Falciparum Asexual Blood Stages Research Articles

Plasmodium falciparum African PfCRT Mutant Isoforms Conducive to Piperaquine Resistance are Infrequent and Impart a Major Fitness Cost.

Piperaquine, used in combination with dihydroartemisinin, has been identified as a promising partner drug for uncomplicated treatment and chemoprevention of Plasmodium falciparum malaria in Africa. In light of the earlier spread of piperaquine resistance in Southeast Asia, mediated primarily by mutations in the drug efflux transporter PfCRT, we have explored whether PfCRT mutations would represent a probable path to piperaquine resistance becoming established in Africa. We edited PfCRT mutations known to mediate piperaquine resistance in Southeast Asia into P. falciparum asexual blood stage parasites expressing three prevalent African mutant PfCRT haplotypes. Gene-edited clones were profiled in antimalarial concentration-response and competitive fitness assays. pfcrt-edited parasites expressing the contemporary Southeast Asian T93S or I218F mutations added to the GB4 and Cam783 haplotypes common in Africa did not mediate piperaquine resistance, with partial survival only at low drug concentrations. In contrast, parasites expressing these mutations on the rare PfCRT FCB haplotype, observed mostly in North-East Africa, acquired a moderate level of piperaquine resistance. Dd2GB4, Dd2Cam783, and Dd2FCB lines edited to express the T93S or I218F mutations showed increased susceptibility to chloroquine. Piperaquine-resistant African PfCRT isoforms conferred a substantial fitness cost, manifesting as reduced asexual blood stage parasite growth rates. These findings suggest that piperaquine-resistant PfCRT mutations that emerged in Southeast Asia mediate resistance only in a limited subset of African PfCRT haplotypes, with fitness costs that we suspect would likely preclude dissemination in high-transmission malaria-endemic African regions.

A multistage Plasmodium CRL4WIG1 ubiquitin ligase is critical for the formation of functional microtubule organization centers in microgametocytes.

Malaria is a mosquito-borne infectious disease caused by unicellular eukaryotic parasites of the Plasmodium genus. Protein ubiquitination by E3 ligases is a critical post-translational modification required for various cellular processes during the lifecycle of Plasmodium parasites. However, little is known about the repertoire and function of these enzymes in Plasmodium. Here, we show that Plasmodium expresses a conserved cullin RING E3 ligase (CRL) complex that is functionally related to CRL4 in other eukaryotes. In P. falciparum asexual blood stages, a cullin-4 scaffold interacts with the RING protein RBX1, the adaptor protein DDB1, and a set of putative receptor proteins that may determine substrate specificity for ubiquitination. These receptor proteins contain WD40-repeat domains and include WD-repeat protein important for gametogenesis 1 (WIG1). This CRL4-related complex is also expressed in P. berghei gametocytes, with WIG1 being the only putative receptor detected in both the schizont and gametocyte stages. WIG1 disruption leads to a complete block in microgamete formation. Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with impaired the formation of the microtubule organization centers that coordinate mitosis with axoneme formation and altered DNA replication during microgametogenesis. This work identifies a CRL4-related ubiquitin ligase in Plasmodium that is critical for the formation of microgametes by regulating proteostasis of ciliary and DNA replication proteins.IMPORTANCEPlasmodium parasites undergo fascinating lifecycles with multiple developmental steps, converting into morphologically distinct forms in both their mammalian and mosquito hosts. Protein ubiquitination by ubiquitin ligases emerges as an important post-translational modification required to control multiple developmental stages in Plasmodium. Here, we identify a cullin RING E3 ubiquitin ligase (CRL) complex expressed in the replicating asexual blood stages and in the gametocyte stages that mediate transmission to the mosquito. WIG1, a putative substrate recognition protein of this ligase complex, is essential for the maturation of microgametocytes into microgametes upon ingestion by a mosquito. More specifically, WIG1 is required for proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. This requirement is linked to DNA replication and microtubule organization center formation, both critical to the development of flagellated microgametes.

Structure-Activity Relationship of a Pyrrole Based Series of PfPKG Inhibitors as Anti-Malarials.

Controlling malaria requires new drugs against Plasmodium falciparum. The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the parasite's life cycle. We defined the structure-activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.

