The Activities of Current Antimalarial Drugs on the Life Cycle Stages of Plasmodium: A Comparative Study with Human and Rodent Parasites

Michael Delves and colleagues compare the activity of 50 current and experimental antimalarials against liver, sexual blood, and mosquito stages of selected human and nonhuman parasite species, including Plasmodium falciparum, Plasmodium berghei, and Plasmodium yoelii.


Introduction
Malaria remains one of the most widespread infectious diseases of our time. The latest estimates reveal that ,250 million people are infected with malaria across the globe, of whom ,800,000 die every year [1], the vast majority being young children. In 2007, the malaria eradication agenda was adopted by many researchers in the antimalarial community and target product profiles for new antimalarial medicines were defined [2]. Most available antimalarials were designed to target the pathogenic blood stages in humans and to address the constant threat of drug resistance [3]. However, to meet the objective of malaria eradication, medicines that block parasite transmission [4] and eliminate the asymptomatic and sometimes cryptic hepatic forms also need to be developed. The bottleneck populations of liver and sexual stage parasites [5] represent potential pathogen vulnerabilities that could be targeted by small molecules; the first such bottleneck is at the liver stage. Within minutes of being released by the bite of an infected female Anopheles mosquito, Plasmodium sporozoites reach the mammalian liver, where they invade hepatocytes and either lie dormant or develop over several days, eventually forming the schizonts that are the prelude to a blood stage infection [6]. Molecules that efficiently target the parasite stages in the liver would offer protection from infection and could theoretically eliminate the cryptic hypnozoite (dormant parasite) infection reservoirs formed by P. vivax and P. ovale. Because only 100 or so sporozoites may be introduced by a bite, there are likely to be many orders of magnitude fewer parasites at this stage than in an active blood stage infection, reducing the possibility of resistance arising. A second bottleneck occurs during sexual development. At each round of schizogony ,1% of merozoites differentiate into gametocytes [7], and it is these developmentally arrested cells that are transmitted to the mosquito. Mature gametocytes are sexually dimorphic, forming microgametocytes and macrogametocytes that escape the red blood cell (RBC) and produce male and female gametes in the blood meal of the mosquito by processes known as exflagellation [8] and activation, respectively. Following fertilization the zygote differentiates into a motile and invasive ookinete within which the briefly diploid genome undergoes meiosis. These processes occur within an environment almost totally derived from host blood, which can therefore provide a novel and ideal conduit for the delivery of drugs to inhibit parasite transmission to the mosquito. Having crossed the mosquito midgut wall [9], the very few surviving ookinetes differentiate into oocysts, which undergo endomitosis, eventually producing thousands of daughter sporozoites. The sporozoites migrate from the midgut of the mosquito to its salivary glands where the lifecycle begins again.
Given that it would be highly desirable for candidate drugs to have activity against hepatic and sexual forms of the malarial parasite, it is surprising that few clinical trials, to date, have examined whether gametocyte carriage can be reduced following drug treatment. The only drugs found to be effective at reducing gametocyte carriage include artemisinin [10], artemisinin combination therapies (ACTs) [11,12], methylene blue [13], and primaquine [12,14]. Additionally, few studies have investigated the impact of drugs on the transmission of parasites from human blood to the mosquito vector [15][16][17][18], nor have many been designed to evaluate antihepatic stage activity. In the context of malaria eradication these gaps in our understanding of the full potential of the drug armoury are problematic.
Here we report the development of a series of novel assays against liver, sexual blood, and mosquito stages of the malaria parasite, using both drug-susceptible and drug-resistant parasite strains. We applied these assays to the current portfolio of schizonticidal compounds, consisting of 50 anti-infectives currently in use or under development..

