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Title: Dissecting the function of histone deacetylases : a three-pronged approach from probes to protein-engineering
Author: Felstead, Robert
ISNI:       0000 0004 2736 9211
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Histone deacetylases (HDACS) are a class of enzymes responsible for deacetylating the lysine residues of histones in chromatin. Inhibitors of these enzymes have shown promise in the treatment of a number of diseases including neurodegenerative diseases such as Alzheimer’s and Huntingdon’s disease, various cancers and most recently as a treatment for acute myocardial infarction. There are 11 zinc-dependent isoforms of HDACs present in humans but to date the majority of inhibitors of these enzymes have shown broad inhibition across all isoforms. This makes determination of the function of individual isoforms via chemical means very difficult. This thesis outlines three chemical methods; namely protein-ligand engineering, isoform-selective inhibitor discovery and activity based protein profiling that have been developed towards dissecting the function of individual histone deacetylase isoforms. A histone deacetylase 1 (HDAC1) mutant protein-ligand pair was designed with the aid of a computational docking program and site-directed mutagenesis used to introduce the mutation into HDAC1, which was expressed in Sf9 cells. A set of potentially selective inhibitors was then synthesised and tested for selectivity compared to the wild-type histone deacetylase 1. A novel series of potential isoform-selective inhibitors were designed based around a cyclotetrapeptide capping group and a 2-phenylamide zinc-binding group and joined together via a ruthenium catalysed cross-metathesis reaction. These were then tested for selectivity against HDAC1 and HDAC2 in a fluorimetric assay. Finally, an activity-based protein profiling probe specific to HDACs was designed and synthesised. The probe was designed to have a reactive chloromethyl ketone head group that would irreversibly bind to HDACs as well as an alkyne group to allow attachment of a fluorescent tag via the copper mediated alkyne-azide ‘click’ reaction. The probe was then tested for its ability to label HDACs in vitro and an in vivo system expressing HDAC1.
Supervisor: Armstrong, Alan ; Mann, David ; Lam, Eric Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral