Exoerythrocytic Forms Research Articles

Exploration and characterization of the antimalarial activity of cyclopropyl carboxamides that target the mitochondrial protein, cytochrome b

Drug resistance against antimalarials is rendering them increasingly ineffective and so there is a need for the development of new antimalarials. To discover new antimalarial chemotypes a phenotypic screen of the Janssen Jumpstarter library against the P. falciparum asexual stage was undertaken, uncovering the cyclopropyl carboxamide structural hit class. Structure-activity analysis revealed that each structural moiety was largely resistant to change, although small changes led to the frontrunner compound, WJM280, which has potent asexual stage activity (EC50 40 nM) and no human cell cytotoxicity. Forward genetics uncovered that cyclopropyl carboxamide resistant parasites have mutations and an amplification in the cytochrome b gene. Cytochrome b was then verified as the target with profiling against cytochrome b drug-resistant parasites and a mitochondrial oxygen consumption assay. Accordingly, the cyclopropyl carboxamide class was shown to have slow-acting asexual stage activity and activity against male gametes and exoerythrocytic forms. Enhancing metabolic stability to attain efficacy in malaria mouse models remains a challenge in the future development of this antimalarial chemotype.

A 3D organoid platform that supports liver-stage P.falciparum infection can be used to identify intrahepatic antimalarial drugs

Malaria, a major public health burden, is caused by Plasmodium spp parasites that first replicate in the human liver to establish infection before spreading to erythrocytes. Liver-stage malaria research has remained challenging due to the lack of a clinically relevant and scalable in vitro model of the human liver. Here, we demonstrate that organoids derived from intrahepatic ductal cells differentiated into a hepatocyte-like fate can support the infection and intrahepatic maturation of Plasmodium falciparum. The P.falciparum exoerythrocytic forms observed expressed both early and late-stage parasitic proteins and decreased in frequency in response to treatment with both known and putative antimalarial drugs that target intrahepatic P.falciparum. The P.falciparum-infected human liver organoids thus provide a platform not only for fundamental studies that characterise intrahepatic parasite-host interaction but can also serve as a powerful translational tool in pre-erythrocytic vaccine development and to identify new antimalarial drugs that target the liver stage infection.

Malaria sporozoites evade host complement attack.

Complement is the first line of the host innate immune response against bacterial and viral infections; however, its role in the development of the malaria liver stage remains undefined. We found that sporozoite infection by either a mosquito bite or intravenous injection activated systemic complement, but neither depletion of C3 nor knockout of C3 had a significant effect on malaria liver stage development. Incubation of mouse serum with trypsin-treated sporozoites, but not naive sporozoites, led to the deposition of a membrane attack complex (MAC) on the surface of sporozoites and greatly reduced the number of exo-erythrocytic forms (EEF). Further studies have shown that the recruitment of complement H factor (CFH) may be associated with the prevention of MAC deposition on the surface of naïve sporozoites. Our data strongly suggest that sporozoites can escape complement attacks and provide us with a novel strategy to prevent malaria infection.

Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity.

Plasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites. Considering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development. Though IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.

Disrupting a Plasmodium berghei putative phospholipase impairs efficient egress of merosomes

Merosome development is a very important part of the Plasmodium life cycle. It constitutes the last step of liver stage development after which merozoites egress to the bloodstream and invade red blood cells. Many parasite proteins have been shown to play key roles in this process. Phospholipases are known to be actively involved in membrane formation and remodeling. At least one phospholipase A2, localized to the parasitophorous vacuolar membrane, is known to be directly important for parasitophorous vacuolar membrane (PVM) rupture and subsequent release of merozoites. Here, we characterize a Plasmodium berghei phosphatidic acid (PA) preferring phospholipase A1 (PbPla1) homolog. C-terminal 3XHA-mCherry tagging revealed that PbPla1 is expressed in all life cycle stages except sporozoites and is present in the parasite’s cytoplasm. Targeted disruption of PbPla1 revealed its non-essentiality for the blood and mosquito stages. Pla1− sporozoites were found to be late in their ability to establish successful blood stage infection in mice. While exoerythrocytic form development was found to be normal in vitro, a decrease in the number of merosomes was observed. PVM rupture in late exo-erythrocytic forms (EEFs) was quantified by counting the number of parasites with intact PVMs, and was found to be significantly higher in Pla1−. Our findings indicate the role of PbPla1 in merosome release.