Ethics Statement
All work involving laboratory animals for the host-to-mosquito transmission studies was performed in accordance with the European Union (EU) regulations ''EU Directive 86/609/EEC'' and within the regulations of the United Kingdom Animals (Scientific Procedures) Act 1986, sanctioned by UK Home Office Licence PLL70/6347 awarded in January 2008. Protocol design and implementation was guided by the principle of the three Rs (reduction, refinement, and replacement) and are of mild-tomoderate severity. Protocols are regularly reviewed and revised following approval by the Imperial College Ethics Review Committee. and incubated at 37uC for 24 h. The parasite pellets were then resuspended in fresh RBCs and human serum treated with the compounds to a 50% haematocrit and immediately fed to mosquitoes as described above and then maintained at 27uC/ 60%RH for 10-12 d before dissection and counting.
Liver Stage Assay 7.5610 3 HepG2 cells in 50 ml of medium (1.5610 5 cells/ml) were seeded in 384 well plates (Aurora 384 IQ-EB Black/Clear Plates) 20-26 h prior to the actual infection [31]. 2 h prior to infection, 50 nl of compound in DMSO (0.1% final DMSO concentration per well) were transferred with a PinTool (GNF Systems) into the assay plates (10 mM final concentration). P. yoelii sporozoites were freshly dissected from infected A. stephensi mosquito salivary glands and filtered twice with a 40-mm strainer. The HepG2 cells were then infected with 8610 3 sporozoites per well. After infection and 1-h incubation at 37uC, the cultures were washed, new media and compound added, and further incubated with 5-fold increased concentration of penicillin/streptomycin for 48 h at 37uC before exoerythrocytic forms (EEFs) quantification of infected cells by immunofluorescence.
EEF immunofluorescence quantification. After washing with 16PBS and fixation of the cells with 4% paraformaldehyde solution (EMS), membranes were permeabilised with 0.5% Triton-X-100 (Thermo Fisher Scientific) and EEFs were stained using a mouse polyclonal serum raised against the Plasmodium heat shock protein 70 (HSP70), an Alexa goat antimouse IgG, Fca-specific DyLight 649 secondary antibody (Invitrogen), and the Hoechst 33342 nucleic acid dye (Invitrogen). Stained EEFs were then quantified using the Opera Confocal High Content Screening system (PerkinElmer). Images were collected using a 206 magnification at a binning of 2 using a 365 Xeon arc lamp illumination to detect the nuclei and 635-nm laser line to excite DyLight649.
High-content imaging of infected HepG2-CD81 cells was performed as described in Meister et al. [31]. Wells were analysed using a custom Acapella (PerkinElmer) script parameterized for this assay. In brief, images from fields inside the well were first discarded as out of focus when the intensity in the nuclear area was too low. Then hepatic cells were counted by detecting the nuclei labelled with Hoechst 33342 and parasites were segmented using the HSP70 immuno-labelling. Morphology-based (e.g., area, roundness) and intensity-based features were calculated for each object detected including the hepatocyte nuclei and the parasites. Parasitemia was set as the ratio between parasite number (Alexa fluor positive) and the hepatocyte nuclei count, determined at the same time.

Schizonticidal Activities of Compounds against Strains of P. falciparum with Known Drug Resistance Markers
A collection containing all antimalarials approved for use in humans and those in clinical development, anti-infectives, and other controls (Figure 1) was profiled simultaneously on asexual blood stage parasites in a standardized growth inhibition assay (GIA) [25] using seven strains of P. falciparum exhibiting diversity in the molecular causes of resistance and geographical origins (Table   S1). The half-maximal inhibitory concentrations (IC 50 ) were determined for each molecule (Table S2). Known mutations in pfcrt, pfmdr1, pfdhfr, and pfdhps correlated with a loss of potency of at least 10-fold for the relevant drugs. As expected the 4aminoquinolines (4-AQs) chloroquine and hydroxychloroquine showed a 6-100-fold reduction in potency against all drugresistant strains containing the mutated chloroquine transporter (PfCRT) ( Figure 2B). Amodiaquine, tert-butyl-isoquine pyronaridine, piperaquine, and naphthoquine were potent against all parasite lines (IC 50 = 2-10 nM), as were the endoperoxides including the natural (artemisinin), the semi-synthetic (artesunate), or the fully synthetic peroxides (ozonides), all with IC 50 values of 1-15 nM (Figure 2A).
