Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance.     

Treatment of Murine Cerebral Malaria by Artemisone in Combination with Conventional Antimalarial Drugs: Antiplasmodial Effects and Immune Responses

The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response.     

Artemisinin Resistance at the China-Myanmar Border and Association with Mutations in the K13 Propeller Gene

Artemisinin resistance in Plasmodium falciparum parasites in Southeast Asia is a major concern for malaria control. Its emergence at the China-Myanmar border, where there have been more than 3 decades of artemisinin use, has yet to be investigated. Here, we comprehensively evaluated the potential emergence of artemisinin resistance and antimalarial drug resistance status in P. falciparum using data and parasites from three previous efficacy studies in this region. These efficacy studies of dihydroartemisinin-piperaquine combination and artesunate monotherapy of uncomplicated falciparum malaria in 248 P. falciparum patients showed an overall 28-day adequate clinical and parasitological response of >95% and day 3 parasite-positive rates of 6.3 to 23.1%. Comparison of the 57 K13 sequences (24 and 33 from day 3 parasite-positive and -negative cases, respectively) identified nine point mutations in 38 (66.7%) samples, of which F446I (49.1%) and an N-terminal NN insertion (86.0%) were predominant. K13 propeller mutations collectively, the F446I mutation alone, and the NN insertion all were significantly associated with day 3 parasite positivity. Increased ring-stage survival determined using the ring-stage survival assay (RSA) was highly associated with the K13 mutant genotype. Day 3 parasite-positive isolates had ∼10 times higher ring survival rates than day 3 parasite-negative isolates. Divergent K13 mutations suggested independent evolution of artemisinin resistance. Taken together, this study confirmed multidrug resistance and emergence of artemisinin resistance in P. falciparum at the China-Myanmar border. RSA and K13 mutations are useful phenotypic and molecular markers for monitoring artemisinin resistance.     

The MSPDBL2 Codon 591 Polymorphism Is Associated with Lumefantrine In Vitro Drug Responses in Plasmodium falciparum Isolates from Kilifi,Kenya

The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem).     

A Class of Tricyclic Compounds Blocking Malaria Parasite Oocyst Development and Transmission

Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.     

Modulation of PF10_0355 (MSPDBL2) Alters Plasmodium falciparum Response to Antimalarial Drugs

Malaria''s ability to rapidly adapt to new drugs has allowed it to remain one of the most devastating infectious diseases of humans. Understanding and tracking the genetic basis of these adaptations are critical to the success of treatment and intervention strategies. The novel antimalarial resistance locus PF10_0355 (Pfmspdbl2) was previously associated with the parasite response to halofantrine, and functional validation confirmed that overexpression of this gene lowered parasite sensitivity to both halofantrine and the structurally related antimalarials mefloquine and lumefantrine, predominantly through copy number variation. Here we further characterize the role of Pfmspdbl2 in mediating the antimalarial drug response of Plasmodium falciparum. Knockout of Pfmspdbl2 increased parasite sensitivity to halofantrine, mefloquine, and lumefantrine but not to unrelated antimalarials, further suggesting that this gene mediates the parasite response to a specific class of antimalarial drugs. A single nucleotide polymorphism encoding a C591S mutation within Pfmspdbl2 had the strongest association with halofantrine sensitivity and showed a high derived allele frequency among Senegalese parasites. Transgenic parasites expressing the ancestral Pfmspdbl2 allele were more sensitive to halofantrine and structurally related antimalarials than were parasites expressing the derived allele, revealing an allele-specific effect on drug sensitivity in the absence of copy number effects. Finally, growth competition experiments showed that under drug pressure, parasites expressing the derived allele of Pfmspdbl2 outcompeted parasites expressing the ancestral allele within a few generations. Together, these experiments demonstrate that modulation of Pfmspdbl2 affects malaria parasite responses to antimalarial drugs.     

