| الوصف: |
The MDM2 oncoprotein and the p53 tumour suppressor form an autoregulatory loop in normal cells. The MDlvI2 gene is a target for direct transcriptional activation by p53, while the MDM2 protein binds and negatively regulates the transcriptional activity of p53, as well as targeting it for proteasomal degradation. Inactivation of the p53 tumour suppressor by elevated MDM2 expression and/or activity has been implicated in a range of cancers (Momand et al. 1998). Inhibitors of the MDM2-p53 interaction are therefore candidates for activation of the p53 pathway in tumours expressing wild type p53. The investigations presented in this thesis were aimed at the discovery and validation of small molecular weight inhibitors of the MDM2-p53 interaction. The studies included (i) structure-based drug design using the isoindolinone scaffold, (ii) high throughput screening of the Cancer Research UK compound library and (iii) in silica virtual screening of the ChemDiv and Specs commercial compound collection. These studies were carried out in collaboration with De Novo pharmaceuticals in Cambridge and the the University of Oxford. Results from the structure-based drug design revealed a set of isoindolinones which were able to disrupt the MDM2-p53 binding interaction in a cell free ELISA. Nuclear Magnetic Resonance (NMR) structural data provided direct evidence for the interaction of NU8165 (ICso 16 ± 1 IlM) with the MDM2 hydrophobic pocket. A binding mode based on the NMR chemical shift changes indicated an interaction of NU8l65 with the p53 binding pocket on MDM2, with similarities to the MDM2-p53 peptide and MDM2-nutlin 2 complexes as observed in their respective x-ray crystal structures (Kussie et al. 1996; Vassi1ev et al. 2004). Cell based studies indicated that NU8165 was able to release p53 transcriptional activity, which was evidenced by the induction of the p53 responsive gene . products MDM2 and p21 WAF!, observed in the paired wild type p53 cell line A2780 but not in the mutant cell line CP70. The High throughput screening (HTS) of the CR UK 800 compound pilot library using the ELISA and in silica virtual screening led to the identification of the SACH series of compounds. Structure activity Relationship (SAR) studies and in silica modelling were used to explore the interaction of SACH 1596 (ICso 120 ± 20 nM) with the p53 binding pocket on MDM2. The cell based studies with these compounds indicated selective induction of the p53 responsive gene products MDM2 and p21WAFI in wt p53 paired and isogenic cell lines. Furthermore, the induction of these proteins was found to be significantly greater in MDlvJ2 amplified cell lines compared to cell lines with normal MDA12 gene copy number. A plausible explanation for this has been presented. Additional experiments including a gene reporter assay provided further evidence that SACH 1596 was able to release p53 transcriptional activity, and this was found to be independent of a DNA damaging stress pathway activation. In conclusion, the studies described in thesis have resulted in the identification of novel small molecular weight compounds that have both potent cell free and cell based activity, which are consistent with the successful disruption of the MDM2-p53 interaction, and are comparable to the best published inhibitors in literature. |
