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A series of tetrahydro-β-carboline derivatives of a lead compound known to target the heat shock 90 protein of Plasmodium falciparum were synthesized and assayed for both potency against the parasite and toxicity against a human cell line. Using a rationalized structure based design strategy, a new lead compound with a potency two orders of magnitude greater than the original lead compound was found. Additional modeling of this new lead compound suggests multiple avenues to further increase potency against this target, potentially paving the path for a therapeutic with a mode of action different than any current clinical treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to several antimalarial drugs such as chloroquine (CQ) or piperaquine (PPQ), a partner molecule in current artemisinin-based combination therapies. As a member of the Drug/Metabolite Transporter (DMT) superfamily, the vacuolar transporter PfCRT may translocate substrate molecule(s) across the membrane of the digestive vacuole (DV), a lysosome-like organelle. However, the physiological substrate(s), the transport mechanism and the functional regions of PfCRT remain to be fully characterized. Here, we hypothesized that identification of evolutionary conserved sites in a tertiary structural context could help locate putative functional regions of PfCRT. Hence, site-specific substitution rates were estimated over Plasmodium evolution at each amino acid sites, and the PfCRT tertiary structure was predicted in both inward-facing (open-to-vacuole) and occluded states through homology modeling using DMT template structures sharing <15% sequence identity with PfCRT. We found that the vacuolar-half and membrane-spanning domain (and especially the transmembrane helix 9) of PfCRT were more conserved, supporting that its physiological substrate is expelled out of the parasite DV. In the PfCRT occluded state, some evolutionary conserved sites, including positions related to drug resistance mutations, participate in a putative binding pocket located at the core of the PfCRT membrane-spanning domain. Through structural comparison with experimentally-characterized DMT transporters, we identified several conserved PfCRT amino acid sites located in this pocket as robust candidates for mediating substrate transport. Finally, in silico mutagenesis revealed that drug resistance mutations caused drastic changes in the electrostatic potential of the transporter vacuolar entry and pocket, facilitating the escape of protonated CQ and PPQ from the parasite DV.

Malaria is still a leading cause of mortality among children in the developing world, and despite the immense progress made in reducing the global burden, further efforts are needed if eradication is to be achieved. In this context, targeting transmission is widely recognized as a necessary intervention toward that goal. After carrying out a screen to discover new transmission-blocking agents, herein we report our medicinal chemistry efforts to study the potential of the most robust hit, DDD01035881, as a male-gamete targeted compound. We reveal key structural features for the activity of this series and identify analogues with greater potency and improved metabolic stability. We believe this study lays the groundwork for further development of this series as a transmission blocking agent.

Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors., (Copyright © 2018. Published by Elsevier Ltd.)

In Plasmodium falciparum the bifunctional enzyme glucose-6-phosphate dehydrogenase‒6-phosphogluconolactonase (PfG6PD‒6PGL) is involved in the catalysis of the first reaction of the pentose phosphate pathway. Since this enzyme has a key role in parasite development, its unique structure represents a potential target for the discovery of antimalarial drugs. Here we describe the first 3D structural model of the G6PD domain of PfG6PD‒6PGL. Compared to the human enzyme (hG6PD), the 3D model has enabled the identification of a key difference in the substrate-binding site, which involves the replacement of Arg365 in hG6PD by Asp750 in PfG6PD. In a prospective validation of the model, this critical change has been exploited to rationally design a novel family of substrate analog-based inhibitors that can display the necessary selectivity towards PfG6PD. A series of glucose derivatives featuring an α-methoxy group at the anomeric position and different side chains at position 6 bearing distinct basic functionalities has been synthesized, and their PfG6PD and hG6PD inhibitory activities and their toxicity against parasite and mammalian cells have been assessed. Several compounds displayed micromolar affinity (K i up to 23 μM), favorable selectivity (up to > 26-fold), and low cytotoxicity. Phenotypic assays with P. falciparum cultures revealed high micromolar IC 50 values, likely as a result of poor internalization of the compounds in the parasite cell. Overall, these results endorse confidence to the 3D model of PfG6PD, paving the way for the use of target-based drug design approaches in antimalarial drug discovery studies around this promising target., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: Malaria remains a global health problem despite availability of effective tools. For malaria elimination, drugs targeting sexual stages of Plasmodium falciparum need to be incorporated in treatment regimen along with schizonticidal drugs to interrupt transmission. Primaquine is recommended as a transmission blocking drug for its effect on mature gametocytes but is not extensively utilized because of associated safety concerns among glucose-6-phosphate dehydrogenase (G6PD) deficient patients. In present work, methylene blue, which is proposed as an alternative to primaquine is investigated for its gametocytocidal activity amongst Indian field isolates. An effort has been made to establish Indian field isolates of P. falciparum as in vitro model for gametocytocidal drugs screening., Methods: Plasmodium falciparum isolates were adapted to in vitro culture and induced to gametocyte production by hypoxanthine and culture was enriched for gametocyte stages using N-acetyl-glucosamine. Gametocytes were incubated with methylene blue for 48 h and stage specific gametocytocidal activity was evaluated by microscopic examination., Results: Plasmodium falciparum field isolates RKL-9 and JDP-8 were able to reproducibly produce gametocytes in high yield and were used to screen gametocytocidal drugs. Methylene blue was found to target gametocytes in a concentration dependent manner by either completely eliminating gametocytes or rendering them morphologically deformed with mean IC 50 (early stages) as 424.1 nM and mean IC 50 (late stages) as 106.4 nM. These morphologically altered gametocytes appeared highly degenerated having shrinkage, distortions and membrane deformations., Conclusions: Field isolates that produce gametocytes in high yield in vitro can be identified and used to screen gametocytocidal drugs. These isolates should be used for validation of gametocytocidal hits obtained previously by using lab adapted reference strains. Methylene blue was found to target gametocytes produced from Indian field isolates and is proposed to be used as a gametocytocidal adjunct with artemisinin-based combination therapy. Further exploration of methylene blue in clinical studies amongst Indian population, including G6PD deficient patients, is recommended.

