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Title: Synthesis and biological evaluation of novel inhibitors of protein biogenesis at the endoplasmic reticulum
Author: Williams, Helen Mary
ISNI:       0000 0004 2719 081X
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2012
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Biosynthesis and folding of proteins entering the secretory pathway of eukaryotic cells is coordinated at the endoplasmic reticulum (ER). Any terminally misfolded proteins are sorted for the endoplasmic reticulum associated degradation (ERAD) pathway for destruction. Eeyarestatin I (ESI) is a potent inhibitor of certain stages of protein biogenesis at the ER including protein translocation across the ER and deubiquitination, a late stage of the ERAD process. Here several structurally related Eeyarestatins (ES) were synthesised based on key structural requirements shown in ESI and the structural activity relationship of these compounds both in vivo and in vitro was investigated. Experiments conducted included: cytotoxicity to determine toxicity of ES compounds at several concentrations; cell microscopy to identify vacuole formation frequently exhibited by ESI; SDS page electrophoresis and western blotting for ubiquitin to ascertain the extent of accumulation of ubiquitinated protein and indicator of inhibition of deubiquitination; a translocation block assay looking to determine the inhibitory effect the ES compounds have on translocation of selected proteins across the ER membrane; and finally a secretion assay to determine the ES compound's ability to block overall secretion of proteins from cells. Whilst none of the 'new' analogues seem to be equipotent to ESI in all inhibitory activities, inhibitory activity of compounds ES40, ES24 Click meta and ES24 Click para appear to be more specific towards DUBs, with ES40 being the most active.
Supervisor: Whitehead, Roger; Flitsch, Sabine; High, Stephen; Swanton, Lisa Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available