The development and evaluation of small molecule inhibitors of the MDM2-p53 interaction

Abstract

As a principal gatekeeper, p53 is inactivated mutationally in over 50% of human cancer (1). However, in approximately 7% of tumours, especially of the glia, bone, and soft tissues, loss of wild-type p53 function results from amplification and over-expression of the MDM2 gene, causing transformation and uncontrolled tumour growth (2-7). Several research reports have revealed that p53 and MDM2 proteins form an auto-regulatory feedback loop (8-10). Inhibition of the p53-MDM2 interaction has been validated as an attractive target for anti-cancer therapy. Several different scaffold small-molecule inhibitors have been discovered by both structure-based design and compound library screening (11-16). The first chapter of this thesis contains a general overview of research carried out that lead to the discovery and optimization of published potent p53-MDM2 interaction inhibitors, together with an overview of the p53-MDM2 antagonists development project carried out in our group, the progress obtained so far and future directions. After the presentation of general materials and methods of experiments applied in my thesis in chapter two, four chapters are formed for presenting the research results I obtained within more than three years laboratory work. Chapter three demonstrates the evaluation (ELISA) and optimization of the in vitro activity of isoindolin-1-one compounds, which is a group of potent lead small-molecule inhibitors of the MDM2-p53 interaction developed by our research group in Newcastle. The results indicate the impressive progress of this series of compounds, from NU8260 (IC50=2.4±0.2 μM) to the most recently developed lead compound, NCL-00016149 (IC50=15.8±2.6 nM), which is about 150-fold more potent. III The followed chapter (chapter 4) presents the evaluation (Western blot, SRB and Caspase-Glo 3/7 assay) of cellular activity of potent isoindolin-1-one compounds, especially the comparison of pure enantiomers, by using Nutlin-3 as the positive control. The result show that isoindolin-1-one compounds can induce p53 and its down stream target MDM2 and p21 accummulation and activation, corresponding to their in vitro potencies; both the in vitro and cellular activities presented are mainly for the potent enantiomer. Chapter five is aimed at obtaining a better understanding of MDM2/MDMX inhibitor design and screening procedures. As part of the FP6 DePPICT project, more than 3800 compounds that were selected as possible MDM2 and/or MDMX inhibitors by in silico screening millions of commercially available compounds, together with some in-house pyrrole scaffold compounds and p53 peptides analogs were evaluated by ELISA. A number of lead compounds showed dose-dependent activity, and 7 scaffold series were selected for further SAR exploration by using synthetic chemistry technology. With the progress of the isoindolin-1-ones and the development of Nutlin-3 and MI-63 series of MDM2 antagonists, several drug candidates may enter clinical trials soon. As a result, relapse caused by drug resistance may need to be considered as early as possible. Therefore, in chapter six, the research of MDM2 antagonists resistant cell clones is demonstrated. Several resistant cell clones were selected by treating SJSA-1 and NGP tumour cell lines with Nutlin-3 and MI-63. Resistance mechanisms were explored by using a range of bioassays, and one of the key mechanisms was confirmed to be p53 dysfunctional missense mutation. Finally, the achievements and future work of my research are discussed in chapter seven.