Malaria parasites contain a relict plastid, the apicoplast, which is considered an excellent drug target due to its bacterial-like ancestry. Numerous parasiticidals have been proposed to target the apicoplast, but few have had their actual targets substantiated. Isopentenyl pyrophosphate (IPP) production is the sole required function of the apicoplast in the blood stage of the parasite life cycle, and IPP supplementation rescues parasites from apicoplast-perturbing drugs. Hence, any drug that kills parasites when IPP is supplied in culture must have a nonapicoplast target. Here, we use IPP supplementation to discriminate whether 23 purported apicoplast-targeting drugs are on- or off-target. We demonstrate that a prokaryotic DNA replication inhibitor (ciprofloxacin), several prokaryotic translation inhibitors (chloramphenicol, doxycycline, tetracycline, clindamycin, azithromycin, erythromycin, and clarithromycin), a tRNA synthase inhibitor (mupirocin), and two IPP synthesis pathway inhibitors (fosmidomycin and FR900098) have apicoplast targets. Intriguingly, fosmidomycin and FR900098 leave the apicoplast intact, whereas the others eventually result in apicoplast loss. Actinonin, an inhibitor of bacterial posttranslational modification, does not produce a typical delayed-death response but is rescued with IPP, thereby confirming its apicoplast target. Parasites treated with putative apicoplast fatty acid pathway inhibitors could not be rescued, demonstrating that these drugs have their primary targets outside the apicoplast, which agrees with the dispensability of the apicoplast fatty acid synthesis pathways in the blood stage of malaria parasites. IPP supplementation provides a simple test of whether a compound has a target in the apicoplast and can be used to screen novel compounds for mode of action., (Copyright © 2017 American Society for Microbiology.)

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

While significant advances have been made in the prevention and treatment of malaria in recent years, these successes continue to fall short of the World Health Organization (WHO) goals for malaria control and elimination. For elimination strategies to be effective, limited disease transmission, achieved through rapid reduction in the infectious parasite reservoir and decreased gametocyte carriage, will be critical. Artemisinin-based combination therapy (ACT) forms the cornerstone of WHO-recommended treatment for uncomplicated Plasmodium falciparum malaria, and in combination with other effective interventions will undoubtedly play a vital role in elimination programmes. The gametocytocidal properties of artemisinins are a bonus attribute; there is epidemiological evidence of reductions in malaria incidence and transmission in African regions since the introduction of these agents. Many studies and analyses have specifically investigated the effects of the ACT, artemether-lumefantrine (AL) on gametocyte carriage. In this systematic review of 62 articles published between 1998 and January 2014, the effects of AL on gametocyte carriage and malaria transmission are compared with other artemisinin-based anti-malarials and non-ACT. The impact of AL treatment of asymptomatic carriers on population gametocyte carriage, and the potential future role of AL in malaria elimination initiatives are also considered. Despite the inherent difficulties in comparing data from a range of different studies that also utilized different diagnostic approaches to assess baseline gametocyte counts, the gametocytocidal effect of AL was proportionately consistent across the studies reviewed, suggesting that AL will continue to play a vital role in the treatment of malaria and contribute to clearing the path towards malaria elimination. However, the specific place of AL is the subject of much ongoing research and will undoubtedly be dependent on different demographic and geographical scenarios. Utilizing ACT, such as AL, within malaria elimination strategies is also associated with a number of other challenges, such as balancing potential increased use of ACT (e g, treatment of asymptomatic carriers and home-based treatment) with rational use and avoidance of drug resistance development.