Compared to the 4-AQs, the 8-AQs (8-AQs) primaquine, diethylprimaquine, and bulaquine were less potent (IC 50 = 0.5-2.5 mM) ( Figure 2C) against both drug-resistant and -sensitive strains. Tafenoquine and NPC-1161B exhibited IC 50 values in the 500-nM range against NF54 and in the 50-nM range against 7G8. IC 50 values of both racemic mefloquine and the +RS isomer were below 10 nM. Halofantrine and lumefantrine displayed potencies below 4 nM against the sensitive strains NF54 and D6 and in the case of halofantrine, 0.3 nM against the multi-drug-resistant strains K1, W2, and 7G8. Resistance to pyrimethamine was verified in all drug-resistant strains ( Figure 2D). Atovaquone a drug active in the subnanomolar range against P. falciparum blood stages showed at most a 10-fold difference in potency between the strains D6 and V1/S. In contrast, a $100-fold loss of potency was observed for cycloguanil between sensitive and resistant strains. The antibiotics azithromycin and trimethoprim, protein synthesis inhibitors, and other molecules such as dehydroepiandrosterone (DHEA), riboflavin, doxycyclin, and the prodrug proguanil showed IC 50 values in the 1-10-mM range with no major differences between strains.

The Identification of Drugs That Additionally Block Transmission from Human Host to Mosquito Vector
Eradicating malaria will require medicines that prevent transmission of the parasite between humans and mosquitoes. Potentially the severe population bottleneck experienced as the parasite progresses from the mature gametocyte in the human host through gametogenesis and fertilization in the mosquito blood meal to the oocyst in the mosquito haemocoele offers the most vulnerable target for intervention. We developed assays for each of these events ( Figure 3A). To integrate both the early sexual stages (gametocyte maturation and gametogenesis) and the late vector stage (sporogony) into the drug-testing cascade, we measured the exflagellation of male gametes in vitro (P. falciparum), ookinete formation in vitro (P.berghei), and the production of oocysts in A. stephensi (P. berghei and P. falciparum). Of these assays, we found analysis of P. berghei ookinete production in vitro was the most robust approach to identify molecules potentially targeting the early development of Plasmodium parasites in the mosquito. Fortysix molecules were tested at a concentration of 10 mM ( Figure 3B). The most potent molecules were cycloheximide (blood stage IC 50 of 25 nM) and atovaquone (IC 50 = 65 nM). Thiostrepton (IC 50 = 8 mM) and pyronaridine (IC 50 = 6 mM) were less potent ( Figure 3C). The latter two molecules and pyrimethamine also inhibited P. falciparum exflagellation by more than 80%, as did The chemical structures of all the main classes of antimalarials and other therapeutic and control molecules are assembled according to either the chemical classes they belong to (endoperoxides, 4-and 8-AQs, amino-alcohols) or their function (antifolate, antibiotics), or both (e.g., sulfonamides, a chemical class of antibiotic used in combined antimalarial therapies). The colour code associated with each class is consistent in all the figures in this report. doi:10.1371/journal.pmed.1001169.g001 Figure 2. The potencies of selected antimalarials against asexual blood stages. Main classes of antimalarials were tested against the blood stage of seven Plasmodium falciparum strains in the [ 3 H]hypoxanthine incorporation assay as described in Vennerstrom et al. [25]. The strains tested here (obtained from MR4) were two drug-sensitive strains NF54 and D6 and five drug-resistant strains: K1, resistant to chloroquine (CQ) and pyrimethamine (PYR), origin Thailand, carries mutations in genes pfmdr1, pfcrt, pfdhfr, pfdhps; W2, resistant to CQ, PYR, quinine, cycloguanil, and sulfadoxine, origin Vietnam; 7G8, resistant to CQ and PYR, origin Brazil; TM90C2A, resistant to CQ, PYR, and MFQ, origin Thailand; and V1/S, resistant to CQ, PYR and cycloguanil, origin Vietnam. Results are expressed as the concentration resulting in 50% growth inhibition (IC 50   All antimalarials were screened at 10 mM. (B) The biological content of the in vitro P. berghei ookinete assay spans gamete formation, fertilization, zygote development, and ookinete formation. Ookinete formation was insensitive to most of the antimalarials tested. Atovaquone, cycloheximide, sulfamethoxazole and mefloquine (+RS). While displaying insignificant activity in the P. berghei ookinete formation assay ( Figure 3C), all endoperoxides, with the sole exception of artemether, inhibited P. falciparum exflagellation by .65% ( Figure 3D). Similarly all 4-AQs inhibited this event by .60%, with the exception of hydroxychloroquine and chloroquine, which enhanced exflagellation by at least 20% [16].
