Franka Teuscher

Artemisinin-Induced Dormancy in Plasmodium falciparum: Duration, Recovery Rates, and Implications in Treatment Failure

BACKGROUND 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. METHODS 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. RESULTS 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 approximately 10-fold reduction in total recovery. Strains with different genetic backgrounds appeared to vary in their capacity to recover. CONCLUSIONS 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.

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.

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

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

BackgroundArtemisinin-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.MethodsIn this study, an existing P. falciparum infection model is modified to incorporate the hypothesis of dormancy. Published in vitro 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.ResultsThe 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.ConclusionsAlthough further studies are required to confirm dormancy in vivo, 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.

The m18 aspartyl aminopeptidase of the human malaria parasite Plasmodium falciparum

A member of the M18 family of aspartyl aminopeptidases is expressed by all intra-erythrocytic stages of the human malaria parasite Plasmodium falciparum (PfM18AAP), with highest expression levels in rings. Functionally active recombinant enzyme, rPfM18AAP, and native enzyme in cytosolic extracts of malaria parasites are 560-kDa octomers that exhibit optimal activity at neutral pH and require the presence of metal ions to maintain enzymatic activity and stability. Like the human aspartyl aminopeptidase, the exopeptidase activity of PfM18AAP is exclusive to N-terminal acidic amino acids, glutamate and aspartate, making this enzyme of particular interest and suggesting that it may function alongside the malaria cytosolic neutral aminopeptidases in the release of amino acids from host hemoglobin-derived peptides. Whereas immunocytochemical studies using transgenic P. falciparum parasites show that PfM18AAP is expressed in the cytosol, immunoblotting experiments revealed that the enzyme is also trafficked out of the parasite into the surrounding parasitophorous vacuole. Antisense-mediated knockdown of PfM18AAP results in a lethal phenotype as a result of significant intracellular damage and validates this enzyme as a target at which novel antimalarial drugs could be directed. Novel phosphinic derivatives of aspartate and glutamate showed modest inhibition of rPfM18AAP but did not inhibit malaria growth in culture. However, we were able to draw valuable observations concerning the structure-activity relationship of these inhibitors that can be employed in future inhibitor optimization studies.

Phenotypic changes in artemisinin resistant Plasmodium falciparum lines in vitro: evidence for decreased sensitivity to dormancy and growth inhibition

ABSTRACT 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.

Fatty Acid Synthesis and Pyruvate Metabolism Pathways Remain Active in Dihydroartemisinin-Induced Dormant Ring Stages of Plasmodium falciparum

ABSTRACT 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 have been implicated as possible causes 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 downregulation 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 of genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, was also maintained. Additions of inhibitors for biotin acetyl-coenzyme A (CoA) carboxylase 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 that most metabolic pathways are downregulated 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.

From the Cover: Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

Structural basis for the inhibition of the essentialPlasmodium falciparumM1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

The aspartyl aminopeptidase of Plasmodium falciparum

By utilizing the enormous chemical diversity of natural products new compounds can be found targeting the malaria parasite Plasmodium falciparum, following precedent set by the isolation of quinine and artemisinin from plant biota. A subset of a natural product extract library was screened for antimalarial activity using the 3H-hypoxanthine radioisotopic assay. The library contained 794 plant extracts from biota collected in Papua New Guinea and tropical Queensland encompassing 123 plant families. Ninety-three of these extracts displayed >40% inhibition against 3D7 parasites at 312μge/mL extract concentration. By retesting these extracts in dose response, antimalarial activity was confirmed in 48 of these extracts against both 3D7 and Dd2 parasites at concentrations between 78 and 390μge/mL. Some of the extracts displayed IC50 values of <20μge/mL against 3D7 and Dd2 and were 12 – 30 times more selective for the parasite than the HeLa-WT mammalian cell line. Progress on the isolation of active compounds from PNG extracts will be reported.Poster presented at MAM 2008, the 3rd Molecular Approaches to Malaria Meeting. The Trichomonas vaginalis TrxR was analysed to assess the efficacy of the organotellurides against a low Mr TrxR, which act by a very different mechanism to that of the high Mr TrxR. The results that will be presented show that: (a) Selectivity of inhibition between hTrxR and PfTrxR is possible (b) Selective inhibition between high Mr and low Mr TrxR can be achieved (c) The generation of inhibitors that specifically affect parasite viability is feasible.Aspartyl aminopeptidases are exopeptidases of the M18 family that specifically remove acidic amino acids (Glu and Asp) from the unblocked N-termini of peptides and proteins. Little is known about the general function of this family of proteases although a role in protein catabolism or processing has been suggested. Investigation of their function is hampered by the lack of potent inhibitors. We have shown that a P. falciparum aspartyl aminopeptidase gene is transcribed and translated in intra- erythrocytic stage parasites and its activity can be detected in soluble extracts of malaria parasites. Immunolocalisation and immunoblotting experiments show that the P. falciparum aspartyl aminopeptidase is expressed in the cytosol and also exported to the parasitophorous vacuole of the parasite and is, therefore, likely to play a role other than in protein catabolism. Using antisense RNA technology to knockdown gene function we have shown that the aspartyl aminopeptidase activity identified in parasite extracts is the product of the PFI1570c gene (Plasmodb.org). Significant morphological differences were also seen in antisense-mediated knockdown parasites, consistent with a lethal phenotype. These results suggest that the PfM18 aspartyl aminopeptidase is suitable target for chemotherapeutic intervention.