Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.     

Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) β5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax. They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

Each year, Plasmodium falciparum (Pf) malaria infects at least 200 million people and causes more than 400,000 deaths (1). Alarmingly, after a decade of gains, progress in tackling malaria has plateaued (1). A major concern is that current antimalarial control is highly dependent on artemisinin-based combination therapies, which are associated with a cure failure rate of ∼50% in some regions in Southeast Asia (2). A rise in resistance-associated mutations in Rwanda (3) may presage spread of resistance to Africa. New compounds with potent activity against all stages of the parasite cycle are needed to feed the antimalarial drug development pipeline.As an organism that grows rapidly in oxidatively stressed niches, malaria parasites are particularly susceptible to compounds that compromise proteostasis. The Plasmodium proteasome is an experimentally validated drug target, with a number of studies showing that proteasome inhibitors rapidly kill parasites at different stages of development (4–12). Importantly, proteasome inhibitors strongly synergize artemisinin-mediated killing of Pf cultures (in vitro) and murine Plasmodium parasites (in vivo) (9–11).The proteasome plays an important role in cellular homeostasis—degrading abnormal, damaged, and short-lived proteins. The inside of the barrel-shaped 20S proteasome complex has six proteolytic active sites, comprising two copies each of three distinct types, termed β1 (caspase-like), β2 (trypsin-like), and β5 (chymotrypsin-like). Previous work showed that inhibition of Pf β5 activity is crucial for parasite killing (11, 13). By contrast, inhibition of human constitutive β5 activity is tolerated in nonmalignant cells, if β2c activity is maintained, thereby providing a therapeutic window (7, 13, 14).Medicines for Malaria Venture (MMV) has published Target Product Profiles for antimalarial compounds. They recommend that new compound classes show activity against all parasite stages and against drug-resistant parasites, preferably with a mechanism of action that is different from currently deployed compounds (15). New drugs should preferably act rapidly and exhibit pharmacokinetic (PK) properties that will enable complete parasite clearance, ideally with a single oral dose (15).We previously reported a screen of a library of peptidyl boronic acids inhibitors of the human proteasome and identified hits that inhibit parasite growth. These dipeptide inhibitors show a range of selectivities for inhibition of the growth of Pf compared with human cell lines (11) and exhibit similar physicochemical properties to bortezomib (VELCADE), which is dosed weekly, intravenously, or subcutaneously, in the clinical treatment of multiple myeloma (16, 17). While dipeptide boronates are considered to have favorable safety and efficacy profiles in a cancer setting, enhanced selectivity and oral bioavailability is desired for the treatment of malaria.Here, we describe a medicinal chemistry program that, coupled with structural biology and biochemical approaches, explores the potential of a series of amino-amide boronates (containing a single amide bond in the backbone) for development as antimalarials. We have identified compounds that exhibit rapid, potent, and selective action against Pf, including drug-resistant strains, and display oral efficacy in a mouse model of Pf malaria.     

Variations in frequencies of drug resistance in Plasmodium falciparum

Continual exposure of malarial parasite populations to different drugs may have selected not only for resistance to individual drugs but also for genetic traits that favor initiation of resistance to novel unrelated antimalarials. To test this hypothesis, different Plasmodium falciparum clones having varying numbers of preexisting resistance mechanisms were treated with two new antimalarial agents: 5-fluoroorotate and atovaquone. All parasite populations were equally susceptible in small numbers. However, when large populations of these clones were challenged with either of the two compounds, significant variations in frequencies of resistance became apparent. On one extreme, clone D6 from West Africa, which was sensitive to all traditional antimalarial agents, failed to develop resistance under simple nonmutagenic conditions in vitro. In sharp contrast, the Indochina clone W2, which was known to be resistant to all traditional antimalarial drugs, independently acquired resistance to both new compounds as much as a 1,000 times more frequently than D6. Additional clones that were resistant to some (but not all) traditional antimalarial agents acquired resistance to atovaquone at high frequency, but not to 5-fluoroorotate. These findings were unexpected and surprising based on current views of the evolution of drug resistance in P. falciparum populations. Such new phenotypes, named accelerated resistance to multiple drugs (ARMD), raise important questions about the genetic and biochemical mechanisms related to the initiation of drug resistance in malarial parasites. Some potential mechanisms underlying ARMD phenotypes have public health implications that are ominous.     