While recognising that the drugs being evaluated are not subject to metabolic degradation/activation by the mammalian hosts, among the 8-AQs, NPC-1161B and diethylprimaquine inhibited exflagellation by more than 70%. In marked contrast primaquine and the amino alcohol halofantrine unexpectedly stimulated exflagellation by 15% and 45%, respectively. All main chemical classes were then evaluated specifically against the vector stages by analysing the inhibition of P. berghei oocyst formation in vivo. Only NPC-1161B and lumefantrine exhibited .90% inhibition when tested at 10 mM ( Figure S1). The transmission-blocking potential of molecules that were active against exflagellation and/or P. berghei sporogony was then assessed against the production of P. falciparum oocysts, which is the most difficult and lowest throughput, yet highest content analysis by encompassing all vector stages from gametocyte uptake to sporogony (see Figure 3A). In this assay, most endoperoxides inhibited oocyst production by .75%; NPC-1161B and mefloquine (+RS) totally blocked transmission at this stage ( Figure 3E). Strikingly, the Coartem component, lumefantrine, and halofantrine impaired sporogony in both P. berghei and P. falciparum, while both mediated only moderate or no inhibition of exflagellation. This finding suggests that these molecules might act specifically on oocysts and not on gametogenesis, a behaviour that could be relevant to transmission reduction in the field given the long half-life of these molecules. As exflagellation is a component process of development within the gut of the mosquito in the P. falciparum oocyst assay (see Figure S2), these results, not unexpectedly, show at least partial concordance with the P. falciparum exflagellation assay.

The Identification of Drugs That Suppress Transmission from the Mosquito to the Human Host
When an infected mosquito bites a host, ,100 sporozoites may be injected into the dermis from where they rapidly invade liver cells [32,33]. This infective step represents the second bottleneck during transmission and therefore another potentially vulnerable point for intervention. In the absence of a practical liver stage assay measuring the formation of P. falciparum/P. vivax liver schizonts, an equivalent assay was developed in P. yoelii and used to assess the activity of the collection of molecules against this specific stage. Specifically, P. yoelii sporozoites were dissected from the salivary glands of infected mosquitoes and were allowed to invade human hepatocarcinoma cells expressing the CD81 protein. The development of the liver schizonts was monitored by immunofluorescence staining using an HSP70 antibody specific to the parasite ( Figure 4A). As only 1% of the hepatocytes are infected in these circumstances, high content imaging was used to quantify growth inhibition of parasite schizonts ( Figure 4B, 4D). Quantification of the total immunofluorescence per well is shown in Figure 4C. Dose response analysis using serially diluted compounds showed that cycloguanil, pyrimethamine, P218, and atovaquone all displayed IC 50 values below 10 nM ( Figure 4B, 4D). Methylene blue and artemisone demonstrated IC 50 values of ,100 nM. All endoperoxides tested in this assay, except artemisinin, exhibited IC 50 values ,3 mM, as did amodiaquine, AQ-13, pyronaridine, and naphthoquine. Of the 8-AQs, NPC-1161B was the only drug active in the submicromolar range against P. yoelii liver stages. Deferoxamine, thiostrepton, trimethoprim, and quinidine exhibited submicromolar potencies. Cycloheximide and thiostrepton, although showing IC 50 values below 200 nM (Table 1), retarded the growth of HepG2-CD81 cells.

