Artemisinin-induced parasite dormancy: a plausible mechanism for treatment failure

<p>Abstract</p> <p>Background</p> <p>Artemisinin-combination therapy is a highly effective treatment for uncomplicated falciparum malaria but parasite recrudescence has been commonly reported following artemisinin (ART) monotherapy. The dormancy recovery hypothesis has been proposed to explain this phenomenon, which is different from the slower parasite clearance times reported as the first evidence of the development of ART resistance.</p> <p>Methods</p> <p>In this study, an existing <it>P. falciparum </it>infection model is modified to incorporate the hypothesis of dormancy. Published <it>in vitro </it>data describing the characteristics of dormant parasites is used to explore whether dormancy alone could be responsible for the high recrudescence rates observed in field studies using monotherapy. Several treatment regimens and dormancy rates were simulated to investigate the rate of clinical and parasitological failure following treatment.</p> <p>Results</p> <p>The model output indicates that following a single treatment with ART parasitological and clinical failures occur in up to 77% and 67% of simulations, respectively. These rates rapidly decline with repeated treatment and are sensitive to the assumed dormancy rate. The simulated parasitological and clinical treatment failure rates after 3 and 7 days of treatment are comparable to those reported from several field trials.</p> <p>Conclusions</p> <p>Although further studies are required to confirm dormancy <it>in vivo</it>, this theoretical study adds support for the hypothesis, highlighting the potential role of this parasite sub-population in treatment failure following monotherapy and reinforcing the importance of using ART in combination with other anti-malarials.</p

A Plasmodium falciparum S33 proline aminopeptidase is associated with changes in erythrocyte deformability

Infection with the apicomplexan parasite Plasmodium falciparum is a major cause of morbidity and mortality worldwide. One of the Striking features of this parasite is its ability to remodel and decrease the deformability of host red blood cells, a process that contributes to disease. To further understand the virulence of Pf we investigated the biochemistry and function of a putative Pf S33 proline aminopeptidase (PJPAP). Unlike other P. falciparum aminopeptidases, PJPAP contains a predicted protein export element that is non-syntenic with other human infecting Plasmodium species. Characterization of PJPAP demonstrated that it is exported into the host red blood cell and that it is a prolyl aminopeptidase with a preference for N-terminal proline substrates. In addition genetic deletion of this exopeptidase was shown to lead to an increase in the deformability of parasite-infected red cells and in reduced adherence to the endothelial cell receptor CD36 under flow conditions. Our studies suggest that PJPAP plays a role in the rigidification and adhesion of infected red blood cells to endothelial surface receptors, a role that may make this protein a novel target for anti-disease interventions strategies. (C) 2016 Elsevier Inc. All rights reserved

Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development

Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs

Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin induced dormant ring stages of Plasmodium falciparum

Artemisinin (ART) based combination therapy (ACT) is used as the first line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART induced ring stage dormancy and recovery has been implicated as possible cause of recrudescence; however, little is known about the characteristics of dormant parasites including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA) induced dormancy and recovery. Transcription analysis showed an immediate down regulation for 10 genes following exposure to DHA, but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, were also maintained. Additions of inhibitors for biotin acetyl CoA carbozylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively following DHA treatment. Our results demonstrate most metabolic pathways are down regulated in DHA induced dormant parasites. In contrast fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment

Artemisinin-induced dormancy in Plasmodium falciparum: Duration, recovery rates, and implications in treatment failure

*This article is free to read on the publisher's website*\ud \ud Background\ud \ud Despite the remarkable activity of artemisinin and its derivatives, monotherapy with these agents has been associated with high rates of recrudescence. The temporary arrest of the growth of ring-stage parasites (dormancy) after exposure to artemisinin drugs provides a plausible explanation for this phenomenon. \ud \ud Methods\ud \ud Ring-stage parasites of several Plasmodium falciparum lines were exposed to different doses of dihydroartemisinin (DHA) alone or in combination with mefloquine. For each regime, the proportion of recovering parasites was determined daily for 20 days. \ud \ud Results\ud \ud Parasite development was abruptly arrested after a single exposure to DHA, with some parasites being dormant for up to 20 days. Approximately 50% of dormant parasites recovered to resume growth within the first 9 days. The overall proportion of parasites recovering was dose dependent, with recovery rates ranging from 0.044% to 1.313%. Repeated treatment with DHA or with DHA in combination with mefloquine led to a delay in recovery and an ∼10-fold reduction in total recovery. Strains with different genetic backgrounds appeared to vary in their capacity to recover. \ud \ud Conclusions\ud \ud These results imply that artemisinin-induced arrest of growth occurs readily in laboratory-treated parasites and may be a key factor in P. falciparum malaria treatment failure

Phenotypic Changes in Artemisinin-Resistant Plasmodium falciparum Lines In Vitro: Evidence for Decreased Sensitivity to Dormancy and Growth Inhibition

The appearance of Plasmodium falciparum parasites with decreased in vivo sensitivity but no measurable in vitro resistance to artemisinin has raised the urgent need to characterize the artemisinin resistance phenotype. Changes in the temporary growth arrest (dormancy) profile of parasites may be one aspect of this phenotype. In this study, we investigated the link between dormancy and resistance, using artelinic acid (AL)-resistant parasites. Our results demonstrate that the AL resistance phenotype has (i) decreased sensitivity of mature-stage parasites, (ii) decreased sensitivity of the ring stage to the induction of dormancy, and (iii) a faster recovery from dormancy

Two new cyclopentanoids from the endophytic fungus Aspergillus sydowii associated with the marine alga Acanthophora spicifera

A marine-derived endophytic fungus, identified as Aspergillus sydowii was isolated from the inner tissue of the marine red alga Acanthophora spicifera. After mass cultivation of the fungus, chemical investigation afforded two new chlorinated 2,5-diarylcyclopentenones, which were named sydowin A and B. The structures of the new compounds were established on the basis of NMR spectroscopic (1H, 13C, 1H detected direct, and long range 1H-13C correlations) and mass spectrometric (ESI and FT-ICR-MS) data. Structurally related fungal compounds have hitherto only been described from higher basidiomycetes, but are characterized for the first time from an ascomycete in this study