Additional PfCRT mutations driven by selective pressure for improved fitness can result in the loss of piperaquine resistance and altered Plasmodium falciparum physiology.

Our study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.

The claudin-like apicomplexan microneme protein is required for gliding motility and infectivity of Plasmodium sporozoites.

Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the micronemes and the rhoptries. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during invasion by Toxoplasma gondii tachyzoites and in Plasmodium falciparum asexual blood stages. CLAMP is also expressed in Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, but its role in this stage is still unknown. CLAMP is essential for Plasmodium blood stage growth and is refractory to conventional gene deletion. To circumvent this obstacle and study the function of CLAMP in sporozoites, we used a conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. We successfully deleted clamp gene in P. berghei transmission stages and analyzed the functional consequences on sporozoite infectivity. In mosquitoes, sporozoite development and egress from oocysts was not affected in conditional mutants. However, invasion of the mosquito salivary glands was dramatically reduced upon deletion of clamp gene. In addition, CLAMP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes. This severe phenotype was associated with major defects in gliding motility and with reduced shedding of the sporozoite adhesin TRAP. Expansion microscopy revealed partial colocalization of CLAMP and TRAP in a subset of micronemes, and a distinct accumulation of CLAMP at the apical tip of sporozoites. Collectively, these results demonstrate that CLAMP is essential across invasive stages of the malaria parasite, and support a role of the protein upstream of host cell invasion, possibly by regulating the secretion or function of adhesins in Plasmodium sporozoites.

Potent acyl-CoA synthetase 10 inhibitors kill Plasmodium falciparum by disrupting triglyceride formation

Identifying how small molecules act to kill malaria parasites can lead to new “chemically validated” targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

Synthesis and Testing of Analogs of the Tuberculosis Drug Candidate SQ109 against Bacteria and Protozoa: Identification of Lead Compounds against Mycobacterium abscessus and Malaria Parasites.

SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we synthesized 18 analogs of SQ109 and tested them against M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis, and Escherichia coli, as well as against the protozoan parasites Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana, and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ∼4-8-fold more active than SQ109 against M. abscessus, including a highly drug-resistant strain harboring an A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl, and benzyl analogs had 4-10× increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new antimalarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3 and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, which is expected to lead to enhanced uncoupler activity.

Evaluation of Chiral Organosulfur Compounds on Their Activity against the Malaria Parasite Plasmodium falciparum.

Malaria is one of the deadliest tropical diseases, especially causing havoc in children under the age of five in Africa. Although the disease is treatable, the rapid development of drug resistant parasites against frontline drugs requires the search for novel antimalarials. In this study, we tested a series of organosulfur compounds from our internal library for their antiplasmodial effect against Plasmodium falciparum asexual and sexual blood stages. Some active compounds were also obtained in enantiomerically pure form and tested individually against asexual blood stages of the parasite to compare their activity. Out of the 23 tested compounds, 7 compounds (1, 2, 5, 9, 15, 16, and 17) exhibited high antimalarial activity, with IC50 values in the range from 2.2 ± 0.64 to 5.2 ± 1.95 µM, while the other compounds showed moderate to very low activity. The most active compounds also exhibited high activity against the chloroquine-resistant strain, reduced gametocyte development and were not toxic to non-infected red blood cells and Hela cells, as well as the hematopoietic HEL cell line at concentrations below 50 µM. To determine if the enantiomers of the active compounds display different antimalarial activity, enantiomers of two of the active compounds were separated and their antimalarial activity compared. The results show a higher activity of the (-) enantiomers as compared to their (+) counterparts. Our combined data indicate that organosulfur compounds could be exploited as antimalarial drugs and enantiomers of the active compounds may represent a good starting point for the design of novel drugs to target malaria.