Modes of Action of Major Classes of Antimalarials beyond the Asexual Blood Stage
The IC 50 values of the known schizonticides against the asexual blood stages determined in this study correlate well with those reported in recent work examining 185 culture-adapted parasite strain lines treated with seven antimalarials [34].
Many schizonticidal drugs are hypothesised to interfere with haemoglobin metabolism. Our study shows that some of these drugs have activities against parasite stages that lack haemoglobin metabolism, e.g., the liver schizont, mature gametocyte, and sporogonic stages. This finding raises interesting questions about the mode(s) of action of these compounds beyond the asexual blood stage and our understanding of parasite metabolism. For example, natural, semi-synthetic and synthetic endoperoxides (artemisinin, DHA, artesunate, OZ277, and OZ439) are not only fast-acting molecules [35], but are among the most potent antimalarials currently used against the asexual blood stages [36]. They are thought to act by alkylating haem and other vital biomolecules [37,38] (e.g., Pf TCTP) [39], and degrading phospholipids in parasite membranes [40]. The latter mechanism would be expected to have a major impact on all vegetative/ replicating stages of Plasmodium's life cycle, e.g., asexual blood stage, liver schizont, oocyst, and microgametogenesis; and this is consistent with our results showing endoperoxide activity directly or indirectly against P. falciparum exflagellation, oocyst production, and P. yoelii liver schizont development. The lesser impact of this chemical class on ookinete and oocyst development in P. berghei might suggest species to species differences. It is also worth noting that because drugs are applied in human blood containing mature gametocytes prior to triggering exflagellation, inhibition of exflagellation by these molecules could be the direct consequence of a gametocytocidal property rather than solely a specific effect on gamete formation. Our data confirmed that 4-AQs are highly active against asexual blood-stage parasites in vitro, while the 8-AQs are not, indicating that subtle changes in the AQ core structure can result in major differences in the mode of action of pyronaridine, pyrimethamine, and thiostrepton all strongly inhibited ookinete formation (p,0.03 by Student's t-test), while tafenoquine, cismirincamycin, and fosmidomycin gave an enhancement of ookinete formation that was not statistically significant. (C) The medium throughput fluorescent ookinete assay determined IC 50 values for pyronaridine, thiostrepton, cycloheximide, and atovaquone of 6 mM, 8 mM, 25 nM, and 65 nM, respectively. (D) The in vitro P. falciparum exflagellation assay exposes mature gametocytes to antimalarials for 24 h before triggering exflagellation. 16 out of 29 antimalarials tested, including all but one of the endoperoxides and 4-AQs, showed a statistically significant .50% inhibition of exflagellation (p,0.05). Pyronaridine, tert-butyl isoquine, NPC-1161B, OZ277, and cycloheximide all inhibited exflagellation totally at 10 mM. All experiments in triplicate. (E) The in vivo P. falciparum oocyst assay differs from the P. berghei oocyst assay in that mature gametocytes were exposed to the antimalarials in culture for 24 h before feeding to mosquitoes. The endoperoxides all strongly reduced transmission. NPC1161-B, lumefantrine, halofantrine, and mefloquine +RS isomer were also active.  some key antimalarials, although our data cannot address the possibility that the 8-AQ metabolites could be more active. Interestingly, molecules such as amodiaquine, pyronaridine, and tert-butyl-isoquine that target inter alia haem degradation also inhibited P. falciparum exflagellation. We are unaware of any prior data suggesting that haem degradation is essential to male gamete formation. This finding raises fascinating questions as to whether their modes of action in the mosquito blood meal are targeting the same, or different, molecular mechanisms. One working hypothesis could be that the major targets mediating the effect of these molecules in the sexual stages might also contribute to the effect seen in the asexual stages.
