Genome wide distribution of G-quadruplexes and their impact on gene expression in malaria parasites.

Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.

Prevalence of mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, and association with ex vivo susceptibility to common anti-malarial drugs against African Plasmodium falciparum isolates.

BACKGROUND: The Plasmodium falciparum chloroquine transporter gene (pfcrt) is known to be involved in chloroquine and amodiaquine resistance, and more particularly the mutations on the loci 72 to 76 localized within the second exon. Additionally, new mutations (T93S, H97Y, C101F, F145I, M343L, C350R and G353V) were recently shown to be associated with in vitro reduced susceptibility to piperaquine in Asian or South American P. falciparum strains. However, very few data are available on the prevalence of these mutations and their effect on parasite susceptibility to anti-malarial drugs, and more particularly piperaquine in Africa. METHODS: A molecular investigation of these mutations was performed in 602 African P. falciparum parasites collected between 2017 and 2018 on malaria patients hospitalized in France after a travel in African countries. Associations between genotypes and in vitro susceptibilities to piperaquine and standard antimalarial drugs were assessed. RESULTS: None of the mutations, previously described as associated with piperaquine resistance, was found in the 602 P. falciparum African isolates. The K76T mutation is associated with resistance to chloroquine (p < 0.0002) and desethylamodiaquine (p < 0.002) in Africa. The K76T mutation is not associated with in vitro reduced susceptibility to piperaquine. The mutation I356T, identified in 54.7% (n = 326) of the African isolates, was significantly associated with reduced susceptibility to quinine (p < 0.02) and increased susceptibility to mefloquine (p < 0.04). The K76T and I356T mutations were significantly associated in West African isolates (p = 0.008). CONCLUSION: None of the mutations in pfcrt found to be associated with piperaquine reduced susceptibility in Asia or South America (T93S, H97Y, C101F, F145I, M343L C350R and G353V) were found in the 602 African isolates including the three isolates with reduced susceptibility to piperaquine. The K76T mutation, involved in resistance to chloroquine and amodiaquine, and the I356T mutation were not associated with in vitro reduced susceptibility to piperaquine. Differences in mefloquine susceptibility between I356 and 356T isolates were, while statistically different, minimal. Further analyses are needed with a more important sample size from the same geographic area to confirm the role of the I356T mutation on quinine susceptibility.

Evaluation of amidoxime derivatives as prodrug candidates of potent bis-cationic antimalarials.

Plasmodium falciparum is responsible for most of the cases of malaria and its resistance to established antimalarial drugs is a major issue. Thus, new chemotherapies are needed to fight the emerging multi-drug resistance of P. falciparum malaria, like choline analogues targeting plasmodial phospholipidic metabolism. Here we describe the synthesis of amidoxime derivatives as prodrug candidates of reverse-benzamidines and hybrid compounds able to mimic choline, as well as the design of a new series of asymmetrical bis-cationic compounds. Bioconversion studies were conducted on amidoximes in asymmetrical series and showed that amidoxime prodrug strategy could be applied on C-alkylamidine moieties, like benzamidines and that N-substituents did not alter the bioconversion of amidoximes. The antimalarial activity of the three series of compounds was evaluated in vitro against P. falciparum and in vivo against P. vinckei petteri in mice.

Toxoplasma gondii chromosomal passenger complex is essential for the organization of a functional mitotic spindle: a prerequisite for productive endodyogeny.

The phylum Apicomplexa encompasses deadly pathogens such as malaria and Cryptosporidium. Apicomplexa cell division is mechanistically divergent from that of their mammalian host, potentially representing an attractive source of drug targets. Depending on the species, apicomplexan parasites can modulate the output of cell division, producing two to thousands of daughter cells at once. The inherent flexibility of their cell division mechanisms allows these parasites to adapt to different niches, facilitating their dissemination. Toxoplasma gondii tachyzoites divide using a unique form of cell division called endodyogeny. This process involves a single round of DNA replication, closed nuclear mitosis, and assembly of two daughter cells within a mother. In higher Eukaryotes, the four-subunit chromosomal passenger complex (CPC) (Aurora kinase B (ARKB)/INCENP/Borealin/Survivin) promotes chromosome bi-orientation by detaching incorrect kinetochore-microtubule attachments, playing an essential role in controlling cell division fidelity. Herein, we report the characterization of the Toxoplasma CPC (Aurora kinase 1 (Ark1)/INCENP1/INCENP2). We show that the CPC exhibits dynamic localization in a cell cycle-dependent manner. TgArk1 interacts with both TgINCENPs, with TgINCENP2 being essential for its translocation to the nucleus. While TgINCENP1 appears to be dispensable, interfering with TgArk1 or TgINCENP2 results in pronounced division and growth defects. Significant anti-cancer drug development efforts have focused on targeting human ARKB. Parasite treatment with low doses of hesperadin, a known inhibitor of human ARKB at higher concentrations, phenocopies the TgArk1 and TgINCENP2 mutants. Overall, our study provides new insights into the mechanisms underpinning cell cycle control in Apicomplexa, and highlights TgArk1 as potential drug target.