Emergence and spread of multidrug resistant strains of Plasmodium falciparum has severely limited the antimalarial chemotherapeutic options. In order to overcome the obstacle, a set of new side-chain modified 4-aminoquinolines were synthesized and screened against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum. The key feature of the designed molecules is the use of methylpiperazine linked α, β(3)- and γ-amino acids to generate novel side chain modified 4-aminoquinoline analogues. Among the evaluated compounds, 20c and 30 were found more potent than CQ against K1 and displayed a four-fold and a three-fold higher activity respectively, with a good selectivity index (SI=5846 and 11,350). All synthesized compounds had resistance index between 1.06 and >14.13 as against 47.2 for chloroquine. Biophysical studies suggested that this series of compounds act on heme polymerization target., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A library comprising 44 diversely substituted aurones derivatives was synthesized by straightforward aldol condensation reactions of benzofuranones and the appropriately substituted benzaldehydes. Microwave enhanced synthesis using palladium catalyzed protocols was introduced as a powerful strategy for extending the chemical space around the aurone scaffold. Additionally, Mannich-base derivatives, containing a 7-aminomethyl-6-hydroxy substitution pattern at ring A, were also prepared. Screening against the chloroquine resistant Plasmodium falciparum W2 strain identified novel aurones with IC50 values in the low micromolar range. The most potent compounds contained a basic moiety, with the ability to accumulate in acidic digestive vacuole of the malaria parasite. However, none of those aurones revealed significant activity against hemozoin formation and falcipain-2, two validated targets expressed during the blood stage of P. falciparum infection and functional in digestive vacuole of the parasite. Overall, this study highlight (i) the usefulness of aurones as platforms for synthetic procedures using palladium catalyzed protocols to rapidly deliver lead compounds for further optimization and (ii) the potential of novel aurone derivatives as promising antimalarial compounds., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

