Development of novel WWP2 ubiquitin ligase inhibitors using structural and biochemical approaches

Watt, Jessica (2018) Development of novel WWP2 ubiquitin ligase inhibitors using structural and biochemical approaches. Doctoral thesis, University of East Anglia.

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Abstract

A fundamental modification called ubiquitination controls the fate of many proteins. The third enzyme in the ubiquitination pathway, the E3 ubiquitin ligase, has been implicated in a variety of cancers. WWP2 is a HECT E3 ligase associated with the progression of prostate cancer, alongside its closest relative WWP1. WWP2 selectively targets substrates via the interaction of WW domains with PPxY motifs. Ubiquitin is consequently added to the substrate using the catalytic cysteine in the HECT domain. The three isoforms of WWP2 with different WW domain compositions have shown selective preferences towards substrates of the TGF-b signalling pathway and PTEN, a negative regulator of AKT signalling. Understanding more about the WW domain interactions and the activity of the HECT domain can lead to the discovery and development of novel therapeutics.

The aim of this thesis is to find novel inhibitors of WWP2 by utilising different approaches. The initial approach was to structurally solve WWP2 WW domains that reside within different isoforms and investigate substrate-based peptide interactions. Using structural analysis by NMR spectroscopy, the solved structure of WW4 is reported here. NMR titrations were used for interaction analysis of WW1 and WW1-2 domains, with substrate-based peptides, to aid peptide-based inhibitor design and characterise the selectivity of the WW domains. The other strategy involved small molecule screening to find inhibitors of WWP2-FL activity. Results presented here identify potential inhibitors with IC50 values below 3 μM. Counter screening against other enzymes in the ubiquitination cascade was also carried out to investigate the selectivity of the hit compounds. A total of 5 final compounds were successfully identified as selective, making them attractive for further investigations. Overall, the data collected in this thesis can be used in further understanding WWP2 interactions and for structure-based/hit-to-lead development for novel inhibitory molecules as potential prostate cancer therapeutics.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Jennifer Whitaker
Date Deposited: 07 Jun 2019 10:43
Last Modified: 07 Jun 2019 10:43
URI: https://ueaeprints.uea.ac.uk/id/eprint/71264
DOI:

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