SCREENING AND IDENTIFICATION OF NATURAL PRODUCT LEADS FOR RESTORATION OF P53

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The objective of laboratory programme 1 is the detection, characterization, isolation, analysis and production from natural resources or via small scale synthesis of active compounds which restore the function of the tumor suppressor gene, p53. The active compounds will be derived from the Xenova natural product resource which currently consists of 26000 microbes and 7500 plant species. Fermentation conditions and secondary metabolite extraction procedures are designed to reveal the chemical diversity inherent in the respective natural resources. This collection will be screened for active compounds exhibiting one of three activities: i) restoration of normal DNA-binding activity to particular p53 mutants; ii) disruption of the interaction between p53 and the oncogene mdm2; iii) ability to mimic the cell cycle-inhibitory activity of WAF1 (which is induced by p53). These screens will be performed using enzyme-linked immunoassays developed specifically for these purposes by laboratory programme 2. The assay formats supplied by laboratory programme 2 will be adapted and validated to run as high throughput automated screens with microbial fermentation and plant extracts. Active samples, typically generated from in excess of 50,000 microbial fermentation extracts and 7000 plant extracts, will be prioritized by state-of-the-art bioinformatics software, and submitted to bioassay-guided chemical fractionation. Confirmation of lead compound functionality will be evaluated using cell- based assays. Active chemical species will be structurally elucidated using a combination of analytical techniques, principally NMR and mass spectrometry. Following characterization of active compounds, analogue synthesis and scale-up of production will be carried out to provide material for pharmacological and toxicological evaluation in relevant animal models and to explore the mechanisms of drug action on the respective targets (laboratory programme 2). This material will also be used to analyse the crystal structure of the target molecule in association with putative drug, an approach which will serve to elucidate the mechanism of action (Laboratory programme 3).