Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Towards an in vitro model of Plasmodium hypnozoites suitable for drug discovery

Background

Amongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites.

Methodology

P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured.

Results

The non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine.

Conclusion

Resistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs.     

Plasmodium falciparum Carbonic Anhydrase is a Possible Target for Malaria Chemotherapy

Plasmodium falciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the α class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the α-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having α-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.     

Quality of Antimalarial Drugs and Antibiotics in Papua New Guinea: A Survey of the Health Facility Supply Chain

Background

Poor-quality life-saving medicines are a major public health threat, particularly in settings with a weak regulatory environment. Insufficient amounts of active pharmaceutical ingredients (API) endanger patient safety and may contribute to the development of drug resistance. In the case of malaria, concerns relate to implications for the efficacy of artemisinin-based combination therapies (ACT). In Papua New Guinea (PNG), Plasmodium falciparum and P. vivax are both endemic and health facilities are the main source of treatment. ACT has been introduced as first-line treatment but other drugs, such as primaquine for the treatment of P. vivax hypnozoites, are widely available. This study investigated the quality of antimalarial drugs and selected antibiotics at all levels of the health facility supply chain in PNG.

Methods and Findings

Medicines were obtained from randomly sampled health facilities and selected warehouses and hospitals across PNG and analysed for API content using validated high performance liquid chromatography (HPLC). Of 360 tablet/capsule samples from 60 providers, 9.7% (95% CI 6.9, 13.3) contained less, and 0.6% more, API than pharmacopoeial reference ranges, including 29/37 (78.4%) primaquine, 3/70 (4.3%) amodiaquine, and one sample each of quinine, artemether, sulphadoxine-pyrimethamine and amoxicillin. According to the package label, 86.5% of poor-quality samples originated from India. Poor-quality medicines were found in 48.3% of providers at all levels of the supply chain. Drug quality was unrelated to storage conditions.

Conclusions

This study documents the presence of poor-quality medicines, particularly primaquine, throughout PNG. Primaquine is the only available transmission-blocking antimalarial, likely to become important to prevent the spread of artemisinin-resistant P. falciparum and eliminating P. vivax hypnozoites. The availability of poor-quality medicines reflects the lack of adequate quality control and regulatory mechanisms. Measures to stop the availability of poor-quality medicines should include limiting procurement to WHO prequalified products and implementing routine quality testing.     

Effects of novel triple-stage antimalarial ionic liquids on lipid membrane models

Primaquine-based ionic liquids, obtained by acid-base reaction between parent primaquine and cinnamic acids, were recently found as triple-stage antimalarial hits. These ionic compounds displayed significant activity against both liver- and blood-stage Plasmodium parasites, as well as against stage V P. falciparum parasites. Remarkably, blood-stage activity of the ionic liquids against both chloroquine-sensitive (3D7) and resistant (Dd2) P. falciparum strains was clearly superior to those of the respective covalent (amide) analogues and of parent primaquine. Having hypothesized that such behaviour might be ascribed to an enhanced ability of the ionic compounds to permeate into Plasmodium-infected erythrocytes, we have carried out a differential scanning calorimetry-based study of the interactions between the ionic liquids and membrane models. Results provide evidence, at the molecular level, that the primaquine-derived ionic liquids may contribute to an increased permeation of the parent drug into malaria-infected erythrocytes, which has relevant implications towards novel antimalarial approaches based on ionic liquids.     

Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil

Background

Chloroquine (CQ) or sulfadoxine-pyrimethamine (SP) monotherapy for Plasmodium falciparum often leads to therapeutic failure in Indonesia. Combining CQ with other drugs, like SP, may provide an affordable, available and effective option where artemisinin-combined therapies (ACT) are not licensed or are unavailable.

Methods

This study compared CQ (n = 29 subjects) versus CQ + SP (with or without primaquine; n = 88) for clinical and parasitological cure of uncomplicated falciparum malaria in the Menoreh Hills region of southern Central Java, Indonesia. Gametocyte clearance rates were measured with (n = 56 subjects) and without (n = 61) a single 45 mg dose of primaquine (PQ).

Results

After 28 days, 58% of subjects receiving CQ had cleared parasitaemia and remained aparasitaemic, compared to 94% receiving CQ combined with SP (p < 0.001). Msp-2 genotyping permitted reinfection-adjusted cure rates for CQ and CQ combined with SP, 70% and 99%, respectively (p = 0.0006).

Conclusion

Primaquine exerted no apparent affect on cure of asexual stage parasitaemia, but clearly accelerated clearance of gametocytes. CQ combined with SP was safe and well-tolerated with superior efficacy over CQ for P. falciparum parasitaemia in this study.     

Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a γ-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-β- -arabinofuranosyladenine-5′-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2′,3′-dideoxythymidine-5′-triphosphate (IC₅₀>400 μM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase γ. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC₅₀>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).     

