Characterization of antimalarial activity of artemisinin-based hybrid drugs.

In response to the spread of artemisinin (ART) resistance, ART-based hybrid drugs were developed, and their activity profile was characterized against drug-sensitive and drug-resistant Plasmodium falciparum parasites. Two hybrids were found to display parasite growth reduction, stage-specificity, speed of activity, additivity of activity in drug combinations, and stability in hepatic microsomes of similar levels to those displayed by dihydroartemisinin (DHA). Conversely, the rate of chemical homolysis of the peroxide bonds is slower in hybrids than in DHA. From a mechanistic perspective, heme plays a central role in the chemical homolysis of peroxide, inhibiting heme detoxification and disrupting parasite heme redox homeostasis. The hybrid exhibiting slow homolysis of peroxide bonds was more potent in reducing the viability of ART-resistant parasites in a ring-stage survival assay than the hybrid exhibiting fast homolysis. However, both hybrids showed limited activity against ART-induced quiescent parasites in the quiescent-stage survival assay. Our findings are consistent with previous results showing that slow homolysis of peroxide-containing drugs may retain activity against proliferating ART-resistant parasites. However, our data suggest that this property does not overcome the limited activity of peroxides in killing non-proliferating parasites in a quiescent state.

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.

Hybrid Peptide-Alkoxyamine Drugs: A Strategy for the Development of a New Family of Antiplasmodial Drugs.

The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite's food vacuoles, our approach is summarized as "to dig its grave with its fork". However, despite promising sub-micromolar IC50 values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity.

Mutation in the Plasmodium falciparum BTB/POZ Domain of K13 Protein Confers Artemisinin Resistance.

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 (pfk13) gene. Here, we carried out in vitro selection over a 1-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

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.

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.

Polyunsaturated fatty acid metabolites: biosynthesis in Leishmania and role in parasite/host interaction.

Inside the human host, Leishmania infection starts with phagocytosis of infective promastigotes by macrophages. In order to survive, Leishmania has developed several strategies to manipulate macrophage functions. Among these strategies, Leishmania as a source of bioactive lipids has been poorly explored. Herein, we assessed the biosynthesis of polyunsaturated fatty acid metabolites by infective and noninfective stages of Leishmania and further explored the role of these metabolites in macrophage polarization. The concentration of docosahexaenoic acid metabolites, precursors of proresolving lipid mediators, was increased in the infective stage of the parasite compared with the noninfective stage, and cytochrome P450-like proteins were shown to be implicated in the biosynthesis of these metabolites. The treatment of macrophages with lipids extracted from the infective forms of the parasite led to M2 macrophage polarization and blocked the differentiation into the M1 phenotype induced by IFN-γ. In conclusion, Leishmania polyunsaturated fatty acid metabolites, produced by cytochrome P450-like protein activity, are implicated in parasite/host interactions by promoting the polarization of macrophages into a proresolving M2 phenotype.

Endoperoxide-based compounds: cross-resistance with artemisinins and selection of a Plasmodium falciparum lineage with a K13 non-synonymous polymorphism.

Background: Owing to the emergence of multiresistant Plasmodium falciparum parasites in Southeast Asia, along with the impressive decrease in the efficacy of the endoperoxide compound artemisinin and of artemisinin-based combination therapies, the development of novel antimalarial drugs or combinations is required. Although several antiplasmodial molecules, such as endoperoxide-based compounds, are in advanced research or development, we do not know whether resistance to artemisinin derivatives might impact the efficacy of these new compounds. Objectives: To address this issue, the antiplasmodial efficacy of trioxaquines, hybrid endoperoxide-based molecules, was explored, along with their ability to select in vitro resistant parasites under discontinuous and dose-escalating drug pressure. Methods: The in vitro susceptibilities of artemisinin- and trioxaquine-resistant laboratory strains and recent Cambodian field isolates were evaluated by different phenotypic and genotypic assays. Results: Trioxaquines tested presented strong cross-resistance with artemisinin both in the artemisinin-resistant laboratory F32-ART5 line and in Cambodian field isolates. Trioxaquine drug pressure over 4 years led to the in vitro selection of the F32-DU line, which is resistant to trioxaquine and artemisinin, similar to the F32-ART lineage. F32-DU whole genome sequencing (WGS) revealed that resistance to trioxaquine was associated with the same non-synonymous mutation in the propeller domain of the K13 protein (M476I) that was found in the F32-ART lineage. Conclusions: These worrisome results indicate the risk of cross-resistance between artemisinins and endoperoxide-based antiplasmodial drugs in the development of the K13 mutant parasites and question the usefulness of these molecules in the future therapeutic arsenal.

Plasmodium falciparum: multifaceted resistance to artemisinins.

Plasmodium falciparum resistance to artemisinins, the most potent and fastest acting anti-malarials, threatens malaria elimination strategies. Artemisinin resistance is due to mutation of the PfK13 propeller domain and involves an unconventional mechanism based on a quiescence state leading to parasite recrudescence as soon as drug pressure is removed. The enhanced P. falciparum quiescence capacity of artemisinin-resistant parasites results from an increased ability to manage oxidative damage and an altered cell cycle gene regulation within a complex network involving the unfolded protein response, the PI3K/PI3P/AKT pathway, the PfPK4/eIF2α cascade and yet unidentified transcription factor(s), with minimal energetic requirements and fatty acid metabolism maintained in the mitochondrion and apicoplast. The detailed study of these mechanisms offers a way forward for identifying future intervention targets to fend off established artemisinin resistance.

