Synthesis and antiplasmodial activity of 3-furyl and 3-thienylquinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives.

The aim of this study was to identify new compounds active against Plasmodium falciparum based on our previous research carried out on 3-phenyl-quinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives. Twelve compounds were synthesized and evaluated for antimalarial activity. Eight of them showed an IC(50) less than 1 microM against the 3D7 strain. Derivative 1 demonstrated high potency (IC(50)= 0.63 microM) and good selectivity (SI=10.35), thereby becoming a new lead-compound

An exported protein-interacting complex involved in the trafficking of virulence determinants in Plasmodium-infected erythrocytes

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer\u27s clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo

Novel components used for protein export and functionality in Plasmodium falciparum

BACKGROUND: Plasmodium parasites, the causative agents of malaria, amplify in the erythrocytes of their host animals. To thrive the parasites remodel their host cells, to import plasma nutrients and evade the immune system. The most pathogenic human parasite is Plasmodium falciparum (Pf), which causes severe morbidity and mortality due to a surface exposed exported protein, PfEMP1, which allows the infected erythrocytes (IE) to bind endothelial cells and avoid splenic clearance. This can cause severe pathogenesis as IE occlude microvasculature, and when this occurs in the brain can result in coma and death. In order to remodel their host cells the intra-erythrocytic parasites export effector proteins into their host cells. These proteins must first traverse the parasite’s plasma membrane and then the parasitophorous vacuole (PV) space and PV membrane (PVM) which envelop the parasite. Proteins cross the PVM in an unfolded state via a proteinaceous pore termed PTEX. There are five currently known components of PTEX including a Hsp100 chaperone. Hsp100s typically interact with other chaperone components, including Hsp70/Hsp40 pairs that can unfold and refold complex proteins. The aim of my thesis was to identify the co-chaperones and accessory proteins that co-operate with PTEX to facilitate protein export. RESULTS: Interrogation of the genome identified a Pf specific Hsp70 chaperone termed Hsp70-x. Antibodies made to Hsp70-x indicated it was first secreted into the PV and then exported into the IE. Although Hsp70-x lacks a typical export motif, genetic knockdown of PTEX expression greatly reduced Hsp70-x export demonstrating PTEX is its exporter. Microscopy of IE indicated that Hsp70-x co-localised with Hsp40 rich J-dots, and also with PfEMP1 implying that the chaperones aid PfEMP1 to reach the host surface. To further resolve Hsp70-x’s function, its gene was deleted creating a ∆hsp70-x mutant. The mutant, although viable, had a slightly longer cell cycle and proliferated more slowly than the parental line under limiting nutrient conditions. ∆hsp70-x also overexpressed a number of exported and chaperone proteins, perhaps as compensation for the loss of the Hsp70-x chaperone. Some of these included structural proteins and resulted in the IE becoming more rigid. PfEMP1 puncta were reduced during export although surface expression of PfEMP1 ultimately reached normal levels. Despite normal PfEMP1 surface display, ∆hsp70-x parasites were deficient at binding to endothelial cell receptors under flow conditions. To identify other proteins associated with PTEX, radiolabelled pulse chase and immunoprecipitation of PTEX identified PV1, which is a 50 kDa essential protein with no homology suggestive of function. A number of additional binding experiments indicated PV1 associated with PTEX and also with certain exported cargoes suggesting that it may aid the export of cargo proteins through PTEX. CONCLUSIONS: Hsp70-x is present in the PV where it could interact with PTEX to aid protein export, and in the host where it could help refold proteins that have been exported via PTEX in an unfolded state. Parasites lacking Hsp70-x grow more slowly, and the IE are more rigid and poorer at binding endothelial cell receptors. Some of these changes could be in response to altered transcription and translation of various exported proteins. The function of Hsp70-x appears to be diverse probably because it aids in the transport and efficient folding of many exported proteins required for nutrient acquisition and cytoadherance. Inhibition of Hsp70-x could therefore result in parasites with greatly reduced in vivo fitness and hence it is a potential drug target that would reduce parasite virulence. Identification of PV1, a novel member of PTEX, has increased our understanding of the mechanism of protein export, and this could also ultimately open new avenues for therapeutics

Novel components used for protein export and functionality in Plasmodium falciparum