In vitro dual activity of Aloe marlothii roots and its chemical constituents against Plasmodium falciparum asexual and sexual stage parasites

Ethnopharmacological relevanceAloe marlothii A.Berger (Xanthorrhoeaceae) is indigenous to southern African countries where its aqueous preparations are used in traditional medicine to treat several ailments including hypertension, respiratory infections, venereal diseases, chest pain, sore throat and malaria. Aim of the studyThe aims of this study were as follows: (i) isolate and identify the antiplasmodial active compounds in A. marlothii roots. As the water extract was previously inactive, the dichloromethane:methanol (DCM:MeOH) (1:1) was used, (ii) examine the activity of the isolated compounds against Plasmodium falciparum asexual blood stage (ABS) parasites as well as for transmission-blocking activity against gametocytes and gametes, and (iii) to use in silico tools to predict the target(s) of the active molecules. Materials and methodsThe crude DCM:MeOH (1:1) extract of A. marlothii roots was fractionated on a reverse phase C8 column, using a positive pressure solid-phase extraction (ppSPE) workstation to produce seven fractions. The resulting fractions and the crude DCM:MeOH extract were tested in vitro against P. falciparum (NF54) ABS parasites using the malaria SYBR Green I based-fluorescence assay. Flash silica chromatography and mass-directed preparative high-performance liquid chromatography were utilised to isolate the active compounds. The isolated compounds were evaluated in vitro against P. falciparum asexual (NF54 and K1 strains) and sexual (gametocytes and gametes) stage parasites. Molecular docking was then used for the in silico prediction of targets for the isolated active compounds in P. falciparum. ResultsThe crude extract and two SPE fractions displayed good antiplasmodial activity with >97% and 100% inhibition of ABS parasites proliferation at 10 and 20 μg/mL, respectively. Following UPLC-MS analysis of these active fractions, a targeted purification resulted in the isolation of six compounds identified as aloesaponol I (1), aloesaponarin I (2), aloesaponol IV (3), β-sorigenin-1-O-methylether (4), emodin (5), and chrysophanol (6). Aloesaponarin I (2) was the most bioactive, compared to other isolated constituents, against P. falciparum ABS parasites exhibiting equipotency against the drug-sensitive (NF54) (IC50 = 1.54 μg/mL (5 μM)) and multidrug-resistant (K1) (IC50 = 1.58 μg/mL (5 μM)) strains. Aloesaponol IV (3) showed pronounced activity against late-stage (>90% stage IV/V) gametocytes (IC50 = 6.53 μg/mL (22.6 μM)) demonstrating a 3-fold selective potency towards these sexual stages compared to asexual forms of the parasite (IC50 = 19.77 ± 6.835 μg/mL (68 μM)). Transmission-blocking potential of aloesaponol IV (3) was validated by in vitro inhibition of exflagellation of male gametes (94% inhibition at 20 μg/mL). In silico studies identified β-hematin and DNA topoisomerase II as potential biological targets of compounds 2 and 3, respectively. ConclusionThe findings from our study substantiate the traditional use of A. marlothii to treat malaria. To our knowledge, this study has provided the first report on the isolation and identification of antiplasmodial compounds from A. marlothii roots. Furthermore, our study has provided the first report on the transmission-blocking potential of one of the compounds from the genus Aloe, motivating for the investigation of other species within this genus for their potential P. falciparum transmission-blocking activity.

The Plasmodium falciparum ABC transporter ABCI3 confers parasite strain-dependent pleiotropic antimalarial drug resistance

SummaryWidespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.

The RNA structurome in the asexual blood stages of malaria pathogen plasmodium falciparum

ABSTRACT Plasmodium falciparum is a deadly human pathogen responsible for the devastating disease called malaria. In this study, we measured the differential accumulation of RNA secondary structures in coding and non-coding transcripts from the asexual developmental cycle in P. falciparum in human red blood cells. Our comprehensive analysis that combined high-throughput nuclease mapping of RNA structures by duplex RNA-seq, SHAPE-directed RNA structure validation, immunoaffinity purification and characterization of antisense RNAs collectively measured differentially base-paired RNA regions throughout the parasite’s asexual RBC cycle. Our mapping data not only aligned to a diverse pool of RNAs with known structures but also enabled us to identify new structural RNA regions in the malaria genome. On average, approximately 71% of the genes with secondary structures are found to be protein coding mRNAs. The mapping pattern of these base-paired RNAs corresponded to all regions of mRNAs, including the 5ʹ UTR, CDS and 3ʹ UTR as well as the start and stop codons. Histone family genes which are known to form secondary structures in their mRNAs and transcripts from genes which are important for transcriptional and post-transcriptional control, such as the unique plant-like transcription factor family, ApiAP2, DNA-/RNA-binding protein, Alba3 and proteins important for RBC invasion and malaria cytoadherence also showed strong accumulation of duplex RNA reads in various asexual stages in P. falciparum. Intriguingly, our study determined stage-specific, dynamic relationships between mRNA structural contents and translation efficiency in P. falciparum asexual blood stages, suggesting an essential role of RNA structural changes in malaria gene expression programs.