Antiplasmodial activities of gold(I) complexes involving functionalized N-heterocyclic carbenes.

A series of twenty five molecules, including imidazolium salts functionalized by N-, O- or S-containing groups and their corresponding cationic, neutral or anionic gold(I) complexes were evaluated on Plasmodium falciparum in vitro and then on Vero cells to determine their selectivity. Among them, eight new compounds were synthesized and fully characterized by spectroscopic methods. The X-ray structures of three gold(I) complexes are presented. Except one complex (18), all the cationic gold(I) complexes show potent antiplasmodial activity with IC50 in the micro- and submicromolar range, correlated with their lipophilicity. Structure-activity relationships enable to evidence a lead-complex (21) displaying a good activity (IC50=210nM) close to the value obtained with chloroquine (IC50=514nM) and a weak cytotoxicity.

[Plasmodium falciparum resistance to artemisinin-based combination therapies (ACTs): Fears of widespread drug resistance]. / Résistance de l'agent du paludisme, Plasmodium falciparum aux combinaisons thérapeutiques à base d'artémisinine (ACTs): Craintes d'une chimiorésistance généralisée.

Malaria treatment with ACTs (Artemisinin-based Combination Therapies), combining a chemical derivative of artemisinin, and a partner drug has, for more than 15 years, produced a notable decrease in the mortality in tropical and subtropical areas. However, since 2008, a serious threat has emerged in western Cambodia, where the clinical efficacy of artemisinins has significantly declined, with a delayed parasite clearance rate and high recrudescence rates in the following weeks. Resistance of Plasmodium to artemisinins is now reported in several countries in South-East Asia. ACTs remain effective as long as the partner drug retains its activity but more and more clinical failures related to parasite resistance to both resistant parasites in sub-Saharan Africa, the continent most affected by malaria, as was the case in the past with other antimalarial treatments. It is therefore essential to better understand, from phenotypic and genotypic points of view, the mechanisms of resistance developed by the parasite Plasmodium falciparum face artemisinin and its derivatives in order to offer new therapeutic tools.

Antileishmanial pharmacomodulation in 8-nitroquinolin-2(1H)-one series.

An antileishmanial pharmacomodulation at position 4 of 8-nitroquinolin-2(1H)-one was conducted by using the Sonogashira and Suzuki-Miyaura coupling reactions. A series of 25 derivatives was tested in vitro on the promastigote stage of Leishmania donovani along with an in vitro cytotoxicity evaluation on the human HepG2 cell line. Only the derivatives bearing a phenyl moiety at position 4 of the quinoline ring displayed interesting biologic profile, when the phenyl moiety was substituted at the para position by a Br or Cl atom, or by a CF3 group. Among them, molecules 17 and 19 were the most selective and were then tested in vitro on the intracellular amastigote stage of both L. donovani and Leishmania infantum, in parallel with complementary in vitro cytotoxicity assays on the macrophage cell lines THP-1 and J774A.1. Molecule 19 showed no activity on the amastigote stages of the parasites and some cytotoxicity on the J774A.1 cell line while molecule 17, less cytotoxic than 19, showed anti-amastigote activity in L. infantum, being 3 times less active than miltefosine but more active and selective than pentamidine. Nevertheless, hit-molecule 17 did not appear as selective as the parent compound.

Peptide-Alkoxyamine Drugs: An Innovative Approach to Fight Schistosomiasis: "Digging Their Graves with Their Forks".

The expansion of drug resistant parasites sheds a serious concern on several neglected parasitic diseases. Our recent results on cancer led us to envision the use of peptide-alkoxyamines as a highly selective and efficient new drug against schistosome adult worms, the etiological agents of schistosomiasis. Indeed, the peptide tag of the hybrid compounds can be hydrolyzed by worm's digestive enzymes to afford a highly labile alkoxyamine which homolyzes spontaneously and instantaneously into radicals-which are then used as a drug against Schistosome adult parasites. This approach is nicely summarized as digging their graves with their forks. Several hybrid peptide-alkoxyamines were prepared and clearly showed an activity: two of the tested compounds kill 50% of the parasites in two hours at a concentration of 100 µg/mL. Importantly, the peptide and alkoxyamine fragments that are unable to generate alkyl radicals display no activity. This strong evidence validates the proposed mechanism: a specific activation of the prodrugs by the parasite proteases leading to parasite death through in situ alkyl radical generation.

In artemisinin-resistant falciparum malaria parasites, mitochondrial metabolic pathways are essential for survival but not those of apicoplast.

