Synthesis of organelle-specific inhibitor of deubiquitinating enzymes

Abstract

Ubiquitination is a reversible post-translational modification which determines the fate of a protein such as its degradation by the proteasome. The ubiquitin proteasome system comprises a wide-ranging number of deubiquitinating enzymes (DUBs) which prevents ubiquitination by inhibition of enzymes E1 (ubiquitin activiating enzyme), E2 (ubiquitin carrier protein) and E3 (ubiquitin ligase enzyme). Various analogues were synthesised based on the prostaglandin pharmacophore, bis-benzylidenepiperidone structure, which has been previously shown to inhibit DUBs and exhibit anti-cancer properties. Following the synthesis of these compounds, a selected few were conjugated with the left hand side of ESI (a well-established ER associated DUB inhibitor). The Whitehead and Swanton groups have previously shown that the LHS of ESI targets the compound to ER. With this in mind, the aim of the project was to synthesise novel compounds containing the DUB inhibitory domain and an ER targeting domain. The non-conjugated analogues of the prostaglandin pharmacophore and the ES-conjugated compounds were then biologically assayed for their ability to induce the accumulation of polyubiquitinated proteins and the stress response. Ubiquitinated protein localisation was also investigated. All compounds were able to accumulate polyubiquitinated proteins to some extent and induced a stress response according to levels of molecular chaperones BiP and Hsp70. From immunofluorescence studies a redistribution of the ubiquitin signal from the nucleus (DMSO treated sample) to the cytoplasm was seen when treated with all compounds. Some compounds presented specific localisation around the nucleus while other compounds displayed a diffused signal throughout the cytoplasm. Results however, did not correlate with each other suggesting that the relationship between ability to accumulate polyubiquitinated proteins, stress levels and protein localisation is more complicated than expected.

Date of Award	3 Jan 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Roger Whitehead (Supervisor), Stephen High (Supervisor) & Eileithyia Swanton (Supervisor)