View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Carolina Digital Repository A Mechanism of Resistance and Mode of Action for Drugs Against Plasmodium falciparum Anne Elizabeth Purfield A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology. Chapel Hill 2007 Approved By: Steven R. Meshnick Edward Collins Peter Gilligan Marcia Hobbs Richard R. Tidwell ©2007 Anne Elizabeth Purfield ALL RIGHTS RESERVED ii ABSTRACT Anne Elizabeth Purfield: A Mechanism of Resistance and Mode of Action for Drugs Against Plasmodium falciparum (Under the direction of Steven R. Meshnick, M.D., Ph.D.) The need for new drugs to control widespread malaria caused by Plasmodium falciparum is critical. Parasite resistance to currently used drugs is rampant and, in many cases, the drug’s mode of action and/or mechanism of resistance is unknown. The three objectives of this dissertation address issues associated with resistance to the currently used antimalarial drugs, in addition to elucidating the mechanism of action of a novel antimalarial compound in development. First, a real time PCR method was developed to distinguish parasite genotypes associated with mefloquine resistance in vitro. Single nucleotide point mutations in the Plasmodium falciparum multi-drug resistance-1 ( pfmdr1 ) gene are associated with mefloquine resistance in vitro. This method may be applied to clinical malaria samples and used to predict treatment outcome as well as for surveillance of drug resistance. In addition, the mechanism of action for the novel compound, [2,5-bis(4-amidinophenyl) furan], (DB75) was investigated. DB75, the active metabolite of the oral pro-drug DB289, is a broad spectrum antiparasitic agent with impressive antimalarial activity both in vitro and in vivo. It is currently in development for treatment of falciparum malaria, however the mode of action against falciparum parasites is unknown. Results from ultraviolet confocal microscopy showed DB75 localization exclusively in the nucleus of parasites in culture. Further, microscopy studies using blood smears to distinguish morphologies suggested DB75 iii has a life stage-specific mechanism. Parasites must be exposed during the ring stage for effective killing. Finally, real time PCR gene expression assays suggested high concentrations of DB75 may alter the expression pattern in a manner consistent with the delay in maturation. However, DB75 did not inhibit or enhance global nuclear transcription or developmental expression of six select genes. The third objective was to determine potential synergistic interactions of DB75 in combination with current antimalarial drugs to determine a mechanism of action for DB75 and to identify potential partner drugs for use in combination therapy with DB75. Taken together, this work contributes to the arsenal of tools for surveillance of falciparum malaria drug resistance and partially elucidates a mechanism of action for a novel antimalarial diamidine that may be used for malaria therapy. iv ACKNOWLEDGEMENTS I thank my adviser and committee members: Steve Meshnick, Rick Tidwell, Marcia Hobbs, Ed Collins and Peter Gilligan. I also thank Jeff Frelinger, Carrie Barnes, Susan Jones, Sheryl Wilson, Michael Chua, David Klapper, Ivan Rusyan lab, Mike Barrett, Vicki Wingate, Stephanie Wallace, Jaina Patel, Paul Wilson and Dixie Flannery for their technical and administrative support. I especially thank: Jesse Kwiek, Jon Juliano, Charlotte Lanteri, Alisa Alker, Kirk Prutzman, Janette Stender, Paul Kurz, my parents (Donna and Tom Purfield), Toby, Holly Gentry-Bray, Sean Palmer, Heather Palmer, Tiffany Warmowski, Tracy Mazurkiewicz, Bruce Alexander, Leslie Arney and Angie Ponguta for listening. v TABLE OF CONTENTS Page LIST OF TABLES…………………………………………………………………………....ix LIST OF FIGURES…………………………………………………………………………...x LIST OF ABBREVIATIONS………………………………………………………………...xi CHAPTER I. Introduction………………………………………………………………………………...1 Geographical distribution of malaria………………………………………………….2 Life cycle of P. falciparum ……………………………………………………............2 Clinical P. falciparum malaria presentation and diagnosis…………...……………...3 Mechanisms of action and resistance for antimalarial drugs……….…………............5 pfmdr1 is a modifier of drug sensitivity………..……………………………………...8 Molecular surveillance of antimalarial drug resistance.….......……………………...11 Artemisinin combination therapy to prevent resistance………………………...........12 Need for new antimalarial drugs ..………………………...………………………....13 Aromatic diamidines have antimalarial activity ……...……...……………………...14 Diamidine mechanisms of actions for antimicrobial activity………...………..