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Targeting Plasmodium Falciparum Heat Shock Protein 90 (Pfhsp90): a Strategy to Reverse Antimalarial Resistance

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Targeting Plasmodium Falciparum Heat Shock Protein 90 (Pfhsp90): a Strategy to Reverse Antimalarial Resistance TARGETING PLASMODIUM FALCIPARUM HEAT SHOCK PROTEIN 90 (PFHSP90): A STRATEGY TO REVERSE ANTIMALARIAL RESISTANCE by Dea Shahinas A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Dea Shahinas (2012) TARGETING PLASMODIUM FALCIPARUM HEAT SHOCK PROTEIN 90 (PFHSP90): A STRATEGY TO REVERSE ANTIMALARIAL RESISTANCE Dea Shahinas Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto 2012 Abstract Drug resistance is one of the major impediments to control Plasmodium falciparum malaria worldwide. Heat shock protein 90 (Hsp90) is an essential component of the buffering capacity of eukaryotic cells as a part of the stress response. P. falciparum is no different and requires Hsp90 to chaperone proteins essential for cell cycle progression and drug resistance. Inhibition of P. falciparum Hsp90 (PfHsp90) may be able to not only cripple the parasite but also serve as an adjunctive antimalarial by circumventing drug resistance. The results presented in this thesis identify novel Hsp90 inhibitors that synergize with conventional antimicrobials, such as chloroquine (CQ), when used in combination. The objectives were to identify specific malaria Hsp90 inhibitors, the mechanism of the synergistic phenotype, and whether the strategy translates in vivo. To this end, the antimalarial activity of the purine analog PU-H71, and novel PfHsp90 inhibitors was tested. PU-H71 and the novel inhibitors APPA, harmine, and acrisorcin exhibited antimalarial activity in the nanomolar range and displayed synergistic activity with CQ. PU-H71 was able to reverse CQ resistance in a cell-based assay using the CQ-resistant strain W2. PU-H71 caused ring-stage arrest during the intra-erythrocytic cycle. Co- immunoprecipitation studies revealed that PfHsp90 interacts directly with the CQ resistance ii transporter (PfCRT). In the P. berghei mouse model of malaria, PU-H71 and harmine were able to reduce parasitemia and synergize with CQ. The interaction of PfHsp90 with PfCRT may underlie the synergistic phenotype. We conclude that PU-H71 and harmine are effective adjunctive antimalarial drugs that may be useful in combination therapies. iii Acknowledgments I would like to take this opportunity to show my gratitude for the support that I have received during this degree. First and foremost, I am immensely thankful to my co-supervisors Dr. Dylan R. Pillai and Dr. Donald E. Low for giving me the opportunity, resources and support to work on this project as well as other side projects at the Public Health Labs. Dr. Dylan R. Pillai has provided extensive input in the development of ideas and the progress of this project. My PhD has been a rewarding professional journey. Many thanks go to my committee members Dr. Leah E. Cowen, Dr. Cyril Guyard and Dr. Ian Crandall for their prompt support, insight, and expertise. Dr. Ian Crandall has provided indispensable support for the testing of the antimalarial combinations in mouse models of malaria. His company at the Division of Comparative Medicine facilitated the progress of the mouse experiments. I would like to offer special thanks to Dr. Harry Elsholtz and Ferzeen Dharas at the Department of Laboratory Medicine and Pathobiology for prompt administrative support and advice. I am also grateful to the parasitology clinical laboratory staff, the molecular research staff and all the blood donors at the Public Health Labs that made it possible to culture malaria parasites. In particular, I would like to acknowledge Rachel Lau for optimizing the conditions of growing malaria parasites and also for helping me with the optimization of the flow cytometry protocols. I would also like to acknowledge the support of two summer students Michael Liang and Christan Benedict, the research assistant Gregory MacMullin and the two post-doctoral fellows: Dr. Krishna Khairnar and Dr. Gitanjali Arya. Special thanks also to Dr. Walid A. Houry, Dr. John Glover and Elisa Leung for providing me access to the Biophysics facility at the Department of Biochemistry and for sharing their protocols in order to achieve biochemical characterization of PfHsp90. The work presented here has been possible as a result of collaborations with: Dr. Alessandro Datti at the SMART high throughput screening facility at Mount Sinai Hospital, Dr. Gabriella Chiosis and Dr. Tony Taldone at the Memorial Sloan Kettering Cancer Centre and Dr. David Fidock, Dr. Jerome Clain who provided us with insight for the drug interaction analysis. iv Last, but not least, thanks go to my husband Peter Siegler, our parents and my brother for the support, patience and love that they have shown throughout the years as well as for the appreciation that they have for my career. v Table of Contents Chapter 1: Introduction ................................................................................. 