Artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome

Bridgford, Jessica L.; Xie, Stanley C.; Cobbold, Simon A.; Pasaje, Charisse Flerida A.; Herrmann, Susann; Yang, Tuo; Gillett, David L.; Dick, Lawrence R.; Ralph, Stuart A.; Dogovski, Con; Spillman, Natalie J.; Tilley, Leann

doi:10.1038/s41467-018-06221-1

Cited by 230 publications

(262 citation statements)

References 52 publications

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“…Several mechanisms have been proposed to explain the role of K13 in ART resistance, including an enhanced cell stress response leading to upregulation of the unfolded protein response, decreased ubiquitination, and increased cell survival (32). Consistent with this model, inhibitors of the proteasome synergize with artemisinin (33, 34). However, other studies have reported the development of delayed clearance or dormancy phenotypes in P. falciparum -infected patients without associated K13 mutations (35, 36).…”

Section: Introductionmentioning

confidence: 59%

Evolution of resistance in vitro reveals a novel mechanism of artemisinin activity in Toxoplasma gondii

Rozenberg

Luth

Winzeler

et al. 2019

Preprint

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250)Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance in Plasmodium falciparum, including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability of T. gondii, a related apicomplexan parasite that shows moderate sensitivity to artemisinin. Resistant populations of T. gondii were selected by culture in increasing drug concentrations and whole genome sequencing identified several non-conservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acids changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild type parasites in the presence of drug, despite not changing EC 50 values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondrial genome, including genes encoding cytochrome b and cytochrome oxidase I. Consistent with prior studies in yeast and mammalian tumor cells that implicate the mitochondrion as a target of artemisinins, treatment of wild type parasites with artemisinin decreased mitochondrial membrane potential, and resistant parasites showed altered morphology and decreased membrane potential. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition in T. gondii. Significance (120)Artemisinins provide important therapeutic agents for treatment of malaria and have potential for use in other infections and in cancer. Their use is threatened by the potential for resistance development, so understanding their mechanism of action and identifying genetic changes that alter sensitivity are important for improving clinical outcomes. Our findings suggest that mutations in novel targets can contribute to the emergence of parasites with increased tolerance to artemisinin treatment and that such mutations can confer a fitness advantage even in the absence of a notable shift in EC 50 . Our findings also support the idea that inhibition of mitochondrial function may be an important target in T.gondii, as previously suggested by studies in yeast and human cancer cells.P. falciparum. The ART mechanism of action against tumor cells is also uncertain, but has been linked to oxidative stress, DNA damage, decreased cell replication, and activation of cell death pathways including apoptosis (41, 42). Studies in Baker's yeast (S. cerevisiae) implicate the mitochondria as a target of ART, and consistent with this model, growth on fermentable carbon sources results in genome sequence, as its assembly has been complicated by th...

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Section: Introductionmentioning

confidence: 59%

Evolution of resistance in vitro reveals a novel mechanism of artemisinin activity in Toxoplasma gondii

Rozenberg

Luth

Winzeler

et al. 2019

Preprint

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“…Responses to oxidative stress and protein damage are shown to mediate emergence of artemisinin resistance in malaria parasites [15, 28, 29, 45, 46]. Interestingly, 775 new PfGCN5 bound sites were acquired under artemisinin stress conditions as indicated by ChIP-sequencing of PfGCN5 (Fig 4A).…”

Section: Resultsmentioning

confidence: 99%

PfGCN5, a global regulator of stress responsive genes, modulates artemisinin resistance inPlasmodium falciparum