Emergence and spread of parasite resistance to artemisinins, the first-line antimalarial therapy, threaten the malaria eradication policy. To identify therapeutic targets to eliminate artemisinin-resistant parasites, the functioning of the apicoplast and the mitochondrion was studied, focusing on the fatty acid synthesis type II (FASII) pathway in the apicoplast and the electron transfer chain in the mitochondrion. A significant enrichment of the FASII pathway among the up-regulated genes in artemisinin-resistant parasites under dihydroartemisinin treatment was found, in agreement with published transcriptomic data. However, using GC-MS analyzes of fatty acids, we demonstrated for the first time that the FASII pathway is non-functional, ruling out the use of FASII inhibitors to target artemisinin-resistant parasites. Conversely, by assessing the modulation of the oxygen consumption rate, we evidenced that mitochondrial respiration remains functional and flexible in artemisinin-resistant parasites and even at the quiescent stage. Two novel compounds targeting electron transport chain (ELQ300, ELQ400) efficiently killed quiescent artemisinin-resistant parasites. Therefore, mitochondrial respiration represents a key target for the elimination of artemisinin-resistant persistent Plasmodium falciparum parasites.

Targeting the human parasite Leishmania donovani: discovery of a new promising anti-infectious pharmacophore in 3-nitroimidazo[1,2-a]pyridine series.

We report herein the discovery of antileishmanial molecules based on the imidazo[1,2-a]pyridine ring. In vitro screenings of imidazopyridines belonging to our chemical library, toward the promastigotes stage of Leishmania donovani, J774A.1 murine and HepG2 human cells, permitted to identify three selective hit-compounds (12, 20 and 28). New derivatives were then synthesized to allow structure-activity and -toxicity relationships analyses, enabling to characterize a lead-compound (44) displaying both a high potency (IC50=1.8 µM) and a good selectivity index, in comparison with three antileishmanial reference drug-compounds (amphotericin B, miltefosine and pentamidine). Moreover, lead-compound 44 also exhibits good in vitro activity against the intracellular amastigote stage of L. donovani. Thus, the 6-halo-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine scaffold appears as a new promising selective antileishmanial pharmacophore, especially when substituted at position 8 by a bromine atom.

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.

2-Phenoxy-3-Trichloromethylquinoxalines Are Antiplasmodial Derivatives with Activity against the Apicoplast of Plasmodium falciparum.

The malaria parasite harbors a relict plastid called the apicoplast. Although not photosynthetic, the apicoplast retains unusual, non-mammalian metabolic pathways that are essential to the parasite, opening up a new perspective for the development of novel antimalarials which display a new mechanism of action. Based on the previous antiplasmodial hit-molecules identified in the 2-trichloromethylquinoxaline series, we report herein a structure-activity relationship (SAR) study at position two of the quinoxaline ring by synthesizing 20 new compounds. The biological evaluation highlighted a hit compound (3i) with a potent PfK1 EC50 value of 0.2 µM and a HepG2 CC50 value of 32 µM (Selectivity index = 160). Nitro-containing (3i) was not genotoxic, both in the Ames test and in vitro comet assay. Activity cliffs were observed when the 2-CCl3 group was replaced, showing that it played a key role in the antiplasmodial activity. Investigation of the mechanism of action showed that 3i presents a drug response by targeting the apicoplast and a quick-killing mechanism acting on another target site.

A New Thienopyrimidinone Chemotype Shows Multistage Activity against Plasmodium falciparum, Including Artemisinin-Resistant Parasites.

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. For decades, the research for novel antimalarials focused on the high-throughput screening of molecules that only targeted the asexual blood stages. In a search for new effective compounds presenting a triple action against erythrocytic and liver stages in addition to the ability to block the transmission of the disease via the mosquito vector, 2-amino-thienopyrimidinone derivatives were synthesized and tested for their antimalarial activity. One molecule, named gamhepathiopine (denoted as "M1" herein), was active at submicromolar concentrations against both erythrocytic (50% effective concentration [EC50] = 0.045 µM) and liver (EC50 = 0.45 µM) forms of Plasmodium falciparum. Furthermore, gamhepathiopine efficiently blocked the development of the sporogonic cycle in the mosquito vector by inhibiting the exflagellation step. Moreover, M1 was active against artemisinin-resistant forms (EC50 = 0.227 µM), especially at the quiescent stage. Nevertheless, in mice, M1 showed modest activity due to its rapid metabolization by P450 cytochromes into inactive derivatives, calling for the development of new parent compounds with improved metabolic stability and longer half-lives. These results highlight the thienopyrimidinone scaffold as a novel antiplasmodial chemotype of great interest to search for new drug candidates displaying multistage activity and an original mechanism of action with the potential to be used in combination therapies for malaria elimination in the context of artemisinin resistance. IMPORTANCE This work reports a new chemical structure that (i) displays activity against the human malaria parasite Plasmodium falciparum at 3 stages of the parasitic cycle (blood stage, hepatic stage, and sexual stages), (ii) remains active against parasites that are resistant to the first-line treatment recommended by the World Health Organization (WHO) for the treatment of severe malaria (artemisinins), and (iii) reduces transmission of the parasite to the mosquito vector in a mouse model. This new molecule family could open the way to the conception of novel antimalarial drugs with an original multistage mechanism of action to fight against Plasmodium drug resistance and block interhuman transmission of malaria.