Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-08-22
2001-06-05
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S326000, C530S328000, C514S002600, C514S013800, C514S016700
Reexamination Certificate
active
06242201
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the area of cancer therapeutics. More particularly, the present invention relates to the identification of the region of p21
WAF1
responsible for binding to proliferating cell nuclear antigen (PCNA) and substances, fragments and mimetics based on this region. The present invention also relates to pharmaceutical compositions comprising these molecules and their use in therapeutic applications for inhibiting DNA replication or binding of PCNA, for example in tumour and other hyperproliferative cells.
BACKGROUND OF THE INVENTION
p21
WAF1
is a protein that may be transcriptionally induced by the tumour suppression protein p53 and acts as a potent inhibitor of cyclin dependent kinases (Cdks) in G1 and S phases of the cell cycle. Thus, p21
WAF1
acts as a regulator of the cell cycle in response to activation of the p53 checkpoint pathway at least in part by inhibiting Cdk activity (1-3). p21
WAf1
is also known as p21
CIP1
(9) p21
pic
(33) p20
CAP
(34) and Sdi1 (35).
Complexes between p21
WAF1
and Cdks can exist in both catalytically active and inactive forms, suggesting that the regulation is a subtle effect (4). p21
WAF1
has also been reported to bind to PCNA at high concentration in vitro and block DNA replication (5). PCNA is a processivity factor for polymerase &dgr; which plays an essential role in DNA replication and repair (6, 7). In transformed cell lines, p21
WAF1
expression is depressed, and cyclin dependent kinases are found in a Cdk/cyclin binary state, rather than in Cdk/Cyclin/p21
WAF1
/PCNA complexes, although the stoichiometry of these complexes is not clear (1, 8-10). Thus, it appears that during p53-mediated suppression of cell proliferation, p21
WAF1
is important for co-ordinating cell cycle progression, DNA replication and repair of damaged DNA.
SUMMARY OF THE INVENTION
We have now found that p21
WAF1
interacts with PCNA in vivo and at concentrations far lower than those reported previously. The mapping of the region of p21
WAF1
that is responsible for the interaction with PCNA is also disclosed. In particular, the applicants have found that peptides derived from the C-terminal region of p21
WAF1
bind to PCNA and have shown that this accounts for the inhibition of DNA replication. The interaction of p21
WAF1
with cyclin-Cdks and PCNA provides the possibility of using p21
WAF1
to co-ordinate cell proliferation and cell cycle control.
The applicants used a yeast two hybrid screening technique to establish an in vivo interaction between the C-terminal part of p21
WAF1
and PCNA. In particular, analysis of a series of overlapping peptides representing the protein sequence of p21
WAF1
identified a 20 amino acid peptide (residues 141-160) which showed high affinity and selectivity of binding to PCNA, and also inhibited SV40 DNA replication in vitro in a concentration-dependent manner. Further experiments have shown that residues essential for PCNA binding and inhibition lie within the motif defined by the sequence QTSMTDFY (SEQ ID NO:1).
This high affinity of the interaction between PCNA (from both normal and tumour cells) and the p21
WAF1
peptide means that it is possible to use these peptides, or fragments or mimetics thereof, in tumour therapy and in the treatment of hyperproliferative diseases in which PCNA is implicated, eg cancer or psoriasis.
Accordingly, in one aspect, the present invention provides a substance which has the property of binding to PCNA, said substance comprising:
(i) a fragment of the p21
WAF1
protein including residues 141 to 160 of the p21
WAF1
amino acid sequence, or an active portion or derivative thereof; or,
(ii) a functional mimetic of said protein fragment.
In the present invention, “an active portion” means a peptide which is less than said full length p21
WAF1
amino acid sequence, but which retains the property of binding to PCNA.
In the present invention, “functional mimetic” means a substance which may not contain an active portion of the p21
WAF1
amino acid sequence, and probably is not a peptide at all, but which has the property of binding to PCNA.
In the present invention, “a derivative” means a fragment of the p21
WAF1
protein modified by varying the amino acid sequence of the protein, eg 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 protein.
Preferably, the fragment of the p21
WAF1
protein includes residues 144 to 151 of the p21
WAF1
amino acid sequence these residues defining a sequence motif QTSMTDFY (SEQ ID NO:1), where residues shown in bold are critical for PCNA binding, and those underlined are important.
Thus, in one embodiment, the present invention provides a class of peptides based on the C-terminal region of p21
WAF1
. These compounds may be useful in the preparation of pharmaceuticals for treating conditions in which PCNA is implicated, including hyperproliferative diseases, such as cancer and psoriasis. These peptides preferably include the sequence motif KRRQTSMTDFYHSKRRLIFS (SEQ ID NO:2), as shown in
FIG. 4
b
, or still more preferably, the sequence motif QTSMTDFY (SEQ ID NO:1), and functional variants or mimetics of these peptides.
In a further aspect, the present invention provides pharmaceutical compositions for inhibiting DNA replication and/or binding PCNA comprising one or more of the above peptides or mimetics. Optionally, the pharmaceutical compositions comprise one or more of the above substances in combination with a physiologically acceptable carrier.
In a further aspect, the present invention provides the peptides, mimetics and compositions described above for use in methods of medical treatment, such as inactivating or functionally depleting PCNA in cells, especially tumour cells.
In a further aspect, the present invention uses a method of screening polypeptides for binding to PCNA comprising:
(i) transforming yeast cells with a vector capable of expressing a fusion of the DNA binding domain of Gal4 and PCNA, the yeast cells being capable of expressing one or more reporter constructs under the control of the Gal1 promoter;
(ii) transforming said yeast cells with one or more vectors capable of expressing a fusion of the activation domain of Gal4 and a given candidate polypeptide; and,
(iii) detecting the production of the reporter constructs caused when the candidate polypeptide binds to PCNA, thereby reconstituting the Gal4 transcriptional activity.
This method is used below to detect the interaction between artificially synthesised peptides and PCNA to define the PCNA binding site in p21
WAF1
more specifically. However, it could readily be used to screen candidate peptides, eg mimetics, for PCNA binding activity.
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a “lead” compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, eg by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its “pharmacophore”.
Once the pharmacophore has been found, its structure is modelled according to its physical propertie
Cox Lynne Suzanne
Glover David Moore
Lane David Philip
Warbrick Emma
Cyclacel Limited
DeConti, Jr. Esq. Giulio A.
Huff Sheela
Lahive & Cockfield LLP
Mandragouras Esq. Amy E.
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