In Vivo Curative and Protective Potential of Orally Administered 5-Aminolevulinic Acid plus Ferrous Ion against Malaria

5-Aminolevulinic acid (ALA) is a naturally occurring amino acid present in diverse organisms and a precursor of heme biosynthesis. ALA is commercially available as a component of cosmetics, dietary supplements, and pharmaceuticals for cancer diagnosis and therapy. Recent reports demonstrated that the combination of ALA and ferrous ion (Fe²⁺) inhibits the in vitro growth of the human malaria parasite Plasmodium falciparum. To further explore the potential application of ALA and ferrous ion as a combined antimalarial drug for treatment of human malaria, we conducted an in vivo efficacy evaluation. Female C57BL/6J mice were infected with the lethal strain of rodent malaria parasite Plasmodium yoelii 17XL and orally administered ALA plus sodium ferrous citrate (ALA/SFC) as a once-daily treatment. Parasitemia was monitored in the infected mice, and elimination of the parasites was confirmed using diagnostic PCR. Treatment of P. yoelii 17XL-infected mice with ALA/SFC provided curative efficacy in 60% of the mice treated with ALA/SFC at 600/300 mg/kg of body weight; no mice survived when treated with vehicle alone. Interestingly, the cured mice were protected from homologous rechallenge, even when reinfection was attempted more than 230 days after the initial recovery, indicating long-lasting resistance to reinfection with the same parasite. Moreover, parasite-specific antibodies against reported vaccine candidate antigens were found and persisted in the sera of the cured mice. These findings provide clear evidence that ALA/SFC is effective in an experimental animal model of malaria and may facilitate the development of a new class of antimalarial drug.     

The Nonartemisinin Sesquiterpene Lactones Parthenin and Parthenolide Block Plasmodium falciparum Sexual Stage Transmission

Parthenin and parthenolide are natural products that are closely related in structure to artemisinin, which is also a sesquiterpene lactone (SQL) and one of the most important antimalarial drugs available. Parthenin, like artemisinin, has an effect on Plasmodium blood stage development. We extended the evaluation of parthenin as a potential therapeutic for the transmissible stages of Plasmodium falciparum as it transitions between human and mosquito, with the aim of gaining potential mechanistic insight into the inhibitory activity of this compound. We posited that if parthenin targets different biological pathways in the parasite, this in turn could pave the way for the development of druggable compounds that could prevent the spread of artemisinin-resistant parasites. We examined parthenin''s effect on male gamete activation and the ookinete-to-oocyst transition in the mosquito as well as on stage V gametocytes that are present in peripheral blood. Parthenin arrested parasite development for each of the stages tested. The broad inhibitory properties of parthenin on the evaluated parasite stages may suggest different mechanisms of action between parthenin and artemisinin. Parthenin''s cytotoxicity notwithstanding, its demonstrated activity in this study suggests that structurally related SQLs with a better safety profile deserve further exploration. We used our battery of assays to test parthenolide, which has a more compelling safety profile. Parthenolide demonstrated activity nearly identical to that of parthenin against P. falciparum, highlighting its potential as a possible transmission-blocking drug scaffold. We discuss the context of the evidence with respect to the next steps toward expanding the current antimalarial arsenal.     

