Drug resistance remains a pressing concern for antimalarial chemotherapies. Management of resistance has largely been a reactive process, but here we propose a proactive approach that exploits predicted evolutionary trajectories to maintain a drug-sensitive parasite population. Our strategy is to design new drugs such that the fitness cost of resistance restricts the ability of mutant parasites to survive combination therapy or to persist in the absence of selection. Through whole organism high-throughput screening we have discovered many new chemotypes that act on mitochondrial targets and either in combination or in a single molecule target both wild-type and mutant parasites and suppress the emergence of resistance in vitro. Here we focus on the Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) target and fully explore the structural, functional, and fitness consequences of drug resistance pathways. We extend the findings from our current award to fully explore mutually incompatible resistance, establishing an in vivo model system to understand the impact of the host environment on the parasite's ability to escape drug pressure. PfDHODH is clinically relevant and has multiple structural classes of inhibitors providing a substantial toolset with which to study our combination strategy in depth. We will identify chemical inhibitors that target wild-type and mutant forms of DHODH and optimize efficacy for in vivo studies. Using an approach that has proven highly productive, we will prioritize the molecules based on cross-resistance screening against a panel of resistant mutants and focus on compounds demonstrating validated potency against both the wild-type and mutants. We will validate the resistance mutations and assess the structural and biological consequences of these mutations by determining the structures of the most prevalent mutant forms, measuring the effects of the mutations on the enzyme, on mitochondrial function, and on the growth of the organism. We will develop a protocol for using a humanized mouse model of P. falciparum infection to measure the most relevant in vivo resistance pathways and test the suppression of drug resistance. This has implications not just for this work, but also for others in the field who are developing methods to prevent the emergence of drug resistance.
In this project, we will use evolutionary biology principles to anticipate and then block the most fit resistance pathways and in doing so protect the efficacy of antimalarial therapies. We will validate combinations of or single dual-active PfDHODH inhibitors that kill both the wild-type and resistant mutant-type parasites and assess the biochemical, structural, and fitness consequences of resistance mutations to these inhibitors. Finally, we seek to develop a protocol for using a humanized mouse model of P. falciparum infection to measure the most relevant in vivo resistance pathways and test the suppression of drug resistance providing a platform that can be used by others who are developing methods to prevent the emergence of drug resistance.
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|Sakata-Kato, Tomoyo; Wirth, Dyann F (2016) A Novel Methodology for Bioenergetic Analysis of Plasmodium falciparum Reveals a Glucose-Regulated Metabolic Shift and Enables Mode of Action Analyses of Mitochondrial Inhibitors. ACS Infect Dis 2:903-916|
|Kato, Nobutaka; Comer, Eamon; Sakata-Kato, Tomoyo et al. (2016) Diversity-oriented synthesis yields novel multistage antimalarial inhibitors. Nature 538:344-349|
|Lukens, Amanda K; Heidebrecht Jr, Richard W; Mulrooney, Carol et al. (2015) Diversity-oriented synthesis probe targets Plasmodium falciparum cytochrome b ubiquinone reduction site and synergizes with oxidation site inhibitors. J Infect Dis 211:1097-103|
|Ross, Leila S; Gamo, Francisco Javier; Lafuente-Monasterio, Maria José et al. (2014) In vitro resistance selections for Plasmodium falciparum dihydroorotate dehydrogenase inhibitors give mutants with multiple point mutations in the drug-binding site and altered growth. J Biol Chem 289:17980-95|
|Comer, Eamon; Beaudoin, Jennifer A; Kato, Nobutaka et al. (2014) Diversity-oriented synthesis-facilitated medicinal chemistry: toward the development of novel antimalarial agents. J Med Chem 57:8496-502|
|Lukens, Amanda K; Ross, Leila Saxby; Heidebrecht, Richard et al. (2014) Harnessing evolutionary fitness in Plasmodium falciparum for drug discovery and suppressing resistance. Proc Natl Acad Sci U S A 111:799-804|