e High-throughput phenotypic screening of chemical libraries has resulted in the identification of thousands of compounds with potent antimalarial activity, although in most cases, the mechanism(s) of action of these compounds remains unknown. Here we have investigated the mode of action of 90 antimalarial compounds derived from the Malaria Box collection using high-coverage, untargeted metabolomics analysis. Approximately half of the tested compounds induced significant metabolic perturbations in in vitro cultures of Plasmodium falciparum. In most cases, the metabolic profiles were highly correlated with known antimalarials, in particular artemisinin, the 4-aminoquinolines, or atovaquone. Select Malaria Box compounds also induced changes in intermediates in essential metabolic pathways, such as isoprenoid biosynthesis (i.e., 2-C-methyl-D-erythritol 2,4-cyclodiphosphate) and linolenic acid metabolism (i.e., traumatic acid). This study provides a comprehensive database of the metabolic perturbations induced by chemically diverse inhibitors and highlights the utility of metabolomics for triaging new lead compounds and defining specific modes of action, which will assist with the development and optimization of new antimalarial drugs. Malaria remains a major global health problem, and there is a pressing need to discover and develop new antimalarials. Traditional antimalarial drugs have been severely undermined by the emergence of drug-resistant strains and current treatments rely heavily on artemisinin-based combination therapies. Alarmingly, artemisinin resistance has arisen in Southeast Asia during the last decade, highlighting the urgent need for new antimalarials (1). Extensive efforts to produce a malaria vaccine have met limited success and a pipeline of new antimalarial drugs will be required to mitigate extensive morbidity and mortality from malaria for the foreseeable future (2).High-throughput phenotypic screening of chemical libraries against the malaria parasite, Plasmodium falciparum, has provided a major step forward in the discovery of novel antimalarial compounds. Almost 30,000 compounds that selectively inhibit growth of cultured P. falciparum asexual red blood cell (RBC) stages have been identified in these screens, providing excellent starting points for the discovery of new antimalarial drugs (3-5). A prioritized collection of these "hit" compounds, known as the Malaria Box, has been assembled by the Medicines for Malaria Venture (MMV) and provided free to the research community to facilitate this drug development pipeline (6).A key limitation to the further optimization of many of these compounds is the lack of information on their mode of action. While not essential for registration, information on the mode of action of inhibitors discovered in phenotypic screens will significantly accelerate drug development by allowing structure-based drug design, monitoring of activity, toxicity and resistance, and facilitation of rational clinical usage in combination with other medicines. Furthermore, in...

Plasmodium falciparum, which causes malaria, extensively remodels its human host cells, particularly erythrocytes. Remodelling is essential for parasite survival by helping to avoid host immunity and assisting in the uptake of plasma nutrients to fuel rapid growth. Host cell renovation is carried out by hundreds of parasite effector proteins that are exported into the erythrocyte across an enveloping parasitophorous vacuole membrane (PVM). The Plasmodium translocon for exported (PTEX) proteins is thought to span the PVM and provide a channel that unfolds and extrudes proteins across the PVM into the erythrocyte. We show that exported reporter proteins containing mouse dihydrofolate reductase domains that inducibly resist unfolding become trapped at the parasite surface partly colocalizing with PTEX. When cargo is trapped, loop-like extensions appear at the PVM containing both trapped cargo and PTEX protein EXP2, but not additional components HSP101 and PTEX150. Following removal of the block-inducing compound, export of reporter proteins only partly recovers possibly because much of the trapped cargo is spatially segregated in the loop regions away from PTEX. This suggests that parasites have the means to isolate unfoldable cargo proteins from PTEX-containing export zones to avert disruption of protein export that would reduce parasite growth.

Plasmodium falciparum exports hundreds of virulence proteins within infected erythrocytes, a process that requires cleavage of a pentameric motif called Plasmodium export element or vacuolar transport signal by the endoplasmic reticulum (ER)-resident protease plasmepsin V. We identified plasmepsin V-binding proteins that form a unique interactome required for the translocation of effector cargo into the parasite ER. These interactions are functionally distinct from the Sec61-signal peptidase complex required for the translocation of proteins destined for the classical secretory pathway. This interactome does not involve the signal peptidase (SPC21) and consists of PfSec61, PfSPC25, plasmepsin V and PfSec62, which is an essential component of the post-translational ER translocon. Together, they form a distinct portal for the recognition and translocation of a large subset of Plasmodium export element effector proteins into the ER, thereby remodelling the infected erythrocyte that is required for parasite survival and pathogenesis.

