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Target-Based Discovery of Novel Inhibitors of Enzymatic Targets from Wolbachia Endosymbionts and Evaluation As Antifilarial Drug Candidates

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Target-Based Discovery of Novel Inhibitors of Enzymatic Targets from Wolbachia Endosymbionts and Evaluation As Antifilarial Drug Candidates Target-based discovery of novel inhibitors of enzymatic targets from Wolbachia endosymbionts and evaluation as antifilarial drug candidates Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von: Christian Stephan Lentz aus Prüm Bonn, Februar 2013 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. med. Achim Hörauf 2. Gutachter: Prof. Dr. rer. nat. Michael Famulok Tag der Promotion: 29.05.2013 Erscheinungsjahr: 2013 Erratum On page 25 of this dissertation (Chapter 2. Materials, Methods & Preparative Experiments) the entry for the composition of the PPDK activity assay buffer was erroneous. The correct specification for this buffer is as follows: PPDK activity assay buffer 1X PPDK-PBS Buffer: 1X PBS, 20 mM NH4Cl, 1 mM MgCl2, pH 7.1 Contents List of abbreviations 5 1 Introduction 7 1.1 Infections with filarial nematodes: Past and present . 7 1.2 Life cycle of filarial nematodes and pathogenesis . 7 1.3 An introduction to Wolbachia endobacteria . 8 1.4 Mutualism between Wolbachia and filarial nematodes . 10 1.5 Model systems of filarial nematodes . 11 1.5.1 Litomosoides sigmodontis .................... 11 1.5.2 Acanthocheilonema viteae .................... 12 1.6 Antifilarial drugs . 12 1.6.1 Classical anthelmintics . 12 1.6.2 Wolbachia-depleting antibiotics . 13 1.6.3 A call for the development of novel antifilarial drugs . 14 1.7 Heme biosynthesis in filarial nematodes . 14 1.8 δ-Aminolevulinic Acid Dehydratase (ALAD) . 17 1.9 Pyruvate Phosphate Dikinase (PPDK) . 19 1.10 Aim of this work . 20 2 Materials, Methods & Preparative Experiments 21 2.1 Materials . 21 2.1.1 Instruments and Equipment . 21 2.1.2 Reagents and Solutions . 22 2.2 Methods.................................. 26 2.2.1 Agarose gel electrophoresis . 26 2.2.2 Polymerase Chain Reactions (PCR) . 26 2.2.3 Cloning . 29 2.2.4 Plasmid and genomic DNA preparations . 30 2.2.5 DNA sequencing reactions . 30 2.2.6 Recombinant protein expression . 30 2.2.7 SDS-PAGE ............................ 32 2.2.8 Coomassie blue protein staining . 33 2.2.9 Native PAGE . 33 2.2.10 In-gel ALAD activity assay . 33 2.2.11 Dialysis of proteins . 33 2.2.12 High-Throughput screening . 34 2.2.13 Chemical compounds and compound handling . 34 2.2.14 PPDK functional assays . 34 2.2.15 Pyruvate Kinase assay . 35 2.2.16 wALAD Assay and High-Throughput Screening . 35 2.2.17 ALAD IC50 assays of different orthologs . 36 2.2.18 Solubility measurements . 36 1 CONTENTS 2.2.19 Thermal Shift Assay . 37 2.2.20 Cell culture . 37 2.2.21 Cytotoxicity assays . 38 2.2.22 Animal handling . 38 2.2.23 Infection experiments with filarial nematodes . 38 2.2.24 Ex vivo co-culture assay with filarial nematodes . 39 2.2.25 wALADin1 in vivo experiments on L. sigmogontis infected mice 40 2.2.26 Toxoplasma gondii culture.................... 40 2.2.27 Plasmodium falciparum culture................. 41 2.2.28 Sequence alignments . 42 2.2.29 Basic Local Alignment Search Tool (BLAST) searches . 42 2.2.30 Data and statistical analyses & Software . 42 2.3 Preparativeexperiments . 43 2.3.1 Cloning of P. aeruginosa hemB gene into pET-21b expression vector ............................... 