Primaquine Administration after Falciparum Malaria Treatment in Malaria Hypoendemic Areas with High Incidence of Falciparum and Vivax Mixed Infection: Pros and Cons

Mixed infections of Plasmodium falciparum and Plasmodium vivax is high (~30%) in some malaria hypoendemic areas where the patients present with P. falciparum malaria diagnosed by microscopy. Conventional treatment of P. falciparum with concurrent chloroquine and 14 days of primaquine for all falciparum malaria patients may be useful in areas where mixed falciparum and vivax infections are high and common and also with mild or moderate G6PD deficiency in the population even with or without subpatent vivax mixed infection. It will be possibly cost-effective to reduce subsequent vivax illness if the patients have mixed vivax infection. Further study to prove this hypothesis may be warranted.     

Imidazolidin-4-one peptidomimetic derivatives of primaquine: synthesis and antimalarial activity

The synthesis of imidazolidin-4-one derivatives of primaquine containing the five-membered ring at the C-terminus of a dipeptide backbone coupled to the parent drug is described. These peptidomimetic derivatives were active against a chloroquine-resistant Plasmodium falciparum strain and inhibited the development of the sporogonic cycle of Plasmodium berghei, affecting the appearance of oocysts in the midguts of the mosquitoes. The novel imidazolidin-4-ones are extremely stable, both in human plasma and in pH 7.4 buffer, as a result of N¹-acylation. Thus, ‘internal’ imidazolidin-4-ones derived from dipeptidyl 8-aminoquinolines represent a new entry in antimalarial structure–activity relationships.     

Antigenic Relationship Between Plasmodium falciparum and Babesia bovis: Reactivity with Antibodies to Culture-Derived Soluble Exoantigens1

Antigenic similarities between Plasmodium and Babesia parasites of the phylum Apicomplexa have been previously demonstrated primarily by the serological cross reactivity observed in the indirect fluorescent antibody (IFA) test. We have now studied the antigenic relationship between the human malaria parasite, Plasmodium falciparum, and the hemoparasitic agent of cattle, Babesia bovis, using rabbit monospecific antibodies produced against individual culture-derived P. falciparum polypeptides and bovine polyspecific antibodies to B. bovis exoantigens. These respective antibodies were found to be distinctly cross reactive in the IFA test using infected erythrocytes (squirrel monkey—P. falciparum; bovine—B. bovis) as antigen substrates. Immunofluorescence was shown to be highly specific for parasite surfaces. Additionally, the degree of reactivity with soluble exoantigens contained in Plasmodium and Babesia culture supernatants was monitored by a two-site enzyme immunoassay employing the cross-reactive antibodies. Further evidence for antigenic cross reactivity between P. falciparum and B. bovis parasites was shown with the in vitro inhibition assay. Antibodies to P. falciparum and B. bovis were found to be highly inhibitory for the in vitro growth of P. falciparum in human erythrocytes.     

Plasmodium falciparum: Association with Erythrocytic Superoxide Dismutase1

Levels of superoxide dismutase (SOD) activity and its properties in Plasmodium falciparum-infected erythrocytes, isolated parasites, and noninfected erythrocytes were studied. A higher specific activity was found in P. falciparum-infected erythrocytes compared to noninfected erythrocytes, resulting from the lower protein content of infected cells and not enzyme synthesis by the parasite, as the superoxide dismutase activity expressed per number of cells was decreased. Superoxide dismutase from noninfected erythrocytes and isolated P. falciparum parasites showed similar sensitivities to various inhibitors and had identical molecular weights and electrophoretic mobilities. These results support the hypothesis of uptake and use of the erythrocytic SOD enzyme by the parasite as a possible mechanism of defense against oxidative stress.     

Comparative Studies on Dihydrofolate Reductases from Plasmodium falciparum and Aotus trivirgatus*

SYNOPSIS Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus). were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCI and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower K_i for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethaminesensitive strains of P. falciparum were found to be similar.     

Further Studies on the Effects of Antipantothenates on Malaria Parasites (Plasmodium coatneyi and P. falciparum) in vitro*

SYNOPSIS. Plasmodium coatneyi and P. falciparum were maintained in host erythrocyte suspensions in vitro under conditions supporting one cycle of development with reinvasion occurring after 2 days. Inhibitory effects were then assessed for a series of analogs of pantothenic acid. In compounds of the general formula R-sulfamoylethyl-2,4-dihydroxy-3,3'-dimethyl butyramide antimalarial activity was strongly influenced by the nature of the R group. Most active was the compound with R = methoxy-quinolyl; this material had an in vitro activity approaching that of primaquine. If R was chloro- or dichlorophenyl, activity was much greater than if it was fluoro- or difluorophenyl. Small changes at the other end of the molecule had less effect; addition of a methyl group to give the valeramide, or omission of the 2-hydroxy did not change activity. Sulfonylethyl and thio-ethyl compounds were less active than the sulfamoylethyl compounds.     