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

Most current antimalarials for treatment of clinical Plasmodium falciparum malaria fall into two broad drug families and target the food vacuole of the trophozoite stage. No antimalarials have been shown to target the brief extracellular merozoite form of blood-stage malaria. We studied a panel of 12 drugs, 10 of which have been used extensively clinically, for their invasion, schizont rupture, and growth-inhibitory activity using high-throughput flow cytometry and new approaches for the study of merozoite invasion and early intraerythrocytic development. Not surprisingly, given reported mechanisms of action, none of the drugs inhibited merozoite invasion in vitro. Pretreatment of erythrocytes with drugs suggested that halofantrine, lumefantrine, piperaquine, amodiaquine, and mefloquine diffuse into and remain within the erythrocyte and inhibit downstream growth of parasites. Studying the inhibitory activity of the drugs on intraerythrocytic development, schizont rupture, and reinvasion enabled several different inhibitory phenotypes to be defined. All drugs inhibited parasite replication when added at ring stages, but only artesunate, artemisinin, cycloheximide, and trichostatin A appeared to have substantial activity against ring stages, whereas the other drugs acted later during intraerythrocytic development. When drugs were added to late schizonts, only artemisinin, cycloheximide, and trichostatin A were able to inhibit rupture and subsequent replication. Flow cytometry proved valuable for in vitro assays of antimalarial activity, with the free merozoite population acting as a clear marker for parasite growth inhibition. These studies have important implications for further understanding the mechanisms of action of antimalarials, studying and evaluating drug resistance, and developing new antimalarials.     

Complex Polymorphisms in the Plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial Response

Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite''s in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.     

In Vitro and In Vivo Antimalarial Efficacies of Optimized Tetracyclines

With increasing resistance to existing antimalarials, there is an urgent need to discover new drugs at affordable prices for countries in which malaria is endemic. One approach to the development of new antimalarial drugs is to improve upon existing antimalarial agents, such as the tetracyclines. Tetracyclines exhibit potent, albeit relatively slow, action against malaria parasites, and doxycycline is used for both treatment (with other agents) and prevention of malaria. We synthesized 18 novel 7-position modified tetracycline derivatives and screened them for activity against cultured malaria parasites. Compounds with potent in vitro activity and other favorable drug properties were further tested in a rodent malaria model. Ten compounds inhibited the development of cultured Plasmodium falciparum with a 50% inhibitory concentration (IC₅₀) after 96 h of incubation of <30 nM, demonstrating activity markedly superior to that of doxycycline (IC₅₀ at 96 h of 320 nM). Most compounds showed little mammalian cell cytotoxicity and no evidence of in vitro phototoxicity. In a murine Plasmodium berghei model, 13 compounds demonstrated improved activity relative to that of doxycycline. In summary, 7-position modified tetracyclines offer improved activity against malaria parasites compared to doxycycline. Optimized compounds may allow lower doses for treatment and chemoprophylaxis. If safety margins are adequate, dosing in children, the group at greatest risk for malaria in countries in which it is endemic, may be feasible.     

Stearylamine Liposomal Delivery of Monensin in Combination with Free Artemisinin Eliminates Blood Stages of Plasmodium falciparum in Culture and P. berghei Infection in Murine Malaria

The global emergence of drug resistance in malaria is impeding the therapeutic efficacy of existing antimalarial drugs. Therefore, there is a critical need to develop an efficient drug delivery system to circumvent drug resistance. The anticoccidial drug monensin, a carboxylic ionophore, has been shown to have antimalarial properties. Here, we developed a liposome-based drug delivery of monensin and evaluated its antimalarial activity in lipid formulations of soya phosphatidylcholine (SPC) cholesterol (Chol) containing either stearylamine (SA) or phosphatidic acid (PA) and different densities of distearoyl phosphatidylethanolamine-methoxy-polyethylene glycol 2000 (DSPE-mPEG-2000). These formulations were found to be more effective than a comparable dose of free monensin in Plasmodium falciparum (3D7) cultures and established mice models of Plasmodium berghei strains NK65 and ANKA. Parasite killing was determined by a radiolabeled [³H]hypoxanthine incorporation assay (in vitro) and microscopic counting of Giemsa-stained infected erythrocytes (in vivo). The enhancement of antimalarial activity was dependent on the liposomal lipid composition and preferential uptake by infected red blood cells (RBCs). The antiplasmodial activity of monensin in SA liposome (50% inhibitory concentration [IC₅₀], 0.74 nM) and SPC:Chol-liposome with 5 mol% DSPE-mPEG 2000 (IC₅₀, 0.39 nM) was superior to that of free monensin (IC₅₀, 3.17 nM), without causing hemolysis of erythrocytes. Liposomes exhibited a spherical shape, with sizes ranging from 90 to 120 nm, as measured by dynamic light scattering and high-resolution electron microscopy. Monensin in long-circulating liposomes of stearylamine with 5 mol% DSPE-mPEG 2000 in combination with free artemisinin resulted in enhanced killing of parasites, prevented parasite recrudescence, and improved survival. This is the first report to demonstrate that monensin in PEGylated stearylamine (SA) liposome has therapeutic potential against malaria infections.     

