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
Reexamination Certificate
1998-07-31
2003-09-02
Borin, Michael (Department: 1631)
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
C530S326000, C514S013800, C514S015800, C514S016700, C435S007100
Reexamination Certificate
active
06613878
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the area of cancer therapeutics. More particularly, the present invention relates to fragments of Fen1 that interact with PCNA, and the use of such fragments or mimetics of Fen1 in methods of screening for compounds useful in treating disorders in which PCNA is implicated.
BACKGROUND OF THE INVENTION
Maintenance of genomic integrity within the cell requires a co-ordination between cell-cycle regulated DNA replication, and DNA repair. In the presence of damaged DNA, proliferating cells must cease DNA replication, so that lesions do not become fixed, and repair all damage before replication can recommence. Therefore, the co-ordination of these two processes is critical to avoid mutation and genomic instability. One protein known to be involved in both in DNA replication and in nucleotide excision repair is proliferating cell nuclear antigen (PCNA).
In solution, PCNA from
Saccharomyces cerevisiae
is thought to exist as a trimer. Each monomer has two structurally similar domains separated by a central loop, and so the trimer shows overall six-fold symmetry, as determined by X-ray crystallographic analysis (Kong et al., 1992; Krishna et al., 1994). Despite variation at the amino acid level, human PCNA is thought to be highly homologous at the structural level to budding yeast PCNA (Krishna et al., 1994). These structural studies have shown that trimeric PCNA forms a toroidal structure around DNA, confirming earlier biochemical studies that suggested that PCNA acts as a sliding clamp around double stranded DNA (reviewed by Kuriyan & O'Donnell, 1993), holding the DNA replication machinery onto its template and thereby greatly enhancing its processivity (Bravo et al., 1987; Prelich et al., 1987b). PCNA is localised to sites of DNA synthesis within the nucleus (eg Bravo & MacDonald-Bravo, 1985), and is required to reconstitute SV40 DNA replication in vitro from purified proteins (Prelich et al., 1987a), clearly demonstrating a requirement for the protein in DNA replication. Similarly, the
Schizosaccharomyces pombe
PCNA gene pcn1 is essential, with cells showing a phenotype characteristic of a defect in DNA replication when pcn1 is deleted (Waseem et al., 1992). In addition to its replication role, PCNA is also required for nucleotide excision repair in cell-free systems (Shivji et al., 1992). However, the way in which PCNA carries out these two separate roles is as yet unclear.
SUMMARY OF THE INVENTION
The present invention is based on the finding that a human protein Fen1 interacts with PCNA. This was shown using a yeast two hybrid screen for proteins encoded by a human cDNA library that interact with human PCNA in a cellular environment.
Fen1 has previously been described as a structure-specific endonuclease (Harrington & Lieber, 1994a) with 5′→3′ exonuclease activity (Robins et al., 1994) that shares homology with putative nucleotide excision repair factors including human xeroderma pigmentosum complementation G group protein (Harbraken et al., 1994; O'Donovan et al., 1994),
S. pombe
rad 2 and rad13 (Carr et al., 1993; Murray et al., 1994), and
S. cerevisiae
RAD27/YKL510 and RAD2 (Jacquier et al., 1992; Siede & Friedberg, 1992). The same protein has, however, been identified as an essential DNA replication factor MF1 (Waga et al., 1994a).
The present invention further relates to the characterisation of the interaction between Fen1 and PCNA at the amino acid level by mapping of the mutual binding sites of each protein. This revealed that p21
Cip1
(also known as p21
WAF
1 or Sdi1), the cyclin-kinase inhibitor that also blocks PCNA's function in DNA replication (Flores et al., 1994; Waga et al., 1994b; Warbrick et al., 1995) but not repair (Li et al., 1994; Shivji et al., 1994), binds to the same site on PCNA as does Fen1. The regions of Fen1 and p21
Cip1
that interact with PCNA are shown to be homologous, and p21
Cip1
peptides are found to compete with Fen1 for binding to PCNA.
The finding that p21
Cip1
, or fragments thereof, compete with Fen1 for PCNA, in particular the region of p21
Cip1
identified in our copending application number PCT/GB95/02583 as being responsible for PCNA binding, leads to the possibility of using Fen1 in the screening of mimetics for p21
Cip1
, in particular those which may block or inhibit cellular DNA replication.
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 Fen1 protein containing a peptide of 89 amino acids from the C-terminal region or an active portion thereof; or,
(ii) a fragment of the Fen1 protein containing the sequence motif QGRLDxFF (SEQ ID NO:1); or
(iii) a functional mimetic of said protein fragments.
We have found that “x” may be S, D or G, but probably other amino acids will be tolerated as well.
In the present invention, “an active portion” means a peptide which is less than said full length Fen1 amino acid sequence, but which retains the property of binding to PCNA.
In the present invention, “functional mimetic” means a substance which may not be a peptide at all, but which has the property of binding to PCNA, excluding the p21
Cip1
fragments disclosed in our earlier application.
In a further aspect, the present invention provides assays using a binding agent which is a fragment or mimetic of Fen1 as described above. In particular, the present invention provides a method of screening for Fen1 or p21
Cip1
mimetics comprising exposing Fen1 or a fragment or mimetic thereof which binds PCNA (herein referred to as “the Fen1 component”) and a candidate mimetic to PCNA or an active fragment thereof (herein referred to as “the PCNA component”), so that the candidate mimetic and the Fen1 component compete to bind the PCNA component, and detecting the extent of binding of the PCNA component to the candidate mimetic and/or the Fen1 component. Candidate mimetics which are found to bind to PCNA can then be further screened for biological activity, especially inhibition of DNA synthesis or inhibition of (tumour) cell growth.
Conveniently, the screening method can be carried out by immobilising the fragment or mimetic of Fen1 on a solid support, and exposing the immobilised Fen1 component to PCNA and various concentrations of the candidate mimetic. The extent of PCNA binding to immobilised Fen1 can be measured using an antibody which detects PCNA. Alternatively, interaction of radiolabelled PCNA with immobilised Fen1 component in the presence of candidate mimetic can be measured in a scintillation proximity assay. Other assay formats and screening techniques using Fen1 fragments or mimetics can be readily determined by the skilled person and used to screen candidate mimetics.
In a further aspect, the present invention includes mimetics obtained by using the above screening method.
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 may be unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
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 conferring 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 properties, eg stereochemistry, bonding, size and/or charge, using data from a range of sources, eg spectroscopic techniques such as N
Cox Lynne S.
Glover David M.
Lane David P.
Warbrick Emma
Borin Michael
Cyclacel Limited
DeConti, Jr. Giulio A.
Kanik Cynthia L.
LaHive & Cockfield, LLP
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