The in vitro blood stage antiplasmodial activity of a series of allylated chalcones based on the licochalcone A as lead molecule was investigated against chloroquine (CQ) sensitive Pf3D7 and CQ resistant PfINDO strains of Plasmodium falciparum using SYBR Green I assay. Of the forty two chalcones tested, eight showed IC50 ≤ 5 μM. Structure-activity relationship (SAR) studies revealed 9 {1-(4-Chlorophenyl)-3-[3-methoxy-4-(prop-2-en-1-yloxy)phenyl]-prop-2-en-1-one} as the most potent (IC50: 2.5 μM) against Pf3D7 with resistance indices of 1.2 and 6.6 against PfDd2 and PfINDO strains, respectively. Later on, the synergistic effects 9 with standard antimalarials {artemisinin (ART) and chloroquine (CQ)} were studied in order to provide the basis for the selection of the best partner drug. In vitro combinations of 9 with ART showed strong synergy against PfINDO (ΣFIC50: 0.31-0.72) but additive to slight antagonistic effects (ΣFIC50: 1.97-2.64) against Pf3D7. ΣFIC50 0.31 of ART+9 combination corresponded to a 320 fold and 3 fold reduction in IC50 of 9 and ART, respectively. Similar combinations of 9 with CQ showed synergy to additivity to mild antagonism against the two strains {ΣFIC50: 0.668-2.269 (PfINDO); 1.45-2.83 (Pf3D7)}. Drug exposure followed by drug withdrawal indicated that 9 taken alone at IC100 killed rings, trophozoites and schizonts of P. falciparum. The combination of ART and 9 (1X ΣFIC100) selectively inhibited the growth of rings while the 2X ΣFIC100 combination of the same caused killing of rings without affecting trophozoites and schizonts. In contrast, the 1X combination of CQ and 9 (ΣFIC100: 0.5) killed rings and trophozoites. DNA fragmentation and loss of mitochondrial membrane potential (ΔΨm) in the 9 treated P. falciparum culture indicated apoptotic death in malaria parasites. Prediction of ADME properties revealed that most of the molecules did not violate Lipinski's parameters and have low TPSA value suggesting good absorption. The results suggest the promising drug-like properties of 9 against CQ resistant Pf and propensity for synergy with classical antimalarial drugs together with easy and economical synthesis., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Discovering the mechanisms by which cell signaling controls the cell cycle of the human malaria parasite Plasmodium falciparum is fundamental to designing more effective antimalarials. To better understand the impacts of melatonin structure and function on the cell cycle of P. falciparum, we have synthesized two families of structurally-related melatonin compounds (7-11 and 12-16). All synthesized melatonin analogs were assayed in P. falciparum culture and their antimalarial activities were measured by flow cytometry. We have found that the chemical modification of the carboxamide group attached at C-3 position of the indole ring of melatonin (6) was crucial for the action of the indole-related compounds on the P. falciparum cell cycle. Among the melatonin derivatives, only the compounds 12, 13 and 14 were capable of inhibiting the P. falciparum growth in low micromolar IC50. These results open good perspectives for the development of new drugs with novel mechanisms of action., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: With malaria drug resistance increasing in prevalence and severity, new technologies are needed to aid and improve the accuracy and clinical relevance of laboratory or field testing for malaria drug resistance. This study presents a method based on simple and reagentless spectroscopic measurements coupled with comprehensive spectral interpretation analysis that provides valuable quantitative information on the morphological and compositional responses of Plasmodium falciparum and infected red blood cells (IRBCs) to anti-malarial treatment., Methods: The changes in the size, internal structure, nucleotide and haemozoin composition of the parasites as well as the morphology (size and shape) and haemoglobin composition of the IRBCs treated with dihydroartemisinin (DHA) and mefloquine (MFQ) were investigated using a spectral interpretation analysis., Results: DHA treatment reduced the sizes of the parasites and their structural organelles. The haemoglobin composition of the host IRBCs determined from spectroscopic analysis changed negligibly following DHA treatment. MFQ treated parasites grew to the same size as those from parallel non-treated cultures but lacked haemozoin. Lesser deformation of the cell shape and no haemoglobin depletion were detected for the IRBCs of MFQ treated cultures., Conclusions: The spectroscopic analysis method proved to be sensitive for recognition of the effects of anti-malarial treatment on the structure and composition of the parasites and IRBCs. The method can have significant potential for research and clinical applications such as evaluating patient specimens for drug action, drug effects or for therapeutic monitoring.

The current state of antimalarial drug resistance emphasizes the need for new therapies with novel modes of action that will add a significant benefit compared with current standards. In this regard, high throughput phenotypic whole-cell screening aids the discovery of novel antiplasmodial scaffolds that are inherently suited to hit-to-lead and lead-optimization efforts. The aminothiazoles and aminopyridines exemplify two such compound classes stemming from whole-cell screening. Respective structure-activity relationship determinations and subsequent optimization around these scaffolds led to frontrunner compounds in each series, which possess the desired antimalarial efficacy, bioavailability and metabolic stability to further progress medicinal chemistry programs.

The discovery of new chemical starting points with the ability to inhibit Plasmodium falciparum sexual stages, and therefore block the disease transmission, is urgently required. These will form the basis for the development of new therapeutic combinations for the treatment and elimination of malaria and the ultimate goal of global eradication. Recent screening of large chemical libraries against the parasite asexual stages has resulted in the public availability of focused subsets of known antimalarial actives, which represent an excellent starting point for the identification of new gametocytocidal compounds. New stage-specific methodologies aimed at increasing the throughput for assessing compound activity against in vitro cultured gametocytes have recently been published. This article discusses the challenges of assay-oriented large-scale gametocyte culturing and reviews the state-of-the art in gametocytocidal assay development and outcomes.

Background: Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes., Methods and Findings: Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV-V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs., Conclusions: The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Plasmodium falciparum has for some time been developing resistance against known anti-malarial drugs, and therefore a new drug is urgently needed. Selenium (Se), an essential trace element, in the form of inorganic Se, selenite (SeO32-), has been reported to have an anti-plasmodial effect, but its mechanism is still unclear. In the present study, we evaluated the anti-plasmodial effect of several Se compounds against P. falciparum in vitro. The anti-plasmodial effect of several Se compounds was analysed and their apoptosis-inducing activity was evaluated by morphological observation, DNA fragmentation assay and mitochondrial function analysis. SeO32-, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial effects with 50% inhibition concentration at 9, 10, 45, and 65 μm, respectively, while selenate and methylselenocysteine up to 100 μm have no effect on parasite growth. The effective Se compounds caused the parasites to become shrunken and pyknotic and significantly increased mitochondrial damage against P. falciparum compared to the untreated control. In conclusion, SeO32-, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial activities that induce apoptosis-like cell death in P. falciparum, and the anti-plasmodial effects of Se seem to be based on its chemical forms. The apoptosis-like cell-death mechanism in P. falciparum can be beneficial to respond to the growing problem of drug resistance.