Field-Based Flow Cytometry for Ex Vivo Characterization of Plasmodium vivax and P. falciparum Antimalarial Sensitivity

Ex vivo antimalarial sensitivity testing in human malaria parasites has largely depended on microscopic determination of schizont maturation. While this microscopic method is sensitive, it suffers from poor precision and is laborious. The recent development of portable, low-cost cytometers has allowed us to develop and validate a simple, field-optimized protocol using SYBR green and dihydroethidium for the accurate and objective determination of antimalarial drug sensitivity in freshly isolated Plasmodium vivax and Plasmodium falciparum.     

Novel pfdhps Haplotypes among Imported Cases of Plasmodium falciparum Malaria in the United Kingdom

Treatment of acute malaria caused by Plasmodium falciparum may include long-half-life drugs, such as the antifolate combination sulfadoxine-pyrimethamine (SP), to provide posttreatment chemoprophylaxis against parasite recrudescence or delayed emergence from the liver. An unusual case of P. falciparum recrudescence in a returned British traveler who received such a regimen, as well as a series of 44 parasite isolates from the same hospital, was analyzed by PCR and direct DNA sequencing for the presence of markers of parasite resistance to chloroquine and antifolates. The index patient harbored a mixture of wild-type and resistant pfdhfr and pfdhps alleles upon initial presentation. During his second malaria episode, he harbored only resistant parasites, with the haplotypes IRNI (codons 51, 59, 108, and 164) and SGEAA (codons 436, 437, 540, 581, and 613) at these two loci, respectively. Analysis of isolates from 44 other patients showed that the pfdhfr haplotype IRNI was common (found in 81% of cases). The SGEAA haplotype of pfdhps was uncommon (found only in eight cases of East African origin [17%]). A previously undescribed mutation, I431V, was observed for seven cases of Nigerian origin, occurring as one of two haplotypes, VAGKGS or VAGKAA. The presence of this mutation was also confirmed in isolates of Nigerian origin from the United Kingdom Malaria Reference Laboratory. The presence of the pfdhps haplotype SGEAA in P. falciparum parasites of East African origin appears to compromise the efficacy of treatment regimens that include SP as a means to prevent recrudescence. Parasites with novel pfdhps haplotypes are circulating in West Africa. The response of these parasites to chemotherapy needs to be evaluated.The United Kingdom health system treats 1,000 to 2,000 cases of imported malaria each year, more than 70% of which are caused by Plasmodium falciparum (12). Infections from all over the globe are represented among these cases, but the majority are from sub-Saharan Africa. Parasite resistance to antimalarial drugs has an impact both on the effectiveness of chemoprophylaxis used by travelers (13) and on the efficacy of treatment regimens such as atovaquone-proguanil (AP), widely used to treat cases of malaria in hospitals in the United Kingdom (14).Quinine remains an effective regimen for treating P. falciparum malaria and is still used in the United Kingdom for the treatment of both uncomplicated and severe malaria (9). At the Hospital for Tropical Diseases (HTD), patients with P. falciparum infections are treated with quinine until parasites are undetectable in peripheral blood; patients are then given a full dose of the fixed-combination antifolate sulfadoxine-pyrimethamine (SP). The long half-lives of both active components of SP in serum provide prophylaxis against the subsequent recrudescence of parasites surviving quinine therapy, or any parasites newly emerging from hepatic schizonts after the cessation of primary therapy (10, 15). This policy appears to have provided efficacious treatment for malaria patients, but there is no active follow-up of malaria patients at present.The efficacy of quinine does not appear to have been significantly diminished over time by the evolution of parasite resistance, although studies of in vitro susceptibility of malaria parasites show some variability in sensitivity to quinine among South American isolates (6). In contrast, SP treatment failure due to high-level parasite resistance is widespread in Asia and common in East Africa (3), although SP retains good efficacy in West Africa (4, 17). Failure of SP therapy is associated with the accumulation of point mutations in two parasite genes, pfdhfr and pfdhps, encoding the folate biosynthesis pathway enzymes dihydrofolate reductase and dihydropteroate synthetase (DHPS), respectively. The continued efficacy of SP in West Africa is probably due to the absence or rarity of some of these mutations, particularly those at codon 164 of pfdhfr (widespread in Asia) and at codon 540 of pfdhps (widely reported from East and Southern Africa). Therefore, ongoing surveillance of the geographic distribution of mutations in pfdhfr and pfdhps is of great importance for informing treatment policy both in countries where malaria is endemic and in those where it is not, such as the United Kingdom, where a large number of imported P. falciparum malaria cases are treated each year.In March 2007, a P. falciparum malaria patient treated at the HTD with quinine plus SP returned with recrudescent malaria and was confirmed as a case of therapeutic failure. It was hypothesized that quinine, a drug rapidly cleared from host circulation, had failed to eradicate all the parasites and that subsequent administration of long-lasting SP had selected a subpopulation of parasites resistant to antifolates that survived and subsequently caused a recrudescent infection. We report here a parasitological evaluation of this particular case and a concurrent survey of molecular markers of drug resistance in parasites from 44 HTD patients presenting with confirmed P. falciparum malaria over 12 months prior to the presentation of this case and in a further 39 isolates from P. falciparum malaria cases referred from all over the United Kingdom to the Health Protection Agency (HPA) Malaria Reference Laboratory (MRL) at the London School of Hygiene and Tropical Medicine.     

