Identification of inhibitors that dually target the new permeability pathway and dihydroorotate dehydrogenase in the blood stage of Plasmodium falciparum

Dickerman, Benjamin K.; Elsworth, Brendan; Cobbold, Simon A.; Nie, Catherine Q; McConville, Malcolm J.; Crabb, Brendan S.; Gilson, Paul R.

doi:10.1038/srep37502

Cited by 41 publications

(46 citation statements)

References 42 publications

(68 reference statements)

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“…Nevertheless, the specific metabolic profile observed in both the RhopH2 knockdown and furosemide-treated parasites (i.e decreased folate and phosphoenolpyruvate, increased threonine, histidine, asparagine, serine and argininosuccinate) supports a common impact on parasite biochemistry. This metabolic profile was not observed following treatment with 100 other antimalarial compounds using the same metabolomics methodology (Creek et al, 2016), suggesting this profile is specific for NPP inhibition and it is consistent with the increased threonine and histidine levels reported for other NPP inhibitors (where folate, phosphoenolpyruvate, serine and argininosuccinate were not assayed) (Dickerman et al, 2016). …”

Section: Discussionsupporting

confidence: 72%

“…In contrast, we saw an increase in the levels of N-carbamoyl aspartate in furosemide-treated parasites, and it was not expected that levels of this intermediate of de novo pyrimidine synthesis would directly depend on NPPs. Interestingly, other NPP inhibitors have been shown to also inhibit dihydroorotate dehydrogenase (DHODH) (Dickerman et al, 2016), an essential enzyme in this pathway that would modulate N-carbamoyl aspartate concentration, and it is likely that the furosemide treatment (at a concentration of 500 µM for ~24 hr) also has secondary effects on metabolism that differ from the RhopH2 knockdown. Nevertheless, the specific metabolic profile observed in both the RhopH2 knockdown and furosemide-treated parasites (i.e decreased folate and phosphoenolpyruvate, increased threonine, histidine, asparagine, serine and argininosuccinate) supports a common impact on parasite biochemistry.…”

Section: Discussionmentioning

confidence: 99%

“…A value of 0% lysis is defined as the Nluc activity of parasites in PBS containing nanoglo substrate. The rate of lysis was derived from a kinetic assay measuring the increase in RLU per minute (Dickerman et al, 2016). Data was analysed for statistical significance using an unpaired student’s t-test.…”

Section: Methodsmentioning

confidence: 99%

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Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

Counihan

Chisholm

Bullen

et al. 2017

eLife

107

135

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Plasmodium falciparum parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium-infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival.DOI: http://dx.doi.org/10.7554/eLife.23217.001

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Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

Counihan

Chisholm

Bullen

et al. 2017

eLife

107

135

View full text Add to dashboard Cite

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“…Here, we have utilized a trappable nanoluciferase (Nluc) reporter construct containing the murine dihydrofolate reductase (mDHFR) enzyme which is refractory to unfolding in the presence of WR99210 (WR) to further probe PTEX function . Nluc reporter proteins are highly quantifiable and have been used previously to examine the export of PEXEL proteins, resistance to sorbitol lysis, merozoite egress and invasion and other aspects of the parasite's life cycle . Here, we generated multiple exported constructs containing both Nluc and mDHFR, but with different additional epitope and affinity tags and followed their fates.…”

Section: Introductionmentioning

confidence: 99%

Spatial organization of protein export in malaria parasite blood stages

Charnaud

Jonsdottir

Sanders

et al. 2018

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Plasmodium falciparum, which causes malaria, extensively remodels its human host cells, particularly erythrocytes. Remodelling is essential for parasite survival by helping to avoid host immunity and assisting in the uptake of plasma nutrients to fuel rapid growth. Host cell renovation is carried out by hundreds of parasite effector proteins that are exported into the erythrocyte across an enveloping parasitophorous vacuole membrane (PVM). The Plasmodium translocon for exported (PTEX) proteins is thought to span the PVM and provide a channel that unfolds and extrudes proteins across the PVM into the erythrocyte. We show that exported reporter proteins containing mouse dihydrofolate reductase domains that inducibly resist unfolding become trapped at the parasite surface partly colocalizing with PTEX. When cargo is trapped, loop-like extensions appear at the PVM containing both trapped cargo and PTEX protein EXP2, but not additional components HSP101 and PTEX150. Following removal of the block-inducing compound, export of reporter proteins only partly recovers possibly because much of the trapped cargo is spatially segregated in the loop regions away from PTEX. This suggests that parasites have the means to isolate unfoldable cargo proteins from PTEX-containing export zones to avert disruption of protein export that would reduce parasite growth.

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“…Nevertheless, the long-held view that the NPPs could serve as an antimalarial drug target was recently confirmed using known inhibitors of the pathway (e.g. furosemide) (Pillai et al, 2012;Dickerman et al, 2016).…”

Section: (A) New Permeation Pathwaysmentioning

confidence: 99%

The transportome of the malaria parasite

Martin

2019

Biological Reviews

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Membrane transport proteins, also known as transporters, control the movement of ions, nutrients, metabolites, and waste products across the membranes of a cell and are central to its biology. Proteins of this type also serve as drug targets and are key players in the phenomenon of drug resistance. The malaria parasite has a relatively reduced transportome, with only approximately 2.5% of its genes encoding transporters. Even so, assigning functions and physiological roles to these proteins, and ascertaining their contributions to drug action and drug resistance, has been very challenging. This review presents a detailed critique and synthesis of the disruption phenotypes, protein subcellular localisations, protein functions (observed or predicted), and links to antimalarial drug resistance for each of the parasite's transporter genes. The breadth and depth of the gene disruption data are particularly impressive, with at least one phenotype determined in the parasite's asexual blood stage for each transporter gene, and multiple phenotypes available for 76% of the genes. Analysis of the curated data set revealed there to be relatively little redundancy in the Plasmodium transportome; almost two‐thirds of the parasite's transporter genes are essential or required for normal growth in the asexual blood stage of the parasite, and this proportion increased to 78% when the disruption phenotypes available for the other parasite life stages were included in the analysis. These observations, together with the finding that 22% of the transportome is implicated in the parasite's resistance to existing antimalarials and/or drugs within the development pipeline, indicate that transporters are likely to serve, or are already serving, as drug targets. Integration of the different biological and bioinformatic data sets also enabled the selection of candidates for transport processes known to be essential for parasite survival, but for which the underlying proteins have thus far remained undiscovered. These include potential transporters of pantothenate, isoleucine, or isopentenyl diphosphate, as well as putative anion‐selective channels that may serve as the pore component of the parasite's ‘new permeation pathways’. Other novel insights into the parasite's biology included the identification of transporters for the potential development of antimalarial treatments, transmission‐blocking drugs, prophylactics, and genetically attenuated vaccines. The syntheses presented herein set a foundation for elucidating the functions and physiological roles of key members of the Plasmodium transportome and, ultimately, to explore and realise their potential as therapeutic targets.

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Identification of inhibitors that dually target the new permeability pathway and dihydroorotate dehydrogenase in the blood stage of Plasmodium falciparum

Cited by 41 publications

References 42 publications

Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

Spatial organization of protein export in malaria parasite blood stages

The transportome of the malaria parasite

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