…….14 DB289, the orally active prodrug of the novel antimicrobial agent, DB75………….17 Dissertation objectives and significance……………………………………………..18 II. A new method for detection of pfmdr1 mutations associated with mefloquine resistance……………………………………………………………………………………..27 vi Introduction…………………………………………………………………………..29 Materials and Methods……………………………………………………………….30 Results……………………………………………………………………...…...........32 Real-time PCR is sensitive and specific ……………………………..……...32 Comparison of SNPs in paired pre- and post-culture DNA.…………............33 Novel His 86 mutation identified………...………………….…………… …..34 Discussion……………………………………………………………………............34 III. Target and mechanism of action for [2,5-bis(4-amidinophenyl)furan] in P. falciparum ………………………………………………...…………………….…………43 Introduction………………………………………………………………....………..45 Materials and Methods……………………………………………………………….46 Results………………………………………………………………………..............52 DB75 inhibits parasite growth.………………………………………............52 Fluorescent localization of DB75 in P. falciparum parasites...……….. ……52 DB75 is stage specific…………………………………………………..........54 DB75 effect on P. falciparum gene transcription………………….…...........56 Discussion…………………………………………………………………..………..59 IV. DB75 interaction with other antimalarial compounds…………………………..………74 Introduction…………………………………………………………………..………76 Materials and Methods……………………………………………………….............77 Results…………………………………………………………………………..........80 Discussion……………………………………………………………………………83 IV. Summary and Future Directions…………………………………………………………92 V. References…………………………………………………………………...…………...97 vii LIST OF TABLES Table 1.1 Important mutations associated with decreased antimalarial susceptibility....24 1.2 Pfmdr1 single nucleotide polymorphisms are geographically linked to antimalarial treatment failure in clinical studies………..………….25 2.1 Primer and probe sequences used in real time PCR assay…………………...38 2.2 Prevalence of mutations in monoclonal culture samples detected by traditional PCR and sequencing (gold standard) and real time PCR, and sensitivity and specificity of real time PCR assay…………………….……………………………………………...40 2.3 Genotype results of DNA from pre-culture and post-culture DNA by real time PCR……….……………………………………………...41 2.4 Prevalence of mutations in patient blood (gold standard) and post-culture samples, and sensitivity and specificity of PCR on culture-derived parasites………………………………………………….42 3.1 Subcellular localization dyes used for confocal microscopy………………...64 3.2 Developmentally expressed P. falciparum genes…………………………....65 4.1 Results for antimalarial agents in combination with DB75………………….90 4.2 DB75 IC50 decreases with extended exposure………………………...….....91 viii LIST OF FIGURES Figure 1.1 Geographic distribution of P. falciparum malaria risk…………………...….20 1.2 Life cycle of P. falciparum parasite………………………………………….21 1.3 Worldwide distribution of P. falciparum drug resistance……………………22 1.4 Model of p-glycoprotein homologue-1 protein encoded by pfmdr1 …............23 1.4 Chemical structures of dicationic diamidines with antimalarial activity….…26 2.1 Schematic representation showing sources of samples used in study…….....37 2.2 Fluorescence intensity of the FAM reporter dye compared to VIC reporter dye as PCR amplification cycles proceed……………………...…...39 3.1 DB75 inhibition of P. falciparum ……………………………………………66 3.2 DB75 subcellular distribution in different life stages upon immediate cell entry (<4 hours)……………………………………………………………...67 3.3 DB75 subcellular distribution following long term drug exposure………….68 3.4 DB75 distribution in an erythrocyte infected with multiple parasites following 72 hours exposure……………………………………………...….69 3.5 Parasite morphology with 96 hour continuous DB75 exposure ………...…..70 3.6 Parasite growth with 96 hour drug exposure ………………………………..71 3.7 Parasitemia over time when DB75 drug pressure is removed after 48 hours of continuous exposure……………………………..………...……….72 3.8 Effect of DB75 on peak gene expression and intensity …...………………...73 4.1 DB75-partner drug interactions with 42-hour co-incubation at various concentration ratios…………………………………………………………..87 4.2 DB75-partner drug interaction with 66-hour or 96-hour co-incubation…..…88 4.3 Clindamycin effect on DB75 IC50 ………………………………………….89 ix LIST OF ABBREVIATIONS AFRIMS Armed Forces Research Institute for the Medical Sciences Asn Asparagine Asp Aspartic Acid CT Cycle threshold FIC Fractional inhibitory concentration dhfr dihydrofolate reductase dhps dihydropteroate synthase HAT Human African trypanosomiasis IC50 Half maximal inhibitory concentration In vitro In cultured parasite-infected erythrocytes In vivo In malaria patients Lys Lysine MDR1 Multi-drug resistance One NPP New permeation pathway Parasitemia