Colorimetric High-Throughput Screen for Detection of Heme Crystallization Inhibitors

Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay''s reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.Malaria poses an enormous public health burden, causing over 1 million fatalities annually worldwide, with the majority of morbidity and mortality attributed to Plasmodium falciparum malaria. Chloroquine (CQ) served as the main chemotherapeutic for several decades, but the emergence and spread of drug resistance has limited its current usefulness. After the introduction of every new antimalarial drug, with the exception of artemisinin, resistant malaria parasites have emerged (25). Hence, the continued development of new antimalarial drugs is necessary to continue to successfully treat malaria infection.The malaria parasite cycles between two hosts, namely, mosquitoes (Anopheles sp.) and humans. In the human host, after a brief liver stage, P. falciparum resides exclusively inside red blood cells, where it feeds on hemoglobin, reproduces, and then releases progeny, which invade new red blood cells. During this intraerythrocytic stage, proteases digest hemoglobin within the food vacuole, a lysosome-like organelle (9). As hemoglobin is digested, heme molecules, containing redox-active iron centers, are released. The parasite overcomes the oxidative stress thus produced through the crystallization of free heme molecules into hemozoin (20). Hemozoin crystals are formed in an enzyme-independent reaction that is essential for parasite survival and therefore an excellent target for antimalarial chemotherapy. CQ, the most successful antimalarial agent to date, accumulates in the food vacuole, where it inhibits heme crystallization and prevents parasite proliferation (18). Small-molecule disruption of hemozoin formation has been proposed as a mechanism of action of many other antimalarial agents, including mefloquine (MQ), amodiaquine (AMQ), quinine, and quinidine, since each of these drugs successfully inhibits heme crystallization in in vitro assays, and they are all structurally related to CQ.We believe that the development of antimalarial agents based on the physicochemical process of heme crystallization could identify molecules that are less likely to generate resistance, like CQ. Drug resistance usually entails expression changes or mutations in the target protein or similar changes in pumps in order to expel the antimalarial agent (22, 26). Since heme crystallization inhibitors do not target a protein but a physicochemical process, resistance can occur only through the latter approach. For example, CQ resistance is achieved through multiple mutations in the P. falciparum CQ resistance transporter 1 (pfcrt1) allele, which encodes a transmembrane protein that, when mutated, significantly reduces the concentration of CQ in the food vacuole (8). Since the heme crystallization pathway remains unaltered in resistant parasites, it is still possible to exploit parasite heme crystallization while avoiding cross-resistance with CQ.In this study, we have adapted the pyridine hemichrome inhibition of β-hematin (Phiβ) assay described by Ncokazi and Egan for high-throughput screening (HTS) (17). This assay recapitulates in vivo heme crystallization by solubilizing hematin and then allowing it to spontaneously form crystalline β-hematin (synthetically identical to hemozoin) within a 384-well plate (1). Pyridine is used as a developing reagent to monitor heme crystallization, as pyridine molecules coordinated with the iron centers of free heme molecules produce a concentration-dependent color change, with a strong absorption at 405 nm. The optimized cell-free heme crystallization screen (CFHCS) was used to verify the mode of action of known antimalarials and to identify new chemical entities that inhibit hemozoin crystal formation.     

