This thesis describes the development of chemical tools that inhibit the sialidases NanA and NanB from Streptococcus pneumonia. The primary focus was on the discovery of allosteric inhibitors of NanA and NanB, however, promising inhibitors that act by binding at the active site of these enzymes were also investigated. Chapter 1 gives an overview of the use of chemical tools in the field of chemical biology. It focuses in particular on chemical tools that function by the allosteric regulation of their target proteins. The uses, advantages and methods of discovery of allosteric tools are discussed. Finally this chapter introduces the use of serendipitous binders for the discovery of allosteric sites. In particular, the use of CHES to identify novel allosteric sites on the sialidase NanB is proposed. Chapter 2 describes how the ‘hits' from a series of high throughput screens were reanalysed using a wide range of secondary assays to eliminate any false positives that were contaminating the results. This process removed eight of the eleven ‘hits'. Two of the remaining three compounds were then analysed further in an attempt to characterise their binding mode to NanA and/or NanB using modelling and X-ray crystallographic studies. Whilst, it was not possible to confirm the binding mode by X-ray crystallography modelling studies using the modelling software GOLD generated possible binding modes for these inhibitors. A structure activity relationship study was conducted for both compounds in an attempt to generate more potent inhibitors. Chapter 3 moves from the use of high throughput screens to identify hits against NanA and NanB to the use of the serendipitous binding of N-cyclohexyl-2-aminoethanesulfonic acid in the active site of NanB for the development of selective NanB inhibitors. First taurine was identified as the minimum unit of N-cyclohexyl-2-aminoethanesulfonic acid required to bind to the active site of NanB. Taurine was then used as the basis of an optimisation study. This chapter concludes with the identification of 2-(benzylammonio)ethanesulfonate as the next key intermediate in the development of N-cyclohexyl-2-aminoethanesulfonic acid based active site inhibitors of NanB. Chapter 4 follows on from Chapter 3 with the optimisation of 2-(benzylammonio)ethanesulfonate describing the design and synthesis of a wide range of analogues. From these compounds 2-[(3-chlorobenzyl)ammonio]ethanesulfonate was identified as the most potent and selective inhibitor. Detailed analysis of the binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to NanB gave a rationale for its improved inhibitory activity. The increase in inhibition occurred because on binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to the active site of NanB a well coordinated water molecule was displaced. The displacement of this water caused an increase in the flexibility of the enzyme's 352 loop. A detailed study of the flexibility of this loop in response to various N-cyclohexyl-2-aminoethanesulfonic acid based chemical tools was then conducted. The research in chapters 2 and 3 has recently been published. In Chapter 5 a molecule of N-cyclohexyl-2-aminoethanesulfonic acid that binds serendipitously in a previously unmentioned secondary site is elaborated into a ligand, known as Optactin, that binds strongly and selectively at this secondary site. It was then shown that Optactin inhibited NanB by binding at this secondary site. It was therefore concluded that this secondary site was in fact an allosteric site that could be used for the regulation of NanB. Chapter 6 describes the development of a rationalisation for the inhibition of NanB by Optactin. This study included the X-ray crystallographic analysis of the apo-NanB structure and the NanB-Optactin complex under a range of conditions. This was followed by mechanistic studies that identified the point in the catalytic cycle at which Optactin was inhibiting NanB. This chapter concludes with a hypothesis for the mechanism of inhibition of NanB by Optactin.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:569029 |
| Date | January 2013 |
| Creators | Brear, Paul |
| Publisher | University of St Andrews |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10023/3451 |
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