In vitro evaluation of ganaplacide/lumefantrine combination against Plasmodium falciparum in a context of artemisinin resistance.

BACKGROUND: Ganaplacide, also known as KAF156, is among the new antimalarial drug candidates that have successfully reached Phase III clinical trials, and is proposed in combination with lumefantrine. This combination could replace the current front-line artemisinin-based combination therapies (ACTs) in case of Plasmodium falciparum resistance to both artemisinins and partner drugs. Indeed, the African continent, where the malaria burden is the highest, is currently experiencing worrying multiple emergences and spread of artemisinin resistance, which urges for the exploration of the antiparasitic properties of KAF156 in this context. OBJECTIVES AND METHODS: The objectives of this work were firstly to evaluate the risk of cross-resistance between artemisinins and KAF156 alone, and in combination with lumefantrine, using a panel of artemisinin-resistant strains carrying different pfk13 mutations and markers of other antiplasmodial drug resistances; secondly to explore in vitro the relevance of combining KAF156 and lumefantrine with artemisinins, based on the model of triple ACTs. RESULTS: Our results highlighted that KAF156 activity was not impaired by mutations in pfk13, pfcrt, pfmdr1, pfmdr2, pfdhps and pfdhfr genes or by pfmdr1 amplification. Moreover, we demonstrated that KAF156 alone and in combination with lumefantrine was active against artemisinin-resistant parasites, including when they are quiescent. CONCLUSIONS: All these in vitro results evidence that multi-drug resistant parasites currently in circulation in the field might not affect KAF156 efficacy, and are encouraging signs for KAF156 use in a triple ACT to preserve the use of artemisinins for as long as possible.

Epigenetic regulation as a therapeutic target in the malaria parasite Plasmodium falciparum.

Over the past thirty years, epigenetic regulation of gene expression has gained increasing interest as it was shown to be implicated in illnesses ranging from cancers to parasitic diseases. In the malaria parasite, epigenetics was shown to be involved in several key steps of the complex life cycle of Plasmodium, among which asexual development and sexual commitment, but also in major biological processes like immune evasion, response to environmental changes or DNA repair. Because epigenetics plays such paramount roles in the Plasmodium parasite, enzymes involved in these regulating pathways represent a reservoir of potential therapeutic targets. This review focuses on epigenetic regulatory processes and their effectors in the malaria parasite, as well as the inhibitors of epigenetic pathways and their potential as new anti-malarial drugs. Such types of drugs could be formidable tools that may contribute to malaria eradication in a context of widespread resistance to conventional anti-malarials.

Epidrugs as Promising Tools to Eliminate Plasmodium falciparum Artemisinin-Resistant and Quiescent Parasites.

The use of artemisinin and its derivatives has helped reduce the burden of malaria caused by Plasmodium falciparum. However, artemisinin-resistant parasites are able, in the presence of artemisinins, to stop their cell cycles. This quiescent state can alter the activity of artemisinin partner drugs leading to a secondary drug resistance and thus threatens malaria eradication strategies. Drugs targeting epigenetic mechanisms (namely epidrugs) are emerging as potential antimalarial drugs. Here, we set out to evaluate a selection of various epidrugs for their activity against quiescent parasites, to explore the possibility of using these compounds to counter artemisinin resistance. The 32 chosen epidrugs were first screened for their antiplasmodial activity and selectivity. We then demonstrated, thanks to the specific Quiescent-stage Survival Assay, that four epidrugs targeting both histone methylation or deacetylation as well as DNA methylation decrease the ability of artemisinin-resistant parasites to recover after artemisinin exposure. In the quest for novel antiplasmodial drugs with new modes of action, these results reinforce the therapeutic potential of epidrugs as antiplasmodial drugs especially in the context of artemisinin resistance.

Alkoxyamines Designed as Potential Drugs against Plasmodium and Schistosoma Parasites.

Malaria and schistosomiasis are major infectious causes of morbidity and mortality in the tropical and sub-tropical areas. Due to the widespread drug resistance of the parasites, the availability of new efficient and affordable drugs for these endemic pathologies is now a critical public health issue. In this study, we report the design, the synthesis and the preliminary biological evaluation of a series of alkoxyamine derivatives as potential drugs against Plasmodium and Schistosoma parasites. The compounds (RS/SR)-2F, (RR/SS)-2F, and 8F, having IC50 values in nanomolar range against drug-resistant P. falciparum strains, but also five other alkoxyamines, inducing the death of all adult worms of S. mansoni in only 1 h, can be considered as interesting chemical starting points of the series for improvement of the activity, and further structure activity, relationship studies. Moreover, investigation of the mode of action and the rate constants kd for C-ON bond homolysis of new alkoxyamines is reported, showing a possible alkyl radical mediated biological activity. A theoretical chemistry study allowed us to design new structures of alkoxyamines in order to improve the selectivity index of these drugs.

Identification of compounds active against quiescent artemisinin-resistant Plasmodium falciparum parasites via the quiescent-stage survival assay (QSA).

BACKGROUND: Quiescence is an unconventional mechanism of Plasmodium survival, mediating artemisinin resistance. This phenomenon increases the risk of clinical failures following artemisinin-based combination therapies (ACTs) by slowing parasite clearance and allowing the selection of parasites resistant to partner drugs. OBJECTIVES: To thwart this multiresistance, the quiescent state of artemisinin-resistant parasites must be taken into consideration from the very early stages of the drug discovery process. METHODS: We designed a novel phenotypic assay we have named the quiescent-stage survival assay (QSA) to assess the antiplasmodial activity of drugs on quiescent parasites. This assay was first validated on quiescent forms from different artemisinin-resistant parasite lines (laboratory strain and field isolates), using two reference drugs with different mechanisms of action: chloroquine and atovaquone. Furthermore, the efficacies of different partner drugs of artemisinins used in ACTs were investigated against both laboratory strains and field isolates from Cambodia. RESULTS: Our results highlight that because of the mechanism of quiescence and the respective pharmacological targets of drugs, drug efficacies on artemisinin-resistant parasites may be different between quiescent parasites and their proliferating forms. CONCLUSIONS: These data confirm the high relevance of adding the chemosensitivity evaluation of quiescent parasites by the specific in vitro QSA to the antiplasmodial drug development process in the current worrisome context of artemisinin resistance.

Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development.

Malaria pathogenesis results from the asexual replication of Plasmodium falciparum within human red blood cells, which relies on a precisely timed cascade of gene expression over a 48-h life cycle. Although substantial post-transcriptional regulation of this hardwired program has been observed, it remains unclear how these processes are mediated on a transcriptome-wide level. To this end, we identified mRNA modifications in the P. falciparum transcriptome and performed a comprehensive characterization of N6-methyladenosine (m6A) over the course of blood-stage development. Using mass spectrometry and m6A RNA sequencing, we demonstrate that m6A is highly developmentally regulated, exceeding m6A levels known in any other eukaryote. We characterize a distinct m6A writer complex and show that knockdown of the putative m6A methyltransferase, PfMT-A70, by CRISPR interference leads to increased levels of transcripts that normally contain m6A. In accordance, we find an inverse correlation between m6A methylation and mRNA stability or translational efficiency. We further identify two putative m6A-binding YTH proteins that are likely to be involved in the regulation of these processes across the parasite's life cycle. Our data demonstrate unique features of an extensive m6A mRNA methylation programme in malaria parasites and reveal its crucial role in dynamically fine-tuning the transcriptional cascade of a unicellular eukaryote.