The 8-AQs are known to be active on the relapsing ''hypnozoite'' liver form of P. vivax following metabolic activation of the parent compound by liver enzymes [41]. In our assay where we do not anticipate any metabolism of any drugs, we note that the 8-AQ NPC-1161B was additionally shown to inhibit exflagellation in vitro and oocyst production in the mosquito vector. This result suggests either that NPC-1161B does not require metabolic activation or the drug exhibits poly-pharmacology by acting through another metabolite-independent mechanism. The antifolates P218 and pyrimethamine were found to be potent against rapidly replicating blood and liver stages and much less so against early vector stages, observations consistent with the essential role of the folate pathway in DNA synthesis [42].
When mature gametocytes are ingested, exflagellation is activated by a reduction in temperature and the presence of xanthurenic acid in the gut of the mosquito [43]. Inhibition of exflagellation by thiostrepton and cycloheximide is consistent with the observation that protein synthesis is a key component of these dramatic morphological changes [27], and all vegetative stages in the life cycle. Similarly our data confirm previous studies [44][45][46] showing the electron transport chain can be efficiently targeted by atovaquone in both the vector and the liver. For such stage-transcendent pathways, it could be hypothesised that the much lower parasite burden observed in the vector and the liver would increase the probability that treatments would eliminate infections when compared to targeting the abundant blood stage parasites. An important consequence would be that drugs specifically targeting the small ''bottleneck'' populations might delay significantly the selection of drug-resistant parasites.

''Management'' of Drug Delivery to the Mosquito Blood Meal
While both our studies and the comprehensive treatise of Peters [47] show unequivocally that for many compounds effective delivery into the blood meal of the mosquito can be achieved, management of such delivery in the field is not trivial. In this context we note that the C max following administration of therapeutic doses for many antimalarials overlap the predicted potencies of the same antimalarials determined here against early vector stages (0.5-10 mM). For instance, in reported clinical trials, C max was shown to vary from 0.1 mM to 13.9 mM for artemether and lumefantrine administered at therapeutic doses, respectively. In the case of new molecules such as OZ439, C max values obtained in blood at relevant doses ranged between 1 and 2.5 mM (Table  S3) [48][49][50][51][52][53][54][55].

Combination Therapies
To support the eradication agenda, new combination therapies will have to address three major issues: transmission of the pathogen, radical cure of P. vivax malaria, and the emergence of drug resistance. Ideally, new drug combinations should contain both schizonticidal and transmission-blocking components. Preferably fast-acting and long-lasting schizonticides would be combined with another agent that would target the parasite either at the sexual/vector stages, liver stage, or both. In the case of the liver stage, a co-or post-treatment prophylaxis would be provided as an end game scenario. From a pharmacokinetic perspective, when considering P. falciparum, some might speculate that this additional transmission-blocking component should be stable enough to exert its inhibitory capacity over several days against vector stages and/or liver schizont development to protect against a re-infection. However, considering that the schizonticidal component would kill both asexual blood stages and young gametocytes (,6-d old), a second coadministered drug efficiently eliminating mature (.6-d) gametocytes could potentially clear the host of all parasites; i.e., a long half-life would not be required [56]. A sustained stability would however be required for drugs targeting the parasite exclusively in the mosquito. Our study suggests that molecules such as atovaquone that inhibit the electron transport chain in the parasite mitochondria could be suitable candidates, but ideally should lack delayed onset of action and be difficult to raise resistance against [57]. To avoid triple therapies and reduce the risk of drug resistance, dual-activity molecules like amodiaquine, which inhibits haemoglobin digestion in the asexual blood stages and potentially inhibits gametocyte maturation/gamete exflagellation by a different mechanism, could be used in combination with a second antimalarial. Such polyvalent multistage activity has significant benefit to overall drug impact. Our study highlights that molecules such as amodiaquine, naphthoquine, tert-butyl-isoquine, and piperaquine do not lose potency when tested against chloroquine resistant strains. Therefore, to mitigate or defer the risk of drug resistance these molecules might be proposed as potential candidates for partnering new antimalarials such as OZ439. An important consideration would be to favour molecules that have never been used as monotherapy to avoid facing parasites that have previously acquired drug resistance. NPC-1161B inhibited both exflagellation and oocyst production; new molecules with similar properties but devoid of haemolytic liability of the 8-AQs in glucose-6-phosphate dehydrogenase (G6PD)-deficient patients could be interesting candidates as specific transmission-blocking agents.