Having previously characterized chloroquine (CQ)-induced programmed cell death (PCD) hallmarks in the malaria parasite Plasmodium falciparum and delineating a pathway linking these features, the roles of non-classical mediators were investigated in this paper. It was shown that the later stages of this pathway are Ca(2+)-dependent and transcriptionally regulated. Moreover, it was demonstrated for the first time that micromolar concentrations of CQ partially permeabilized the parasite's digestive vacuole (DV) membrane and that this important upstream event appears to precede mitochondrial dysfunction. This permeabilization of the DV occurred without rupture of the DV membrane and was reminiscent of lysosome-mediated cell death in mammalian cells. As such micromolar concentrations of CQ are found in the patient's plasma after initial CQ loading, this alludes to a clinically relevant antimalarial mechanism of the drug which has yet to be recognized. Furthermore, other 'non-antimalarial' lysosomotropic compounds were also shown to cause DV permeabilization, triggering PCD in both CQ-sensitive and -resistant parasites. These findings present new avenues for antimalarial developments, which induce DV destabilization to kill parasites.

Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for "delayed death" compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC(50) in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.

Background: Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates., Results: In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype., Conclusions: The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.

4-Aminoquinolines were hybridized with artemisinin and 1,4-naphthoquinone derivatives via the Ugi-four-component condensation reaction, and their biological activities investigated. The artemisinin-containing compounds 6a-c and its salt 6c-citrate were the most active target compounds in the antiplasmodial assays. However, despite the potent in vitro activities, they also displayed cytotoxicity against a mammalian cell-line, and had lower therapeutic indices than chloroquine. Morphological changes in parasites treated with these artemisinin-containing hybrid compounds were similar to those observed after addition of artemisinin. These hybrid compounds appeared to share mechanism(s) of action with both chloroquine and artemisinin: they exhibited potent β-hematin inhibitory activities; they caused an increase in accumulation of hemoglobin within the parasites that was intermediate between the increase observed with artesunate and chloroquine; and they also appeared to inhibit endocytosis as suggested by the decrease in the number of transport vesicles in the parasites. No cross-resistance with chloroquine was observed for these hybrid compounds, despite the fact that they contained the chloroquinoline moiety. The hybridization strategy therefore appeared to be borrowing the best from both classes of antimalarials., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Multi-drug resistant Plasmodium falciparum is a major obstacle to malaria control and is emerging as a complex phenomenon. Mechanisms of drug evasion based on the intracellular extrusion of the drug and/or modification of target proteins have been described. However, cellular mechanisms related with metabolic activity have also been seen in eukaryotic systems, e.g. cancer cells. Recent observations suggest that such mechanism may occur in P. falciparum., Methodology/principal Findings: We therefore investigated the effect of mefloquine exposure on the cell cycle of three P. falciparum clones (3D7, FCB, W2) with different drug susceptibilities, while investigating in parallel the expression of four genes coding for confirmed and putative drug transporters (pfcrt, pfmdr1, pfmrp1 and pfmrp2). Mefloquine induced a previously not described dose and clone dependent delay in the intra-erythrocytic cycle of the parasite. Drug impact on cell cycle progression and gene expression was then merged using a non-linear regression model to determine specific drug driven expression. This revealed a mild, but significant, mefloquine driven gene induction up to 1.5 fold., Conclusions/significance: Both cell cycle delay and induced gene expression represent potentially important mechanisms for parasites to escape the effect of the antimalarial drug.

The plasmodial surface anion channel (PSAC) is an unusual small-conductance ion channel induced on erythrocytes infected with plasmodia, including parasites responsible for human malaria. Although broadly available inhibitors produce microscopic clearance of parasite cultures at high concentrations and suggest that PSAC is an antimalarial target, they have low affinity for the channel and may interfere with other parasite activities. To address these concerns, we developed a miniaturized assay for PSAC activity and carried out a high-throughput inhibitor screen. Approximately 70,000 compounds from synthetic and natural product libraries were screened, revealing inhibitors from multiple structural classes including two novel and potent heterocyclic scaffolds. Single-channel patch-clamp studies indicated that these compounds act directly on PSAC, further implicating a proposed role in transport of diverse solutes. A statistically significant correlation between channel inhibition and in vitro parasite killing by a family of compounds provided chemical validation of PSAC as a drug target. These new inhibitors should be important research tools and may be starting points for much-needed antimalarial drugs.

