Development of novel active site and allosteric inhibitors of enzymes associated with cancer, neurodegenerative diseases and bacterial infections
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The sirtuins are a family of NAD⁺-dependent deacetylase enzymes which are implicated in various illnesses including cancer and neurodegenerative diseases. Part I of this thesis describes the synthesis and biological evaluation of inhibitors of the SIRT1 and SIRT2 isoforms of this important family of enzymes. Chapter 1 gives an overview of sirtuin biology and the physiological roles of these enzymes. In particular the link between SIRT1 and cancer and SIRT2 and its role in the onset of neurodegenerative diseases is discussed. A review of the most potent and selective inhibitors of SIRT1 and SIRT2 is given including an introduction to the tenovin and cambinol classes of inhibitor. Chapter 2 describes various issues relating to the structure of the important chemical tool tenovin-6. The synthesis of analogues to improve the solubility, determine the preferred conformation and verify the products of metabolism of tenovin-6 is presented including their evaluation by in vitro and in cell methods. Part II of this chapter reports the design and use of a ¹H NMR method used to monitor the sirtuin-mediated deacetylation reaction. This was particularly relevant due to concerns raised about the possibility of false positive results obtained with the commercially available assay kit commonly used by the sirtuin community. This new ¹H NMR method was used to validate the inhibition of SIRT2 by tenovin-6. Chapter 3 describes the parallel synthesis and evaluation of tenovin analogues as inhibitors of SIRT1 and SIRT2. This study identified that replacement of the t-butyl substituent of tenovin-6 with the 3,5-dihalogen-4-alkoxy substitution pattern led to a variety of analogues having SIRT2 selectivity. As well as the collection of valuable SAR data, in cell data is also presented for the analogues. Chapter 4 provides attempts to rationalise the SAR data collected in Chapters 2 and 3 through a computational study. The molecular docking software GOLD was used to predict the binding site of the tenovin scaffold and hence rationalise the observed potencies of various analogues. Chapter 5 reports the synthesis and biological evaluation of triazole and cambinol analogues as SIRT1 and SIRT2 inhibitors. Part I details the synthesis and in vitro testing of a series of ring constrained tenovin analogues based on the 1,4-disubstituted triazole using click chemistry. A series of 1,5-disubstituted analogues were also synthesised. Part II describes the synthesis of S-alkylated cambinol analogues and the effect of N3-methylation upon activity and selectivity towards SIRT1. Part II of this thesis details the synthesis and biological testing of novel potent allosteric inhibitors of RmlA. RmlA is the first enzyme in the L-rhamnose biosynthetic pathway in bacteria. L-rhamnose is an important component of the bacterial cell wall and as such RmlA is therefore an important target in the discovery of novel anti-bacterial compounds. Chapter 7 provides an overview of the RmlA enzyme including its role in L-rhamnose biosynthesis and why it is an attractive target for anti-bacterial drug discovery. No small molecule inhibitors of RmlA have been reported previously. Chapter 8 describes the design and synthesis of pyrimidine-2,4-dione analogues as novel allosteric inhibitors of RmlA. SAR data is generated and rationalised by X-ray crystallographic techniques to study the structures of complexes of RmlA with various analogues. Analogues were also tested for their ability to inhibit the growth of the important human pathogen Mycobacterium tuberculosis.
Thesis, PhD Doctor of Philosophy
Embargo Date: 2017-11-22
Embargo Reason: Thesis restricted in accordance with University regulations. Print and electronic copy restricted until 22nd November 2017
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