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Plasmodium Falciparum Plasmepsins IX and X: Structure- Function Analysis and the Discovery of New Lead Antimalarial Drugs

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Plasmodium Falciparum Plasmepsins IX and X: Structure- Function Analysis and the Discovery of New Lead Antimalarial Drugs Plasmodium falciparum Plasmepsins IX and X: Structure- Function Analysis and the Discovery of New Lead Antimalarial drugs Author McGeorge, Rachael Dawn Published 2014 Thesis Type Thesis (PhD Doctorate) School School of Biomolecular and Physical Sciences DOI https://doi.org/10.25904/1912/610 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/365553 Griffith Research Online https://research-repository.griffith.edu.au Plasmodium falciparum Plasmepsins IX and X: Structure- function analysis and the discovery of new lead antimalarial drugs Rachael Dawn McGeorge Bachelor of Forensic Science Bachelor of Criminology and Criminal Justice Bachelor of Science (Hons 1) School of Biomolecular and Physical Sciences Griffith Sciences Griffith University Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy April, 2014 Abstract Malaria is a deadly parasitic infection that poses an enormous threat to global health. While drugs are available, all of our current antimalarials are being gradually rendered ineffective by spreading drug resistance. Reports of increasing tolerance to artemisinin combination therapies, our most potent antimalarial treatments, are particularly concerning. To combat this problem there is an urgent need to identify new and unique targets within the malaria parasite against which novel chemotherapeutics can be developed. Studies investigating the antimalarial activity of HIV protease inhibitors (HIV PIs) have shown that these drugs can inhibit the growth of Plasmodium in vitro, in vivo and ex vivo at clinically relevant concentrations. While the anti-parasitic action of these drugs is not fully understood, it is believed these agents inhibit the growth of parasites by targeting an essential malarial aspartic protease or plasmepsin (PM) and that these enzymes may represent new targets for drug development. The aim of this thesis was to investigate the Plasmodium falciparum aspartic proteases PM IX and X as potential new targets for antimalarial development. To determine if PfPM IX is a viable drug target and to substantially contribute to existing knowledge on this enzyme, I sought to determine its function and location in asexual parasites. While the function of PfPM IX remains unknown, expression and localisation data generated during this work, together with data suggesting that PfPM IX is important to parasite growth, support the hypothesis that it is a feasible drug target. Data showing that PfPM IX is transported outside of the parasite into the infected red cell suggest this enzyme has one or more essential function/s distinct from other malaria parasite aspartic proteases. Expression data also indicated that PfPM IX is required throughout the asexual life cycle and that drugs targeting this enzyme will be active throughout asexual development. Additionally, transgenic parasites over-expressing PfPM IX have been shown to have decreased sensitivity to selected HIV PIs. Although sufficient quantities of active recombinant enzyme could not be produced for in-depth analysis, inhibitor assessment and X-ray crystal structure determination, my work in this area has been of benefit to others who are now pursuing further optimisation and expression trials. Given the complexity of PfPM IX and the lack of suitable templates for in silico modelling tools to accurately predict the structure of this enzyme, it is clear that recombinant enzyme will be required to accurately determine the structure of this PfPM. The availability of recombinant enzyme will also permit further characterisation and inhibitor studies to be performed. i Data examining the role of PfPM X in asexual parasites also supports the hypothesis that this enzyme is a good drug target for chemotherapeutic development. Similar to PfPM IX, PfPM X appears to perform a unique function within malaria parasites. Unlike PfPM IX, PfPM X is not exported into the host red cell. However, it also does not locate to the digestive vacuole or the endoplasmic reticulum, suggesting that it has a distinct function from the remaining PfPMs expressed in asexual parasite stages. Data describing that GFP-tagged and purified PfPM X is sensitive to HIV PIs, together with data demonstrating that parasites over-expressing PfPM X are less sensitive to selected HIV PIs, suggests this enzyme may be a target of these inhibitors. In silico modelling are also supportive of this hypothesis, with top binding HIV PIs interacting with the model’s catalytic aspartic residues. As the vast majority of PfPM X transcription occurs during later schizont-stages, this enzyme may have a role in egress and/or invasion. The slow growth, as compared to wild-type parents, of parasites transfected with an antisense construct of PfPM X which transcribed less PfPM X, together with the inability of this work to generate a population of parasites with a PfPM X targeted gene disruption, provides further evidence that this protein plays a vital role in asexual parasite stages. Whilst the current body of research was not successful in identifying the function of PfPM IX and PfPM X, our data supports the hypothesis that these enzymes play an important role in parasite growth and development. The tools and resources developed during this project will continue to be useful for further studies investigating the role of these and other PMs in malaria parasites. They may also aid in the identification of more potent specific inhibitors. ii Statement of Originality This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. _____________________________ Rachael Dawn McGeorge iii Table of Contents Abstract ............................................................................................................................ i Statement of Originality ................................................................................................ iii Table of Contents ........................................................................................................... iv List of Figures and Tables ............................................................................................ xi List of Appendices ........................................................................................................ xv Ethics ............................................................................................................................. xv List of Abbreviations ................................................................................................... xvi Acknowledgements .................................................................................................... xviii Publications ................................................................................................................... xx Presentations: ................................................................................................................ xx Chapter 1: Introduction and Literature Review ......................................................... 2 1.1 Malaria: ................................................................................................................... 4 1.1.1 A Lethal Parasitic Infection: ............................................................................ 4 1.1.2 Plasmodium falciparum: ................................................................................. 5 1.1.2.1 Life Cycle: ................................................................................................ 5 1.1.2.2 Falciparum Malaria: ................................................................................. 5 1.1.2.3 Global Distribution: .................................................................................. 7 1.1.2.4 Disease Demographics: ............................................................................ 7 1.1.2.5 Disease Interactions: ................................................................................. 8 1.1.3 Vector Control: ................................................................................................ 9 1.1.4 Vaccine Development: .................................................................................... 9 1.1.5 Chemotherapy: .............................................................................................. 10 1.1.5.1 Prevention: .............................................................................................. 10 1.1.5.2 Current Treatment Guidelines for Falciparum Malaria: ......................... 11 1.1.5.2.1 Quinoline Antimalarials: ................................................................. 11 1.1.5.2.2 Antifolates: ...................................................................................... 14 iv 1.1.5.2.3 Artemisinin and derivatives: (Sesquiterpene lactones and metabolites) .................................................................................................... 15 1.2 Identification of new Plasmodium drug targets .................................................... 16 1.2.1 Haemoglobin digestion by P. falciparum ...................................................... 16 1.2.2 P. falciparum Plasmepsins ............................................................................ 17 1.2.3 PfPM IX and X: ............................................................................................
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