Characteristics of primary and recrudescent Plasmodium falciparum infections were evaluated in 25 children who did not recover after amodiaquine (AQ) treatment. Recrudescence was detected by a thick blood smear and confirmed by polymerase chain reaction. Over half of recrudescent events occurred after 14 days of initiation of treatment and were associated with relatively low asexual parasitaemia. We examined the gametocyte sex ratio (GSR) in these children and in age and gender-matched controls that had AQ-sensitive (AQ-S) infections (n = 50). In both AQ-S and AQ-resistant (AQ-R) infections, the GSR was female-biased pre-treatment and became male-biased by the third day after treatment initiation. However, gametocyte males persisted after this period in children with AQ-R infections. AQ-recrudescent infections are relatively low (25 of 612.4%) in children from this endemic area.

Background: Plasmodium vivax is a major cause of malaria and is still primarily treated with chloroquine. Chloroquine inhibits the polymerization of haem to inert haemozoin. Free haem monomers are thought to catalyze oxidative damage to the Plasmodium spp. trophozoite, the stage when haemoglobin catabolism is maximal. However preliminary in vitro observations on P. vivax clinical isolates suggest that only ring stages (early trophozoites) are sensitive to chloroquine. In this study, the stage specific action of chloroquine was investigated in synchronous cryopreserved isolates of P. vivax., Methods: The in vitro chloroquine sensitivity of paired ring and trophozoite stages from 11 cryopreserved P. vivax clinical isolates from Thailand and two Plasmodium falciparum clones (chloroquine resistant K1 and chloroquine sensitive FC27) was measured using a modified WHO microtest method and fluorometric SYBR Green I Assay. The time each stage was exposed to chloroquine treatment was controlled by washing the chloroquine off at 20 hours after the beginning of treatment., Results: Plasmodium vivax isolates added to the assay at ring stage had significantly lower median IC50s to chloroquine than the same isolates added at trophozoite stage (median IC50 12 nM vs 415 nM p < 0.01). Although only 36% (4/11) of the SYBR Green I assays for P. vivax were successful, both microscopy and SYBR Green I assays indicated that only P. vivax trophozoites were able to develop to schizonts at chloroquine concentrations above 100 nM., Conclusion: Data from this study confirms the diminished sensitivity of P. vivax trophozoites to chloroquine, the stage thought to be the target of this drug. These results raise important questions about the pharmacodynamic action of chloroquine, and highlight a fundamental difference in the activity of chloroquine between P. vivax and P. falciparum.

The effects of quinoline antimalarials on endocytosis by Plasmodium falciparum was investigated by measuring parasite hemoglobin levels, peroxidase uptake, and transport vesicle content. Mefloquine, quinine, and halofantrine inhibited endocytosis, and chloroquine inhibited vesicle trafficking, while amodiaquine shared both effects. Protease inhibitors moderated hemoglobin perturbations, suggesting a common role for heme binding.

A P-glycoprotein homologue (Pgh1) is believed to play a role in modulating levels of chloroquine resistance in Plasmodium falciparum. To study the role of Pgh1 in the mechanism of chloroquine (CQ) resistance, antisera were raised against this protein. There was no direct association between the level of Pgh1 expression and chloroquine sensitivity. We also failed to detect phosphorylation of Pgh1 in the food vacuole (FV), suggesting that other mechanisms regulate the chloroquine-resistant (CQR) phenotype. Therefore, high levels of expression of Pgh1 or phosphorylation of this protein in the FV could not account for CQ sensitivity. In addition, the lack of inhibition of CQ accumulation by anti-Pgh1 antibodies suggests that Pgh1 is not involved as a CQ transporter in the plasma membrane of P. falciparum. Furthermore, resistance reversers do not appear to act at the plasma membrane level.

A panel of sulfated oligosaccharides was tested for antimalarial activity and inhibition of adhesion to the placental malaria receptor chondroitin-4-sulfate (CSA). The heparan sulfate mimetic PI-88, currently undergoing phase II anticancer trials, displayed the greatest in vitro antimalarial activity against Plasmodium falciparum (50% inhibitory concentration of 7.4 microM) and demonstrated modest adhesion inhibition to cell surface CSA.