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration–response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate N-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development.

UDP-N-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and N- and O-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate N-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual Plasmodium falciparum parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from Cryptosporidium parvum, an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for P. falciparum GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria.IMPORTANCE Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite's ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate N-acetyltransferase (GNA1), required for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), is essential for P. falciparum asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite C. parvum, used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization of Plasmodium falciparum and Plasmodium knowlesi asexual blood-stage parasites.

During the course of the asexual erythrocytic stage of development, Plasmodium spp. parasites undergo a series of morphological changes and induce alterations in the host cell. At the end of this stage, the parasites egress from the infected cell, after which the progeny invade a new host cell. These processes are rapid and occur in a time-dependent manner. Of particular importance, egress and invasion of erythrocytes by the parasite are difficult to capture in an unsynchronized culture, or even a culture that has been synchronized within a window of one to several hours. Therefore, precise synchronization of parasite cultures is of paramount importance for the investigation of these processes. Here we describe a method for synchronizing Plasmodium falciparum and Plasmodium knowlesi asexual blood stage parasites with ML10, a highly specific inhibitor of the cGMP-dependent protein kinase (PKG) that arrests parasite growth approximately 15 minutes prior to egress. This inhibitor allows parasite cultures to be synchronized so that all parasites are within a window of development of several minutes, with a simple wash step. Furthermore, we show that parasites remain viable for several hours after becoming arrested by the compound and that ML10 has advantages, owing to its high specificity and low EC50, over the previously used PKG inhibitor Compound 2. Here, we demonstrate that ML10 is an invaluable tool for the study of Plasmodium spp. asexual blood stage biology and for the routine synchronization of P. falciparum and P. knowlesi cultures.

Activity of Epigenetic Inhibitors against Plasmodium falciparum Asexual and Sexual Blood Stages.

Earlier genetic and inhibitor studies showed that epigenetic regulation of gene expression is critical for malaria parasite survival in multiple life stages and a promising target for new antimalarials. We therefore evaluated the activity of 350 diverse epigenetic inhibitors against multiple stages of Plasmodium falciparum We observed ≥90% inhibition at 10 μM for 28% of compounds against asexual blood stages and early gametocytes, of which a third retained ≥90% inhibition at 1 μM.

Discovery of a new predominant cytosine DNA modification that is linked to gene expression in malaria parasites.

DNA cytosine modifications are key epigenetic regulators of cellular processes in mammalian cells, with their misregulation leading to varied disease states. In the human malaria parasite Plasmodium falciparum, a unicellular eukaryotic pathogen, little is known about the predominant cytosine modifications, cytosine methylation (5mC) and hydroxymethylation (5hmC). Here, we report the first identification of a hydroxymethylcytosine-like (5hmC-like) modification in P. falciparum asexual blood stages using a suite of biochemical methods. In contrast to mammalian cells, we report 5hmC-like levels in the P. falciparum genome of 0.2–0.4%, which are significantly higher than the methylated cytosine (mC) levels of 0.01–0.05%. Immunoprecipitation of hydroxymethylated DNA followed by next generation sequencing (hmeDIP-seq) revealed that 5hmC-like modifications are enriched in gene bodies with minimal dynamic changes during asexual development. Moreover, levels of the 5hmC-like base in gene bodies positively correlated to transcript levels, with more than 2000 genes stably marked with this modification throughout asexual development. Our work highlights the existence of a new predominant cytosine DNA modification pathway in P. falciparum and opens up exciting avenues for gene regulation research and the development of antimalarials.

Screen of traditional soup broths with reported antipyretic activity towards the discovery of potential antimalarials

ObjectiveThe global impact of artemisinin-based combination therapies on malaria-associated mortality and their origins in ancient Chinese medicine has heightened interest in the natural discovery of future antimalarials.MethodsA double-blind study to...