Little Polymorphism at the K13 Propeller Locus in Worldwide Plasmodium falciparum Populations Prior to the Introduction of Artemisinin Combination Therapies

The emergence and spread of artemisinin-resistant Plasmodium falciparum is of huge concern for the global effort toward malaria control and elimination. Artemisinin resistance, defined as a delayed time to parasite clearance following administration of artemisinin, is associated with mutations in the Pfkelch13 gene of resistant parasites. To date, as many as 60 nonsynonymous mutations have been identified in this gene, but whether these mutations have been selected by artemisinin usage or merely reflect natural polymorphism independent of selection is currently unknown. To clarify this, we sequenced the Pfkelch13 propeller domain in 581 isolates collected before (420 isolates) and after (161 isolates) the implementation of artemisinin combination therapies (ACTs), from various regions of endemicity worldwide. Nonsynonymous mutations were observed in 1% of parasites isolated prior to the introduction of ACTs. Frequencies of mutant isolates, nucleotide diversity, and haplotype diversity were significantly higher in the parasites isolated from populations exposed to artemisinin than in those from populations that had not been exposed to the drug. In the artemisinin-exposed population, a significant excess of dN compared to dS was observed, suggesting the presence of positive selection. In contrast, pairwise comparison of dN and dS and the McDonald and Kreitman test indicate that purifying selection acts on the Pfkelch13 propeller domain in populations not exposed to ACTs. These population genetic analyses reveal a low baseline of Pfkelch13 polymorphism, probably due to purifying selection in the absence of artemisinin selection. In contrast, various Pfkelch13 mutations have been selected under artemisinin pressure.     

Evaluation of Antimalarial Resistance Marker Polymorphism in Returned Migrant Workers in China

Imported malaria has been a great challenge for public health in China due to decreased locally transmitted cases and frequent exchange worldwide. Plasmodium falciparum has been mainly responsible for the increasing impact. Currently, artesunate plus amodiaquine, one of the artemisinin combination therapies recommended by the World Health Organization, has been mainly used against uncomplicated P. falciparum malaria in China. However, drug resistance marker polymorphism in returning migrant workers has not been demonstrated. Here, we have evaluated the prevalence of pfmdr1 and pfcrt polymorphisms, as well as the K13 propeller gene, a molecular marker of artemisinin resistance, in migrant workers returned from Ghana to Shanglin County, Guangxi Province, China, in 2013. A total of 118 blood samples were randomly selected and used for the assay. Mutations of the pfmdr1 gene that covered codons 86, 184, 1034, and 1246 were found in 11 isolates. Mutations at codon N86Y (9.7%) were more frequent than at others, and Y₈₆Y₁₈₄S₁₀₃₄D₁₂₄₆ was the most prevalent (63.6%) of the four haplotypes. Mutations of the pfcrt gene that covered codons 74, 75, and 76 were observed in 17 isolates, and M₇₄N₇₅T₇₆ was common (70.6%) in three haplotypes. Eight different genotypes of the K13 propeller were first observed in 10 samples in China, 2 synonymous mutations (V487V and A627A) and 6 nonsynonymous mutations. C580Y was the most prevalent (2.7%) in all the samples. The data presented might be helpful for enrichment of molecular surveillance of antimalarial resistance and will be useful for developing and updating antimalarial guidance in China.     