ATG Ubiquitination Is Required for Circumsporozoite Protein to Subvert Host Innate Immunity Against Rodent Malaria Liver Stage.

Although exo-erythrocytic forms (EEFs) of liver stage malaria parasite in the parasitophorous vacuole (PV) are encountered with robust host innate immunity, EEFs can still survive and successfully complete the infection of hepatocytes, and the underlying mechanism is largely unknown. Here, we showed that sporozoite circumsporozoite protein (CSP) translocated from the parasitophorous vacuole into the hepatocyte cytoplasm significantly mediated the resistance to the killing of EEFs by interferon-gamma (IFN-γ). Attenuation of IFN-γ-mediated killing of EEFs by CSP was dependent on its ability to reduce the levels of autophagy-related genes (ATGs) in hepatocytes. The ATGs downregulation occurred through its enhanced ubiquitination mediated by E3 ligase NEDD4, an enzyme that was upregulated by CSP when it translocated from the cytoplasm into the nucleus of hepatocytes via its nuclear localization signal (NLS) domain. Thus, we have revealed an unrecognized role of CSP in subverting host innate immunity and shed new light for a prophylaxis strategy against liver-stage infection.

Histone deacetylase inhibitor AR-42 and achiral analogues kill malaria parasites in vitro and in mice

Malaria is caused by infection with Plasmodium parasites and results in significant health and economic impacts. Malaria eradication is hampered by parasite resistance to current drugs and the lack of a widely effective vaccine. Compounds that target epigenetic regulatory proteins, such as histone deacetylases (HDACs), may lead to new therapeutic agents with a different mechanism of action, thereby avoiding resistance mechanisms to current antimalarial drugs. The anticancer HDAC inhibitor AR-42, as its racemate (rac-AR-42), and 36 analogues were investigated for in vitro activity against P. falciparum. Rac-AR-42 and selected compounds were assessed for cytotoxicity against human cells, histone hyperacetylation, human HDAC1 inhibition and oral activity in a murine malaria model. Rac-AR-42 was tested for ex vivo asexual and in vitro exoerythrocytic stage activity against P. berghei murine malaria parasites. Rac-AR-42 and 13 achiral analogues were potent inhibitors of asexual intraerythrocytic stage P. falciparum 3D7 growth in vitro (IC50 5–50 nM), with four of these compounds having >50-fold selectivity for P. falciparum versus human cells (selectivity index 56–118). Rac-AR-42 induced in situ hyperacetylation of P. falciparum histone H4, consistent with PfHDAC(s) inhibition. Furthermore, rac-AR-42 potently inhibited P. berghei infected erythrocyte growth ex vivo (IC50 40 nM) and P. berghei exoerythrocytic forms in hepatocytes (IC50 1 nM). Oral administration of rac-AR-42 and two achiral analogues inhibited P. berghei growth in mice, with rac-AR-42 (50 mg/kg/day single dose for four days) curing all infections. These findings demonstrate curative properties for HDAC inhibitors in the oral treatment of experimental mouse malaria.