Bioinformatics analysis for structure and function of CPR of Plasmodium falciparum

ObjectiveTo analyse the structure and function of NADPH-cytochrome p450 reductase (CYPOR or CPR) from Plasmodium falciparum (Pf), and to predict its' drug target and vaccine target.MethodsThe structure, function, drug target and vaccine target of CPR from Plasmodium falciparum were analyzed and predicted by bioinformatics methods.ResultsPfCPR, which was older CPR, had close relationship with the CPR from other Plasmodium species, but it was distant from its hosts, such as Homo sapiens and Anopheles. PfCPR was located in the cellular nucleus of Plasmodium falciparum. 335aa–352aa and 591aa – 608aa were inserted the interior side of the nuclear membrane, while 151aa–265aa was located in the nucleolus organizer regions. PfCPR had 40 function sites and 44 protein–protein binding sites in amino acid sequence. The teriary structure of 1aa–700aa was forcep–shaped with wings. 15 segments of PfCPR had no homology with Homo sapien CPR and most were exposed on the surface of the protein. These segments had 25 protein–protein binding sites. While 13 other segments all possessed function sites.ConclusionsThe evolution or genesis of Plasmodium falciparum is earlier than those of Homo sapiens. PfCPR is a possible resistance site of antimalarial drug and may involve immune evasion, which is associated with parasite of sporozoite in hepatocytes. PfCPR is unsuitable as vaccine target, but it has at least 13 ideal drug targets.     

Small-molecule histone methyltransferase inhibitors display rapid antimalarial activity against all blood stage forms in Plasmodium falciparum

Epigenetic factors such as histone methylation control the developmental progression of malaria parasites during the complex life cycle in the human host. We investigated Plasmodium falciparum histone lysine methyltransferases as a potential target class for the development of novel antimalarials. We synthesized a compound library based upon a known specific inhibitor (BIX-01294) of the human G9a histone methyltransferase. Two compounds, BIX-01294 and its derivative TM2-115, inhibited P. falciparum 3D7 parasites in culture with IC₅₀ values of ∼100 nM, values at least 22-fold more potent than their apparent IC₅₀ toward two human cell lines and one mouse cell line. These compounds irreversibly arrested parasite growth at all stages of the intraerythrocytic life cycle. Decrease in parasite viability (>40%) was seen after a 3-h incubation with 1 µM BIX-01294 and resulted in complete parasite killing after a 12-h incubation. Additionally, mice with patent Plasmodium berghei ANKA strain infection treated with a single dose (40 mg/kg) of TM2-115 had 18-fold reduced parasitemia the following day. Importantly, treatment of P. falciparum parasites in culture with BIX-01294 or TM2-115 resulted in significant reductions in histone H3K4me3 levels in a concentration-dependent and exposure time-dependent manner. Together, these results suggest that BIX-01294 and TM2-115 inhibit malaria parasite histone methyltransferases, resulting in rapid and irreversible parasite death. Our data position histone lysine methyltransferases as a previously unrecognized target class, and BIX-01294 as a promising lead compound, in a presently unexploited avenue for antimalarial drug discovery targeting multiple life-cycle stages.     

Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design

Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, signal peptide peptidase (SPP), have high selectivity and potency for P. falciparum. By using a variety of methodologies, we validate that SPP inhibitors target P. falciparum SPP in parasites, disrupt the protein’s ability to facilitate degradation of unstable proteins, and inhibit its proteolytic activity. These compounds also show low nanomolar activity against liver-stage malaria parasites and are also equipotent against a panel of pathogenic protozoan parasites. Collectively, these data suggest ER quality control as a vulnerability of protozoan parasites, and that SPP inhibition may represent a suitable transmission blocking antimalarial strategy and potential pan-protozoan drug target.     

Cysteine proteinase inhibitors block early steps in hemoglobin degradation by cultured malaria parasites

Erythrocytic malaria parasites degrade hemoglobin as a source of amino acids for parasite protein synthesis. Cysteine proteinase inhibitors have been shown to block the hydrolysis of globin by cultured parasites, indicating that a malarial cysteine proteinase is required for this process. In the present study, we have evaluated the role of parasite proteinases in earlier steps of hemoglobin degradation, namely the disassociation of the hemoglobin tetramer and the separation of heme from globin. Hemoglobin did not spontaneously denature or release heme under the pH and reducing conditions of the malarial food vacuole, suggesting that parasite enzymatic activity is necessary for early steps in hemoglobin degradation. The incubation of cultured parasites with cysteine proteinase inhibitors inhibited the denaturation of hemoglobin and the release of heme from globin. These results suggest that, in addition to its role in globin hydrolysis, a malarial cysteine proteinase participates in the dissociation of the hemoglobin tetramer and the release of heme from globin. Thus, the malarial cysteine proteinase is a promising target for antimalarial chemotherapy.     

