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University of Southampton Research Repository Eprints Soton University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES School of Chemistry Novel Antibiotics from DNA Adenine Methyltransferase Inhibitors by Jennifer Claire McKelvie Thesis for the degree of Doctor of Philosophy October 2011 Abstract The re-emergence of plague as a world-wide health concern and the potential risk posed by bioterrorism has led to an increased interest in available treatments for the disease. The bacterial DNA adenine-N6 methyltransferase, Dam, is involved in the regulation of a range of pathogenic bacteria and has been validated as a target for the development of antimicrobial agents with activity against Yersinia pestis, the causative agent of plague. The lack of a functionally similar enzyme in mammals suggests that highly selective Dam inhibitors could be developed. A coupled, real-time break light Dam activity assay has been optimised for HTS, and assays for the validation and characterisation of screening hits have also been developed. Screening of random and in silico enriched compound libraries, and the subsequent application of counter- screening and hit confirmation assays, resulted in the identification of a single viable lead, (4-(N-(2-hydroxyethyl)sulfamoyl)phenyl) stibonic acid (13776). Screening of compounds analogous to 13776 identified a series of arylstibonic acids with activity against Dam. Kinetic characterisation of the most potent arylstibonic acid, 4-stibonobenzenesulfonic acid (13746), revealed a DNA-competitive mode of action, and a K of 6.46 ± 0.07 nM. However, selectivity assays have revealed a potentially non- i specific mode of action for the stibonic acids, which have shown activity against a range of DNA and protein binding enzymes. Yersinia cell culture experiments have shown a single compound, (3-((2-hydroxyethyl)carbamoyl)phenyl)stibonic acid (13782), to be capable of penetrating Yersinia cells and partially inhibiting methylation, and mRNA profiling experiments have shown 13782 to induce a statistically significant change in several genes involved in the pathogenicity of Y. pestis. Attempts at resynthesising 13782 have proved challenging, with only a fraction of the activity of the original sample reproduced. HPLC analysis of the original and resynthesised samples has shown the former to comprise two components, with only one present in both samples. The in vitro evaluation of a series of bisubstrate analogues designed to mimic both the methyl donor S-adenosylmethionine (AdoMet), and the methylation target (adenine) has shown that substitution of the AdoMet sulfur for nitrogen results in a significant but not total loss of activity. Furthermore, the addition of a bicyclic heteroaromatic adenine analogue mimic to this scaffold led to an increase in potency and selectivity for Dam over the human cytosine methyltransferase DNMT1 but a reduction in selectivity for Dam over the restriction enzyme DpnI. These results suggest that a selective and potent Dam inhibitor can be obtained by carefully modifying both components of the bisubstrate analogue inhibitor. i ii Contents Abstract ....................................................................................................................... i Contents .................................................................................................................... iii List of Figures ............................................................................................................ ix List of Tables ............................................................................................................ xv List of Equations ...................................................................................................... xix Declaration of Authorship ........................................................................................ xxi Acknowledgements ................................................................................................ xxiii Abbreviations .......................................................................................................... xxv 1 Introduction .......................................................................................................... 1 1.1 Antibiotics and the emergence of resistant bacteria ....................................... 1 1.1.1 Discovery and development of antibiotics ............................................... 1 1.1.2 Antibiotic resistance ............................................................................... 3 1.1.3 Antibiotic resistance in Yersinia pestis .................................................... 7 1.2 Methyltransferases ...................................................................................... 10 1.2.1 Overview .............................................................................................. 10 1.2.2 DNA methyltransferases ....................................................................... 12 1.2.3 DNA adenine-N6 methyltransferase ...................................................... 15 1.2.4 Structural and kinetic characterisation of DNA adenine-N6 methyltransferase ............................................................................................... 18 1.2.5 Methyltransferase inhibitors ................................................................. 22 1.2.6 Methyltransferase assays ...................................................................... 25 1.3 Drug discovery ............................................................................................ 28 1.4 Aims of the project ...................................................................................... 30 2 Primary Assay Development ................................................................................ 33 2.1 Introduction ................................................................................................ 33 2.1.1 Enzyme assays ..................................................................................... 33 2.1.2 The real-time break light DNA adenine-N6 methyltransferase activity assay 36 2.2 Protein expression and purification ............................................................. 38 2.2.1 Expression and purification of DpnI ...................................................... 39 2.2.2 Expression and purification of Y. pestis Dam......................................... 42 2.3 Optimisation ............................................................................................... 47 2.3.1 Buffer conditions .................................................................................. 48 2.3.2 Assay temperature ................................................................................ 51 2.3.3 Enzyme concentration and stability ....................................................... 52 2.3.4 Effect of compound library solvent........................................................ 56 2.3.5 Assay volume ....................................................................................... 57 iii 2.3.6 Substrate concentrations ...................................................................... 60 2.3.7 Assay optimisation for the kinetic characterisation of Dam inhibitors .... 66 2.4 Summary ..................................................................................................... 70 3 Secondary Assay Development ............................................................................ 73 3.1 Introduction ................................................................................................ 73 3.2 Counter-screening ....................................................................................... 74 3.2.1 DpnI activity assay ................................................................................ 74 3.2.2 Fluorescent intercalator displacement assay ......................................... 76 3.3 Gel-based Dam methylation assay ............................................................... 78 3.3.1 Optimisation ........................................................................................ 79 3.3.2 Validation ............................................................................................. 84 3.4 Real-time break light cytosine methyltransferase activity assay .................... 86 3.4.1 Optimisation ........................................................................................ 87 3.4.2 Validation ............................................................................................. 95 3.5 Fluorescence anisotropy assay ..................................................................... 96 3.5.1 Optimisation .......................................................................................
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