Contribution of the precursors and interplay of the pathways in the phospholipid metabolism of the malaria parasite.

The malaria parasite, Plasmodium falciparum, develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d9, ethanolamine-d4, and serine-d3) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches.

High Accumulation and In Vivo Recycling of the New Antimalarial Albitiazolium Lead to Rapid Parasite Death.

Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei-infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei-infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs.

Uncovering new structural insights for antimalarial activity from cost-effective aculeatin-like derivatives.

A series of new aculeatin-like analogues were synthesized in two steps by combining two sets of building blocks. Many compounds showed inhibitory activities in vitro against Plasmodium falciparum and have helped to gain more insight into structure-activity relationships around the spirocyclohexadienone pharmacophoric scaffold. Plasmodium falciparum thioredoxin reductase (PfTrxR) has been investigated as a putative cellular target. Moreover, a new aculeatin-like scaffold without Michael acceptor properties, efficient at 0.86 µM against P. falciparum 3D7, was identified and raises the prospect of developing a new antimalarial agent.

New insight into the mechanism of accumulation and intraerythrocytic compartmentation of albitiazolium, a new type of antimalarial.

Bis-thiazolium salts constitute a new class of antihematozoan drugs that inhibit parasite phosphatidylcholine biosynthesis. They specifically accumulate in Plasmodium- and Babesia-infected red blood cells (IRBC). Here, we provide new insight into the choline analogue albitiazolium, which is currently being clinically tested against severe malaria. Concentration-dependent accumulation in P. falciparum-infected erythrocytes reached steady state after 90 to 120 min and was massive throughout the blood cycle, with cellular accumulation ratios of up to 1,000. This could not occur through a lysosomotropic effect, and the extent did not depend on the food vacuole pH, which was the case for the weak base chloroquine. Analysis of albitiazolium accumulation in P. falciparum IRBC revealed a high-affinity component that was restricted to mature stages and suppressed by pepstatin A treatment, and thus likely related to drug accumulation in the parasite food vacuole. Albitiazolium also accumulated in a second high-capacity component present throughout the blood cycle that was likely not related to the food vacuole and also observed with Babesia divergens-infected erythrocytes. Accumulation was strictly glucose dependent, drastically inhibited by H+/K+ and Na+ ionophores upon collapse of ionic gradients, and appeared to be energized by the proton-motive force across the erythrocyte plasma membrane, indicating the importance of transport steps for this permanently charged new type of antimalarial agent. This specific, massive, and irreversible accumulation allows albitiazolium to restrict its toxicity to hematozoa-infected erythrocytes. The intraparasitic compartmentation of albitiazolium corroborates a dual mechanism of action, which could make this new type of antimalarial agent resistant to parasite resistance.

Potent in vivo anti-malarial activity and representative snapshot pharmacokinetic evaluation of artemisinin-quinoline hybrids.