Changing ideas on chloroquine in Plasmodium falciparum

For 40 years scientists have hotly debated the questions of how chloroquine kills malarial parasites and how resistance to this once first-line antimalarial drug has evolved. While an end to these debates is not in sight, as a result of the complexity of the subject, new findings have come forward that give the discussion a new direction. In this paper we will summarize current knowledge on chloroquine's antimalarial mode of action and the genesis of the resistant phenotype in the human malarial parasite Plasmodium falciparum, with special emphasis on the most recent developments in this field.     

The hexose transporter of Plasmodium falciparum is a worthy drug target

Despite substantial efforts at control over several decades, malaria is still a major global health problem as chemotherapy of malaria parasites is limited by established drug resistance and lack of novel treatment options. Intraerythrocytic stages of these parasites are wholly dependent upon host glucose for energy and malarial proteins involved in hexose permeation are therefore attractive new drug targets. For Plasmodium falciparum, the causative agent of severe malaria, a facilitative hexose transporter (PfHT), encoded by a single-copy gene mediates glucose uptake. We first established heterologous expression in Xenopus laevis to allow functional characterisation of PfHT. This review describes the value of using Xenopus oocytes in heterologous studies of P. falciparum-encoded proteins and summarises the properties of PfHT. Comparisons between Gluts (mammalian facilitative hexose transporters) and PfHT using this expression system have highlighted important mechanistic and structural differences between parasite and host proteins. Certain O-methyl derivatives of glucose proved particularly useful discriminators between mammalian transporters and PfHT. We exploited this selectivity and synthesised a long-chain O-3-hexose derivative (compound 3361) that potently inhibits PfHT expressed in oocytes and also kills P. falciparum when it is cultured in medium containing either glucose or fructose as a carbon source. To extend our observations to the second most important human malarial pathogen, we have cloned and expressed the Plasmodium vivax orthologue of PfHT, and demonstrate inhibition of glucose uptake by compound 3361. These findings validate malarial hexose transporters as a novel target. We now aim to design a new class of antimalarials by the discovery of highly specific inhibitors which could act with a broad spectrum of action on different Plasmodium spp. infections.     

Malaria Risk Mapping for Control in the Republic of Sudan

Evidence shows that malaria risk maps are rarely tailored to address national control program ambitions. Here, we generate a malaria risk map adapted for malaria control in Sudan. Community Plasmodium falciparum parasite rate (PfPR) data from 2000 to 2010 were assembled and were standardized to 2–10 years of age (PfPR_2–10). Space-time Bayesian geostatistical methods were used to generate a map of malaria risk for 2010. Surfaces of aridity, urbanization, irrigation schemes, and refugee camps were combined with the PfPR_2–10 map to tailor the epidemiological stratification for appropriate intervention design. In 2010, a majority of the geographical area of the Sudan had risk of < 1% PfPR_2–10. Areas of meso- and hyperendemic risk were located in the south. About 80% of Sudan''s population in 2011 was in the areas in the desert, urban centers, or where risk was < 1% PfPR_2–10. Aggregated data suggest reducing risks in some high transmission areas since the 1960s.     

