Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-07-12
2004-06-22
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C424S094500, C514S002600, C514S012200, C530S350000, C530S352000
Reexamination Certificate
active
06753158
ABSTRACT:
The present invention relates to screening methods, peptides, mimetics, and methods of use based on the surprising discovery and characterisation of an interaction between known proteins and the establishment that such interaction plays a key role in DNA repair, and thus numerous cellular processes of interest in therapeutic contexts. Two proteins in question are XRCC4 and DNA ligase IV. Interaction between XRCC4 and DNA-PKcs/Ku is also indicated.
The invention has arisen on the basis of the work of the present inventors establishing for the first time crucial information about XRCC4. Some information was available on the physiological function of this protein, it having been implicated in the Ku-associated DNA double-strand break repair (KADR) apparatus. However, very little was known about its biological activity and what its role in the KADR apparatus actually is. Prior to the making of the present invention it was not feasible to provide assays useful as primary screens for inhibitors of XRCC4.
Furthermore, the inventors' new cloning work has identified a yeast homologue of mammalian DNA ligase IV. No physiological function has previously been assigned to mammalian DNA ligase IV, but the inventors' yeast work, including analysis of the effect of knock-out mutation in yeast, now establishes the physiological relevance of DNA ligase IV and thus provides indication of therapeutic contexts in which modulation of its function can be effected.
The work disclosed herein establishing interaction between XRCC4 and DNA ligase IV, interaction between XRCC4 and DNA-PKcs/Ku, and also a biological role for such interactions, now gives rise to screening methods for identifying compounds which affect the interaction, particularly those which interfere with it, and which may affect or modulate particular aspects of cellular DNA repair activity, useful in a therapeutic context, for example in the treatment of proliferative disorders, cancers and tumours, disorders involving retroviruses such as AIDS, human adult T-cell leukemia/lymphoma, Type I diabetes and multiple sclerosis, and also in radiotherapy. Furthermore it gives rise to the rational design of peptides or mimetics or functional analogues which fulfil this function.
One of the most dangerous forms of damage that can befall a cell is the DNA double-strand break (DSB), which is the principal lethal lesion induced by ionizing radiation and by radiomimetic agents. Consequently, cells have evolved highly effective and complex systems for recognizing this type of DNA damage and ensuring that it is repaired efficiently and accurately. Two major pathways have evolved to repair DNA DSBs in eukaryotes, homologous recombination and DNA non-homologous end-joining (NHEJ).
Much of what is currently known about DNA NHEJ in mammalian systems has been obtained through studies of a series of mutant rodent cell lines that were identified originally as being hypersensitive towards ionizing radiation and which display severe defects in DNA DSB repair (reviewed in
Jeggo
et al., 1995
; Roth
et al., 1995). Characterisation of these cell lines has revealed that they fall into three complementation groups, termed. IR4, IR5 and IR7. The hamster cell line XR-1 defines IR4, IR5 consists of a number of independently isolated hamster cell mutants, and IR7 contains the hamster cell line V3 and cells derived from the severe combined immune-deficient (scid) mouse. Various studies have shown that IR4, IR5 and IR5 cells are defective in antibody and T-cell receptor V(D)J recombination.
Considerable effort has been directed towards establishing the nature of the gene-products defective in cells of IR4, IR5 and IR7, and determining how they function in DNA NHEJ. As a result of such studies, it was shown that cells of IR5 and IR7 are deficient in components of the DNA-dependent protein kinase (DNA-PK) (Ku80 and DNA-PKcs, respectively). DNA-PK is a nuclear protein Ser/Thr kinase that displays the unusual property of being activated upon binding to DNA DSBs or other perturbations of the DNA double-helix (Jackson, 1997). In light of the biochemical properties of DNA-PK which have been established, an attractive hypothesis is that this enzyme serves as a DNA damage sensor in vivo.
In contrast to cells of IR5 and IR7, XR-1 cells of IR4 are not deficient in a DNA-PK component, as evidenced by the fact that extracts of these cells have normal DNA end-binding activity (Getts and Stamato, 1994; Rathmell and Chu, 1994; Finnie et al., 1996) and DNA-PK activity (Blunt et al., 1995), and that expression of neither Ku80 nor DNA-PKcs complements the V(D)J recombination or radiosensitivity defects of XR-1 cells (Taccioli et al., 1994; Blunt et al., 1995). Instead, it has been shown that DNA from human chromosome region 5q13-14 complements XR-1 cells, the complementing gene being termed XRCC4 (Otevrel and Stamato, 1995).
Furthermore, (Li et al., 1995) have identified the XRCC4 gene recently through its ability to confer normal V(D)J recombination activity and partially restore the DSB repair defect on XR-1 cells, and have demonstrated that the XRCC4 locus is deleted in XR-1 cells.
Interestingly, XRCC4 encodes a small 334 amino acid residue protein of calculated molecular weight of 38 kDa, and the human and mouse homologues of this protein have been shown to be approximately 75% identical (Li et al., 1995). Perhaps surprisingly, however, sequence analyses reveal that XRCC4 is not significantly related to any previously-characterized proteins. Therefore, although it is clear that XRCC4 plays a crucial role in DNA DSB repair and V(D)J recombination, the cloning and sequencing of the cDNA for this factor has so far provided little clue to its mechanism of action.
The Li et al. paper is the only paper published on the XRCC4 protein as such prior to the priority date of the present invention. It reports that XRCC4 is not related to any other proteins and so its sequence gives no clear clues as to its function. Prior to the present work, therefore, the only assays available for XRCC4 were cellular radiosensitivity and cellular V(D)J recombination—assays that cannot be used as primary screens for inhibitors. Consequently, it was impossible to conceive of any biochemical screen for the activity of this factor.
It should be noted too that the Li et al. paper does not provide any evidence that XRCC4 is a nuclear protein (shown herein) and discusses on page 1084 that XRCC4 has putative sites for cytoplasmic protein tyrosine kinases. Thus, it is clear that there really was nothing known about how this protein might act.
The present inventors have shown that XRCC4 exists, at least in part, in the cell nucleus and demonstrated convincingly that it interacts with DNA ligase IV, and also DNA-PKcs/Ku. Evidence is provided herein in the experimental section, with confirmation being provided also by Mizuta et al., 1997. Grawunder et al, 1997 has also provided evidence of interaction between XRCC4 and DNA ligase IV. See also the inventors' publications Teo and Jackson, 1997 and Critchlow et al. 1997.
DNA ligases are catalysts which join together Okazaki fragments during lagging strand DNA synthesis, complete exchange events between homologous duplex DNA molecules, and seal single- or double-strand breaks in the DNA that are produced either by the direct action of a DNA damaging agents or by DNA repair enzymes removing DNA lesions (for review, see Lindahl and Barnes, 1992). In contrast to prokaryotic and yeast systems, where only a single species of DNA ligase has been previously been described (Johnston and Nasmyth, 1978), four biochemically distinct DNA ligases have been identified in mammalian cells (Tomkinson et al., 1991; Wei et al., 1995; Robins and Lindahl, 1996). In vitro assays, and studies of yeast and human cells containing mutated alleles of DNA ligase I suggest that this enzyme joins Okazaki fragments during DNA replication (Henderson et al., 1985; Malkas et al., 1990; Tomkinson et al., 1991; Barnes et al., 1992; Li et al., 1994; Prigent et al., 1994; Waga et al., 1994). Furth
Critchlow Susan Elizabeth
Jackson Stephen Philip
Bozicevic Field & Francis LLP
Kudos Pharmaceuticals Limited
Liu Samuel W.
Low Christopher S. F.
Sherwood Pamela J.
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