Inactivation of the p53 tumour suppressor is a frequent event in human neoplasia. The inactivation can occur by mutation of the p53 gene or through binding to viral or cellular oncogene proteins, such as the SV40 large T antigen and MDM2. While the mechanism through which wild-type p53 suppresses tumour cell growth is as yet poorly defined it is clear that one key feature of the growth suppression is the property of p53 to act as a transcription factor (Farmer, G., et al. (1992). Nature, 358, 83-86; Funk, W. D. et al. (1992). Mol. Cell. Biol., 12, 2866-2871; Kern, S. E., et al. (1992). Science, 256, 827-830). Currently, considerable effort is being made to identify growth control genes that are regulated by p53 binding to sequence elements near or within these genes. A number of such genes have been identified. In cases such as the muscle creatine kinase gene (Weintraub, H., et al. (1991). Proc. Natl. Acad. Sci. U.S.A., 88, 4570-4571; Zambetti, G. P., et al. (1992). Genes Dev., 6, 1143-1152) and a GLN retroviral element (Zauberman, A., et al. (1993). Embo J., 12, 2799-2808) the role these genes might play in the suppression of growth control is unclear. Yet there are other examples, namely mdm2 (Barak, Y., et al. (1993). Embo J., 12, 461-468; Wu, X., et al. (1993). Genes Dev., 7, 1126-1132) GADD 45 (Kastan, M. B., et al. (1992). Cell, 71, 587-597) and WAF1 or CIP1 (El-Beiry, W. S., et al. (1993). Cell, 75, 817-825; Harper, J. W., et al. (1993). Cell., 75, 805-816) where their involvement in the regulation of cell growth is better understood.
In the present text "mdm2" refers to the oncogene and "MDM2" refers to the protein obtained as a result of expression of that gene.
Mdm2, a known oncogene, was originally found on mouse double minute chromosomes (Cahilly-Snyder., L., et al. (1987) Somatic Cell Mol. Genet. 13, 235-244). Its protein product was subsequently found to form a complex with p53, which was first observed in a rat fibroblast cell line (Clone 6) previously transfected with a temperature sensitive mouse p53 gene (Michalovitz, D., et al. (1990). Cell, 62, 671-680). The rat cell line grew well at 37.degree. C. but exhibited a G1 arrest when shifted down to 32.degree. C., which was entirely consistent with an observed temperature dependent switch in p53 conformation and activity. However, the p53-MDM2 complex was only observed in abundance at 32.degree. C., at which temperature p53 was predominantly in a functional or "wild-type" form (Barak, Y. et al. (1992). Embo J., 11, 2115-2121 and Oren, 1992; Momand, J., et al. (1992). Cell, 69, 1237-1245). By shifting the rat cell line down to 32.degree. C. and blocking de novo protein synthesis it was shown that only "wild-type" p53 induced expression of the mdm2 gene, thereby accounting for the differential abundance of the complex in terms of p53 transcriptional activity (Barak, Y., et al. (1993). Embo J., 12, 461-468) The explanation was further developed by the identification of a DNA binding site for wild-type p53 within the first intron of the mdm2 gene (Wu, X., et al. (1993). Genes Dev., 7, 1126-1132). Reporter constructs employing this p53 DNA binding site revealed that they were inactivated when wild-type p53 was co-expressed with MDM2.
This inhibition of the transcriptional activity of p53 may be caused by MDM2 blocking the activation domain of p53 and/or the DNA binding site. Consequently, it was proposed that mdm2 expression is autoregulated, via the inhibitory effect of MDM2 protein on the transcriptional activity of wild-type p53. This p53-mdm2 autoregulatory feedback loop provided a novel insight as to how cell growth might be regulated by p53. Up to a third of human sarcomas are considered to overcome p53-regulated growth control by amplification of the mdm2 gene (Oliner, J. D., et al. (1992). Nature, 358, 80-83). Hence the interaction between p53 and MDM2 represents a key potential therapeutic target.
The cDNA sequence encoding the human MDM2 protein (which is also referred to as "HDM2" in the art) is known from WO93/20238. This application also discloses that human MDM2 protein binds with human p53 and it has been suggested that molecules which inhibit the binding of MDM2 to p53 would be therapeutic by alleviating the sequestration of p53. However it is also suggested that the p53 and MDM2 binding site is extensive, including amino acid residues 13-41 of p53 as well an additional nine to thirteen residues at either the amino or carboxyl terminal side of the peptide are also involved. This would indicate that a large polypeptide or other large molecule would be required in order to significantly interfere with the binding.
The applicants have therefore sought to immunochemically characterize the p53-MDM2 complex, and also determine in fine detail the MDM2 binding site on p53.
Surprisingly, it has been found that only a relatively small number of amino acids within the p53 protein are involved in binding to MDM2.