Effects of piperaquine, chloroquine, and amodiaquine on drug uptake and of these in combination with dihydroartemisinin against drug-sensitive and -resistant Plasmodium falciparum strains.

Piperaquine is being developed as a long-acting component in artemisinin combination therapies. It was highly active in vitro and drug interaction studies showed that dihydroartemisinin combinations with piperaquine, chloroquine, and amodiaquine were indifferent tending toward antagonism. Competitive uptake of radiolabeled chloroquine and dihydroartemisinin in combination with other antimalarials was observed

Improved synchronous production of Plasmodium falciparum gametocytes in vitro.

The sexual stages of the Plasmodium falciparum life cycle are attractive targets for vaccines and transmission blocking drugs. Difficulties in culturing and obtaining large amounts of sexual stage P. falciparum parasites, particularly early stages, have often limited research progress in this area. We present a new protocol which simplifies the process of stimulating gametocytogenesis leading to improved synchronous gametocyte production. This new method can be adapted to enrich for early stage gametocytes (I and II) with a higher degree of purity than has previously been achieved, using MACS magnetic affinity columns. The protocol described lends itself to large scale culturing and harvesting of synchronous parasites suitable for biochemical assays, northern blots, flow cytometry, microarrays and proteomic analysis

Drug uptake, sensitivity, interaction studies and resistance associated mutations in Plasmodium falciparum

Evidence is accumulating that polymorphisms in plmdr 1 and plcrt are involved in resistance to the quinoline based blood schizontocides and possibly artemisinin derivatives; and mutations in the cytochrome b gene (cytb) are strongly associated with resistance to atovaquone. With this in mind, we studied sensitivity, drug interactions and uptake in a range of Plasmodium lalciparum lines, including pfmdr 1 transfectants, plcrt mutated lines 106/1 and K76I, and an atovaquone-resistant isolate (NGATVOl) with a unique point mutation (tyr268asn). In vitro susceptibility studies and genetic characterisation supported the role of plmdr 1 and plert polymorphisms in the sensitivity of parasite lines to a range of structurally diverse antimalarials. Mutations in plcrt conferred chloroquine resistance modulated by changes in plmdr 1, while the wild-type genes were associated with reduced sensitivity to mefloquine, halofantrine, lumefantrine and dihydroartemisinin. Drug interaction studies in vitro using a modified isobologram method showed that dihydroartemisinin in combination with chloroquine, amodiaquine or the new bisquinoline piperaquine was antagonistic in all parasite lines examined. The response was synergistic when the drug was combined with mefloquine, halofantrine or lumefantrine against chloroquine-sensitive (wild-type plmdrl and plcrt), but additive against chloroquine-resistant parasite lines. However, in the 7G8-m<fIP10 transfectant (wild-type plmdrl and mutated plert), synergy was shown between each of these three drugs and dihydroartemisinin. These results suggest that the interaction profile of dihydroartemisinin with arylaminoa1cohols depends on mutations in plmdrl in the presence of other chloroquine resistance mutations. In the D 1 0_mdr7G8 transfectant, the introduction of the mutated plmdr 1 converted the synergy between lumefantrine and dihydroartemisinin or artemether to addition; supporting the idea that interactions between artemisinin-derivatives and lumefantrine are dependent on mutations within pfmdr 1 alone. An exception was seen when dihydroartemisinin was combined with arylaminoa1cohol quinine. It showed a strain-specific effect not related to resistance polymorphisms, although a similar change to synergy in the 7G8-md~10 transfectant as for the other arylaminoalcohols was seen. A verapamil-reversible change in the quinine and dihydroartemisinin interaction from additive to synergistic was also noted with the introduction of a mutated PfCRT codon 76 in K76I. An antagonistic effect of dihydroartemisinin with pyrimethamine, was observed in all pyrimethamine-sensitive parasites, but the effect was additive in pyrimethamine-resistant parasites. In contrast to the quinolines, this was not associated with inhibition of eH]-dihydroartemisinin uptake. The interaction between atovaquone and proguanil was strongly synergistic in atovaquone-sensitive lines Kl and T996 and moderately synergistic in the atovaquoneresistant NGATVOI isolate. While the interaction between atovaquone and dihydroartemisinin was additive in the NGATVOI isolate, the interaction was antagonistic in the atovaquone-sensitive strains tested. Mutations in plmdr 1 and plert had no effect on uptake of eH]-dihydroartemisinin, although eH]-chloroquine accumulation was strongly affected by these polymorphisms. Uptake studies in combination with other antimalarials indicated that some of the quinoline-related drugs and arternisinin-derivatives competed with the uptake of the two radiolabelled drugs. This work highlights the important role of drug resistance polymorphisms in sensitivity, interaction and uptake of structurally diverse antimalarials. The unusual pattern of antagonism between dihydroartemisinin and pyrimethamine seen in vitro is supported by observations in vivo with artemisinin in rodent parasites. These and other differing interactions between dihydroartemisinin and antimalarials could have particularly important implications for the design of drug combinations in the future

Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria

We report the first in vitro and genetic confirmation of Malarone(®) (GlaxoSmithKline; atovaquone and proguanil hydrochloride) resistance in Plasmodium falciparum acquired in Africa. On presenting with malaria two weeks after returning from a 4-week visit to Lagos, Nigeria without prophylaxis, a male patient was given a standard 3-day treatment course of Malarone(®). Twenty-eight days later the parasitaemia recrudesced. Parasites were cultured from the blood and the isolate (NGATV01) was shown to be resistant to atovaquone and the antifolate pyrimethamine. The cytochrome b gene of isolate NGATV01 showed a single mutation, Tyr268Asn which has not been seen previously

Structure and non-essential function of glycerol kinase in <i>Plasmodium</i> <i>falciparum</i> blood stages

Malaria pathology is caused by multiplication of asexual parasites within erythrocytes, whereas mosquito transmission of malaria is mediated by sexual precursor cells (gametocytes). Microarray analysis identified glycerol kinase (GK) as the second most highly upregulated gene in Plasmodium falciparum gametocytes with no expression detectable in asexual blood stage parasites. Phosphorylation of glycerol by GK is the rate-limiting step in glycerol utilization. Deletion of this gene from P. falciparum had no effect on asexual parasite growth, but surprisingly also had no effect on gametocyte development or exflagellation, suggesting that these life cycle stages do not utilize host-derived glycerol as a carbon source. Kinetic studies of purified PfGK showed that the enzyme is not regulated by fructose 1,6 bisphosphate. The high-resolution crystal structure of P. falciparum GK, the first of a eukaryotic GK, reveals two domains embracing a capacious ligand-binding groove. In the complexes of PfGK with glycerol and ADP, we observed closed and open forms of the active site respectively. The 27° domain opening is larger than in orthologous systems and exposes an extensive surface with potential for exploitation in selective inhibitor design should the enzyme prove to be essential in vivo either in the human or in the mosquito

Gametogenesis in Malaria Parasites Is Mediated by the cGMP-Dependent Protein Kinase

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle

Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis

Phosphodiesterase (PDE) and guanylyl cyclase (GC) enzymes are key components of the cGMP signalling pathway and are encoded in the genome of Plasmodium falciparum. Here we investigate the role of specific GC and PDE isoforms in gamete formation – a process that is essential for malaria transmission and occurs in the Anopheles mosquito midgut following feeding on an infected individual. Details of the intracellular signalling events controlling development of the male and female gametes from their precursors (gametocytes) remain sparse in P. falciparum. Previous work involving the addition of pharmacological agents to gametocytes implicated cGMP in exflagellation – the emergence of highly motile, flagellated male gametes from the host red blood cell. In this study we show that decreased GC activity in parasites having undergone disruption of the PfGCβ gene had no significant effect on gametogenesis. By contrast, decreased cGMP-PDE activity during gametocyte development owing to disruption of the PfPDEδ gene, led to a severely reduced ability to undergo gametogenesis. This suggests that the concentration of cGMP must be maintained below a threshold in the developing gametocyte to allow subsequent differentiation to proceed normally. The data indicate that PfPDEδ plays a crucial role in regulating cGMP levels during sexual development

Plasmodium falciparum Malaria and Atovaquone-Proguanil Treatment Failure

We noticed overrepresentation of atovaquone-proguanil therapeutic failures among Plasmodium falciparum–infected travelers weighing >100 kg. We report here 1 of these cases, which was not due to resistant parasites or impaired drug bioavailability. The follow-up of such patients should be strengthened

Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations

The widespread introduction of artemisinin-based combination therapy has contributed to recent reductions in malaria mortality. Combination therapies have a range of advantages, including synergism, toxicity reduction, and delaying the onset of resistance acquisition. Unfortunately, antimalarial combination therapy is limited by the depleting repertoire of effective drugs with distinct target pathways. To fast-track antimalarial drug discovery, we have previously employed drug-repositioning to identify the anti-amoebic drug, emetine dihydrochloride hydrate, as a potential candidate for repositioned use against malaria. Despite its 1000-fold increase in in vitro antimalarial potency (ED50 47 nM) compared with its anti-amoebic potency (ED50 26±32 uM), practical use of the compound has been limited by dose-dependent toxicity (emesis and cardiotoxicity). Identification of a synergistic partner drug would present an opportunity for dose-reduction, thus increasing the therapeutic window. The lack of reliable and standardised methodology to enable the in vitro definition of synergistic potential for antimalarials is a major drawback. Here we use isobologram and combination-index data generated by CalcuSyn software analyses (Biosoft v2.1) to define drug interactivity in an objective, automated manner. The method, based on the median effect principle proposed by Chou and Talalay, was initially validated for antimalarial application using the known synergistic combination (atovaquone-proguanil). The combination was used to further understand the relationship between SYBR Green viability and cytocidal versus cytostatic effects of drugs at higher levels of inhibition. We report here the use of the optimised Chou Talalay method to define synergistic antimalarial drug interactivity between emetine dihydrochloride hydrate and atovaquone. The novel findings present a potential route to harness the nanomolar antimalarial efficacy of this affordable natural product