1 1 HISTORY OF MALARIA.............................................................................................1 2 AN OVERVIEW OF THE DISEASE............................................................................5 3 THE MALARIA LIFE CYCLE.....................................................................................7 4 ANTIMALARIALS AND THE EMERGENCE OF RESISTANCE..........................11 5 HEAT SHOCK PROTEIN 90......................................................................................14 6 HEAT SHOCK PROTEIN 90 INHIBITORS ..............................................................19 7 PLASMODIUM FALCIPARUM HEAT SHOCK PROTEIN 90 .................................20 References............................................................................................................................22 Chapter 2: ARTESUNATE MISUSE BY RETURNING TRAVELLER WITH PLASMODIUM FALCIPARUM INFECTION.......................... 28 1 THE PATIENT.............................................................................................................28 References............................................................................................................................34 Chapter 3: IDENTIFICATION OF NOVEL AND SELECTIVE PFHSP90 INHIBITORS WITH DRUG POTENTIAL......................... 35 1 INTRODUCTION........................................................................................................35 2 MATERIALS AND METHODS .................................................................................38 2.1 Cloning and Protein Purification..................................................................................38 2.2 4,40-dianalino-1,10-binaphthyl-5,50-disulfonic acid Dipotassium salt (bis-ANS) Binding Assay with the PfHsp90 ATP-Binding Domain.............................................38 2.3 P. falciparum Culture Methods....................................................................................39 2.4 Antimalarial Drug Screening Cell Assay and in vitro susceptibility testing................39 2.5 Entamoeba histolytica cultures ....................................................................................40 3 RESULTS.....................................................................................................................41 3.1 Robotic High Throughput Screen with Recombinant Hsp90 ATP-Binding Domains 41 3.2 EhHsp90 and PfHsp90 inhibitor effect on cell culture.................................................44 vi 3.3 APPA, Harmine and Acrisorcin show Antimalarial Potency in P. falciparum Drug Resistant Strains ...........................................................................................................49 3.4 APPA, harmine and acrisorcin act synergistically with chloroquine...........................53 3.5 Harmine displays synergistic activity with known antimalarials in vitro ....................56 4 DISCUSSION ..............................................................................................................58 References............................................................................................................................64 Chapter 4: BIOCHEMICAL CHARACTERIZATION OF PLASMODIUM FALCIPARUM HEAT SHOCK PROTEIN 90.......... 71 1 INTRODUCTION........................................................................................................71 2 MATERIALS AND METHODS .................................................................................81 2.1 Multiple sequence alignment........................................................................................81 2.2 Sequencing of the PfHsp90 ATP-Binding Domain .....................................................81 2.3 PU-H71 and Geldanamycin (GA) docking in the PfHsp90 GHKL domain structure .81 2.4 Cloning and Protein Purification..................................................................................81 2.5 Site-directed mutagenesis.............................................................................................82 2.6 Surface Plasmon Resonance (SPR) Measurements......................................................82 2.7 ATPase Activity Assay ................................................................................................83 2.8 P. falciparum culture methods and preparation of protein extracts .............................83 3 RESULTS.....................................................................................................................84 3.1 The PfHsp90 ATP binding domain is structurally conserved......................................84
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