Chemogenomic Interrogation of the Adaptive Proline Response to Halofuginone in Plasmodium falciparum

Abstract

With 220 million infections and 435,000 deaths in 2017, malaria is an infectious disease of significant global impact. This devastating disease is caused by eukaryotic parasites of the genus Plasmodium and transmitted to humans by the bite of infected Anopheles mosquitoes. Over the last decade, great progress has been made in decreasing the prevalence of malaria in endemic countries worldwide. However, recent indicators imply that this progress has slowed or even stalled. This is likely attributable to a number of factors, but of particular concern are increasing incidence of reduced clinical efficacy of the frontline antimalarial drugs. Malaria parasites have been able to evolve resistance to every frontline therapy deployed, including the latest artemisinin combination therapies (ACTs). This dire reality highlights the urgent need for new drugs that exploit new parasite targets. Aminoacyl tRNA synthetases (aaRSs) are attractive targets for next generation chemotherapeutic development in malaria. With numerous replicative cycles and massive population expansion in the human host, Plasmodium parasites depend on efficient protein translation to establish an infection. In their canonical role of charging tRNAs for use in the translational machinery, aaRSs play an essential cellular function and as such can be exploited for therapeutic intervention. Disruption of translation, and aaRSs specifically, are validated antibacterial drug targets and studies in Plasmodium show promise to similarly advance this class of enzymes for antimalarial drug development. Halofuginone (HFG) is a derivative of the active component of the Chinese medicinal plant Dichroa febrifuga and a potent inhibitor of the Plasmodium falciparum cytoplasmic prolyl tRNA synthetase (PfcPRS). Structural studies show that HFG binds PfcPRS and occupies the proline binding site which correlates with our observation that HFG activity can be affected in a competitive manner by the addition of excess proline. Treatment of parasites with HFG induces phosphorylation of eIF2α, which is generally considered as a sensitive biomarker for the activation of the integrated stress response (ISR), a cellular stress response mechanism in the context of amino acid starvation. In vitro resistance evolution studies revealed that parasites treated with HFG exhibit a novel mechanism of drug tolerance wherein intracellular proline levels are increased by 20- to 30-fold with a corresponding reduction in HFG efficacy. This metabolic adaptation, termed the Adaptive Proline Response (APR), persists after drug withdrawal and renders the parasite tolerant to HFG treatment. I explored the molecular basis of the APR by probing the role of eIF2α signaling in its activation and by identifying the source of the increased proline. Using parasites lacking eIK1, the Plasmodium eIF2α kinase that responds to amino acid limitation, I investigated a functional link between the ISR pathway and activation of the APR. We observed that HFG-treated eIK1 knockout parasites acquired phenotypic resistance to the drug with a concomitant increase in intraparasitic proline levels, suggesting that the modulation of proline homeostasis in response to drug treatment is independent of the AAR and eIF2α signaling. We further sought to identify the source of the increased intracellular proline in the HFG-tolerant parasite using a multiplexed high-resolution mass-spectrometry based assay. Metabolomic analysis of HFG-treated parasites implicated arginine metabolism in the activation of the APR. We used CRISPR/Cas9 technology to disrupt arginine biosynthesis of proline by inactivating the ornithine δ-aminotransferase (PfOAT) gene. These PfOAT knockout parasites were unable to activate the APR following HFG-treatment, demonstrating that arginine metabolism is required for the APR.