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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.

Cutaneous squamous cell carcinoma (cSCC) is a type of non-melanoma skin cancer that is the 4th most common cancer registration in Scotland after BCC, lung and breast cancer. Over 30,000 cSCC incidences are reported each year in the United Kingdom. In addition, around 1 in 4 skin cancer deaths in the UK are due to cSCCs. Amongst those highly prone to developing cSCCs include organ transplant recipient, immunosuppressed, recessive dystrophic epidermolysis bullosa (RDEB) and Xeroderma Pigmentosum (XP) patients. cSCC patients that display regional metastasis have a 5-year survival rate of 25-50%, whilst this rate is close to 0% in RDEB patients with multiple cSCCs. Wild-type p53 (wt-p53) has been shown to prevent cSCC development and induce tanning and sunburn responses in skin cells. However, TP53 mutations are found in over half of all human cancers and cSCC is no exception as TP53 mutational frequency in cSCCs is around 64-87.5% (Durinck et al, 2011; South et al, 2014). The majority of TP53 mutations in cSCCs are UV-signature missense mutations, highlighting UV-radiation as one of the main risk factors for cSCC development. Mutant p53 proteins can lose wt-p53 functions, have dominant-negative effects against wt-p53 and acquire gain of function (GOF) activities. Mutant p53 GOF activity is induced by the accumulation of mutant p53 in tumour cells. Mutant p53 accumulation is not due to intrinsic properties of the mutants but requires other cellular events, possibly those known to stabilise wt-p53 under cellular stress. It is known that the TP53 mutations and mutant p53 accumulation are early steps in cSCC development. This makes skin an excellent system to investigate the early changes to p53. We have investigated the potential of targeting mutant p53 for cSCC therapy and mechanisms that promote mutant p53 accumulation in cSCCs. We selected low-passage cSCC cell lines that express hotspot mutant p53 proteins, in cSCCs and in general, by analysing TP53 mutational data from the IARC database and next generation sequencing studies performed on cSCC primary tumours by Dr South at Ninewells Hospital, Dundee. cSCC cell lines were generated from immunocompetent, transplant and RDEB patients by Dr South’s group at Ninewells Hospital, Dundee. We found that: 1. PRIMA-1MET, a small molecule reported to restore wt-p53 activity, lacked tumour selectivity as it is able to reduce cell viability in both normal skin and cSCC cells with similar potency. cSCC cell lines are relatively resistant to PRIMA-1MET compared to cell lines derived from other tumour types. 2. Mutant p53 knockdown studies performed on cSCC cell lines suggest that some p53 mutants play a pro-proliferative role. However, there is no evidence for a pro-migratory role of mutant p53 in cSCC. 3. There are no clear alterations in DNA-damage response pathways or the general ubiquitin proteasome system that could contribute to mutant p53 stabilisation in cSCC. 4. Heat shock factor 1 (HSF-1) is upregulated in cSCC compared to normal human keratinocytes (NHK). HSP90 inhibitors, 17-AAG and 17-DMAG, reduce mutant p53 protein levels suggesting that HSP90 plays a role in stabilising mutant p53 in cSCCs. 5. PR-619, a broad range deubiquitinating enzyme (DUB) inhibitor, reduces mutant p53 protein levels in a range of cSCC cell lines. This is rescued by the addition of bortezomib suggesting that DUBs can play a role in protecting mutant p53 from proteasomal degradation. Expression of HAUSP and USP10, which have been shown to stabilise wild-type p53, is generally elevated in cSCC compared to NHK. However, knockdown of these DUBs does not reduce protein levels of mutant p53 in cSCC cell lines. 6. A potential isoform of MDMX (51 kDa) is strongly upregulated in all cSCC cell lines examined. There is an association between the ability of MDMX siRNAs to deplete the 51 kDa protein and reduce mutant p53 protein levels and stability. Furthermore we show that the protein can form complexes with MDM2 in vitro and in cSCC cells. We propose that the MDMX isoform is able to stabilise mutant p53 in cSCC cells through this interaction with MDM2.

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. ARF binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild type conformation and allows p53-MDM2-SUV39H1 complex to bind DNA. These results provide further evidence for an ubiquitination-independent mechanism of p53 regulation by MDM2, and reveal how MDM2-interacting repressors gain access to p53 target promoters and repress transcription. Furthermore, we show that the MDM2 inhibitor Nutlin cooperates with the proteasome inhibitor Bortezomib by stimulating p53 DNA binding and transcriptional activity, providing a rationale for combination therapy using proteasome and MDM2 inhibitors.

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