While being strong inhibitors of blood-stage parasites, some molecules such as chloroquine reportedly enhance gametocytogenesis-a property that might have facilitated the spread of drug- resistant parasites [15,58]. The panel of assays that we applied in this study has confirmed such ''collateral'' activity, in this case one that could prejudice the rational implementation in any elimination/eradication strategy.

Other Human Malarias
Although many have argued for the use of drug screens using nonhuman malarias [47], we recognize that to some, including assays with rodent malaria species might be considered suboptimal. Recent observations identifying interspecific variations include cysteine proteases in rodent plasmodia that show subtle active site differences to those in P. falciparum, leading to questions about the use of these models [59]. A critical role of amino acid 23 mediates activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites [60]. P. yoelii 17X strain is intrinsically partially resistant to chloroquine and is therefore a poor model for studying acquisition of P. falciparum chloroquine resistance. Nevertheless we must recall that there are five species of Plasmodium that infect man and their biologies are patently different, therefore detecting drugs that may be active against multiple species in initial screens may offer long-term potential. Our assays provide, to our knowledge, the most comprehensive global overview of antimalarial drug action to date within the constraints imposed by the current state of culture methodologies for all life stages of all mammalian malaria parasites. Ideally, antimalarials developed against P. falciparum would have an even broader clinical usefulness if proven to be as effective against P. vivax [61]. The potencies of some antimalarials against the asexual blood stage of cultivated P. falciparum and P. vivax field isolates show a very good correlation (Table S4) [62][63][64][65][66][67]. This observation suggests that most of the pathways inhibited by antimalarials in P. falciparum are conserved and may offer valid targets in P. vivax. Moreover, the endoperoxide OZ439, which is currently evaluated in phase IIa clinical trials, has recently demonstrated equivalent efficacy in the treatment of P. falciparum and P. vivax patients (personal communication, MMV).

Drug Gaps and Future Steps
Our work has revealed previously unforeseen opportunities in the current discovery and development pipeline for new antimalarials. We demonstrated that drugs in the current portfolio, like pyronaridine and atovaquone, can also target liver and sexual stages in addition to asexual blood stages. Safe and stable drugs with similar multistage potential are now required. Developing drugs with long half-lives like mefloquine and chloroquine is essential to ensure that in patient blood the exposure of these drugs will remain above the minimum inhibitory concentration for several erythrocytic cycles and should ideally cover the period of gametocyte maturation. Additionally, new chemical scaffolds (e.g., nonendoperoxide) with fast killing potential are needed for a ''one dose cure.'' The search for new drugs would be enhanced by the continued development of P. falciparum and P. vivax culture systems for every parasite life stage. It is critical to the malaria eradication agenda that these assays are able to identify drugs, such as the 8-AQs, with the capacity to safely eliminate P. vivax hypnozoites from the liver: this objective will also require the implementation and validation of in vitro and in vivo G6PD deficiency-dependent hemolysis assays. In parallel with efforts to discover innovative drugs for radical cure (elimination of P. vivax hypnozoites from the liver), new molecules blocking the onward development of mature (stage V) gametocytes are the other major priority in antimalarial discovery. There is therefore an urgent need to develop and validate high throughput screening assays allowing new libraries to be tested against P. falciparum and P. vivax gametocytes/ transmission. These assays could then prioritise compounds for examination in preclinical studies in small mammals and then in standard membrane feeding assays (SMFA) using patient blood to find drugs blocking transmission in the field/clinical situation. Fields studies will remain essential to carefully examine any correlation between activities of new molecules against stabilized laboratory parasite strains and against field isolates.