RNase L and RNA-dependent protein kinase (PKR) are effectors of the interferon antiviral response that share homology in their pseudokinase and protein kinase domains, respectively. Sunitinib is an orally available, ATP-competitive inhibitor of VEGF and PDGF receptors used clinically to suppress angiogenesis and tumor growth. Sunitinib also impacts IRE1, an endoplasmic reticulum protein involved in the unfolded protein response that is closely related to RNase L. Here, we report that sunitinib is a potent inhibitor of both RNase L and PKR with IC 50 values of 1.4 and 0.3 M, respectively. In addition, flavonol activators of IRE1 inhibited RNase L. Sunitinib treatment of wild type (WT) mouse embryonic fibroblasts resulted in about a 12-fold increase in encephalomyocarditis virus titers. However, sunitinib had no effect on encephalomyocarditis virus growth in cells lacking both PKR and RNase L. Furthermore, oral delivery of sunitinib in WT mice resulted in 10-fold higher viral titers in heart tissues while suppressing by about 2-fold the IFN-␤ levels. In contrast, sunitinib had no effect on viral titers in mice deficient in both RNase L and PKR. Also, sunitinib reduced mean survival times from 12 to 6 days in virus-infected WT mice while having no effect on survival of mice lacking both RNase L and PKR. Results indicate that sunitinib treatments prevent antiviral innate immune responses mediated by RNase L and PKR.RNase L and PKR 2 are host enzymes of higher vertebrates that participate in innate immunity against viral infections (1-4). Activation of both RNase L and PKR is triggered by the viral pathogen-associated molecular pattern, double-stranded RNA (dsRNA). However, whereas dsRNA directly binds to and activates PKR, in the case of RNase L activation is indirect. Interferon (IFN) treatment of cells induces PKR that, upon binding to dsRNA, phosphorylates first itself and then EIF2␣ thus blocking protein synthesis among other effects. RNase L degrades single-stranded RNA resulting in pleiotropic antiviral effects (5). Short 5Ј-triphosphorylated, 2Ј,5Ј-oligoadenylates (2-5A) are produced from ATP when viral dsRNA stimulates IFN-inducible oligoadenylate synthetases. 2-5A binds ankyrin repeats 2 and 4 in the N-terminal region of RNase L causing its dimerization and activation (6). RNase L is also pseudokinase with amino acid sequence homology to the PKR kinase domains (7).IRE1, a kinase and endoribonuclease involved in the unfolded protein response, is another relative of RNase L (8). IRE1 spans the endoplasmic reticulum (ER) membrane. The intralumenal domains of IRE1 directly or indirectly sense unfolded proteins in the ER leading to autophosphorylation and ribonuclease activities in the cytoplasmic domains. IRE1 excises an intron from pre-mRNA for a transcription factor (HAC1 in yeast and XBP1 in mammals) leading to splicing and translation (9). HAC1/XBP1 drives expression of ER chaperones and protein folding enzymes that re-establish ER function. The kinase-extension-nuclease (KEN) domains of RNase L and IRE1 ar...

Plasmodium parasites are responsible for the devastating disease malaria that affects hundreds of millions of people each year. Blood stage parasites establish new permeability pathways (NPPs) in infected red blood cell membranes to facilitate the uptake of nutrients and removal of parasite waste products. Pharmacological inhibition of the NPPs is expected to lead to nutrient starvation and accumulation of toxic metabolites resulting in parasite death. Here, we have screened a curated library of antimalarial compounds, the MMV Malaria Box, identifying two compounds that inhibit NPP function. Unexpectedly, metabolic profiling suggested that both compounds also inhibit dihydroorotate dehydrogense (DHODH), which is required for pyrimidine synthesis and is a validated drug target in its own right. Expression of yeast DHODH, which bypasses the need for the parasite DHODH, increased parasite resistance to these compounds. These studies identify two potential candidates for therapeutic development that simultaneously target two essential pathways in Plasmodium, NPP and DHODH.