibility that the binding of cdks by substances comprising a peptide based on this region of p16 could be used in tumour suppression by inhibiting the phosphorylation of Rb protein.
Small peptides can sometimes be powerful tools to identify regions of proteins involved in protein-protein interactions and biological activity (16-19). In this work, we synthesised a series of overlapping 20 amino acid (aa) peptides that spanned the p16 amino acid sequence, and tested the capacity of each biotinylated peptide to interact with
35
S-labelled cdk4 and cdk6 expressed in rabbit reticulocyte lysates.
These experiments identified a 20 amino acid synthetic peptide corresponding to residues 84 to 103 of p16 that interacts with cdk4 and cdk6, and inhibits cdk4-cyclin D1 mediated phosphorlotion of Rb protein in vitro. An alanine substitution series defined amino acid residues important for the cdk4 and cdk6 interaction and for the inhibition of Rb phosphorylation. In this application, residues 84 to 103 of p16 correspond to the sequence sets out in
FIG. 1C
, i.e. DAAREGFLDTLVVLHRAGAR (SEQ ID NO:1).
Further, when coupled to a small peptide carrier molecule and applied directly to tissue culture medium, the p16-derived peptide blocked cell cycle entrance into S-phase in both serum starved human HaCaT cells and other types of cells that are cycling normally. This was associated with an inhibition of pRb phosphorylation in vivo. These results demonstrate that a p16-derived synthetic peptide coupled to a small carrier molecule can mimic the G1-phase arrest associated with overexpression of full length p16 protein. This provides a route to the restoration of the p16 suppressor gene function in human tumours.
Accordingly, in a first aspect, the present invention provides a substance having the property of binding to cyclin dependent kinase (cdk) comprising:
(i) a peptide including amino acid residues 84 to 103 of full length p16 protein, or an active portion or derivative thereof; or,
(ii) a functional mimetic of the fragment, active portion or derivative;
wherein the substance excludes full length p16, p15, p18 and p19 proteins;
Preferably, the cyclin dependent kinase (cdk) is cdk4 or cdk6. The substance preferably also has the property of inhibiting the phosphorylation of Rb protein which is mediated by a complex formed between cdks and cyclin D. This in turn can be used to block cellular differentiation by preventing the entry of cells into the S-phase. As the substances bind cyclin dependent kinases, they can also be used to prevent the formation of the complex between cdks and cyclin D, having the additional biological effect of increasing cyclin D levels in cells. As well as blocking cdk4 and cdk6 dependent phosphorylation of pRb, the substances described herein could be used to target other cellular substrates, including the pRb family members p107 and p130, or other substances that are targets for cdk4 and cdk6 mediated regulation.
In the present invention, “an active portion” means a portion of the peptide which is less than the full amino acid sequence of the fragment above, but which retains the property of binding to a cyclin dependent kinase (cdk). Preferably, the peptide also has the property of inhibiting pRb phosphorylation.
In the present invention, a “derivative” is a protein modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, without fundamentally altering the essential activity of the proteins. As an example, a derivative of peptide 6 in which aspartic acid 92 was substituted for alanine was found to be more potent than the peptide 6 (residues 84 to 103) in binding to cdk4 and cdk6, and having a greater inhibition of pRb phosphorylation. Other derivatives include inserting one or more amino acid residues between amino acid motifs FLD ard LVVL.
In the present invention, “functional mimetic” means a substance which may not contain a fragment or active portion of the p16 amino acid sequence, and probably is now a peptide at all, but which has some or all of the properties of the p16 fragment, in particular the property of binding to a cyclin dependent kinase and/or inhibiting pRb phosphorylation.
In a preferred embodiment, the peptide includes residues 89 to 97 of full length p16 protein. More preferably, the peptide includes the peptide motif FLD, corresponding to amino acids 90 to 92 of full length p16 protein, and/or the peptide motif LVVL (SEQ ID NO:2), corresponding to amino acids 94 to 97 of full length p16 protein. We have also found that both the D and L isoforms of the peptides share the property of binding to cdk and/or inhibiting pRb phosphorylation.
In a further aspect, the present invention provides compounds comprising any of the above substances coupled to carrier molecules, enabling the compounds to be delivered to cells in vivo. In one embodiment, the carrier molecule is a 16 aa peptide sequence derived from the homeodomain of Antennapedia (e.g. as sold under the name “Penetratin”), which can be coupled to one of the above substances via a terminal Cys residue. The “Penetratin” molecule and its properties are described in WO 91/18981.
In a further aspect, a substance comprising one of the above peptides can be stabilised by coupling to another peptide sequence. Preferably, this allows the peptide to adopt a conformation more closely resembling that of full length p16, typically having the advantage of increasing the activity of the peptide relative to the uncoupled fragment, e.g. so that the peptide fragment has an activity more closely approac
Fahraeus Robin
Lane David Philip
Cyclacel Limited
DeConti, Jr. Giulio A.
Friend Tomas
Kanik Cynthia L.
Lahive & Cockfield LLP
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