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.Malaria is a global health problem with 214 million new cases of malaria and 438,000 deaths reported in 2015, mostly in sub-Saharan Africa (1). The endoperoxide class of antimalarial drugs, such as artemisinin (ART), is the first line of defense against malaria infection against a backdrop of multidrug-resistant parasites (2) and lack of effective vaccines (3, 4). Given the effectiveness of the ART class, the question arises: how do these drugs kill parasites? A suggested mechanism of action involves the cleavage of the endoperoxide bridge by a source of Fe²⁺ or heme. This cleavage results in the formation of oxyradicals that rearrange into primary or secondary carbon-centered radicals. These radicals have been proposed to alkylate parasite proteins that somehow result in the death of the parasite (5). However, this proposal remains a subject of intense debate (6, 7), while these alkylated proteins are yet to be formally identified. So far, the proposed targets of ART action include a PfATP6 enzyme, the Plasmodium falciparum ortholog of mammalian sarcoendoplasmic reticulum Ca²¹-ATPases (SERCAs) (5), translational controlled tumor protein, and heme (5). Additionally, Haynes et al. (8) proposed that ART may act by impairing parasite redox homeostasis as a consequence of an interaction between the drug and flavin adenine dinucleotide (FADH) and/or other parasite flavoenzymes in the parasite, leading to the generation of reactive oxygen species (ROS). New approaches are required for definitive identification of ART molecular targets. This insight into the drug activation-dependent mechanism of action will be invaluable in the target-led development of more potent drugs with the potential to circumvent the emergence of resistance to current first-line ART-based therapies. The goal of this study was to identify ART-targeted proteins and their interacting partners in P. falciparum. We recently adopted a proteomic approach developed by Speers and Cravatt (9) to synthesize a suite of pyrethroid activity-based protein profiling probes (ABPPs) (10). Using alkyne/azide-coupling partners through “click chemistry,” we identified several cytochrome P450 enzymes that metabolized deltamethrin in rat liver microsomes (10). More recently, a chemical proteomic approach was developed to identify parasite proteins targeted by an albitiazolium antimalarial drug candidate in situ using a photoactivation cross-linking approach (11). However, this generic approach can introduce significant promiscuity in the proteins tagged based on the intracompartmental distribution of drug independent of actual mechanisms.Here, we introduced the design and synthesis of click chemistry-compatible activity-based probes incorporating the endoperoxide scaffold of ART as a warhead to alkylate and identified the ART molecular target(s) in asexual stages of the malaria parasite (Fig. 1). A major advantage of this strategy is that the reporter tags are introduced under “click” reaction conditions performed after the drug has achieved its biological effects, enabling purification, identification, and quantification of alkylated parasite’s proteins and their interacting partners as shown in Fig. 1B. To avoid nonspecific probe-dependent tagging, a common limitation of these approaches, we generated the respective “control” nonperoxide partners to improve the specificity and biological relevance of our resultant tagged protein list.Open in a separate windowFig. 1.Rational design of the ART-ABPPs. (A) Conversion of ART to ART-ABPPs involves the addition of a clickable handle (i.e., an alkyne or azide to the ART drug pharmacophore by the peptide-coupling method illustrated in SI Text). The structures of the alkyne (P1) and azide (P2) probes and respective inactive deoxy controls CP1 and CP2 with in vitro IC₅₀ values are presented. (B) General workflow of copper-catalyzed and copper-free click chemistry approaches used in the identification of alkylated proteins after in situ treatment of P. falciparum parasite with alkyne and azide ART-ABPPs. The azide- and alkyne-modified proteins are tagged with biotin azide and biotin dibenzocyclooctyne (Biotin-DIBO), respectively, via click reactions followed by affinity purification tandem with LC-MS/MS for protein identification.     

Prevailing Plasmodium falciparum dihydrofolate reductase 108-asparagine in Hodeidah,Yemen: A questionable sulfadoxine–pyrimethamine partner within the artemisinin-based combination therapy

Given that the evolution and spread of resistance to sulfadoxine–pyrimethamine (SP) have been documented at a quick pace worldwide, the present study investigated the mutant Plasmodium falciparum dihydrofolate reductase 108-asparagine (dhfr 108N) as a key marker of resistance to the combination among parasite isolates from Hodeidah. The association of parasitologic indices with the dhfr 108N mutant allele was also studied. Ninety patients with microscopically confirmed P. falciparum infection from Hodeidah were included in the present study. Polymerase chain reaction-restriction fragment length polymorphism approach was adopted for the molecular detection of this marker. The dhfr 108N was detected among about 61% of P. falciparum isolates, in its pure and mixed-type forms, from Hodeidah. Age, gender and residence of patients were not significant predictors for the presence of the mutant allele among parasite isolates. In contrast, a history of malaria and antimalarial drug intake in the year preceding the study as well as frequent antimalarial drug intake were significantly associated with this mutant allele. The high frequency of dhfr 108N among parasites isolates makes the role of SP questionable as a partner with outstanding effectiveness within the ACT, at least, in the near future. SP plus artesunate should be monitored for its antimalarial efficacy at regular intervals, preferably through the molecular detection of resistance-associated mutations.     

Gene selective mRNA cleavage inhibits the development of Plasmodium falciparum

Unique peptide-morpholino oligomer (PMO) conjugates have been designed to bind and promote the cleavage of specific mRNA as a tool to inhibit gene function and parasite growth. The new conjugates were validated using the P. falciparum gyrase mRNA as a target (PfGyrA). Assays in vitro demonstrated a selective degradation of the PfGyrA mRNA directed by the external guide sequences, which are morpholino oligomers in the conjugates. Fluorescence microscopy revealed that labeled conjugates are delivered into Plasmodium-infected erythrocytes during all intraerythrocytic stages of parasite development. Consistent with the expression of PfGyrA in all stages of parasite development, proliferation assays showed that these conjugates have potent antimalarial activity, blocking early development, maturation, and replication of the parasite. The conjugates were equally effective against drug sensitive and resistant P. falciparum strains. The potency, selectivity, and predicted safety of PMO conjugates make this approach attractive for the development of a unique class of target-specific antimalarials and for large-scale functional analysis of the malarial genome.     