Beyond Schuh: early studies on the oxidation of LDL and other lipoproteins and its role in atherosclerosis

Abstract

The malaria parasite Plasmodium falciparum is still a major threat to human health in the non-industrialised world mainly due to the increasing incidence of drug resistance. Therefore, there is an urgent need to identify and validate new potential drug targets in the parasite's metabolism that are suitable for the design of new anti-malarial drugs. It is known that infection with P. falciparum leads to increased oxidative stress in red blood cells, implying that the parasite requires efficient antioxidant and redox systems to prevent damage caused by reactive oxygen species. In recent years, it has been shown that P. falciparum possess functional thioredoxin and glutathione systems. Using genetic and chemical tools, it was demonstrated that thioredoxin reductase, the first step of the thioredoxin redox cycle, and γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step of glutathione synthesis, are essential for parasite survival. Indeed, the mRNA levels of γ-GCS are elevated in parasites that are oxidatively stressed, indicating that glutathione plays an important antioxidant role in P. falciparum. In addition to this antioxidant function, glutathione is important for detoxification processes and is possibly involved in the development of resistance against drugs such as chloroquine.     

Homology Modeling of Falcipain-2: Validation,De Novo Ligand Design and Synthesis of Novel Inhibitors

Abstract

Increasing resistance of malaria parasites, in particular Plasmodiun falciparum, demands a serious search for novel targets. Cysteine protease in P. falciparum, encoded by a previously unidentified gene falcipain 2, provides one such target to design chemotherapeutic agents for treatment of malaria. In fact, a few cysteine protease inhibitors have been shown to inhibit growth of cultured malarial parasites. In absence of a crystal structure for this enzyme, homology modeling proved to be a reasonable alternative to study binding requirements of the enzyme. A homology model for falcipain 2 was developed and validated by docking of known vinyl sulfone inhibitors. Further, based on the observations of these studies, novel isoquinoline inhibitors were designed and synthesized, which exhibited in vitro enzyme inhibition at micromolar concentrations.     

Characterization of the Plasmodium falciparum and P. berghei glycerol 3‐phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast

Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti‐malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3‐phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N‐terminal targeting sequence to GFP and 3′ tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site‐directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3‐phosphate acyltransferase in malaria parasites.     

Lipid peroxidation and its repair in malaria parasites

During its life cycle, the human malaria parasite Plasmodium falciparum is subjected to elevated levels of oxidative stress that cause damage to membrane lipids, a process referred to as lipid peroxidation. Control and repair of lipid peroxidation is critical for survival of P. falciparum. Here, we present an introduction into lipid peroxidation and review the current knowledge about the control and repair of the damage caused by lipid peroxidation in P. falciparum blood stages. We also review the recent identification of host peroxiredoxin 6 (PRDX6), as a key lipid-peroxidation-repair enzyme in P. falciparum blood stages. Such critical host factors provide novel targets for development of drugs against malaria.     

Formation of catalytically active cross-species heterodimers of thymidylate synthase from <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> and <Emphasis Type="Italic">Plasmodium vivax</Emphasis>

Thymidylate synthase (TS) of Plasmodium dihydrofolate reductase-thymidylate synthase (DHFR-TS) functions as a homodimeric enzyme with two active sites located near the subunit interface. The dimerization is essential for catalysis, since the active site of each subunit contains amino acid residues contributed from the other TS domain. In P. falciparum DHFR-TS, it has been shown that the active sites require Cys-490 from one domain and Arg-470 donated from the other domain. Mutants of these two series can complement one another giving rise to active enzyme. Here, the potential to form cross-species heterodimers between P. falciparum and P. vivax TS has been explored. Formation of cross-species heterodimer was tested by co-transformation of TS-inactive Cys-490 mutants of P. falciparum or P. vivax with corresponding TS-inactive Arg-486 mutants of P. vivax or P. falciparum into thymidine-requiring Escherichia coli. Active heterodimers were detected by subunit complementation and 6-[³H]-FdUMP binding assays. All combinations of the mutants tested, except for (Pf)R470A+(Pv)C506Y, were able to form catalytically active cross-species heterodimers. The single active site formed by (Pf)R470D+(Pv)C506Y and (Pv)R486D+(Pf)C490A pairs of cross-species heterodimers has k _cat and K _m values similar to those of intra-species heterodimers of P. falciparum and P. vivax. This is the first report to demonstrate that the TS subunit interface between Plasmodium species is sufficiently conserved to allow formation of fully active cross-species heterodimer.