Peptide and peptidomimetic leads for the inhibition of MDM2-mediated ubiquitination of p53
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The tumour suppressor p53 is essential for genome stability and loss of its function can lead to human cancer. The functional roles of p53 are regulated by a variety of mechanisms, some of which are not well understood. However, the murine double minute 2 (MDM2) protein, a major negative regulator of p53, has been found to be overexpressed in many human cancer cell lines in which p53 was not mutated; thus establishing MDM2 as a target for cancer therapeutics. MDM2 is defined as both an oncoprotein and an E3-ubiquitin ligase; its interactions with p53 are controlled through multiple domains, providing different possible pathways to inhibit MDM2 and therefore reactivate p53 function. Previous work undertaken in the Ball laboratory has shown that the MDM2 RING domain plays a critical role in p53 ubiquitination; thus screening for modulation of its activity by small molecules could provide new leads for the inhibition of the E3 ligase activity of MDM2. The MDM2 RING domain was cloned, expressed and purified so that it could be studied using a series of in vitro experiments. The generation of a library of short (12-mer) peptides as potential inhibitors of the MDM2 RING domain was investigated using phage display against His-tagged RING protein to screen the peptide ligands. In order to study the specificity of these peptides towards MDM2 (res. 396-491 and 396-479) compared with MDM4 and BRCA1, the MDM4 RING domain (res. 395-490 and 395-478) and BRCA1 (res. 1-304) domain were expressed and purified for further characterisation. A small selection of peptides was isolated and their binding affinity and activity as MDM2 inhibitors evaluated by in vitro ELISA, affinity chromatography and ubiquitination assays. One peptide in particular, KCCYFETHMPRH, was found to bind to MDM2 and was able to inhibit MDM2-mediated ubiquitination of p53 in vitro. Preliminary optimisation of this peptide by alanine scan revealed a peptide with a 2-fold increased potency. Since peptides provide comparatively weak therapeutic leads due to a combination of poor cellular uptake and susceptibility to cleavage by proteases, cyclic peptidomimetics based upon this lead were developed using side-chain to side-chain cyclisation. These peptidomimetics were successfully generated by the synthesis and incorporation of novel N-propargylated glycine and N-azidoalkyl glycine building blocks into a peptide sequence by Solid Phase Peptide Synthesis (SPPS). Following a Copper-catalysed Azide-Alkyne Cycloaddition (CuAAC) on solid phase or in solution, these peptoid-peptide hybrids were isolated, purified and characterised.