Pre-clinical studies characterizing the evolutionary dynamics of resistance to novel antimalarials in Plasmodium falciparum

Abstract

Malaria, caused by protozoan parasites of the genus Plasmodium, affects millions of people and is a leading cause of childhood mortality. Antimalarial treatments save lives and are an important cornerstone of the global malaria elimination strategy. Unfortunately, drug resistance continuously threatens the efficacy of these important public health tools. While the current antimalarial pipeline is more robust than ever, drug development still has not kept pace with parasite evolution; resistance to frontline artemisinin-based therapies has emerged, and no new classes of drugs have yet been approved which could replace these treatments should resistance become globally widespread. To get ahead, we need to better understand how drug resistance evolves, and ideally, use this knowledge to design novel treatment strategies that delay the emergence and spread of resistant parasites. In this thesis, we characterize the evolution of resistance to novel antimalarials targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). Our group and others have identified many compounds with diverse chemical structures that inhibit this enzyme. Interestingly, we also observed that resistance to one inhibitor often confers increased sensitivity to another DHODH inhibitor of a distinct chemical class. This phenomenon—broadly known as collateral sensitivity—opens the possibility of designing compound combinations that create an ‘evolutionary trap,’ blocking the emergence of resistance pathways. In Chapter 1 of this thesis, we review the biology of Plasmodium parasites and outline the factors that influence the evolution of resistance. We propose a framework to better predict the propensity for resistance to novel antimalarials. In Chapter 2, we assess the evolution of resistance to the DHODH inhibitor DSM265, a clinical antimalarial candidate. We find that resistance emerges readily both in vitro and in a mouse model of in vivo infection, and can be conferred by 13 distinct point mutations in DHODH. Our findings suggested a high risk of resistance for the clinical use of DSM265, a finding which was ultimately corroborated when resistance to DSM265 emerged during Phase 2 clinical trials. Chapter 3 of the thesis explores the landscape of cross-resistance and collateral sensitivity to Plasmodium dihydroorotate dehydrogenase inhibitors. From this work, we identify pairs of compounds exhibiting complementary activity profiles to varying DHODH mutant lines. We hypothesize that treating parasites with these compounds in combination will suppress the emergence of resistant parasites. In Chapters 4 and 5, we directly test this hypothesis. In Chapter 4, we perform in vitro selections with DSM265 and TCMDC-125334, a pan-mutant active inhibitor. Combination treatment selected for cross-resistant parasites that are evolutionarily fit in competitive growth assays. This result emphasized the mutational flexibility of the DHODH enzyme as a key limitation of this treatment strategy. In Chapter 5, we treated parasites with another DHODH inhibitor combination—DSM265 and Genz669178—using both in vitro and in vivo platforms of selection. Interestingly, while cross-resistance to this combination arose readily in vitro, the emergence of resistance was suppressed in the mouse infection model. This finding demonstrated that pharmacokinetics and the in vivo environment can influence the evolution of resistance, highlighting the importance of in vivo models to study these dynamics. Throughout these chapters, we characterize several factors that impact the propensity of resistance evolution. In the final chapter, we discuss common themes that emerged, as well as key take-aways for future drug development efforts.