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Chapter 4 SAR and Antibiotic Activities of Diphenyl

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Chapter 4 SAR and Antibiotic Activities of Diphenyl Structure-Guided Development of Novel LpxC Inhibitors by Chul-Jin Lee Department of Biochemistry Duke University Date:_______________________ Approved: ___________________________ Pei Zhou, Supervisor ___________________________ David C. Richardson ___________________________ Terrence G. Oas ___________________________ Richard G. Brennan ___________________________ Eric J. Toone Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University 2013 i v ABSTRACT Structure-Guided Development of Novel LpxC Inhibitors by Chul-Jin Lee Department of Biochemistry Duke University Date:_______________________ Approved: ___________________________ Pei Zhou, Supervisor ___________________________ David C. Richardson ___________________________ Terrence G. Oas ___________________________ Richard G. Brennan ___________________________ Eric J. Toone An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry in the Graduate School of Duke University 2013 Copyright by Chul-Jin Lee 2013 Abstract The incessant increase of antibiotic resistance among Gram-negative pathogens is a serious threat to public health worldwide. A lack of new antimicrobial agents, particularly those against multidrug-resistant Gram-negative bacteria further aggravates the situation, highlighting an urgent need for development of effective antibiotics to treat multidrug-resistant Gram-negative infections. Past efforts to improve existing classes of antimicrobial agents against drug-resistant Gram-negative bacteria have suffered from established (intrinsic or acquired) resistance mechanisms. Consequently, the essential LpxC enzyme in the lipid A biosynthesis, which has never been exploited by existing antibiotics, has emerged as a promising antibiotic target for developing novel therapeutics against multidrug-resistant Gram-negative pathogens. In Chapter I, I survey the medically significant Gram-negative pathogens, the molecular basis of different resistance mechanisms and highlight the benefits of novel antibiotics targeting LpxC. In Chapter II, I discuss a structure-based strategy to optimize lead compounds for LpxC inhibition, revealing diacetylene-based compounds that potently inhibit a wide range of LpxC enzymes. The elastic diacetylene scaffold of the inhibitors overcomes the resistance mechanism caused by sequence and conformational heterogeneity in the LpxC substrate-binding passage that is largely defined by Insert II of LpxC. In Chapter III, I describe the structural basis of inhibitor specificity of first- generation LpxC inhibitors, including L-161,240 and BB-78485 and show that bulky iv moieties of early inhibitors create potential clashes with the a-b loop of Insert I of non- susceptible LpxC species such as P. aeruginosa LpxC, while these moieties are tolerated by E. coli LpxC containing long and flexible Insert I regions. These studies reveal large, inherent conformational variation of distinct LpxC enzymes, providing a molecular explanation for the limited efficacy of existing compounds and a rationale to exploit more flexible scaffolds for further optimization of LpxC-targeting antibiotics to treat a wide range of Gram-negative infections. In Chapters IV and V, a fragment-based screening and structure-guided ligand optimization approach is presented, which has resulted in the discovery of a difluoro biphenyl diacetylene hydroxamate compound LPC-058 with superior activity in antibacterial spectrum and potency over all existing LpxC inhibitors. In Chapter VI, I describe our efforts to improve the cellular efficacy of LPC-058 by reducing its interaction with plasma proteins, such as human serum albumin (HSA). The binding mode of LPC-058 was captured in the crystal structure of HSA/LPC-058 complex. The acquired structural information facilitated the development of the dimethyl amine substituted compound LPC-088 that displays significantly improved cellular potency in presence of HSA. v Dedication I dedicate this dissertation to my wonderful family, Hyojin Kim and Ian Pius Lee. vi Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................ xii List of Figures ............................................................................................................................ xiii Acknowledgements .................................................................................................................... xv 1. Introduction .............................................................................................................................. 1 1.1 Summary ..................................................................................................................... 2 1.2 Antimicrobial resistance in Gram-negative bacteria ............................................. 3 1.2.1 Growing concern of drug-resistant Gram-negative bacterial infections to public health ............................................................................................................ 3 1.2.2 Mechanisms of antibiotics- resistant Gram-negative bacteria ....................... 8 1.2.3 Clinically important MDR Gram-negative pathogens .................................. 16 1.3 LpxC, novel antibiotic target for Gram-negative bacteria .................................. 20 1.3.1 Lipid A biosynthesis as an unexploited antibiotic target ............................. 20 1.3.2 LpxC as a promising antibiotic target .............................................................. 23 1.3.3 Unique structural features of LpxC ................................................................. 23 1.3.4 Catalytic mechanism of LpxC enzyme ............................................................ 26 1.3.5 Known, potent LpxC inhibitors ........................................................................ 27 2. Species-Specific and Inhibitor-Dependent Conformations of LpxC:Implications for Antibiotic Design .................................................................................................................... 33 2.1 Abstract ..................................................................................................................... 34 2.2 Introduction .............................................................................................................. 34 2.3 Results ....................................................................................................................... 37 vii 2.3.1 Compounds Based on the Diacetylene Scaffold Potently Inhibit a Wide Range of LpxC orthologs ..................................................................................... 37 2.3.2 Overall Structure of EcLpxC in Complex with LPC-009 .............................. 42 2.3.3 Inherent Conformational Differences of LpxC Orthologs ............................ 46 2.3.4 Ligand-induced Conformations of LpxC ........................................................ 51 2.4 Discussion ................................................................................................................. 53 2.5 Materials and methods ............................................................................................ 57 2.5.1 Chemical Synthesis ............................................................................................. 57 2.5.2 Protein Purification ............................................................................................ 57 2.5.3 Enzymatic Inhibition Assay .............................................................................. 58 2.5.4 MIC Tests ............................................................................................................. 59 2.5.5 Crystallization and X-ray Data Collection ...................................................... 60 2.5.6 Model Building and Refinement ...................................................................... 61 2.5.7 Accession Numbers ............................................................................................ 62 3. Structural Basis of Species-Specific Inhibitor Selectivity of the Deacetylase LpxC ....... 63 3.1 Abstract ..................................................................................................................... 64 3.2 Introduction .............................................................................................................. 65 3.3 Results ....................................................................................................................... 66 3.3.1 MIC tests and ITC Experiments to Confirm the Inhibitor-selectivity of LpxC ........................................................................................................................ 66 3.3.2 Structure of EcLpxC in Complex with L-161,240 ........................................... 69 3.3.3 Binding Mode of L-161,240 ............................................................................... 72 3.3.4 Key Contacts of LpxC Underlie Inhibitor-Selectivity .................................... 76 viii 3.4 Discussion ................................................................................................................. 85 3.5 Materials and methods ...........................................................................................
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