PHYSIOLOGY DIRECTED DRUG DISCOVERY IN MULTIDRUG RESISTANT PLASMODIUM FALCIPARUM A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry By Amila Chathuranga Siriwardana, B. S. Washington, DC April 20, 2017 Copyright 2017 by Amila Chathuranga Siriwardana All Rights Reserved ii PHYSIOLOGY DIRECTED DRUG DISCOVERY IN MULTIDRUG RESISTANT PLASMODIUM FALCIPARUM Amila Chathuranga Siriwardana, B. S. Thesis Advisor: Paul D. Roepe, Ph. D. ABSTRACT Resistance to front-line antimalarial therapies is a widespread issue, from early chloroquine failure to the more recent concerns of Artemisinin Combination Therapy (ACT) failure. Traditional methods of quantifying drug resistance focused on growth inhibitory, or IC50, concentrations of drug. In the clinical setting, however, parasites are exposed to much higher concentrations of drug that kill parasites within hours. Defined as cytocidal activity, these concentrations have only recently begun to be explored. Recent reports suggest that, drug targets and molecular mechanisms of drug resistance differ at cytostatic versus cytocidal levels. To investigate these possible targets and mechanisms of resistance at cytocidal levels of drug, immunofluorescence assays were conducted to visualize PfAtg8 localization in chloroquine sensitive versus resistant parasites treated at cytocidal drug concentrations. In addition to understanding chloroquine resistance, the recent emergence of a delayed clearance phenotype (DCP) and piperaquine resistance further warrant novel methods of probing molecular mechanisms of drug resistance. Mutations in the propeller domains of a Kelch domain- containing protein on chromosome 13 (K13) have been linked to DCP and traditional methods of quantifying cytostatic or cytocidal activities of drugs fail to distinguish K13-mutant parasites from wild-type parasites. In 2013, Witkowski et al. developed the ring-stage susceptibility assay (RSA) using dihydroartemisinin to correlate DCP with an in vitro measurement: increased parasite iii survival. This method was modified to investigate other endoperoxide drugs and of the seven compounds tested, OZ439 was only compound that circumvented the endoperoxide cross- resistance pattern. A high-throughput screen identified several potent antimalarials, including phosphatidylinositol 3-kinase (PI3K) inhibitors. These PI3K inhibitors are not only extremely potent antimalarials, but they were also found to inhibit the formation of Atg8 puncta in parasites and were highly synergistic with ART derivatives. Based on recent studies suggesting the involvement of elements in the autophagy cascade in artemisinin resistance, immunofluorescence assays were done with K-13 mutant and wild type parasites and results suggest a dysregulation of an autophagy-like process in the K13-mutant parasites. These results guided me towards conducting single-point Chou-Talalay combination analyses to investigate the efficacy of traditional ACTs in comparison with novel combinations involving an endoperoxide compound plus various PI3K inhibitors. Synergy was observed in OZ439-PI3K combinations in both DCP and wild-type strains. iv I dedicate this work to my family and friends who have been there to help and support me every day and without whom I would not have been able to achieve this. Many thanks, Amila Chathuranga Siriwardana v TABLE OF CONTENTS CHAPTER I INTRODUCTION ..................................................................................................... 1 1.1 General Background ............................................................................................................. 1 1.2 Autophagy in Apicomplexan Parasites ................................................................................. 4 1.2.1 Cytocidal Drug Resistance ............................................................................................. 4 1.2.2 Malaria Cell Death Mechanisms .................................................................................... 6 1.2.3 Autophagy is a Stress Response Mechanism ................................................................. 9 1.2.4 Autophagy as a Drug Target ........................................................................................ 12 1.3 Resistance to Artemisinin Combination Therapies............................................................. 18 1.3.1 Artemisinin Combination Therapies ............................................................................ 18 1.3.2 Delayed Clearance Phenotype ..................................................................................... 19 1.3.3 Ring-Stage Susceptibility Assay .................................................................................. 20 1.3.4 Molecular Marker of Artemisinin Resistance .............................................................. 22 1.4 Drug Combination Analysis ............................................................................................... 28 1.5 Objectives ........................................................................................................................... 31 CHAPTER II MATERIALS AND METHODS ........................................................................... 34 2.1 Materials ............................................................................................................................. 34 2.2 Methods............................................................................................................................... 35 2.2.1 Plasmodium falciparum Culturing ............................................................................... 35 2.2.2 Sample Preparation for Single Cell Photometry .......................................................... 36 2.2.3 In situ Calcium Calibration of Fura-2 .......................................................................... 37 2.2.4 Single Cell Photometry ................................................................................................ 38 2.2.5 Preparation of Affinity Purified ATG8 IgG and ATG8 Monoclonal Antibody 2K19 39 2.2.6 Immunohistochemistry ................................................................................................ 39 2.2.7 Spinning Disk Confocal Microscopy ........................................................................... 40 2.2.8 Antiplasmodial Assays................................................................................................. 44 2.2.9 Drug Combination Assays Using the Chou-Talalay Method ...................................... 45 2.2.10 High-Throughput Screening of Drug Combinations ................................................. 47 2.2.11 QTL Analysis ............................................................................................................. 51 2.2.12 Ring-Stage Susceptibility Assay (RSA) .................................................................... 52 2.2.13 Flow Cytometry ......................................................................................................... 53 CHAPTER III A PROCESS SIMILAR TO AUTOPHAGY IS ASSOCIATED WITH CYTOCIDAL CHOLOQUINE RESISTANCE IN PLASMODIUM FALCIPARUM ............... 54 3.1 Abstract ............................................................................................................................... 54 3.2 Background ......................................................................................................................... 55 vi 3.3 Results ................................................................................................................................. 58 3.4 Discussion ........................................................................................................................... 74 3.5 Appendix A ......................................................................................................................... 82 3.6 Acknowledgements ............................................................................................................. 89 CHAPTER IV HIGH-THROUGHPUT MATRIX SCREENING IDENTIFIES SYNERGISTIC AND ANTAGONISTIC ANTIMALARIAL DRUG COMBINATIONS ................................... 90 4.1 Abstract ............................................................................................................................... 90 4.2 Background ......................................................................................................................... 91 4.3 Results ................................................................................................................................. 95 4.4 Discussion ......................................................................................................................... 110 4.5 Appendix B ....................................................................................................................... 114 4.6 Acknowledgenemts ........................................................................................................... 119 CHAPTER V ENDOPEROXIDE DRUG CROSS RESISTANCE PATTERNS FOR PLASMODIUM FALCIPARUM EXHIBITING A DELAYED CLEARANCE PHENOTYPE ..................................................................................................................................................... 120 5.1 Abstract ............................................................................................................................. 120 5.2 Background