Resistance to antimalarials has been widely reported and constitutes a major problem around the world. In Colombia, resistance of P. falciparum to chloroquine has been reported as 47%-97%, to amodiaquine 3%-7% and to sulfadoxine-pyrimethamine as 9%-13%. The search for new antimalarials is a priority and with this aim we studied the in vitro antimalarial activity of plants used by traditional healers. Incorporation of (3)H-hypoxanthine by the strain FCB-2 of P. falciparum was used to measure the degree of inhibition produced by the steroids SN-1 tumacone A (C(29)H(44)O(5)), SN-2 tumacone B (C(27)H(42)O(4)), SN-3 tumacoside A (C(35)H(54)O(10)), and SN-4 tumacoside B (C(33)H(52)O(9)). All compounds were obtained from the dried stems and leaves of Solanum nudum. The mean growth inhibition of P. falciparum was 71%, 56%, 21% and 12% with each of the compounds SN-1, SN-2, SN-3 and SN-4. These results constitute an important discovery since they may account for the antimalarial properties of extracts of Solanum nudum by a sensitive method. Future work should include study of the in vivo anti-malarial effect of these extracts., (Copyright 2002 John Wiley & Sons, Ltd.)

Objective: To evaluate the clinical efficacy of dihydroartemisinin-mefloquine on acute uncomplicated falciparum malaria., Methods: Fifty-four patients with symptomatic falciparum malaria were allocated to receive oral dihydroartemisinin at a single dose of 120 mg on day 1, followed by mefioquine, 750 mg and 500 mg on days 2 and 3, respectively. Follow-up was performed on days 1, 2, 3, 4, 7, 14, 21, and 28., Results: All patients had a rapid initial response to treatment. The parasite clearance time (PCT) after treatment was 30.7 +/- 3.6 hours. The fever subsidence time (FST) after treatment was 21.2 +/- 2.8 hours. Two patients had a recrudescence 21 and 25 days respectively after the disappearance of parasitemia, hence the recrudescence rate was 3.7% and the cure rate was 96.3%. No serious adverse effects were observed, only mild and transient nausea, vomiting and loss of appetite., Conclusion: A combination of dihydroartemisinin and mefloquine is effective in the treatment of acute uncomplicated falciparum malaria.

Our previous studies have shown that riboflavin has activity against Plasmodium falciparum asexual-stage parasites in vitro. In the present study we examine the gametocytocidal activity of riboflavin and the interaction of riboflavin with some commonly used antimalarial drugs against the asexual forms of P. falciparum in vitro. The addition of riboflavin to P. falciparum cultures killed gametocytes at all stages, even those at late stages (III to V), which are not affected by many of the commonly used antimalarials. Combinations of riboflavin with mefloquine, pyrimethamine, and quinine showed a marked potentiation of the activities of these drugs against asexual-stage parasites in vitro. The combination of riboflavin with artemisinin was additive, while that with chloroquine was mildly antagonistic. High doses of riboflavin are used clinically to treat several inborn errors of metabolism with no adverse side effects. Its efficacy in combination with standard antimalarial drugs in treating and preventing the transmission of P. falciparum malaria can therefore be evaluated in humans.

One hundred and eight children with acute, symptomatic, uncomplicated, falciparum malaria were randomized to receive chloroquine (for 3 days) plus chlorpheniramine alone (for seven days) (CQ-CP group; N = 55) or, in a sequential treatment, chloroquine plus chlorpheniramine for 3 days followed, on the fourth day, by a single oral dose of sulfadoxine-pyrimethamine (25 mg sulfadoxine/kg) (CQ-CP-SP group; N = 53). The mean (S.D.) parasite-clearance time in the CQ-CP group [2.1 (0.7) days; range = 1-5 days] was similar to that in the CQ-CP-SP [2.1 (0.8) days; range = 1-5 days]. The fever-clearance times were also similar: 1.2 (0.1) days (range = 1-3 days) v. 1.1 (0.4) days (range = 1-3 days). The cure rates on days 14, 21 and 28 were 98.2%, 96.3% and 92.7%, respectively in the CQ-CP group, and 100%, 100% and 96.2%, respectively, in the CQ-CP-SP group. The rates of gametocyte carriage were low and similar (5.4% in the CQ-CP group and 3.8% in the CQ-CP-SP group) throughout the duration of the study. Both treatment regimens were relatively well tolerated, the main adverse reactions being similar: sleepiness (on day 1) and pruritus (on days 1-3). No adverse effect was attributable to SP. The results indicate that sequential treatment, for 3 days with CQ and CP, followed by a single dose of SP, is effective and well tolerated in children with acute, uncomplicated, falciparum malaria and may be an alternative treatment for CQ- and/or SP-resistant falciparum malaria. Treatment with a CQ-CP combination (CQ and CP for 3 days and then CP alone for another 4 days) is also effective but requires continuing administration after the signs and symptoms of acute malaria have disappeared.