Preparing for Transmission: Gene Regulation in Plasmodium Sporozoites

Plasmodium sporozoites are transmitted to mammals by anopheline mosquitoes and first infect the liver, where they transform into replicative exoerythrocytic forms, which subsequently release thousands of merozoites that invade erythrocytes and initiate the malaria disease. In some species, sporozoites can transform into dormant hypnozoites in the liver, which cause malaria relapses upon reactivation. Transmission from the insect vector to a mammalian host is a critical step of the parasite life cycle, and requires tightly regulated gene expression. Sporozoites are formed inside oocysts in the mosquito midgut and become fully infectious after colonization of the insect salivary glands, where they remain quiescent until transmission. Parasite maturation into infectious sporozoites is associated with reprogramming of the sporozoite transcriptome and proteome, which depends on multiple layers of transcriptional and post-transcriptional regulatory mechanisms. An emerging scheme is that gene expression in Plasmodium sporozoites is controlled by alternating waves of transcription activity and translational repression, which shape the parasite RNA and protein repertoires for successful transition from the mosquito vector to the mammalian host.

Hepatic Inflammation Confers Protective Immunity Against Liver Stages of Malaria Parasite

Deciphering the mechanisms by which Plasmodium parasites develop inside hepatocytes is an important step toward the understanding of malaria pathogenesis. We propose that the nature and the magnitude of the inflammatory response in the liver are key for the establishment of the infection. Here, we used mice deficient in the multidrug resistance-2 gene (Mdr2−/−)-encoded phospholipid flippase leading to the development of liver inflammation. Infection of Mdr2−/− mice with Plasmodium berghei ANKA (PbANKA) sporozoites (SPZ) resulted in the blockade of hepatic exo-erythrocytic forms (EEFs) with no further development into blood stage parasites. Interestingly, cultured primary hepatocytes from mutant and wild-type mice are equally effective in supporting EEF development. The abortive infection resulted in a long-lasting immunity in Mdr2−/− mice against infectious SPZ where neutrophils and IL-6 appear as key effector components along with CD8+ and CD4+ effector and central memory T cells. Inflammation-induced breakdown of liver tolerance promotes anti-parasite immunity and provides new approaches for the design of effective vaccines against malaria disease.

Immune responses to malaria pre-erythrocytic stages: Implications for vaccine development.

Radiation-attenuated sporozoites induce sterilizing immunity and remain the ‘gold standard’ for malaria vaccine development. Despite practical challenges in translating these whole sporozoite vaccines to large-scale intervention programmes, they have provided an excellent platform to dissect the immune responses to malaria pre-erythrocytic (PE) stages, comprising both sporozoites and exoerythrocytic forms. Investigations in rodent models have provided insights that led to the clinical translation of various vaccine candidates—including RTS,S/AS01, the most advanced candidate currently in a trial implementation programme in three African countries. With advances in immunology, transcriptomics and proteomics, and application of lessons from past failures, an effective, long-lasting and wide-scale malaria PE vaccine remains feasible. This review underscores the progress in PE vaccine development, focusing on our understanding of host-parasite immunological crosstalk in the tissue environments of the skin and the liver. We highlight possible gaps in the current knowledge of PE immunity that can impact future malaria vaccine development efforts.

A Novel Chemically Differentiated Mouse Embryonic Stem Cell-Based Model to Study Liver Stages of Plasmodium berghei.

SummaryAsymptomatic and obligatory liver stage (LS) infection of Plasmodium parasites presents an attractive target for antimalarial vaccine and drug development. Lack of robust cellular models to study LS infection has hindered the discovery and validation of host genes essential for intrahepatic parasite development. Here, we present a chemically differentiated mouse embryonic stem cell (ESC)-based LS model, which supports complete development of Plasmodium berghei exoerythrocytic forms (EEFs) and can be used to define new host-parasite interactions. Using our model, we established that host Pnpla2, coding for adipose triglyceride lipase, is dispensable for P. berghei EEF development. In addition, we also evaluated in-vitro-differentiated human hepatocyte-like cells (iHLCs) to study LS of P. berghei and found it to be a sub-optimal infection model. Overall, our results present a new mouse ESC-based P. berghei LS infection model that can be utilized to study the impact of host genetic variation on parasite development.

A Plasmodium berghei putative serine-threonine kinase 2 (PBANKA_0311400) is required for late liver stage development and timely initiation of blood stage infection.

