A study of the mechanism of action and resistance of artemisinin antimalarials in Plasmodium falciparum Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy Matthew Phanchana November 2016 Liverpool School of Tropical Medicine Abstract A study of the mechanism of action and resistance of artemisinin antimalarials in Plasmodium falciparum Matthew Phanchana Malaria remains a global health and economic issue affecting nearly half of the world's population. In the past decade, effective chemotherapy and vector control have been the major interventions used to control and reduce the burden of malaria. However, resistance to antimalarial drugs and insecticides is compromising the control and treatment strategies and the goal to eliminate malaria. In most malaria endemic countries, artemisinin combination therapies (ACTs) are the first line treatment for uncomplicated Plasmodium falciparum malaria, the most lethal cause of malaria. Despite the widespread use of artemisinin-based therapies, the mechanism of action of this class of drug remains elusive. Emergence of resistance to ACTs in South East Asia is a global concern for drug efficacy. In this thesis, a click chemistry coupled with mass spectrometry (MS) proteomics approach was used to identify the molecular targets of artemisinin in various stages of P. falciparum strain 3D7 and extensively applied to the candidate trioxolanes, a new class of fully-synthetic artemisinin-like drugs. Using artemisinin activity-based probes, a number of biological targets were identified, these targets derive from key biological pathways/process that include; haemoglobin metabolism, glycolysis, nucleic acid and protein biosynthesis, antioxidant defence and oxidative stress response. The identified fingerprint of biological targets was similar between semi synthetic artemisininin and fully synthetic next generation artemisinins. Identified biological targets were enriched with glutathionylated proteins, indicating that these proteins are vulnerable to endoperoxide antimalarial inhibition and loss of function. The shared protein targets or protein pattern of semisynthetic artemisinin and fully synthetic trioxolane suggest that they might share similar mechanism or action and, possibly, mechanism of resistance. This raises the concern of cross resistance between them. The ring stage parasites which showed the least sensitivity to artemisinins and associated with resistance to artemisinins have much less proteins identified, including the absence of proteins in haemoglobin metabolism and reduction in proteins of major pathways. These findings support the working hypothesis that artemisinin is most effective against later stages of the parasite in line with the activity of haemoglobin digestion, the main activator of artemisinins and other endoperoxides. The reduced sensitivity during the ring stage is possibly due to less activation of artemisinin. A whole genome sequence comparative approach was undertaken with parasites displaying phenotypic artemisinin resistance (slow clearance phenotype) derived from an experimental in vivo model of infection. Parasite genes that we correlated to the slow clearance phenotype included genes involved in the unfolded protein response pathway consistent with recent models of parasite resistance to artemisinins. The results presented in this thesis using chemical biology and omics technologies, have contributed to our understanding of the mechanism of action and resistance of endoperoxides and offer future research directions to study this important class of antimalarials. ii Acknowledgements I would like to acknowledge my supervisors, Prof Steve Ward and Prof Giancarlo Biagini, for their tireless guidance and all opportunities they have given. Working with malaria has never been easy but challenging. They have scientifically and personally supported me during the past years helping me to become a better me. I also thank my advisory panel members, Dr Robert Harrison and Dr Simon Wagstaff for their constructive comments and reflections. I would like to extend my thanking to Dr Simon Wagstaff and the bioinformatics team; Dr John Archer, Dr Enrique Salsedo-Sora and Dr Gareth Weedall, for their contribution on genome analysis. I am indebted to Dr Hanafy Ismail for his helping hands and all the click techniques and cautions, Dr Gavin Laing and Dr Gemma Molyneux for their expertise on MS proteomics. Life in the lab would not be this enjoyable without them! Eva, Arturas, Saif, Eilidh, Vera, and Mameow. The past and present members of the SAW/GB lab, Ashley, Paul, Ally, Jill, Grazia, Dave, Gavin, Ricky, Alison, Mary, and Angela, have made the lab became more than just a lab but a great environment and company. I am delighted to be your mate! I owed a huge thank to Prof Mathirut Mungthin and Prof Peerapan Tan-ariya who opened the world of malaria research to me. All at the Parasitology group at PCM, you were and are always good friends and very supportive, especially HaMuay and Bee for laying the first stone of malaria work for me. Furthermore, I am thankful for Mahidol-Liverpool Chamlong Harinasuta PhD scholarship, and the selection panel for their beliefs in me. Another life outside the school was just amazing because of these mates, Gluay, Oey, Yin, Fha, Smart, Mai, Big, Tor, T-family, Gam, Bear, Yayee, Pu, Nham, and the past and present members of Liverpool Thai Society. All the good and bad time I spent with you was precious. I would like to thank Lee for her generosity and all at Chaba Chaba for being good colleagues and sharing the great time with me. Lastly, I would like to express my love and thank to my parents for their love, prayers and supports for all the thing that I do. iii Contents| 1.1 Malaria ................................................................................................................................................. 1 1.1.1 Life cycle ........................................................................................................................................................ 2 1.2 Antimalarial drugs .............................................................................................................................. 5 1.2.1 4-aminoquinolines ....................................................................................................................................... 5 1.2.2 8-aminoquinolines ....................................................................................................................................... 9 1.2.3 Arylaminoalcohols .....................................................................................................................................10 1.2.4 Antifolates ...................................................................................................................................................11 1.2.5 Inhibitors of the respiratory chain ...........................................................................................................13 1.2.6 Antibiotics ...................................................................................................................................................14 1.2.7 Mechanisms of antimalarial resistance ..................................................................................................15 1.3 Development of new antimalarial drugs ...................................................................................... 16 1.4 Prevention and control .................................................................................................................... 19 1.4.1 Vector control .............................................................................................................................................19 1.4.2 Chemoprevention and malaria vaccine ..................................................................................................20 1.4.3 Case management .....................................................................................................................................21 1.5 Malaria eradication program .......................................................................................................... 22 1.6 Artemisinins ...................................................................................................................................... 22 1.6.1 Bioactivation of artemisinins ....................................................................................................................24 1.6.2 Postulated mechanisms of action and resistance of artemisinin ........................................................25 1.7 Approaches toward understanding mode of action ................................................................... 28 1.8 Aims ................................................................................................................................................... 29 2.1 Chemicals .......................................................................................................................................... 30 2.2 Preparation of HEPES ...................................................................................................................... 30 2.3 Preparation of hypoxanthine ......................................................................................................... 30 2.4 Preparation of human serum ........................................................................................................