BACKGROUND: Because Plasmodium falciparum displays increase tolerance against the recommended artemisinin combination therapies (ACT), new classes of anti-malarial drugs are urgently required. Previously synthesized artemisinin-aminoquinoline hybrids were evaluated to ascertain whether the potent low nanomolar in vitro anti-plasmodial activity would carry over in vivo against Plasmodium vinckei. A snapshot pharmacokinetic analysis was carried out on one of the hybrids to obtain an indication of the pharmacokinetic properties of this class of anti-malarial drugs. METHODS: In vitro activity of hybrids 2 and 3 were determined against the 3D7 strain of P. falciparum. Plasmodium vinckei-infected mice were treated with hybrids 1 - 3 for four days at a dosage of 0.8 mg/kg, 2.5 mg/kg, 7.5 mg/kg or 15 mg/kg intraperitoneally (ip), or orally (per os) with 2.7 mg/kg, 8.3 mg/kg, 25 mg/kg or 50 mg/kg. Artesunate was used as reference drug. A snapshot oral and IV pharmacokinetic study was performed on hybrid 2. RESULTS: Hybrids 1 - 3 displayed potent in vivo anti-malarial activity with ED50 of 1.1, 1.4 and <0.8 mg/kg by the ip route and 12, 16 and 13 mg/kg per os, respectively. Long-term monitoring of parasitaemia showed a complete cure of mice (without recrudescence) at 15 mg/kg via ip route and at 50 mg/kg by oral route for hybrid 1 and 2, whereas artesunate was only able to provide a complete cure at 30 mg/kg ip and 80 mg/kg per os. CONCLUSIONS: These compounds provide a new class of desperately needed anti-malarial drug. Despite a short half-life and moderate oral bioavailability, this class of compounds was able to cure malaria in mice at very low dosages. The optimum linker length for anti-malarial activity was found to be a diaminoalkyl chain consisting of two carbon atoms either methylated or unmethylated.

Purine containing carbonucleoside phosphonate analogues as novel chemotype for Plasmodium falciparum Inhibition.

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and ß-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.

Antiplasmodial properties of acyl-lysyl oligomers in culture and animal models of malaria.

Our previous analysis of antiplasmodial properties exhibited by dodecanoyl-based oligo-acyl-lysyls (OAKs) has outlined basic attributes implicated in potent inhibition of parasite growth and underlined the critical role of excess hydrophobicity in hemotoxicity. To dissociate hemolysis from antiplasmodial effect, we screened >50 OAKs for in vitro growth inhibition of Plasmodium falciparum strains, thus revealing the minimal requirements for antiplasmodial potency in terms of sequence and composition, as confirmed by efficacy studies in vivo. The most active sequence, dodecanoyllysyl-bis(aminooctanoyllysyl)-amide (C(12)K-2α(8)), inhibited parasite growth at submicromolar concentrations (50% inhibitory concentration [IC(50)], 0.3 ± 0.1 µM) and was devoid of hemolytic activity (<0.4% hemolysis at 150 µM). Unlike the case of dodecanoyl-based analogs, which equally affect ring and trophozoite stages of the parasite developmental cycle, the ability of various octanoyl-based OAKs to distinctively affect these stages (rings were 4- to 5-fold more sensitive) suggests a distinct antiplasmodial mechanism, nonmembranolytic to host red blood cells (RBCs). Upon intraperitoneal administration to mice, C(12)K-2α(8) demonstrated sustainable high concentrations in blood (e.g., 0.1 mM at 25 mg/kg of body weight). In Plasmodium vinckei-infected mice, C(12)K-2α(8) significantly affected parasite growth (50% effective dose [ED(50)], 22 mg/kg) but also caused mortality in 2/3 mice at high doses (50 mg/kg/day × 4).

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways.

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.

Reliability of antimalarial sensitivity tests depends on drug mechanisms of action.

In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.

Reverse-benzamidine antimalarial agents: design, synthesis, and biological evaluation.

In the frame of the development of bis-cationic choline analogs, the RSA of bis-N-alkylamidines were studied and a new series of reverse-benzamidine derivatives was designed. Contrary to the lipophilicity, the basicity of alkylamidine compounds directly influences their antimalarial potencies.

Exploration of potential prodrug approach of the bis-thiazolium salts T3 and T4 for orally delivered antimalarials.

We report here the synthesis and biological evaluation of a series of 37 compounds as precursors of potent antimalarial bis-thiazolium salts (T3 and T4). These prodrugs were either thioester, thiocarbonate or thiocarbamate type and were synthesized in one step by reaction of an alkaline solution of the parent drug with the appropriate activated acyl group. Structural variations affecting physicochemical properties were made in order to improve oral activity. Twenty-five of them exhibited potent antimalarial activity with IC(50) lower than 7nM against Plasmodium falciparum in vitro. Notably, 3 and 22 showed IC(50)=2.2 and 1.8nM, respectively. After oral administration 22 was the most potent compound clearing the parasitemia in Plasmodium vinckei infected mice with a dose of 1.3mg/kg.

ß-Hydroxy- and ß-Aminophosphonate Acyclonucleosides as Potent Inhibitors of Plasmodium falciparum Growth.