43 2.3.2 Recombinant expression of pALAD protein . 48 2.3.3 Recombinant expression of wALAD and hALAD protein . 50 3 Results 53 3.1 wALAD HT-Screening . 53 3.1.1 Discovery of wALADin-benzimidazole: species-selective in- hibitors of wALAD . 53 3.1.2 Furtherhitstructures . 57 3.2 Functional characterization of wALADin1 . 58 3.2.1 Bindingexperiments ....................... 58 3.2.2 wALADin1 and the oligomeric equilibrium of wALAD . 59 3.2.3 The molecular mode-of-action of wALADin1 . 60 3.2.4 Solubility of wALADin1 . 62 3.3 Antifilarial activity of wALADin1 . 63 3.3.1 Cytotoxicity tests . 63 3.3.2 Efficacy of wALADin1 in L. sigmodontis co-culture assay . 64 3.3.3 Effects of 5-ALA in the L. sigmodontis co-culture . 66 3.3.4 BLAST search for a gene with protoporphyrinogen IX oxidase function in Wolbachia ...................... 66 3.4 Tests for antifilarial activity of wALADin1 in vivo during L. sigmo- dontis infection.............................. 68 3.5 wALADin1 Structure-Activity Relationship studies (SAR) . 71 3.5.1 Inhibitory potency of wALADin1 derivatives in the enzymatic assay................................ 71 3.5.2 Antifilarial activity of wALADin1 derivatives . 74 3.6 wALAD inhibitors with a different chemical scaffold . 75 3.6.1 Inhibitory properties of virtual screening hits . 75 3.6.2 Inhibitory properties of the Peakdale compound set . 76 3.7 ALAD Cross-species SAR . 83 3.7.1 Inhibitory activity of wALADins on different orthologs . 83 3.7.2 The inhibitory mechanism of wALADin1 on P. sativum ALAD 86 3.7.3 Stimulatory vs. inhibitory activity on the P. aeruginosa ALAD 86 3.8 Antiprotozoal effects of wALADin1 . 94 3.8.1 wALADins in P. falciparum culture .............. 94 3.8.2 wALADins in T. gondii culture................. 96 3.9 PPDKHT-Screening........................... 97 3.9.1 Establishment of PPDK enzymatic screening assay . 97 3.9.2 PPDKscreening ......................... 97 3.9.3 Hit validation . 99 2 CONTENTS 3.9.4 Determination of the type-of-inhibition . 103 4 Discussion 105 4.1 The rationale for HTS-based drug discovery of novel antifilarial com- pounds acting on Wolbachia targets . 105 4.2 Targeting Pyruvate Phosphate Dikinase of Wolbachia: Interfering with endobacterial energy metabolism . 106 4.2.1 The biochemistry of PPDK . 106 4.2.2 Previously described inhibitors of PPDK and screening assays 107 4.2.3 PPDKin1, a novel inhibitor of Wolbachia PPDK . 108 4.2.4 Summary PPDK . 109 4.3 Interfering with the heme biosynthesis of Wolbachia endobacteria . 110 4.3.1 Currently known inhibitors, structure analogs . 111 4.4 wALADins as novel species-selective ALAD inhibitors: Structure and Function at the molecular level . 113 4.4.1 A novel Mg-dependent type of inhibition . 113 4.4.2 Metal-cofactor requirements of ALAD orthologs . 114 4.4.3 Mg2+-usage of wALAD . 115 4.4.4 The inhibitory mechanism of wALADin1 discussed at the structurallevel .......................... 116 4.4.5 Mechanistic aspects of the stimulatory/inhibitory dichotomy of wALADin1 . 116 4.4.6 The inhibitory mechanism of wALADin2 . 117 4.4.7 SAR of benzimidazole derivatives . 118 4.4.8 ALAD-inhibitors based on a different molecular scaffold . 119 4.4.9 Summarizing the functional characterization of wALADin in- hibitors at the molecular level . 119 4.5 The antifilarial effect of wALADins . 120 4.5.1 Potential secondary target effects . 121 4.5.2 In vivo activity of wALADin1 . 