ABSTRACTIn Plasmodium, protein kinases govern key biological processes of the parasite life cycle involved in the establishment of infection, dissemination and sexual reproduction. The rodent malaria model Plasmodium berghei encodes for 66 putative eukaryotic protein kinases (ePKs) as identified through modelling domain signatures and are highly conserved in Plasmodium falciparum. We report here the functional characterisation of a putative serine-threonine kinase PBANKA_0311400 identified in this kinome analysis and designate it as Pbstk2. To elucidate its role, we knocked out Pbstk2 locus and performed a detailed phenotypic analysis at different life cycle stages. The Pbstk2 knockout (KO) was not compromised in asexual blood stage propagation, transmission and development in the mosquito vector. The Pbstk2 KO produced viable salivary gland sporozoites that successfully transformed into exo-erythrocytic forms (EEFs) and were morphologically indistinguishable from wild-type GFP (WT GFP) with regard to size and shape until 48 h. An intravenous dose of 1×103 Pbstk2 KO sporozoites in C57BL/6 mice failed to establish blood stage infection and a higher dose of 5X103 showed a 2–3 day delay in prepatency as compared to WT GFP parasites. Consistent with such an observation, analysis of in vitro EEF development at 62 h revealed that the hepatic merozoite numbers were reduced to nearly 40% as compared to WT GFP and showed meagre expression of MSP1. Our studies provide evidence for the role of PbSTK2 in late liver stage development and for the successful establishment of a timely blood stage infection.

Plasmodium berghei sporozoite specific genes- PbS10 and PbS23/SSP3 are required for the development of exo-erythrocytic forms

Plasmodium sporozoites are infective forms of the parasite to mammalian hepatocytes. Sporozoite surface or secreted proteins likely play an important role in recognition, invasion and successful establishment of hepatocyte infection. By approaches of reverse genetics, we report the functional analysis of two Plasmodium berghei (Pb) sporozoite specific genes- PbS10 and PbS23/SSP3 that encode for proteins with a putative signal peptide. The expression of both genes was high in oocyst and salivary gland sporozoite stages as compared to other life cycle stages and PbS23/SSP3 protein was detected in salivary gland sporozoites. Both mutants were indistinguishable to wild-type parasites with regard to asexual growth in RBC, ability to complete sexual reproduction and form sporozoites in vector host. While the sporozoite stage of both mutants were able to glide and invade hepatocytes normally in vitro and in vivo, PbS10 mutants suffered growth attenuation at an early stage while PbS23/SSP3 mutants manifested defect during late exo-erythrocytic form maturation. Interestingly, both mutants gave rare breakthrough infections, suggesting that while both were critical for liver stage development, their depletion did not completely abrogate blood stage infection. These findings have important implications for weakening sporozoites by multiple gene attenuation towards the generation of a safe whole organism vaccine.

Primaquine\u2013quinoxaline 1,4-di-N-oxide hybrids with action on the exo-erythrocytic forms of Plasmodium induce their effect by the production of reactive oxygen species

BackgroundThe challenge in anti-malarial chemotherapy is based on the emergence of resistance to drugs and the search for medicines against all stages of the life cycle of Plasmodium spp. as a therapeutic target. Nowadays, many molecules with anti-malarial activity are reported. However, few studies about the cellular and molecular mechanisms to understand their mode of action have been explored. Recently, new primaquine-based hybrids as new molecules with potential multi-acting anti-malarial activity were reported and two hybrids of primaquine linked to quinoxaline 1,4-di-N-oxide (PQ–QdNO) were identified as the most active against erythrocytic, exoerythrocytic and sporogonic stages.MethodsTo further understand the anti-malarial mode of action (MA) of these hybrids, hepg2-CD81 were infected with Plasmodium yoelii 17XNL and treated with PQ–QdNO hybrids during 48 h. After were evaluated the production of ROS, the mitochondrial depolarization, the total glutathione content, the DNA damage and proteins related to oxidative stress and death cell.ResultsIn a preliminary analysis as tissue schizonticidals, these hybrids showed a mode of action dependent on peroxides production, but independent of the activation of transcription factor p53, mitochondrial depolarization and arrest cell cycle.ConclusionsPrimaquine–quinoxaline 1,4-di-N-oxide hybrids exert their antiplasmodial activity in the exoerythrocytic phase by generating high levels of oxidative stress which promotes the increase of total glutathione levels, through oxidation stress sensor protein DJ-1. In addition, the role of HIF1a in the mode of action of quinoxaline 1,4-di-N-oxide is independent of biological activity.