Malaria is an infectious disease caused by a parasite of the genus Plasmodium, and the emergence of parasites resistant to all current antimalarial drugs highlights the urgency of having new classes of molecules. We developed an effective method for the synthesis of a series of ß-modified acyclonucleoside phosphonate (ANP) derivatives, using commercially available and inexpensive materials (i.e., aspartic acid and purine heterocycles). Their biological evaluation in cell culture experiments and SAR revealed that the compounds' effectiveness depends on the presence of a hydroxyl group, the chain length (four carbons), and the nature of the nucleobase (guanine). The most active derivative inhibits the growth of Plasmodium falciparum in vitro in the nanomolar range (IC50 = 74 nM) with high selectivity index (SI > 1350). This compound also showed remarkable in vivo activity in P. berghei-infected mice (ED50 ∼ 0.5 mg/kg) when administered by the ip route and is, although less efficient, still active via the oral route. It is the first ANP derivative with such potent antimalarial activity and therefore has considerable potential for development as a new antimalarial drug.

Statistical model to evaluate in vivo activities of antimalarial drugs in a Plasmodium cynomolgi-macaque model for Plasmodium vivax malaria.

Preclinical animal models informing antimalarial drug development are scarce. We have used asexual erythrocytic Plasmodium cynomolgi infections of rhesus macaques to model Plasmodium vivax during preclinical development of compounds targeting parasite phospholipid synthesis. Using this malaria model, we accumulated data confirming highly reproducible infection patterns, with self-curing parasite peaks reproducibly preceding recrudescence peaks. We applied nonlinear mixed-effect (NLME) models, estimating treatment effects in three drug studies: G25 (injected) and the bisthiazolium prodrugs TE4gt and TE3 (oral). All compounds fully cured P. cynomolgi-infected macaques, with significant effects on parasitemia height and time of peak. Although all three TE3 doses tested were fully curative, NLME models discriminated dose-dependent differential pharmacological antimalarial activity. By applying NLME modeling treatment effects are readily quantified. Such drug development studies are more informative and contribute to reduction and refinement in animal experimentation.

N-substituted bis-C-alkyloxadiazolones as dual effectors: efficient intermediates to amidoximes or amidines and prodrug candidates of potent antimalarials.

A convenient route to N-substituted bis-C-alkylamidines possessing antiplasmodial activity and their oxadiazolone and amidoxime prodrug candidates, is described. These three families of compounds were available after a key N-alkylation step of the parent oxadiazolone 1a. Testing of the three compound classes in vitro and in vivo is also presented.

Design and synthesis of amidoxime derivatives for orally potent C-alkylamidine-based antimalarial agents.

Within the frame of the design of prodrug candidates to deliver a C-alkylamidine antimalarial agent, we showed that specific O-substitutions were needed on the alkylamidoxime structure. Among the newly synthesized molecules, bis-oxadiazolone and bis-O-methylsulfonylamidoxime derivatives induced a complete clearance of parasitemia in mice after oral administration.

Quantitation of SAR97276 in mouse tissues by rapid resolution liquid chromatography-mass spectrometry.

1,12-Bis[5-(2-hydroxyethyl)-4-methyl-1,3-thiazol-3-ium]dodecane dibromide (SAR97276, T3) is a new antimalarial drug, which is currently being evaluated in clinical trials for severe malaria. Drug accumulation inside the parasite and a dual mechanism of action are a major strength of this compound, as it could help delay the development of resistance. The purpose of this article was to develop a rapid resolution LC-MS method for quantifying SAR97276 in mouse tissues. The LC system consisted of Zorbax Eclipse XDB C8 (1.8 microm, 50 x 4.6 mm, 60 degrees C) column. Elution with a gradient mobile phase consisting of ACN-trimethylamine-formate buffer (pH 3) at a flow rate of 1 mL/min yielded sharp, utmost-resolved peaks within 2 min. Tissue samples were powdered under liquid nitrogen. After protein precipitation with citric acid, SPE using WCX cartridges was used for sample preparation. There was no influence of the matrix on the detection of either SAR97276 or the IS. Assay precision was <13% and accuracy was 90-107%. The lower LOQs were 3.3 microg/kg in brain and 33 microg/kg in liver and heart. This newly developed method was used to study the tissue distribution of SAR97276 in mouse as part of the ongoing development of SAR97276.