BACKGROUND: Plasmodium parasites, the causative agents of malaria, amplify in the erythrocytes of their host animals. To thrive the parasites remodel their host cells, to import plasma nutrients and evade the immune system. The most pathogenic human parasite is Plasmodium falciparum (Pf), which causes severe morbidity and mortality due to a surface exposed exported protein, PfEMP1, which allows the infected erythrocytes (IE) to bind endothelial cells and avoid splenic clearance. This can cause severe pathogenesis as IE occlude microvasculature, and when this occurs in the brain can result in coma and death. In order to remodel their host cells the intra-erythrocytic parasites export effector proteins into their host cells. These proteins must first traverse the parasite’s plasma membrane and then the parasitophorous vacuole (PV) space and PV membrane (PVM) which envelop the parasite. Proteins cross the PVM in an unfolded state via a proteinaceous pore termed PTEX. There are five currently known components of PTEX including a Hsp100 chaperone. Hsp100s typically interact with other chaperone components, including Hsp70/Hsp40 pairs that can unfold and refold complex proteins. The aim of my thesis was to identify the co-chaperones and accessory proteins that co-operate with PTEX to facilitate protein export. RESULTS: Interrogation of the genome identified a Pf specific Hsp70 chaperone termed Hsp70-x. Antibodies made to Hsp70-x indicated it was first secreted into the PV and then exported into the IE. Although Hsp70-x lacks a typical export motif, genetic knockdown of PTEX expression greatly reduced Hsp70-x export demonstrating PTEX is its exporter. Microscopy of IE indicated that Hsp70-x co-localised with Hsp40 rich J-dots, and also with PfEMP1 implying that the chaperones aid PfEMP1 to reach the host surface. To further resolve Hsp70-x’s function, its gene was deleted creating a ∆hsp70-x mutant. The mutant, although viable, had a slightly longer cell cycle and proliferated more slowly than the parental line under limiting nutrient conditions. ∆hsp70-x also overexpressed a number of exported and chaperone proteins, perhaps as compensation for the loss of the Hsp70-x chaperone. Some of these included structural proteins and resulted in the IE becoming more rigid. PfEMP1 puncta were reduced during export although surface expression of PfEMP1 ultimately reached normal levels. Despite normal PfEMP1 surface display, ∆hsp70-x parasites were deficient at binding to endothelial cell receptors under flow conditions. To identify other proteins associated with PTEX, radiolabelled pulse chase and immunoprecipitation of PTEX identified PV1, which is a 50 kDa essential protein with no homology suggestive of function. A number of additional binding experiments indicated PV1 associated with PTEX and also with certain exported cargoes suggesting that it may aid the export of cargo proteins through PTEX. CONCLUSIONS: Hsp70-x is present in the PV where it could interact with PTEX to aid protein export, and in the host where it could help refold proteins that have been exported via PTEX in an unfolded state. Parasites lacking Hsp70-x grow more slowly, and the IE are more rigid and poorer at binding endothelial cell receptors. Some of these changes could be in response to altered transcription and translation of various exported proteins. The function of Hsp70-x appears to be diverse probably because it aids in the transport and efficient folding of many exported proteins required for nutrient acquisition and cytoadherance. Inhibition of Hsp70-x could therefore result in parasites with greatly reduced in vivo fitness and hence it is a potential drug target that would reduce parasite virulence. Identification of PV1, a novel member of PTEX, has increased our understanding of the mechanism of protein export, and this could also ultimately open new avenues for therapeutics

Knockdown of the translocon protein EXP2 in Plasmodium falciparum reduces growth and protein export.

Malaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effector proteins exported from the parasite into the host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment

Synthesis and antiplasmodial activity of 3-furyl and 3-thienylquinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives. Molecules 2008

Initial data presented at the 11 t

Synthesis and structure-activity relationship of 3-phenylquinoxaline 1,4-di-N-oxide derivatives as antimalarial agents.

As a continuation of our research and with the aim of obtaining new antimalarial agents, new series of 3-phenylquinoxaline 1,4-di-N-oxide derivatives have been synthesized following the classical Beirut reaction. Antiplasmodial activity was evaluated in vitro against Plasmodium falciparum by the incorporation of [3H]-hypoxanthine. Cytotoxicity was tested in KB cells by AlamarBlue assay. Twenty-one of the 60 compounds that were assayed against 3D7 (CQ-sensitive) showed enough activity to be also evaluated against K1 (CQ-resistant) strain. Ten of them were shown to be more active than chloroquine in the resistant strain. The most interesting compounds are 7-(methyl or methoxy)-3-(4'-fluoro or chloro)phenylquinoxaline-2-carbonitrile 1,4-di-N-oxides because of their low IC50 and their high SI shown for the K1 strain, making them valid new leads

<i>Plasmodium falciparum</i> Transfected with Ultra Bright NanoLuc Luciferase Offers High Sensitivity Detection for the Screening of Growth and Cellular Trafficking Inhibitors

<div><p>Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in <i>Plasmodium falciparum</i> and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.</p></div