Antimalarial Synergy of Cysteine and Aspartic Protease Inhibitors

It has been proposed that the Plasmodium falciparum cysteine protease falcipain and aspartic proteases plasmepsin I and plasmepsin II act cooperatively to hydrolyze hemoglobin as a source of amino acids for erythrocytic parasites. Inhibitors of each of these proteases have potent antimalarial effects. We have now evaluated the antimalarial effects of combinations of cysteine and aspartic protease inhibitors. When incubated with cultured P. falciparum parasites, cysteine and aspartic protease inhibitors exhibited synergistic effects in blocking parasite metabolism and development. The inhibitors also demonstrated apparent synergistic inhibition of plasmodial hemoglobin degradation both in culture and in a murine malaria model. When evaluated for the treatment of murine malaria, a combination of cysteine and aspartic protease inhibitors was much more effective than higher concentrations of either compound used alone. These results support a model whereby plasmodial cysteine and aspartic proteases participate in the degradation of hemoglobin, and they suggest that combination antimalarial therapy with inhibitors of the two classes of proteases is worthy of further study.Malaria is one of the most important infectious diseases in the world. Infections with Plasmodium falciparum, the most virulent human malaria parasite, are responsible for hundreds of millions of illnesses and over a million deaths per year (22). A major reason for the continued severity of the worldwide malaria problem is the increasing resistance of malaria parasites to available drugs (12). Thus, it is important to identify new targets for antimalarial therapy and to evaluate new modes of therapy directed against these targets.Potential new targets for antimalarial chemotherapy include parasite enzymes required for the degradation of hemoglobin. Erythrocytic malaria parasites degrade hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis (reviewed in references 5 and 16). The food vacuole of P. falciparum contains the cysteine protease falcipain and the aspartic proteases plasmepsin I and plasmepsin II (7, 8, 15). Each of these proteases degrades hemoglobin in vitro, and it has been proposed that the enzymes act in a concerted manner to hydrolyze globin to small peptides or free amino acids (5, 16). In a number of in vitro studies, inhibitors of both cysteine and aspartic proteases had potent effects against cultured malaria parasites (1, 4, 11, 14, 15, 17, 18, 20). In an in vivo study utilizing a murine malaria model, a peptidyl cysteine protease inhibitor cured Plasmodium vinckei-infected mice (14). However, high doses of this inhibitor (200 to 400 mg/kg of body weight/day) were required for a pronounced antimalarial effect.As cysteine and aspartic proteases appear to act cooperatively to degrade hemoglobin, and as inhibitors of both classes of proteases have antimalarial effects, it may be appropriate to use combinations of inhibitors to treat malaria. Such combination therapy might improve efficacy and also slow the development of resistance to new agents. We now report an evaluation of the in vitro and in vivo antimalarial effects of combinations of peptidyl cysteine and aspartic protease inhibitors. These combinations had strong, apparently synergistic inhibitory effects on plasmodial development and hemoglobin degradation in both cultured parasites and in a murine malaria model.     

The Antibiotic Micrococcin Is a Potent Inhibitor of Growth and Protein Synthesis in the Malaria Parasite

The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite.