Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cunningham, E. Articles by Bell, A. Search for Related Content PubMed PubMed Citation Articles by Cunningham, E. Articles by Bell, A.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, September 2008, p. 3221-3228, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.01327-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Chemical Target Validation Studies of Aminopeptidase in Malaria Parasites Using -Aminoalkylphosphonate and Phosphonopeptide Inhibitors

Eithne Cunningham,¹ Marcin Drag,^2, Pawel Kafarski,² and Angus Bell^1*

Department of Microbiology, School of Genetics and Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland,¹ Faculty of Chemistry, Biochemistry, and Biotechnology, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland²

Received 15 October 2007/ Returned for modification 12 December 2007/ Accepted 27 April 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During its intraerythrocytic phase, the most lethal human malarial parasite, Plasmodium falciparum, digests host cell hemoglobin as a source of some of the amino acids required for its own protein synthesis. A number of parasite endopeptidases (including plasmepsins and falcipains) process the globin into small peptides. These peptides appear to be further digested to free amino acids by aminopeptidases, enzymes that catalyze the sequential cleavage of N-terminal amino acids from peptides. Aminopeptidases are classified into different evolutionary families according to their sequence motifs and preferred substrates. The aminopeptidase inhibitor bestatin can disrupt parasite development, suggesting that this group of enzymes might be a chemotherapeutic target. Two bestatin-susceptible aminopeptidase activities, associated with gene products belonging to the M1 and M17 families, have been described in blood-stage P. falciparum parasites, but it is not known whether one or both are required for parasite development. To establish whether inhibition of the M17 aminopeptidase is sufficient to confer antimalarial activity, we evaluated 35 aminoalkylphosphonate and phosphonopeptide compounds designed to be specific inhibitors of M17 aminopeptidases. The compounds had a range of activities against cultured P. falciparum parasites with 50% inhibitory concentrations down to 14 µM. Some of the compounds were also potent inhibitors of parasite aminopeptidase activity, though it appeared that many were capable of inhibiting the M1 as well as the M17 enzyme. There was a strong correlation between the potencies of the compounds against whole parasites and against the enzyme, suggesting that M17 and/or M1 aminopeptidases may be valid antimalarial drug targets.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Malaria remains a widespread and devastating disease, and new drugs effective against the most lethal human parasite, Plasmodium falciparum, are desperately needed. The spread of drug-resistant parasites makes it crucially important to identify chemical classes and parasite targets that have not previously been exploited in antimalarial chemotherapy (9). During its intraerythrocytic cycle, the parasite digests up to 75% of the host cell hemoglobin, incorporating some of the liberated amino acids into parasite protein (19). This pathway of hemoglobin degradation has been studied intensively, and recent work has indicated that the process is needed for parasite survival (2, 25, 33). Inhibitors of aspartyl endopeptidases (plasmepsins I, II, and IV and the closely related histo-aspartyl protease) and cysteinyl endopeptidases (falcipains 2, 2', and 3) active on globin are under investigation as potential antimalarial drugs (10, 31). The initial cleavages take place in a digestive vacuole to which erythrocyte cytosol is transported. The terminal stages of globin degradation are thought to be catalyzed by one or more aminopeptidases that have generally been believed to be cytosolic (16), although recent work suggests that some may be active in the digestive vacuole (6). Aminopeptidase inhibitors, including bestatin, nitrobestatin, and amastatin, have antimalarial activity in culture (16, 27), and different combinations of endo- and aminopeptidase inhibitors show synergism (17).

Aminopeptidases catalyze the hydrolysis of amino acids from the N termini of proteins or peptides. Most are metallopeptidases, and they are classified into families based on sequence similarity (30) (http://merops.sanger.ac.uk). Often, members of the same family will share substrate preferences; e.g., M17 aminopeptidases generally have preferences for leucine at the N terminus and have often been termed leucine aminopeptidases, although many will hydrolyze other similarly hydrophobic residues (29). (We therefore use the MEROPS family designation for the enzymes where possible in this paper and use terms such as "leucyl aminopeptidase" [LAP] to refer to a measured activity on a leucine-containing substrate.) Aminopeptidases are involved in a number of diverse processes, including protein maturation, activation, and stability, and have been found to be associated with many diseases. For example, altered LAP activity is seen in cancer, cataracts, inflammation, and human immunodeficiency virus infection (20).

A number of aminopeptidase genes have been identified in the P. falciparum genome (38). The best studied of these are the metalloaminopeptidases P. falciparum aminopeptidase M1 (PfAP-M1; also referred to as PfA-M1) (1, 14) and PfAP-M17 (also referred to as PfLAP) (35), which have been characterized in blood-stage parasites. Both PfAP-M1 and PfAP-M17 are inhibited by bestatin, but it is not clear whether one or both of these enzymes are the targets for the antimalarial activity of this compound (1, 35). Both were located in the parasite cytosol and were more active at near-neutral pHs. Partially purified PfAP-M1 has been shown to be processed from a 122-kDa protein to two smaller fragments of 96 and 68 kDa. Although M1 family enzymes are generally regarded as "alanyl" aminopeptidases (AAPs), PfAP-M1 preferentially digested lysine substrates in vitro, followed closely by alanine, arginine, and leucine substrates. The AAP and LAP activities of the protein were very similar (1). The PfAP-M17 gene encodes a protein of 68 kDa that appears, like most M17 aminopeptidases, to form catalytically active homohexamers. Recombinant protein had good LAP activity (but very poor AAP activity), and this LAP activity was greatly increased by incubation with divalent metal ions, particularly Co²⁺ or Mn²⁺ (35). As expected, a cytosolic extract from the parasites demonstrates AAP and LAP activities (16). A parasite extract incubated with CoCl₂ and separated by high-performance liquid chromatography showed two peaks of LAP activity at 82 kDa and 320 kDa, presumed to correspond to fractions containing the M1 and M17 enzymes, respectively. The 82-kDa peak also had AAP activity, but the 320-kDa peak did not. The other aminopeptidase genes identified in the P. falciparum genome are not expected to encode products susceptible to bestatin and are not considered further here. Methionine aminopeptidases (M24A family) have been proposed to be good drug targets (5, 39), but they are unlikely to be involved in globin digestion.

So far, no knockouts of either the PfAP-M1 or the PfAP-M17 aminopeptidase gene in P. falciparum have been reported. Transgenic parasites carrying the PfAP-M17 gene on a multicopy plasmid demonstrated reduced susceptibility to bestatin, suggesting that PfAP-M17 might be the major target of this agent (15). The overexpression of PfAP-M17 was not quantified, however, and an indirect effect on PfAP-M1 due to the increased PfAP-M17 copy number could not be ruled out. No line overproducing PfAP-M1 was obtained. Therefore, it is not clear whether one or both of the two enzymes are the targets for the antimalarial activity of bestatin. Although bestatin is generally specific for M1 and M17 family aminopeptidases, the mammalian (M16) endopeptidase nardilysin is also susceptible (32).

The best inhibitors of M17 family aminopeptidases (based primarily on work with the M17 family aminopeptidase from porcine kidney [pkLAP]) are analogues of short peptides, e.g., the well-known bestatin. Amino acid analogues are also good inhibitors, e.g., L-leucinal and the phosphonic acid analogue of L-leucine (LeuP), with the latter group being more specific for metallopeptidases. Both groups of inhibitors act as transition state analogues, binding to the metal ions in the active site of the enzyme (18). In this study, we have assessed the antimalarial and antiaminopeptidase activities of a series of phosphonic derivatives designed against M17 aminopeptidase. In order to assess the validity of the PfAP-M17 enzyme as a target, we employed as chemical tools a structurally related series of -aminoalkylphosphonates and phosphonopeptides designed to target the active site. We used knowledge of the differing substrate specificities and cation dependencies of the M1 and M17 enzymes to dissect the inhibition of the two activities in parasite cytosolic extracts. Our results support the contention that PfAP-M17 may be a valid antimalarial drug target amenable to the design of specific inhibitors. They also suggest that PfAP-M1 is a possible alternative or additional target.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents. All reagents were purchased from Sigma Aldrich, Dublin, Ireland, unless otherwise stated. Stock solutions of inhibitors were prepared by dissolving them in phosphate-buffered saline to concentrations of 1 to 5 mM, followed by filter sterilization.

Synthesis of inhibitors. The compounds used in this study were synthesized as described previously. Racemic 1-aminoalkanephosphonic acids were obtained using either Oleksyszyn or modified Arbuzov methodologies (24, 28). Enantiomerically pure or diastereomer pairs of 1-aminoalkylphosphonates were synthesized using the Hamilton-Walker reaction (22). 2-Amino-1-hydroxyphosphonates were synthesized as described by Drag et al. (8). Dipeptidic analogues of the 1-aminoalkylphosphonates were synthesized as described by Kafarski and Lejczak (23). Compound D36 was obtained by the monoesterification of the diphenyl ester of the 1-aminoalkylphosphonate, as described by Szewczyk et al. (36).

Parasite culture and growth inhibition. P. falciparum clone 3D7 (obtained from M Grainger, National Institute of Medical Research, London, United Kingdom) was cultured in O⁺ erythrocytes as described previously (11). Inhibitor susceptibility assays were carried out on asynchronous, asexual, blood-stage parasites in 96-well plates by using the spectrophotometric parasite lactate dehydrogenase assay described by Makler et al. (26). Dose-response curves were constructed from absorbance readings after 72 h of incubation, and the 50% inhibitory concentrations (IC₅₀) were determined graphically.

Preparation of material for analysis of aminopeptidase activity. Free parasites were harvested from infected erythrocytes according to the method of Zuckerman et al. (40), and cytosolic parasite extracts were prepared by three cycles of freeze-thawing followed by centrifugation at 12,000 x g and 4°C for 20 min, as described previously (16). Recombinant tPfAP-M17, a truncated form of PfAP-M17 lacking the first 82 residues and with the asparagine residues at positions 152, 515, and 546 changed to glutamines, was provided by John P. Dalton, IBID, University of Technology, Sydney, Australia. The recombinant enzyme was produced in baculovirus-infected Sf9 cells and was purified by Ni²⁺ chelate affinity chromatography, as described previously (35). Protein concentrations were determined by the Bradford method (3).

Analysis of aminopeptidase activity and inhibition. Aminopeptidase activity was determined by measurement of the release of the fluorogenic leaving group 7-amino-4-methyl-coumarin (AMC) from peptide substrates (Leu-AMC and Ala-AMC, used to measure LAP and AAP activities, respectively) essentially as described previously (35). The parasite extract (final concentration, 25 µg/ml) or recombinant protein (final concentration, 2.7 µg/ml or 0.05 µM) was incubated in 50 mM Tris-Cl, pH 8.0, with or without CoCl₂ (1 mM) and/or inhibitors (10 or 0.1 µM) as required, for 15 min at 37°C before addition of the substrate to a final concentration of 10 µM. Reactions were measured over a 30-min period and were read using a spectrofluorimeter (LS-50B; Perkin-Elmer) with excitation at 370 nm and emission at 440 nm. The 15-min preincubation was found to be sufficient for maximal inhibition of enzyme activity by the compounds used at the concentrations indicated here. For all compounds, the reactions were linear for the full duration of the experiment, and for a subset of the compounds tested, the reactions were linear for as long as 2 h (see Fig. 1 inset).

View larger version (15K): [in this window] [in a new window]

FIG. 1. Aminopeptidase activities of the P. falciparum cytosolic extract. The extract was incubated in 50 mM Tris-Cl (pH 8.0) with or without CoCl2 (1 mM) at 37°C for 15 min before the addition of the substrate, Ala-AMC or Leu-AMC, to a final concentration of 10 µM. Activity is expressed as a percentage of the AAP activity of the extract in the absence of CoCl2. (Inset) Linear reaction rate observed for the inhibition of the LAP activity of the parasite extract. The fluorescence produced by the cleavage of a Leu-AMC substrate by the parasite extract (preincubated in 1 mM CoCl2, with or without an inhibitor) was observed over a 2-h period. The open circles and the continuous line indicate the LAP activity of the extract alone, while the filled triangles and the dotted line indicate the inhibition of the activity of the extract by D14. Similar profiles were observed for D7 and D12 (data not shown).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antimalarial activities of -aminoalkylphosphonates and phosphonopeptides. The series of -aminoalkylphosphonate and phosphonopeptide compounds were tested against cultured P. falciparum parasites. They had various potencies against the parasites, ranging from inactivity at the highest concentrations possible to IC₅₀ as low as 14 µM (Table 1). The two most active compounds were D14 and D12, alicyclic phosphonates (IC₅₀, 14 µM and 15 µM, respectively). These were closely followed by D7 and D17, phosphonic acid analogues of phenylalanine with extended aliphatic chains of one or three methylene groups, respectively (IC₅₀, 21 and 22 µM, respectively). D36, a monophenyl ester derivative of D7, was synthesized in the hope of improving uptake into parasites. It demonstrated similar but not improved activity. A number of the compounds had very low or negligible antimalarial activity, inhibiting parasite growth by less than 10% at the highest concentration tested (256 µM).

View this table: [in this window] [in a new window]

TABLE 1. Activities of compounds on cultured P. falciparum parasites

Inhibition of the LAP and AAP activities of the parasite extract. We first investigated the abilities of the series of compounds to inhibit the aminopeptidase activities of parasite cytosolic extracts (14, 27). The rationale for this approach was to assess whether the compounds were, like bestatin, capable of inhibiting the entire measurable aminopeptidase activity of asexual blood-stage parasites. The extracts were expected, on the basis of previous work (summarized above), to contain both PfAP-M1 and PfAP-M17 activities. Compounds were tested at two fixed concentrations, 10 µM and 0.1 µM, by incubating them with the cytosolic extract before the addition of the fluorogenic substrate, Leu-AMC or Ala-AMC. A range of activities against LAP activity was seen (Table 2), with the five most potent compounds, D14, D12, D17, D7, and D36, inhibiting LAP activity by 90% when assayed at 10 µM, while the least effective compounds could inhibit LAP activity only by a few percentage points or not at all. The compounds are ordered by decreasing antimalarial activity (i.e., by increasing IC₅₀ [see Table 1]) in Table 2 and demonstrate a corresponding trend of a decreasing percentage of inhibition of enzyme activity, indicating the good correlation between antimalarial and anti-LAP activities. There were a couple of minor exceptions to the general trend of agreement, including D18, which might be expected to have better antimalarial activity (e.g., closer to that of D2 or D13) given its antiaminopeptidase activity. Conversely, D21 might be expected to inhibit the parasite extract to a higher degree given its IC₅₀.

View this table: [in this window] [in a new window]

TABLE 2. Inhibition of the aminopeptidase activities of the parasite cytosolic extract

A similar pattern of inhibition of the AAP activity of the extract, and a similar correlation with antimalarial activity, was observed. This indicated that the compounds might be having some effect on the M1 aminopeptidase of P. falciparum, since previous work has indicated that PfAP-M17 has low AAP activity (35). Inhibition of PfAP-M1 was not entirely unexpected; although these compounds were designed as specific M17 aminopeptidase inhibitors, some of them demonstrated activity against the mammalian M1 family enzyme aminopeptidase N (7).

Contributions of PfAP-M17 and PfAP-M1 to the aminopeptidase activities of the parasite cytosolic extract. In order to investigate which aminopeptidase activity was being inhibited by the compounds, the parasite extract was tested for LAP and AAP activities in the presence of CoCl₂, which was expected, on the basis of previous results (35; also unpublished data), to boost PfAP-M17 activity but reduce PfAP-M1 activity. Following incubation with CoCl₂, the overall LAP activity of the extract was reduced markedly, to 21% of its original activity, but the AAP activity was virtually abolished, down to 4% (Fig. 1). Therefore, we deduced that, providing that the enzymes behave similarly in pure and impure preparations, (i) assaying the parasite extract without CoCl₂ and with Ala-AMC as the substrate should measure PfAP-M1 almost exclusively, while (ii) assaying the extract in the presence of CoCl₂ and with Leu-AMC as the substrate should measure predominantly PfAP-M17. Although the effect of Co²⁺ on the PfAP-M1 holoenzyme preparation of Allary et al. (1) was not measured, other reports of aminopeptidase-containing Plasmodium fractions with characteristics similar to those of the M1 enzyme indicated that their activities were substantially reduced by 1 mM Co²⁺ (4, 21, 37), though not much affected by 0.5 mM Co²⁺ (35).

Inhibition of the LAP activity of the parasite extract in the presence and absence of Co²⁺ ions. The same general trend of inhibition of LAP activity was seen for the parasite extract incubated with CoCl₂ as for the extract without CoCl₂ (Table 3). Overall, there was a strong trend for the compounds to show some increased inhibition of LAP activity in the presence of CoCl₂, i.e., under conditions favoring PfAP-M17 activity. This led us to consider the possibility that most of the inhibitors may (unlike bestatin) be better inhibitors of PfAP-M17 than of PfAP-M1. However, the percentages of inhibition are not comparable because of differences in the K_m between PfAP-M1 (60 µM [1]) and PfAP-M17 (12 µM [35]). Unfortunately, it was not possible to compare the susceptibilities of PfAP-M17 and PfAP-M1 directly, because the latter was not available in recombinant form.

View this table: [in this window] [in a new window]

TABLE 3. Inhibition of the LAP activity of the parasite extract in the absence and presence of CoCl2

Correlation of the antimalarial and antiaminopeptidase activities of inhibitors. A clear linear relationship between the antimalarial activities of the compounds and their antiaminopeptidase activities was seen when the percentages of inhibition (at 10 µM) of the LAP activity (in the presence of CoCl₂) and AAP activity of the extract were plotted against the log IC₅₀ for growth in culture (Fig. 2). The data were analyzed using Spearman nonparametric correlation and were found to have r values of –0.8531 (P = 0.0008) and –0.8671 (P = 0.0005) for LAP and AAP activities, respectively. This suggests a good negative correlation between the log IC₅₀ of the compounds and their percentages of inhibition of the aminopeptidase activities of the extract; i.e., as the antimalarial activity decreases, so too does the inhibition of the aminopeptidase activity of the extract.

View larger version (15K): [in this window] [in a new window]

FIG. 2. Correlation of antimalarial activity and inhibition of the aminopeptidase activity of the parasite extract. The percentages of inhibition of the LAP and AAP activities of the parasite extract by the 12 most active compounds (at 10 µM) were plotted against their log IC50 for parasite growth. The open circles and the continuous line indicate the inhibition of the LAP activity of the parasite extract preincubated in CoCl2 (1 mM) (r = –0.8531; P = 0.0008), while the filled triangles and the dotted line indicate the inhibition of the AAP activity (r = –0.8671; P = 0.0005). Spearman rank correlation was used to analyze the data.

Inhibition of recombinant PfAP-M17 activity. A subset of the compounds was tested for their abilities to inhibit the LAP activity of the recombinant P. falciparum M17 aminopeptidase tPfAP-M17 (Table 4). This recombinant enzyme is a truncated (58-kDa) form of the protein produced in baculovirus-infected insect cells. Very similar profiles of LAP inhibition of the recombinant enzyme and the extract were seen. As a result, there is a correspondingly good correlation between the inhibition of the recombinant enzyme and antimalarial activity, although D17 is a slight exception to this. Considering its good inhibition of the mammalian M1 family enzyme aminopeptidase N relative to that by the other compounds, it is possible that this inhibitor has slightly more activity against PfAP-M1. D14 is the best inhibitor of recombinant tPfAP-M17, and it also has the best antimalarial activity of all the compounds tested. However, it is the poorest inhibitor of pkLAP, with a K_i of 9.57 µM, indicating that it may be somewhat selective for PfAP-M17 over the mammalian enzyme.

View this table: [in this window] [in a new window]

TABLE 4. Inhibition of the LAP activities of the parasite extract and recombinant tPfAP-M17 incubated with CoCl2

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Comparison of the results of the antimalarial and antiaminopeptidase (whole-parasite extract and recombinant enzyme) activity assays with the data previously obtained for mammalian M1 and M17 aminopeptidase inhibition enables the identification of the properties likely to confer potency and selectivity against PfAP-M17. The inhibitors had a range of antimalarial potencies down to low micromolar IC₅₀. The most potent compounds were those with long, hydrophobic side chains (D14, D12, D17, and D7) that might occupy the S1 subsite of the enzyme. As can be seen from the structures of these compounds, replacement of the aromatic ring with the more flexible alicyclic ring does not alter the antimalarial activity greatly. By contrast, the least active group of compounds generally had shorter side chains. These results suggest that the S1 pocket of PfAP-M17 is quite large and is thus able to accommodate compounds with bulky side chains. Also, the surface of the S1 cleft seems to be involved in multiple interactions with the bound inhibitors, especially deep inside the pocket, since effectors with extended side chains were exclusively the most active. Unfortunately, no crystal structure data of PfAP-M17 with which one could unambiguously judge this hypothesis are available. The data outlined here also suggest that the interactions are of a hydrophobic nature, since the presence of the hydroxy group on the compounds tested dramatically decreased their inhibitory potencies. Moreover, these observations correlate precisely with the results obtained in previous studies for mammalian (porcine kidney) LAP, where the inhibitors with bulky substituents were the best inactivators of the enzyme. The weak inhibitory potencies of the phosphonodipeptides equipped with the reactive group at the C terminus indicate that the enzyme is a classic monoexopeptidase that is able to cleave only one amino acid at a time and is not capable of binding large peptides effectively. The presence of the free amine at the position is also indispensable for the effective interaction between inhibitors and the PfAP-M17 aminopeptidase, as observed by the very weak inactivation by compounds with a hydroxy group at this position (D24 to D27).

Within the group of compounds with IC₅₀ in the 50 to 70 µM range (D5, D21, D3, and D2), it seems that the presence of the methoxy group in the para position is important, since increased inhibition of activity was seen with the inhibitors that contained this functional group. Alteration to a free hydroxyl group in this position (D1, D35, D4) and/or another (D6, D35) decreased the antimalarial activity dramatically.

Comparison of the antimalarial and antiaminopeptidase activities of the R enantiomers (corresponding to the natural L-amino acids) tested here (D21, D38, and D10) with those of their S configuration counterparts (D22, D23, and D11, respectively) shows a preference for the R enantiomers. This is in agreement with the expected chiral specificity of enzymes, especially aminopeptidases, which usually favor substrates with natural L chirality.

All of the active compounds appeared to inhibit both the PfAP-M17 and PfAP-M1 enzymes to some degree, inhibiting both the LAP and AAP activities of extracts. The compounds appeared to have slightly higher inhibitory effects against the aminopeptidase activities of extracts under conditions favoring PfAP-M17 over PfAP-M1 (i.e., assaying activity against a leucine substrate after preincubation with 1 mM CoCl₂). However, as already stated, the differences in the K_m values for the two aminopeptidases meant that it was not possible directly to compare the percentages of inhibition of extract aminopeptidase activities. It is possible that apparent differences in susceptibility may in fact be due to differing substrate affinities.

There was good agreement between M17 aminopeptidase inhibition in extracts and in recombinant tPfAP-M17 produced in insect cells. The fact that compound D14, which had marked selectivity for PfAP-M17 over pkLAP, was also the best inhibitor of parasite growth suggests that D14 should be further investigated in the hope of increasing its potency.

Owing to the lack of active recombinant PfAP-M1 and the difficulty and low yield of its purification (1, 14), we did not test this enzyme in complete isolation from PfAP-M17, so our data cannot be used to validate PfAP-M1 as an antimalarial target. Quinoline (13)- and malonic hydroxamate (12)-based inhibitors of PfAP-M1 with antimalarial activity have nevertheless been described. It would be informative to test combinations of representatives of these groups with the best compounds described here so as to determine whether inhibition of both aminopeptidase classes might confer synergistic antimalarial action.

While this paper was under review, Skinner-Adams et al. published work on a much smaller group of phosphinate dipeptide M17 aminopeptidase inhibitors with a range of antimalarial activities similar to those of the compounds described here (34). Their results indicated that two compounds with relatively high affinities for the recombinant M17 aminopeptidase, hPheP(CH₂)Phe and hPheP(CH₂)Tyr, had IC₅₀ in the 13 to 75 µM range and that the former compound was effective in a nonlethal Plasmodium chabaudi chabaudi model of murine malaria.

Taken together, the results indicate that the M17 and/or M1 aminopeptidase of P. falciparum may be a valid antimalarial target and that apparent differences in the S1 subsites of parasite and human M17 enzymes may be exploited to design more-potent and selective inhibitors.

 
We thank John Dalton for the gift of recombinant tPfAP-M17.

This work was supported by grants from Enterprise Ireland (SC/2003/111/B) and the Health Research Board of Ireland (RP/2005/57) to A.B.

 
* Corresponding author. Mailing address: Department of Microbiology, Moyne Institute, Trinity College, Dublin 2, Ireland. Phone: (353 1) 896 1414. Fax: (353 1) 679 9294. E-mail: abell{at}tcd.ie

Published ahead of print on 5 May 2008.

Present address: Burnham Institute for Medical Research, 10901 North Torrey Pines Rd., La Jolla, CA 92037.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Allary, M., J. Schrével, and I. Florent. 2002. Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase. Parasitology 125:1-10.[Medline]
2
2. Bonilla, J. A., T. D. Bonilla, C. A. Yowell, H. Fujioka, and J. B. Dame. 2007. Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function. Mol. Microbiol. 65:64-75.[CrossRef][Medline]
3
3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
4
4. Charet, P., E. Aissi, P. Maurois, S. Bouquelet, and J. Biguet. 1980. Aminopeptidase in rodent Plasmodium. Comp. Biochem. Physiol. B 65:519-524.[CrossRef]
5
5. Chen, X., C. R. Chong, L. Shi, T. Yoshimoto, D. J. Sullivan, Jr., and J. O. Liu. 2006. Inhibitors of Plasmodium falciparum methionine aminopeptidase 1b possess antimalarial activity. Proc. Natl. Acad. Sci. USA 103:14548-14553.[Abstract/Free Full Text]
6
6. Dalal, S., and M. Klemba. 2007. Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum. J. Biol. Chem. 282:35978-35987.[Abstract/Free Full Text]
7
7. Drag, M., J. Grembecka, M. Pawelczak, and P. Kafarski. 2005. -Aminoalkylphosphonates as a tool in experimental optimisation of P1 side chain shape of potential inhibitors in S1 pocket of leucine- and neutral aminopeptidases. Eur. J. Med. Chem. 40:764-771.[CrossRef][Medline]
8
8. Drag, M., R. Latajka, E. Gumienna-Kontecka, H. Kozlowski, and P. Kafarski. 2003. Stereoselective synthesis, solution structure and metal complexes of (1S,2S)-2-amino-1-hydroxyalkylphosphonic acids. Tetrahedron Asymmetry 14:1837-1845.[CrossRef]
9
9. Edwards, G., and G. A. Biagini. 2006. Resisting resistance: dealing with the irrepressible problem of malaria. Br. J. Clin. Pharmacol. 61:690-693.[CrossRef][Medline]
10
10. Ersmark, K., B. Samuelsson, and A. Hallberg. 2006. Plasmepsins as potential targets for new antimalarial therapy. Med. Res. Rev. 26:626-666.[CrossRef][Medline]
11
11. Fennell, B. J., J. A. Naughton, E. Dempsey, and A. Bell. 2006. Cellular and molecular actions of dinitroaniline and phosphorothioamidate herbicides on Plasmodium falciparum: tubulin as a specific antimalarial target. Mol. Biochem. Parasitol. 145:226-238.[CrossRef][Medline]
12
12. Flipo, M., T. Beghyn, V. Leroux, I. Florent, B. P. Deprez, and R. F. Deprez-Poulain. 2007. Novel selective inhibitors of the zinc plasmodial aminopeptidase PfA-M1 as potential antimalarial agents. J. Med. Chem. 50:1322-1334.[CrossRef][Medline]
13
13. Flipo, M., I. Florent, P. Grellier, C. Sergheraert, and R. Deprez-Poulain. 2003. Design, synthesis and antimalarial activity of novel, quinoline-based, zinc metallo-aminopeptidase inhibitors. Bioorg. Med. Chem. Lett. 13:2659-2662.[CrossRef][Medline]
14
14. Florent, I., Z. Derhy, M. Allary, M. Monsigny, R. Mayer, and J. Schrével. 1998. A Plasmodium falciparum aminopeptidase gene belonging to the M1 family of zinc-metallopeptidases is expressed in erythrocytic stages. Mol. Biochem. Parasitol. 97:149-160.[CrossRef][Medline]
15
15. Gardiner, D. L., K. R. Trenholme, T. S. Skinner-Adams, C. M. Stack, and J. P. Dalton. 2006. Overexpression of leucyl aminopeptidase in Plasmodium falciparum parasites. Target for the antimalarial activity of bestatin. J. Biol. Chem. 281:1741-1745.[Abstract/Free Full Text]
16
16. Gavigan, C. S., J. P. Dalton, and A. Bell. 2001. The role of aminopeptidases in haemoglobin degradation in Plasmodium falciparum-infected erythrocytes. Mol. Biochem. Parasitol. 117:37-48.[CrossRef][Medline]
17
17. Gavigan, C. S., S. G. Machado, J. P. Dalton, and A. Bell. 2001. Analysis of antimalarial synergy between bestatin and endoprotease inhibitors using statistical response-surface modelling. Antimicrob. Agents Chemother. 45:3175-3181.[Abstract/Free Full Text]
18
18. Giannousis, P. P., and P. A. Bartlett. 1987. Phosphorus amino acid analogues as inhibitors of leucine aminopeptidase. J. Med. Chem. 30:1603-1609.[CrossRef][Medline]
19
19. Goldberg, D. E. 2005. Hemoglobin degradation. Curr. Top. Microbiol. Immunol. 295:275-291.[Medline]
20
20. Grembecka, J., and P. Kafarski. 2001. Leucine aminopeptidase as a target for inhibitor design. Mini Rev. Med. Chem. 1:133-144.[CrossRef][Medline]
21
21. Gyang, F. N., B. Poole, and W. Trager. 1982. Peptidases from Plasmodium falciparum cultured in vitro. Mol. Biochem. Parasitol. 5:263-273.[CrossRef][Medline]
22
22. Hamilton, R., B. Walker, and B. J. Walker. 1995. A highly convenient route to optically pure -aminophosphonic acids. Tetrahedron Lett. 36:4451-4454.[CrossRef]
23
23. Kafarski, P., and B. Lejczak. 1989. Mixed carboxylic-carbonic anhydride method in phosphonopeptide synthesis. Tetrahedron 45:7387-7396.[CrossRef]
24
24. Kowalik, J., L. Kupczyk-Subotkowska, and P. Mastalerz. 1981. Preparation of dialkyl 1-aminoalkanephosphonates by reduction of dialkyl 1-hydroxyiminoalkanephosphonates with zinc in formic acid. Synthesis 1981:57-58.[CrossRef]
25
25. Liu, J., E. S. Istvan, I. Y. Gluzman, J. Gross, and D. E. Goldberg. 2006. Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems. Proc. Natl. Acad. Sci. USA 103:8840-8845.[Abstract/Free Full Text]
26
26. Makler, M. T., J. M. Ries, J. A. Williams, J. E. Bancroft, R. C. Piper, B. L. Gibbins, and D. J. Hinrichs. 1993. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am. J. Trop. Med. Hyg. 48:739-741.[Abstract/Free Full Text]
27
27. Nankya-Kitaka, M. F., G. P. Curley, C. S. Gavigan, A. Bell, and J. P. Dalton. 1998. Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin. Parasitol. Res. 84:552-558.[CrossRef][Medline]
28
28. Oleksyszyn, J., R. Tyka, and P. Mastalerz. 1978. Direct synthesis of 1-aminoalkanephosphonic and 1-aminoalkanephosphinic acids from phosphorus trichloride or dichlorophosphines. Synthesis 1978:479-480.[CrossRef]
29
29. Rawlings, N. D., and A. J. Barrett. 1993. Evolutionary families of peptidases. Biochem. J. 290:205-218.[Medline]
30
30. Rawlings, N. D., F. R. Morton, and A. J. Barrett. 2006. MEROPS: the peptidase database. Nucleic Acids Res. 34:D270-D272.[Abstract/Free Full Text]
31
31. Rosenthal, P. J. 2004. Cysteine proteases of malaria parasites. Int. J. Parasitol. 34:1489-1499.[CrossRef][Medline]
32
32. Scornik, O. A., and V. Botbol. 2001. Bestatin as an experimental tool in mammals. Curr. Drug Metab. 2:67-85.[CrossRef][Medline]
33
33. Sijwali, P. S., J. Koo, N. Singh, and P. J. Rosenthal. 2006. Gene disruptions demonstrate independent roles for the four falcipain cysteine proteases of Plasmodium falciparum. Mol. Biochem. Parasitol. 150:96-106.[CrossRef][Medline]
34
34. Skinner-Adams, T. S., J. Lowther, F. Teuscher, C. M. Stack, J. Grembecka, A. Mucha, P. Kafarski, K. R. Trenholme, J. P. Dalton, and D. L. Gardiner. 2007. Identification of phosphinate dipeptide analog inhibitors directed against the Plasmodium falciparum M17 leucine aminopeptidase as lead antimalarial compounds. J. Med. Chem. 50:6024-6031.[CrossRef][Medline]
35
35. Stack, C. M., J. Lowther, E. Cunningham, S. Donnelly, D. L. Gardiner, K. R. Trenholme, T. S. Skinner-Adams, F. Teuscher, J. Grembecka, A. Mucha, P. Kafarski, L. Lua, A. Bell, and J. P. Dalton. 2007. Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development. J. Biol. Chem. 282:2069-2080.[Abstract/Free Full Text]
36
36. Szewczyk, J., B. Lejczak, and P. Kafarski. 1982. Transesterification of diphenyl phosphonates using the potassium fluoride/crown ether/alcohol system. Part 2. The use of diphenyl 1-aminoalkanephosphonates in phosphonopeptide synthesis. Synthesis 1982:409-412.[CrossRef]
37
37. Vander Jagt, D. L., B. R. Baack, and L. A. Hunsaker. 1984. Purification and characterization of an aminopeptidase from Plasmodium falciparum. Mol. Biochem. Parasitol. 10:45-54.[CrossRef][Medline]
38
38. Wu, Y., X. Wang, X. Liu, and Y. Wang. 2003. Data-mining approaches reveal hidden families of proteases in the genome of malaria parasite. Genome Res. 13:601-616.[Abstract/Free Full Text]
39
39. Zhang, P., D. E. Nicholson, J. M. Bujnicki, X. Su, J. J. Brendle, M. Ferdig, D. E. Kyle, W. K. Milhous, and P. K. Chiang. 2002. Angiogenesis inhibitors specific for methionine aminopeptidase 2 as drugs for malaria and leishmaniasis. J. Biomed. Sci. 9:34-40.[Medline]
40
40. Zuckerman, A., D. Spira, and J. Hamburger. 1967. A procedure for the harvesting of mammalian plasmodia. Bull. W. H. O. 37:431-436.[Medline]

---

Antimicrobial Agents and Chemotherapy, September 2008, p. 3221-3228, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.01327-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cunningham, E. Articles by Bell, A. Search for Related Content PubMed PubMed Citation Articles by Cunningham, E. Articles by Bell, A.