CXCR4 regulates Plasmodium development in mouse and human hepatocytes.

The liver stage of the etiological agent of malaria, Plasmodium, is obligatory for successful infection of its various mammalian hosts. Differentiation of the rod-shaped sporozoites of Plasmodium into spherical exoerythrocytic forms (EEFs) via bulbous expansion is essential for parasite development in the liver. However, little is known about the host factors regulating the morphological transformation of Plasmodium sporozoites in this organ. Here, we show that sporozoite differentiation into EEFs in the liver involves protein kinase C ζ-mediated NF-κB activation, which robustly induces the expression of C-X-C chemokine receptor type 4 (CXCR4) in hepatocytes and subsequently elevates intracellular Ca2+ levels, thereby triggering sporozoite transformation into EEFs. Blocking CXCR4 expression by genetic or pharmacological intervention profoundly inhibited the liver-stage development of the Plasmodium berghei rodent malaria parasite and the human Plasmodium falciparum parasite. Collectively, our experiments show that CXCR4 is a key host factor for Plasmodium development in the liver, and CXCR4 warrants further investigation for malaria prophylaxis.

Structure-Activity and Structure-Toxicity Relationships of Peptoid-Based Histone Deacetylase Inhibitors with Dual-Stage Antiplasmodial Activity.

Novel malaria intervention strategies are of great importance, given the development of drug resistance in malaria-endemic countries. In this regard, histone deacetylases (HDACs) have emerged as new and promising malaria drug targets. In this work, we present the design, synthesis, and biological evaluation of 20 novel HDAC inhibitors with antiplasmodial activity. Based on a previously discovered peptoid-based hit compound, we modified all regions of the peptoid scaffold by using a one-pot multicomponent pathway and submonomer routes to gain a deeper understanding of the structure-activity and structure-toxicity relationships. Most compounds displayed potent activity against asexual blood-stage P. falciparum parasites, with IC50 values in the range of 0.0052-0.25 μm and promising selectivity over mammalian cells (SIPf3D7/HepG2 : 170-1483). In addition, several compounds showed encouraging sub-micromolar activity against P. berghei exo-erythrocytic forms (PbEEF). Our study led to the discovery of the hit compound N-(2-(benzylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-4-isopropylbenzamide (2 h) as a potent and parasite-specific dual-stage antiplasmodial HDAC inhibitor (IC50 Pf3D7=0.0052 μm, IC50 PbEEF=0.016 μm).

One-pot, multi-component synthesis and structure-activity relationships of peptoid-based histone deacetylase (HDAC) inhibitors targeting malaria parasites

Malaria drug discovery has shifted from a focus on targeting asexual blood stage parasites, to the development of drugs that can also target exo-erythrocytic forms and/or gametocytes in order to prevent malaria and/or parasite transmission. In this work, we aimed to develop parasite-selective histone deacetylase inhibitors (HDACi) with activity against the disease-causing asexual blood stages of Plasmodium malaria parasites as well as with causal prophylactic and/or transmission blocking properties. An optimized one-pot, multi-component protocol via a sequential Ugi four-component reaction and hydroxylaminolysis was used for the preparation of a panel of peptoid-based HDACi. Several compounds displayed potent activity against drug-sensitive and drug-resistant P. falciparum asexual blood stages, high parasite-selectivity and submicromolar activity against exo-erythrocytic forms of P. berghei. Our optimization study resulted in the discovery of the hit compound 1u which combines high activity against asexual blood stage parasites (Pf 3D7 IC50: 4 nM; Pf Dd2 IC50: 1 nM) and P. berghei exo-erythrocytic forms (Pb EEF IC50: 25 nM) with promising parasite-specific activity (SIPf3D7/HepG2: 2496, SIPfDd2/HepG2: 9990, and SIPbEEF/HepG2: 400).

