Targeting Mitochondrial Metabolism as a Key Vulnerability in Artemisinin-Resistant Plasmodium falciparum Malaria

Malaria is one of the highest infectious disease priorities for the U.S. military and the Military Infectious Disease Research Program (MIDRP). Infection by the malaria pathogen Plasmodium falciparum (Pf) begins with the bite of an infected mosquito. Injected parasites migrate to the liver where they develop inside hepatocytes over the course of 1 week. Subsequently, liver stage parasites emerge into the bloodstream and infect red blood cells, with new cycles of infection and intracellular development every 48 hours. These blood-stage parasites cause symptoms that can be lethal, especially in non-immune individuals who are not treated with effective drugs. The majority of malaria deaths occur in Africa as a result of Pf infection. In the early 2000s, artemisinin-based combination therapies (ACTs) were adopted worldwide as first-line therapies to treat Pf malaria. The positive impact was vast, with malaria deaths decreasing annually from one million to ~440,000 at present. Disease rates fell more slowly, from an estimated 260 million cases per year to ~210 million.

The past 3 years, however, have seen malaria efforts stall, as mortality and morbidity rates plateau. Compounding this situation is the rise in artemisinin (ART)-resistant Pf strains in Southeast Asia, which now extend throughout the Greater Mekong Subregion (covering Cambodia, Thailand, Vietnam, Laos, and Myanmar). Clinically, this translates to ART drugs, which are typically very fast-acting but have a short half-life in the body, no longer being able to rapidly clear infections. This results in partner drugs facing a greater parasite biomass, from which resistance can emerge. Indeed, the partner drug piperaquine has failed rapidly since the emergence of ART resistance, and therapeutic efficacy rates of the dihydroartemisinin+piperaquine combination are now below 50% in Cambodia. Historically, drug resistance that arose in Asia has migrated to and across Africa, where malaria exerts its largest toll. One can predict that ART and accompanying ACT resistance could therefore spread to Africa, thus decimating our ability to treat malaria.

Ensuring the future protection of U.S. military personnel from malaria requires the development of new therapeutic strategies that are predicated on understanding existing mechanisms of antimalarial drug resistance and identifying chemical agents that can effectively eliminate these drug-resistant infections. In recent years, ART resistance has been shown to result from mutations in the Pf gene K13, which allow circulating young 'ring-stage' parasites to survive ART action during those few hours when the drug is effective. Biological studies suggest that parasites achieve resistance by being able to temporarily enter a dormant or 'quiescent' state. Competing theories have suggested that changes in protein degradation or lipid transport might produce resistance. However, these have not been independently confirmed. Using a panel of parasite lines modified to differ only in their K13 sequence, we have undertaken a comprehensive profiling of their biological features, including analysis of their protein repertoires, metabolites, RNA expression profiles, and K13-colocalizing proteins. Our findings provide a transformative breakthrough in discovering that these K13 mutations alter multiple features of parasite mitochondria, which is the cellular engine that drives energy production, redox regulation, and synthesis of DNA precursors and heme. These data generate exciting hypotheses about how mutant K13 poises parasites to readily enter a quiescent state upon exposure to ART drugs and thus achieve resistance. We can leverage this information to rationally discover new therapeutic strategies. This work directly supports the mission of the Department of Defense to protect its personnel from drug-resistant malaria.

Our first aim is to identify which pathways in the Pf mitochondria are being modified in a way that achieves ART resistance. Using K13 mutant and 'wild-type' parasites, we will implement biochemical assays to determine whether energy production, redox regulation, and the respiratory process are essential to resistance. We will also test whether parasites reduce their synthesis of heme as a way to limit ART activation in the cell. These studies will extend to exploring whether the salvage or synthesis of DNA precursors might also provide a path to resistance. In our second aim, we will leverage upon our current data and new findings from Aim 1 to identify key vulnerabilities in the ART resistance mechanism, which we will exploit in our screen for chemical inhibitors that can eliminate ART-resistant parasites. This work will use existing antimalarials as well as compounds that could be leads for future antimalarial drug development campaigns. We believe that this Discovery Award has the potential to transform our understanding of ART resistance and provide innovative approaches to effectively and safely prevent and treat malaria in U.S. Military personnel.