In the city of Mumbai (formerly Bombay), chloroquine (CQ) continues to be recommended as the drug of first choice for the treatment of Plasmodium vivax and P. falciparum infections, even though > 50% of local isolates of P. falciparum are resistant to it. Primaquine, an 8-aminoquinoline is also given to patients with falciparum malaria, in a single, 45-mg dose, to kill the gametocytes and so reduce transmission. The gametocytocidal activity of supervised primaquine (45 mg given on day 8) was investigated in 90 patients who had been treated with CQ. Of these, 15 were found to be CQ-sensitive patients, 61 were resistant (49, eight and four considered RI, RII and RIII, respectively) and 14 were lost before completion of the follow-up. The mean (S.D.) baseline gametocytaemias in the CQ-sensitive and RI-resistant cases were 665.1 (411.3) and 1537.4 (1045.5)/microliter, respectively. Despite supervised primaquine treatment, four of the 15 CQ-sensitive patients and 32 of the 49 patients found to be RI-resistant had gametocytes on day 29. There therefore appears to be a need to review the current, gametocytocidal, primaquine-dosage schedule and to re-treat patients who remain gametocytaemic with higher doses of primaquine, as an important, transmission-blocking strategy.

As chemoresistance of Plasmodium falciparum to chloroquine has arisen, new ways of combating the infection are needed. Similarities exist between the multidrug resistance of mammalian cells and chloroquine resistance of P. falciparum, based on the occurrence of internucleosomal deoxyribonucleic acid (DNA) breakdown and the ability of some anticancer drugs and chloroquine to induce apoptosis. Using chloroquine, oligonucleosomal DNA fragmentation was observed with a sensitive strain of P. falciparum, but not with a resistant one. This suggests that apoptosis may be involved in the action of chloroquine on the parasite.

Catalytic RNA (ribozymes) suppressed the growth of the human malarial parasite Plasmodium falciparum in vitro. The phosphorothioated hammerhead ribozymes targeted unique regions of the P. falciparum carbamoyl-phosphate synthetase II gene. The P. falciparum carbamoyl-phosphate synthetase II gene encodes the first and limiting enzyme in the pathway, and its mRNA transcript contains two large insert regions absent in other carbamoyl-phosphate synthetases, including that from humans. These inserts are ideal targets for nucleic acid therapy. Exogenous delivery of ribozymes to cultures reduced malarial viability up to 55% at 0.5 microM ribozyme concentrations, which is significantly greater than control levels (5-15% reduction), suggesting a sequence-specific inhibition. This inhibition was shown to be stage-specific, with optimal inhibitions being detected after 24 h, coincident with maximal production of the carbamoyl-phosphate synthetase enzyme in the course of the life cycle of the parasite. A decrease in total carbamoyl-phosphate synthetase activity was observed only in cultures treated with the ribozymes. The task of developing alternative therapeutic agents against malaria is urgent due to the evolution of drug-resistant strains of P. falciparum, the most virulent of all human malarial parasites. Another critical issue to be addressed is the possibility of eliminating or reducing any systemic toxicity to the host, which can potentially be provided by nucleic acid therapy. This work is the first reported assessment of the ability of ribozymes as antimalarials. Ribozyme inhibition assays can also aid in identifying important antimalarial loci for chemotherapy. The malarial parasite can, in turn, be a useful in vivo host to study the catalysis and function of new ribozyme designs.

Resistance to mefloquine in Plasmodium falciparum has begun to occur along the border of Thailand and Kampuchea. As a means of assessing the natural occurrence of mefloquine resistance, the admission and post-treatment parasite isolates from a mefloquine treatment failure were cloned and characterized. Clones from the admission isolate were susceptible to mefloquine in vitro (ID50 of 3.4 [2-5], G [95% CI] ng/ml) and showed a mixture of isozyme types for glucose phosphate isomerase (GPI types I and II). The post-treatment clones were resistant to mefloquine in vitro (ID50 of 17.3 [13-23] ng/ml) with only one isozyme (GPI type I) detected. These observations suggest that under mefloquine pressure a resistant parasite population was selected in the patient, indicating that the potential for mefloquine resistance already exists in the indigenous P. falciparum gene pool. In addition, the mefloquine-resistant clones showed decreased susceptibility in vitro to halofantrine suggesting possible cross-resistance to this new antimalarial drug currently under development.