High-throughput screening of the Plasmodium falciparum cGMP-dependent protein kinase identified a thiazole scaffold which kills erythrocytic and sexual stage parasites

Antimalarial drug resistance compels the quest for new compounds that target alternative pathways to current drugs. The Plasmodium cyclic GMP-dependent protein kinase (PKG) has essential functions in all of the major life cycle developmental stages. An imidazopyridine PKG inhibitor scaffold was previously shown to clear P. falciparum infection in a rodent model in vivo and blocked transmission to mosquitoes providing proof of concept for this target. To find new classes of PKG inhibitors to serve as alternative chemical starting points, we performed a high-throughput screen of the GSK Full Diversity Collection using recombinant P. falciparum PKG. We developed a robust enzymatic assay in a 1536-well plate format. Promising compounds were then tested for activity against P. falciparum asexual blood stage growth, selectivity and cytotoxicity. By using a scoring system we selected the 66 most promising PKG inhibitors (comprising nine clusters and seven singletons). Among these, thiazoles were the most potent scaffold with mid-nanomolar activity on P. falciparum blood stage and gamete development. Using Kinobeads profiling we identified additional P. falciparum protein kinases targeted by the thiazoles that mediate a faster speed of the kill than PKG-selective compounds. This scaffold represents a promising starting point to develop a new antimalarial.

One-pot, multi-component synthesis and structure-activity relationships of peptoid-based histone deacetylase (HDAC) inhibitors targeting malaria parasites

Malaria drug discovery has shifted from a focus on targeting asexual blood stage parasites, to the development of drugs that can also target exo-erythrocytic forms and/or gametocytes in order to prevent malaria and/or parasite transmission. In this work, we aimed to develop parasite-selective histone deacetylase inhibitors (HDACi) with activity against the disease-causing asexual blood stages of Plasmodium malaria parasites as well as with causal prophylactic and/or transmission blocking properties. An optimized one-pot, multi-component protocol via a sequential Ugi four-component reaction and hydroxylaminolysis was used for the preparation of a panel of peptoid-based HDACi. Several compounds displayed potent activity against drug-sensitive and drug-resistant P. falciparum asexual blood stages, high parasite-selectivity and submicromolar activity against exo-erythrocytic forms of P. berghei. Our optimization study resulted in the discovery of the hit compound 1u which combines high activity against asexual blood stage parasites (Pf 3D7 IC50: 4 nM; Pf Dd2 IC50: 1 nM) and P. berghei exo-erythrocytic forms (Pb EEF IC50: 25 nM) with promising parasite-specific activity (SIPf3D7/HepG2: 2496, SIPfDd2/HepG2: 9990, and SIPbEEF/HepG2: 400).

Shelph2, a bacterial-like phosphatase of the malaria parasite Plasmodium falciparum, is dispensable during asexual blood stage.

During the erythrocytic cycle of the malaria parasite Plasmodium falciparum, egress and invasion are essential steps finely controlled by reversible phosphorylation. In contrast to the growing number of kinases identified as key regulators, phosphatases have been poorly studied, and calcineurin is the only one identified so far to play a role in invasion. PfShelph2, a bacterial-like phosphatase, is a promising candidate to participate in the invasion process, as it was reported to be expressed late during the asexual blood stage and to reside within an apical compartment, yet distinct from rhoptry bulb, micronemes, or dense granules. It was also proposed to play a role in the control of the red blood cell membrane deformability at the end of the invasion process. However, genetic studies are still lacking to support this hypothesis. Here, we take advantage of the CRISPR-Cas9 technology to tag shelph2 genomic locus while retaining its endogenous regulatory regions. This new strain allows us to follow the endogenous PfShelph2 protein expression and location during asexual blood stages. We show that PfShelph2 apical location is also distinct from the rhoptry neck or exonemes. We further demonstrate PfShelph2 dispensability during the asexual blood stage by generating PfShelph2-KO parasites using CRISPR-Cas9 machinery. Analyses of the mutant during the course of the erythrocytic development indicate that there are no detectable phenotypic consequences of Pfshelph2 genomic deletion. As this lack of phenotype might be due to functional redundancy, we also explore the likelihood of PfShelph1 (PfShelph2 paralog) being a compensatory phosphatase. We conclude that despite its cyclic expression profile, PfShelph2 is a dispensable phosphatase during the Plasmodium falciparum asexual blood stage, whose function is unlikely substituted by PfShelph1.