Morphological effects of pyronaridine on malarial parasites

The ultrastructural changes caused by a new antimalarial drug, pyronaridine, were investigated using mice infected with erythrocytic forms of Plasmodium berghei and P. falciparum cultivated in vitro in human erythrocytes. The first changes observed in both parasites after exposure to pyronaridine occurred in the food vacuoles. This suggests that the target organelle of this drug may be the food vacuole of malarial parasites. In addition, rapid alterations were also noted within the pellicular complex of both plasmodia.     

Inhibition of Cytochrome bc1 as a Strategy for Single-Dose,Multi-Stage Antimalarial Therapy

Single-dose therapies for malaria have been proposed as a way to reduce the cost and increase the effectiveness of antimalarial treatment. However, no compound to date has shown single-dose activity against both the blood-stage Plasmodium parasites that cause disease and the liver-stage parasites that initiate malaria infection. Here, we describe a subset of cytochrome bc₁ (cyt bc₁) inhibitors, including the novel 4(1H)-quinolone ELQ-400, with single-dose activity against liver, blood, and transmission-stage parasites in mouse models of malaria. Although cyt bc₁ inhibitors are generally classified as slow-onset antimalarials, we found that a single dose of ELQ-400 rapidly induced stasis in blood-stage parasites, which was associated with a rapid reduction in parasitemia in vivo. ELQ-400 also exhibited a low propensity for drug resistance and was active against atovaquone-resistant P. falciparum strains with point mutations in cyt bc₁. Ultimately, ELQ-400 shows that cyt bc₁ inhibitors can function as single-dose, blood-stage antimalarials and is the first compound to provide combined treatment, prophylaxis, and transmission blocking activity for malaria after a single oral administration. This remarkable multi-stage efficacy suggests that metabolic therapies, including cyt bc₁ inhibitors, may be valuable additions to the collection of single-dose antimalarials in current development.     

Artemisinin resistance or tolerance in human malaria patients

Malaria is a major cause of morbidity and mortality in the developing world. This situation is mainly due to emergence of resistance to most antimalarial drugs currently available. Artemisinin-based combination treatments are now first-line drugs for Plasmodium falciparum (P. falciparum) malaria. Artemisinin (qinghaosu) and its derivatives are the most rapid acting and efficacious antimalarial drugs. This review highlights most recent investigations into the emergence of artemisinin resistance in falciparum malaria patients on the Thai-Cambodian border, a historical epicenter for multidrug resistance spread spanning over 50 years. The study presents the first evidence that highlights the parasites reduced susceptibility to artemisinin treatment by prolonged parasite-clearance times, raising considerable concern on resistance development. Although the exact mechanism of action remains unresolved, development of resistance was proposed based from both in vitro experiments and human patients. Lines of evidence suggested that the parasites in the patients are in dormant forms, presumably tolerate to the drug pressure. The World Health Organization has launched for prevention and/or containment of the artemisinin-resistant malaria parasites. Taken together, the emergence of artemisinin resistance to the most potent antidote for falciparum malaria, poses a serious threat to global malaria control and prompts renewed efforts for urgent development of new antimalarial weapons.     

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC(50)), assessed by parasite incorporation of [(3)H]hypoxanthine, were between 0.2 and 1.1 microM. CLT concentrations of 2 microM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (approximately 48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 microM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.     

Potent antimalarial and transmission-blocking activities of centanamycin, a novel DNA-binding agent

Most treatments for malaria target the blood stage of infection in the human host, although few can also block transmission of the parasite to the mosquito. We show here that the compound centanamycin is very effective against blood-stage malarial infections in vitro and in vivo and has profound effects on sexual differentiation of the parasites in mosquitoes. After drug treatment, parasite development is arrested within the midguts of mosquitoes, failing to produce the infective forms that migrate to the salivary glands. The mechanism of parasite death is associated with modification of Plasmodium genomic DNA. We detected DNA damage in parasites isolated from mice 24 h after treatment with centanamycin, and, importantly, we also detected this DNA damage in parasites within mosquitoes that had fed on these mice 10 days earlier. This demonstrates that damage to parasite DNA during blood-stage infection persists from the vertebrate to the mosquito host and provides a novel biochemical strategy to block malaria transmission.     

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.     

Longitudinal seroepidemiologic study of the 2009 pandemic influenza A (H1N1) infection among health care workers in a children's hospital

Background

The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum.

Methods

A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope.

Results

Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts.

Conclusion

Collectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials.