Overexpression of Leucyl Aminopeptidase in Plasmodium falciparum Parasites

Gardiner, Donald L.; Trenholme, Katharine R.; Skinner‐Adams, Tina S.; Stack, Colin M.; Dalton, John P.

doi:10.1074/jbc.m508955200

Cited by 58 publications

(53 citation statements)

References 37 publications

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“…Our report of the unique active site structure of PfA-M1, in complex with the antimalarial inhibitors bestatin and Co4, provides the groundwork for the de novo discovery of a previously unrecognized class of antimalarials by using high-throughput chemical screening and medicinal chemistry. Co4 also inhibits the second important neutral aminopeptidase of malaria, PfA-M17 (12,26), creating the possibility of developing a 2-target or combination therapy that would be more resilient to the emergence of drug-resistant malaria parasites.…”

Section: Discussionmentioning

confidence: 99%

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

McGowan

Porter

Lowther

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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143

278

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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[CH 2]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. drug design ͉ malaria ͉ structural biology ͉ protease T here are 300-500 million cases of clinical malaria annually, and 1.4-2.6 million deaths. Malaria is caused by apicomplexan parasites of the genus Plasmodium, with Plasmodium falciparum the most lethal of the 4 species that infect humans. Clinical manifestations begin when parasites enter erythrocytes, and most antimalaria drugs, such as chloroquine, exert their action by preventing the parasite development within these cells (1). As a result of the rapid spread of drug-resistant parasites, there is a constant need to identify and validate new antimalarial targets.Intraerythrocytic parasites have limited capacity for de novo amino acid synthesis and rely on degradation of host hemoglobin (Hb) to maintain protein metabolism and synthesis, and an osmotically stable environment within the erythrocyte (1-4). Within the erythrocytes, malaria parasites consume as much as 75% of the cellular Hb (1). Hb is initially degraded by the concerted action of cysteine-, aspartyl-, and metalloendoproteases, and a dipeptidase (cathepsin C) within a digestive vacuole (DV) to di-and tripeptide fragments (5, 6). These fragments are suggested to be exported to the parasite cytoplasm, where further hydrolysis to release free amino acids takes place [supporting information (SI) Fig. S1; see refs. 7 and 8].The release of amino acids involves 2 metallo-exopeptidases: an alanyl aminopeptidase, PfA-M1 (9, 10), and a leucine aminopeptidase, PfA-M17 (7,11,12). We have demonstrated that the aminopeptidase inhibitor bestatin, an antibiotic and natural analogue of the dipeptide Phe-Leu derived from the fungus Streptomyces olivoretticul, prevents P. falciparum malaria growth in culture (13,14). More recently, it was shown not only that synthetic phosphinate dipeptide analogues that inhibit metallo-aminopeptidases prevented the growth of wildty...

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Section: Discussionmentioning

confidence: 99%

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

McGowan

Porter

Lowther

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

143

278

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“…A widely cited model holds that the terminal stages of hemoglobin degradation occur in the cytosol, with peptides exported from the FV for hydrolysis by cytosolic aminopeptidases (11,12). This model is based largely on the apparent lack of aminopeptidase (AP) activity in extracts of enriched FVs (12), on the localization of two P. falciparum APs to the cytosol (13)(14)(15), and on the apparent absence of carboxypeptidase homologs from the annotated parasite genome sequence (16). Support for this model is weakened, however, by the absence of experimental evidence for peptide export from the FV, and by the presence of uncharacterized AP homologs in the P. falciparum genome sequence that could play a role in vacuolar peptide catabolism.…”

mentioning

confidence: 99%

Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum

Dalal

Klemba

2007

Journal of Biological Chemistry

150

186

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During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.With an estimated 2 million deaths annually to its name (1), the human malaria parasite Plasmodium falciparum continues to present an enormous public health challenge. Clinical manifestations of infection appear as the parasite replicates within host erythrocytes. During this replication cycle, the parasite endocytoses and catabolizes large amounts (up to 75%) of host hemoglobin to constituent amino acids (2, 3). This process makes available large quantities of free amino acids for parasite protein synthesis (4) and may also modulate the osmotic environment of the host cell (5) and/or create space inside the red cell for parasite growth (6). Amino acids derived from hemoglobin catabolism are also important for the uptake of isoleucine from the extracellular environment (7). As hemoglobin catabolism is essential for parasite replication and is currently a major focus of anti-malarial drug development efforts, a comprehensive understanding of the biochemistry of this pathway is an urgent priority.Hemoglobin catabolism occurs in an acidic, degradative organelle termed the food vacuole (FV) 2 or digestive vacuole. In the lumen of the FV, endopeptidases of diverse catalytic mechanism and specificity (plasmepsin (PM) I, II, and IV, histo-aspartic protease, falcipain-2, -2Ј, and -3, and falcilysin) contribute to the hydrolysis of the ␣-and ␤-globin chains to oligopeptides (8, 9). These ol...

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“…PfM17LAP was detected as a 68-70 kDa protein in soluble extracts of P. falciparum malaria parasites, immunocytochemical studies located the enzyme to the parasite cytoplasm. 41,52 This location has been confirmed by confocal microscopy of live transfected parasites expressing fluorescently-tagged PfM17LAP 30,41,52 . Using specific inhibitors of PfM17LAP, Harbut et al 45 showed that killing of P. falciparum in culture occurs early in the intraerythrocytic cycle (ring stages) prior to the onset of significant haemoglobin digestion suggesting that this enzyme does not play a part in this process.…”

Section: Do Pfm1aap and Pfm17lap Function In The Same Or Different Cementioning

confidence: 75%

“…30,34 We also generated transgenic P. falciparum parasites that over-expressed functionally active aminopeptidases in the cytoplasm and showed that these parasites were less susceptible to killing by bestatin (1) and hPheP[CH 2 ]Phe (3) when compared to wild-type parasites. 42,52 These data imply that both PfM1AAP and PfM17LAP are targets for the anti-malarial activity of these two broad-spectrum aminopeptidase inhibitors. Since most inhibitors used in the study of the malaria aminopeptidases block the activity of both PfM1AAP and PfM17LAP it remained unclear from reported in vitro and in vivo tests whether one or both enzymes was the targets of the aminopeptidase inhibitor-mediated killing of malaria parasites.…”

Section: Are Pfm1aap and Pfm17lap Targets For Anti-malarial Drug Devementioning

confidence: 98%

Anti-malaria drug development targeting the M1 alanyl and M17 leucyl aminopeptidases

Thivierge¹,

Mathew²,

Nsangou³

et al. 2012

Arkivoc

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The M1 alanyl aminopeptidase and M17 leucyl aminopeptidase are critical to the growth and development of malaria parasites inside host erythrocytes. Potent aminopeptidase inhibitors kill malaria parasites in culture and are also active in vivo against murine malaria. Functional recombinant enzyme studies have been used to decipher the three-dimensional structures of both enzymes that together with new and specific inhibitors are facilitating structure-activityrelationship (SAR) and functional studies. Here we review the progress made in our knowledge of these two enzymes which is bringing them closer to being validated anti-malarial drug targets.

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Overexpression of Leucyl Aminopeptidase in Plasmodium falciparum Parasites

Cited by 58 publications

References 37 publications

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

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

Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum

Anti-malaria drug development targeting the M1 alanyl and M17 leucyl aminopeptidases

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