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Fragments of Microcin B17 As a Source of New Topoisomerase Inhibitors

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Fragments of Microcin B17 As a Source of New Topoisomerase Inhibitors FRAGMENTS OF MICROCIN B17 AS A SOURCE OF NEW TOPOISOMERASE INHIBITORS Frédéric Collin This thesis is submitted in partial fulfillment of the requirements of the degree of Doctor of Philosophy at the University of East Anglia. Biological Chemistry Department John Innes Centre Norwich Research Park Norwich NR4 7UH December 2010 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotation from the thesis, nor any information derived therefrom, may be published without the author's prior, written consent. Statement The research described in this thesis is my own work, except where due reference has been made, and has not been submitted to any university for any degree. Frédéric Collin Norwich, December 2010 - 2 - Frédéric Collin December 2010 Fragments of microcin B17 as a source of new topoisomerase inhibitors Abstract: DNA topoisomerases are a broad class of enzymes responsible for topological changes in DNA. DNA gyrase is a prokaryotic topoisomerase with the essential function of negatively supercoiling closed-circular DNA at the expense of ATP hydrolysis. There are two main known families of gyrase inhibitors: quinolones and aminocoumarins. A few bacterial toxins are also known for their inhibitory properties on gyrase, among them CcdB and microcin B17. Microcin is a peptide produced by strains of Escherichia coli expressing the plasmid-borne mccB17 operon, which is post-translationally modified to convert serine- and cysteine- containing sequences into oxazole, thiazole, and oxazole-thiazole fused rings. Previous work has shown that these heterocycles are critical, and alterations of these residues proved to be detrimental to MccB17’s antibacterial activity. In spite of its attractive antibacterial properties, MccB17’s physico-chemical properties prevent it from being a good drug candidate. In the pursuit of smaller inhibitors derived from MccB17, we describe here how modifications of the whole toxin led to a better understanding of the elements that are critical for its activity. We generated fragments of MccB17 by chemical and biochemical methods and we show that some of these fragments are topoisomerase inhibitors. The active fragments generated, in addition to highlighting features responsible for MccB17 activity, will be valuable starting points for the design of new inhibitors. Additionally, novel small molecules topoisomerase inhibitors sharing structural features with the heterocycles found in MccB17 were identified and characterized. This work had mainly focused on E. coli gyrase as a model target, but we have shown as well that MccB17 and its fragments had potential application on other topoisomerases. - 3 - List of contents Statement ........................................................................................................................... - 2 - Abstract ................................................................................................................................ - 3 - List of contents ..................................................................................................................... - 4 - List of figures ...................................................................................................................... - 12 - List of tables ....................................................................................................................... - 17 - Acknowledgements ............................................................................................................ - 18 - CHAPTER I Introduction......................................................................................................... 19 Overview ................................................................................................................................ 20 1 Antibacterial mode of action and rise of resistance ...................................................... 22 1.1 Milestones of antibacterials history ...................................................................... 22 1.2 Bacteria structure and antibacterial targets .......................................................... 22 1.3 General mechanism of bacterial resistance ........................................................... 24 1.4 Common multi-drug-resistant bacteria ................................................................. 25 1.5 Inhibitors of cell wall synthesis .............................................................................. 25 1.5.1 Cell wall synthesis .......................................................................................... 25 1.5.2 β-lactam antibiotics ....................................................................................... 28 1.5.3 Glycopeptides ................................................................................................ 32 1.5.4 Fosfomycin ..................................................................................................... 34 1.5.5 Lipopeptides ................................................................................................... 36 1.5.6 Polymyxins ..................................................................................................... 36 1.5.7 Isoniazid ......................................................................................................... 37 1.6 Inhibitors of protein synthesis ............................................................................... 37 1.6.1 Protein synthesis by the ribosome................................................................. 37 1.6.2 Mechanism of action...................................................................................... 37 1.6.3 Families .......................................................................................................... 38 1.7 Antibacterials affecting nucleic acids ..................................................................... 43 1.7.1 Nitroimidazoles .............................................................................................. 43 1.7.2 Sulfonamides (Sulfa drugs) and trimethoprim ............................................... 44 1.7.3 Rifamycins ...................................................................................................... 45 1.8 Fluoroquinolones and aminocoumarins ................................................................ 45 - 4 - 2 General information about topoisomerases ................................................................. 46 2.1 Classification of topoisomerases ........................................................................... 46 2.1.1 Type I DNA topoisomerases ........................................................................... 46 2.1.2 Type II DNA topoisomerases .......................................................................... 47 2.2 The occurrence of DNA topoisomerases in different cell types ............................ 48 2.2.1 Eubacteria ...................................................................................................... 48 2.2.2 Archaebacteria ............................................................................................... 48 2.2.3 Yeasts ............................................................................................................. 48 2.2.4 Higher eukaryotes .......................................................................................... 48 3 Topoisomerases as drug targets .................................................................................... 48 3.1 Eukaryotic Topoisomerase I ................................................................................... 49 3.2 Eukaryotic Topoisomerase II .................................................................................. 49 3.3 Prokaryotic topoisomerase II: ................................................................................ 50 4 DNA gyrase and topo IV (in E. coli) ................................................................................ 50 4.1 Biological activity ................................................................................................... 51 4.2 Structure ................................................................................................................ 52 4.2.1 Gyrase ............................................................................................................ 52 4.2.2 Topo IV ........................................................................................................... 56 4.2.3 A word about type II topoisomerases C-terminal domain (CTD) ................... 57 4.3 Gyrase and topo IV Inhibitors ................................................................................ 58 4.3.1 Coumarins ...................................................................................................... 58 4.3.2 Quinolones ..................................................................................................... 60 4.3.3 Quinolone-like compounds ............................................................................ 62 4.3.4 Other gyrase inhibitors .................................................................................. 63 5 Microcin B17 .................................................................................................................. 70 5.1 The microcins ......................................................................................................... 70 5.2 Activity of MccB17 ................................................................................................
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