Rawat

Kanyal

Sahasrabudhe

et al. 2019

Preprint

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2 3 4 PfGCN5, a global regulator of stress responsive genes, modulates artemisinin resistance 5 in Plasmodium falciparum 2 25 Abstract26 Plasmodium falciparum has evolved resistance to almost all front-line drugs including 27 artemisinins, which threatens malaria control and elimination strategies. Oxidative stress and 28 protein damage responses have emerged as key players in the generation of artemisinin 29 resistance. In this study, we show that PfGCN5, a histone acetyltransferase, binds to the stress 30 responsive and multi-variant family genes in poised state and regulates their expression under 31 stress conditions. We have also provided biochemical and cellular evidences that PfGCN5 32 regulates stress responsive genes by acetylation of PfAlba3. Furthermore, we show that upon 33 artemisinin exposure, genome-wide binding sites for PfGCN5 are increased and it is directly 34 associated with the genes implicated in artemisinin resistance generation like BiP and TRiC 35 chaperone. Moreover, inhibition of PfGCN5 in artemisinin resistant parasites, Kelch13 36 mutant, K13I543T and K13C580Y (RSA~ 25% and 6%, respectively) reverses the sensitivity 37 of the parasites to artemisinin treatment indicating its role in drug resistance generation.38 Together, these findings elucidate the role of PfGCN5 as a global chromatin regulator of 39 stress-responses with potential role in modulating artemisinin drug resistance, and identify 40 PfGCN5 as an important target against artemisinin resistant parasites. 41 3 42 Author Summary 43 Malaria parasites are constantly adapting to the drugs we used to eliminate them. Thus, when 44 we use the drugs to kill parasites; with time, we select the parasites with the favourable 45 genetic changes. Parasites develop various strategies to overcome exposure to the drugs by 46 exhibiting the stress responses. The changes specific to the drug adapted parasites can be 47 used to understand the mechanism of drug resistance generation. In this study, we have 48 identified PfGCN5 as a global transcriptional regulator of stress responses in Plasmodium 49 falciparum. Inhibition of PfGCN5 reverses the sensitivity of the parasites to the artemisinin 50 drug and identify PfGCN5 as an important target against artemisinin resistant parasites. 51 52 53 54 55 56 57 58 59 4 60 Introduction 61 Malaria is a life threatening infectious disease caused by parasites from the genus 62 Plasmodium, with an estimated 200 million cases worldwide [1]. The Anopheles mosquito 63 serves as a vector for varied species of the human malaria parasite namely P. falciparum, P. 64 vivax, P. ovale, P. malariae and P. knowlesi. Of these five species, P. falciparum causes most 65 lethal form of malaria. The Plasmodium life cycle consists of two phases, sexual and asexual 66 in mosquitoes and humans, respectively. Since Plasmodium completes its life cycle in two 67 different hosts, it requires mechanisms for coordinated modulation of gene expression [2]. An 68 efficient transcriptional and post-transcriptional regulation of gene ex...

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“…Ubiquitylation is important in mediating the effects of artemisinin. Parasite treatment with artemisinin results in activation of the unfolded protein response and the accumulation of polyubiquitylated substrates, compromising proteasomal function [16,59,60], and the proteostatic dysregulation of parasite phosphatidylinositol 3-kinase (PI3K) [61]. Amino acid substitutions in the K13 Kelch protein are associated with artemisinin resistance, and it has been proposed that K13 is a ubiquitin E3 ligase adaptor for PI3K [61].…”

Section: Discussionmentioning

confidence: 99%

“…K13 and PI3K associate with the parasite ER and potentially involved in quality control of exported and cytoplasmic proteins [62,63]. Interestingly an E1 inhibitor antagonises dihydroartemisinin activity [16] and therefore such compounds would not be useful in combination with artemisinin, but DUB inhibitors such as PR619 may be useful in this regard.…”

Section: Discussionmentioning

confidence: 99%

“…Whilst there has been considerable interest in targeting the proteasome with newly identified drugs [15], until recently [16,17] ubiquitin biology has been largely neglected in malaria research. There is extensive current research to identify inhibitors of the ubiquitin pathway for other therapeutic applications, particularly in the area of cancer chemotherapy [18].…”

Section: Introductionmentioning

confidence: 99%

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Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Encheva

Green

Lasonder

et al. 2019

Preprint

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Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage.These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite's elaboration of a new food vacuole.

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Artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome

Cited by 230 publications

References 52 publications

Evolution of resistance in vitro reveals a novel mechanism of artemisinin activity in Toxoplasma gondii

Evolution of resistance in vitro reveals a novel mechanism of artemisinin activity in Toxoplasma gondii

PfGCN5, a global regulator of stress responsive genes, modulates artemisinin resistance inPlasmodium falciparum

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

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