122 4.6 Applications of wALADin 1 beyond filarial nematodes . 123 4.6.1 Inhibitory spectrum of wALADin and derivatives . 123 4.6.2 An introduction to malaria . 124 4.6.3 ALAD and heme biosynthesis as a potential drug target in apicomplexan parasites . 125 4.6.4 Potent Antiplasmodial activity in vitro . 126 4.7 wALADin inhibitors in light of current trends in drug development . 126 5 Summary 129 Bibliography 131 List of Figures 153 List of Tables 155 A Chemical structure appendix i A.1 wALAD High-Throughput Screening hits . ii A.2 wALADin1derivatives .......................... iii A.3 Virtualscreeninghits .......................... v A.4 Selected compounds from the Peakdale set . vi A.5 Validated PPDK High-Throughput Screening hits . vii B Supplementary videos x 3 4 List of abbreviations 5-ALA 5-Aminolevulinic acid ALB Albendazole BHQ-1 Black Hole Quencher-1 BSA Bovine serum albumin BTP Bis-tris propane CD 2-Hydroxypropyl-β-cyclodextrin cDNA Complementary DNA d Day DEC Diethylcarbamazine DMAB Para-dimethylaminobenzaldehyde DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid dpi Days post infection DTT Dithiothreitol EBSS Earl’s Balanced Salt Solution EDTA Ethylenediaminetetraacetic acid FAM Fluoresceine amidite FC Ferrochelatase Fig. Figure h Hour HEX Hexachloro-fluoresceine HTS High-Throughput Screening i.m. Intramuscular i.p. Intraperitoneal i.t. Intrathoracic i.v. Intravenous IMMIP Institute for Medical Microbiology, Immunology and Parasitology, Bonn, Germany IPTG Isopropyl β-D-1-thiogalactopyranoside IVM Ivermectin LDH Lactic acid dehydrogenase LIMES Institute Life & Medical Sciences Institute, Laboratory of Chemical Biology, Bonn, Germany LF Lymphatic filariasis MDA Mass Drug Administration Program MER ModifiedEhrlich’sReagent MF Microfilariae MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NLR Non-linear regression analysis OD Opticaldensity Oncho Onchocerciasis p.i. Post infection PAGE Polyacrylamide gel electrophoresis PBG Porphobilinogen PCR Polymerase Chain Reaction PDB Protein Data Bank PDR Pyruvate Detection Reagent PEP Phosphoenol pyruvate PK Pyruvate kinase 5 PPDK Pyruvate phosphate dikinase RBC Red blood cell(s) / erythrocyte(s) RD Restriction digested RNA Ribonucleic acid rpm Rotations per minute RT Room temperature SA Succinyl Acetone SAR Structure-Activity Relationship SD Standard deviation SDS Sodium dodecyl sulfate SEM Standard error of the mean TAMRA Tetramethylrhodamine TSA Thermal shift assay Tris Tris(hydroxymethyl)aminomethane VS Virtual screening WHO World Health Organisation 6 Chapter 1 Introduction 1.1 Infections with filarial nematodes: Past and present "SiR,-Permit me to call the attention of your readers, and especially that of fo- reigners, to some recent additions to our knowledge of the prevalence and probable source of various haematozoa infecting mankind and animals." [50]: "Those who take an interest in helminthological studies may remember that in the month of July last I was permitted to announce in THE LANCET the discovery of a new entozoon. In honour of the discoverer I named the worm "Filaria Bancrofti"" [51]. In these words T. Spencer Cobbold addressed the editors and the public readership of The Lancet in the 1870’s and praised the work by Lewis, Wucherer, Bancroft and others who worked on the recent discoveries of human pathogenic filarial nematodes that were assumed as the causative agents of elephantiasis by Manson a few years later [215].
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