Developing Plasmodium vivax Resources for Liver Stage Study in the Peruvian Amazon Region.

To develop new drugs and vaccines for malaria elimination, it will be necessary to discover biological interventions, including small molecules that act against Plasmodium vivax exoerythrocytic forms. However, a robust in vitro culture system for P.vivax is still lacking. Thus, to study exoerythrocytic forms, researchers must have simultaneous access to fresh, temperature-controlled patient blood samples, as well as an anopheline mosquito colony. In addition, researchers must rely on native mosquito species to avoid introducing a potentially dangerous invasive species into a malaria-endemic region. Here, we report an in vitro culture system carried out on site in a malaria-endemic region for liver stage parasites of P.vivax sporozoites obtained from An.darlingi, the main malaria vector in the Americas. P.vivax sporozoites were obtained by dissection of salivary glands from infected An.darlingi mosquitoes and purified by Accudenz density gradient centrifugation. HC04 liver cells were exposed to P.vivax sporozoites and cultured up to 9 days. To overcome low P.vivax patient parasitemias, potentially lower mosquito vectorial capacity, and humid, nonsterile environmental conditions, a new antibiotic cocktail was included in tissue culture to prevent contamination. Culturing conditions supported exoerythrocytic (EEF) P.vivax liver stage growth up to 9 days and allowed for maturation into intrahepatocyte merosomes. Some of the identified small forms were resistant to atovaquone (1 μM) but sensitive to the phosphatidylinositol 4-kinase inhibitor, KDU691 (1 μM). This study reports a field-accessible EEF production process for drug discovery in a malaria-endemic site in which viable P.vivax sporozoites are used for drug studies using hepatocyte infection. Our data demonstrate that the development of meaningful, field-based resources for P.vivax liver stage drug screening and liver stage human malaria experimentation in the Amazon region is feasible.

Protein O-fucosylation in Plasmodium falciparum ensures efficient infection of mosquito and vertebrate hosts

O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.

Design and Synthesis of Terephthalic Acid-Based Histone Deacetylase Inhibitors with Dual-Stage Anti-Plasmodium Activity.

In this work we aimed to develop parasite-selective histone deacetylase inhibitors (HDAC) inhibitors with activity against the disease-causing asexual blood stages of Plasmodium as well as causal prophylactic and/or transmission blocking properties. We report the design, synthesis, and biological testing of a series of 13 terephthalic acid-based HDAC inhibitors. All compounds showed low cytotoxicity against human embryonic kidney (HEK293) cells (IC50 : 8->51 μm), with 11 also having sub-micromolar in vitro activity against drug-sensitive (3D7) and multidrug-resistant (Dd2) asexual blood-stage P. falciparum parasites (IC50 ≈0.1-0.5 μm). A subset of compounds were examined for activity against early- and late-stage P. falciparum gametocytes and P. berghei exo-erythrocytic-stage parasites. While only moderate activity was observed against gametocytes (IC50 >2 μm), the most active compound (N1 -((3,5-dimethylbenzyl)oxy)-N4 -hydroxyterephthalamide, 1 f) showed sub-micromolar activity against P. berghei exo-erythrocytic stages (IC50 0.18 μm) and >270-fold better activity for exo-erythrocytic forms than for HepG2 cells. This, together with asexual-stage in vitro potency (IC50 ≈0.1 μm) and selectivity of this compound versus human cells (SI>450), suggests that 1 f may be a valuable starting point for the development of novel antimalarial drug leads with low host cell toxicity and multi-stage anti-plasmodial activity.