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The Pharmaceutical Journal Vol 265 No 7116 p486
September 30, 2000 The Conference

Science Symposium - Medicines for the new millennium

Gene p53 - a paradigm for drug discovery

Individualisation of therapy should become commonplace as the number of drug targets swells through greater understanding of the molecular basis of disease. So suggested Professor Sir David Lane (department of surgery and medical oncology, University of Dundee) describing some themes concerning the future of drug discovery. Individualisation, he said, would create many challenges in the future for academia, industry and, ultimately, for pharmacists, doctors and patients. More and more drugs would be developed as a result of applied knowledge, but what would distinguish these from the drugs of the past was that they would be precisely effective and non-toxic. As a result, clinical trials would be cheaper and quicker: “I am optimistic,” he commented.
Many of the paradigms for the discovery of new targets and their drugs could be illustrated by the discovery of the tumour suppressor gene, p53 (see Panel), said Professor Lane. First discovered 20 years ago, it took a further 10 years to identify that 50 per cent of tumours carried mutations in the p53 protein, he said.
When considering the role of any gene in cancer, it was necessary to distinguish whether the gene was altered in an inheritable way in the reproductive cells (germ line), or whether changes had arisen in the somatic (any non-reproductive) cells during a lifetime, said Professor Lane. In the case of p53, the vast majority of the mutations were somatic. It was only in very rare family syndromes that inheritable changes were seen in the germ line (eg, Li-Fraumeni syndrome): these syndromes provided great models for research, he said.

P53 explained
p53 is a tumour suppressor protein that acts as a universal sensor of DNA damage, even that resulting from sunburn. The gene coding for the p53 protein is the most commonly mutated gene in all human cancers: 50 per cent of tumours are found to have lesions in the p53 gene, rising to 60-70 per cent in lung and colon carcinomas.

Following DNA damage, p53 promotes the production of other proteins that induce cell cycle arrest and/or apoptosis. Thus, in normal cells, p53 either induces cell death where DNA damage is too extensive or it buys time for DNA repair to take place.

p53 is tightly controlled within the cell by another protein, MDM2. The two proteins form an autoregulatory feedback system where p53 causes MDM2 levels to rise. MDM2 then binds to p53 and triggers its breakdown through a pathway called the ubiquitin system (a common route for protein breakdown). In the presence of DNA damage, the production of MDM2 is reduced, so allowing p53 to initiate its tumour suppressing functions. Mice lacking the MDM2 gene die in the early stages of embryogenesis, confirming its importance in regulating p53 in the absence of DNA damage; mice without the p53 gene die after about nine months. A second protein important in p53 control is p14ARF which inhibits MDM2 and activates p53.

The study of p53 illustrated the extremely rapid changes that were taking place in science and technology, said Professor Lane. Identification of specific p53 mutations required determining the full sequence of the gene in every case: advances in sequencing technology now made this a straightforward task as even the whole Escherichia coli genome, originally sequenced over nine years, could now be sequenced in four days. However, it would not be sufficient just to study the DNA sequence of a gene — it would be necessary to examine and understand the variation in the gene products also. But the advances in studying protein expression were also impressive: “Such advances were inconceivable five years ago,” he said.
One of the less well described challenges in drug development was applying the knowledge of cell biology to the drug discovery process and determining the “nodal point” for intervention in a pathway, said Professor Lane. Detailed structural biology would assist the definition of the type of molecule required, he continued.
As with all drug targets, it was important to understand the detail of the p53 pathway, suggested Professor Lane. Why for example, did half of all tumours harbour a mutant form of p53? In some cases, the pathway had become blocked and its reactivation was a very exciting therapeutic idea. Describing the control of the p53 pathway (see Panel), Professor Lane said that it might also be possible to target regulators of p53 with drug molecules. Already, experiments had investigated the interaction between p53 and the negative regulating protein, MDM2. Using the technique of phage display, a peptide mimicking the MDM2-binding region of p53 was selected which bound to MDM2 20-100 fold more tightly than native p53, said Professor Lane. This peptide was then inserted into the active-site loop of thioredoxin [where thioredoxin is used as a carrier protein to form a TIP, a thioredoxin insert protein]. When introduced into cells expressing low levels of p53, the TIP caused accumulation and induction of p53. This offered the possibility of regulating p53 pharmacologically and the potential to develop new, non-toxic drugs, Professor Lane suggested. The potential of such a therapy might be moderated in cases where MDM2 was deficient. Thus, if tumours could be stained heavily for p53 then this therapy would not be effective or suitable. Such cases illustrated the importance of pathology in the development of new drugs, he said.
Another point of control for p53 and a possible point for therapeutic intervention lay with the positive regulator, p14ARF, said Professor Lane. This protein was frequently lost in tumours retaining normal p53 expression. The design of peptides mimicking the region of p14ARF that bound to the negative regulator MDM2 (see Panel) had successfully led to the inhibition of p53 degradation, he said.