In all kinds of disease models, many proteins involved in protein-protein interactions (PPIs) are mutated and do not function properly. The important role of PPIs in disease makes the design of small molecule inhibition an interesting proposition. This project looks at mouse double minute 2 (MDM2) and mouse double minute X (MDMX) which binds and inhibits the tumour suppressor protein p53. MDM2 and MDMX are therefore attractive therapeutic targets due to their role in tumour progression. The aim is to identify small molecule dual inhibitors that are able to disrupt MDM2 and MDMX from binding to p53. Both N-terminal MDM2 and MDMX were successfully expressed and purified with high purity and decent yield. These proteins were used to develop Fluoresence Polarization (FP) and Capillary Electrophoresis (CE) assays for small molecule inhibitors screening. This work has successfully developed FP and CE assays for detecting weakly interacting fragments. The CE assay is a novel method for detecting weak fragments for protein-protein interactions, which are a challenging target. Two approaches were employed to identify small molecule inhibitors for MDM2- N/p53 interaction. At first, small molecules were identified using in silico screening and these hits were verified using FP and CE assays. Second, analogue exploration was applied to identify fragments from the small molecule inhibitors discovered from the in silico screening. Diphenylamine and oxindole fragments were identified as the most potent. However, diphenylamine fragment was discovered to aggregate MDM2-N and was ranked as a false positive hit. No protein aggregation was found when incubated with the oxindole fragment. Therefore oxindole can provide a good starting point for the design of higher affinity analogues. Studying the interaction of MDMX has only recently been undertaken. MDMX contains a high homology binding site with MDM2. Hence, developing a dual MDM2/MDMX inhibitor has become an attractive target to focus on. FP and CE assays were developed to screen compounds against MDMX-N. In silico screening against MDM2-N and MDMX-N found several hits. One compound was discovered as a dual binder to MDM2-N and MDMX-N with low μM affinity. This novel hit is potentially a good starting point for the design of higher affinity analogues.

A growing body of literature suggests Breast Cancer-Associated Protein 1 (BRCA1) is important not only as a cause, but also as a target in the quest for cancer treatment. BRCA1 deficient cells treated with radiation as well as PARP inhibitors and other chemotherapeutics demonstrate a greater sensitivity than cells with wild type BRCA1. Inhibitors of BRCA1 would take advantage of this synthetic lethality and represent a significant advance in cancer treatment as well as an understanding of the biology of DNA repair. Despite significant study of BRCA1 protein and function, it is a large protein (220 KDa) that is still largely uncharacterized, but its N- and C-terminal domains have been described by significant structural data. The BRCT (BRCA1 C-Terminal) Domain is a phosphoprotein binding domain that is commonly mutated or lost in cancers and has a binding cleft seemingly very suitable for drug design. Small molecule screens have been conducted against this domain, but the resulting hits with moderate affinity have not been shown to induce BRCA1 deficient phenotypes. Phosphopeptides have also been studied as potential BRCA1 inhibitors, yet despite some having affinities in the mid-nanomolar range the presence of a phosphate is not without its pharmacologic challenges. We generated an mRNA display library with 1.3 x 10^13 cyclized peptides covalently attached to the mRNA that encoded them. Eight rounds of selection exposing the library to a GST-BRCT fusion resulted in selection of non-phosphorylated peptides that bind to a BRCT domain of BRCA1. The sequences resulting from the selection have common homologies and initial characterization has shown that these peptides may be the first viable non-phosphoserine containing inhibitors of BRCA1.