Conclusions
For the first time, the main chemical classes of current and future antimalarials have been profiled simultaneously in standardized conditions against three Plasmodium species with respect to every major cellular strategy of the malarial life cycle, e.g., vegetative replication, dispersal, and sex ( Figure 5). The present study provides the antimalarial research community with a reference set of methods and data, which may serve as a benchmark for newly discovered molecules when profiled against the entire life cycle of Plasmodium. This information might guide decisions regarding which molecules could be optimally combined to provide the next generation of drugs that will succeed to artemisinin combination therapies (ACTs) [68] and support the eradication of malaria. This comprehensive approach to drug discovery has potential utility for targeting other pathogens with complex life cycles. Figure S1 The transmission-blocking potential of selected compounds against P. berghei in standard membrane feeding assays. All antimalarials were screened at 10 mM in triplicate in independent experiments. The biological content of this assay spans gamete formation through to occyst development all within the gut of the mosquito. NPC1161-B, lumefantrine, pyrimethamine, and cycloheximide showed most notable transmission-blocking activity. (TIF) Figure S2 Comparative summary of the transmissionblocking potential of selected compounds across vectorstage assays. All antimalarials were screened at 10 mM. By comparing the activities of compounds in assays covering different biological ranges of transmission-stage biology, it is possible to infer the stages at which antimalarial drugs are exerting their effects. Lumefantrine was found to have little activity against exflagellation and ookinete development but showed activity in oocyst assays of P. berghei and P. falciparum. Endoperoxides showed activity as early as exflagellation but not during ookinete development, indicative of action against the mature gametocyte/exflagellation. NPC1161-B showed potency in all assays except the ookinete assay, suggesting that it may have dual actions both in early vector-stage development and later on. (TIF)  Table S2 The numerical potencies of selected antimalarials against asexual blood stages. Data corresponding to   Why Was This Study Done? Most currently available antimalarial drugs primarily target the disease-causing parasites' stages in the human blood system. But to eradicate malaria, new drugs that block transmission of the parasite between the human host and the mosquito vector, and eliminate the various stages of the parasite during its cycle in the human body, are needed. In this laboratory study, the researchers compared the profiles of all available and experimental antimalarials and analyzed each drug for activity against each specific stage in the malaria parasite's life cycle to provide a reference set of methods and data, that might serve as a benchmark to help guide the malaria research community in assessing the potential of newly discovered antimalarials. Furthermore, this analysis could provide insights into which chemical drug classes might provide transmission-blocking capabilities-an essential component of malaria eradication.

Supporting Information
What Did the Researchers Do and Find? The researchers used novel laboratory techniques under standardized conditions to develop a series of novel assays to analyze the activities of 50 antimalarial compounds (current drugs and those under development) against three Plasmodium species encompassing every major cellular strategy of the malarial life cycle including drug resistant parasite strains. In their comparative analysis, the researchers undertook a chemical profiling approach to identify the drugs that block transmission from the host to the mosquito vector and additionally suppress transmission from the mosquito to the human host. The researchers highlighted some encouraging results; for example, the potencies of some antimalarials against the asexual blood stage of cultivated P. falciparum and P. vivax isolates show a very good correlation, suggesting that most of the pathways inhibited by antimalarials in P. falciparum may also be valid targets in P. vivax. The researchers also have shown that approved drugs, such as pyronaridine and atovaquone, can target liver and sexual stages in addition to asexual blood stages. Furthermore, the researchers found promising results for new compounds currently in clinical trials, such as the endoperoxide OZ439, a stable synthetic molecule currently being studied in a phase IIa clinical trial, which seemed to be a strong inhibitor of gametocyte maturation and gamete formation. The new 8-aminoquinoline, NPC-1161B, also inhibited sporogony.
What Do These Findings Mean? The results of this analysis provide a valuable guide to help researchers decide which drugs and compounds show most promise as potential future antimalarial drugs for blocking the transmission of malaria. This study could also help researchers make decisions about which molecules could be best combined to provide the next generation of drugs that will succeed artemisinin compound therapy and support the eradication of malaria. Furthermore, this comprehensive approach to drug discovery could be applied to test drugs against other pathogens with complex life cycles.
Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10. 1371/journal.pmed.1001169.
N The ''malERA -a research agenda for malaria eradication'' sponsored collection, published by PLoS in January 2011, comprises 12 Review articles that discuss agendas in malaria research and development