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

Abstract

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 M. 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.

The influence of α-aminophosphonic acids on the activity of aminopeptidase from barley seeds—an approach to determine the enzyme specificity

“…Its homologue, compound 19, bearing an additional methyl at carbon atom in position 3 also exhibited a significant activity against this enzyme. A similar model of activity was observed for the porcine aminopeptidases (Table 2) and aminopeptidase from Plasmodium falciparum (value of IC 50 for compound 18 is 22 lM; Cunningham et al 2008). This, alongside with significant activity of compound 21, indicates that there is a spacious binding site able to accommodate hydrophobic N-terminal fragments of inhibitors.…”

“…Aminopeptidase M17 prefers both aliphatic and aromatic hydrophobic aminophosphonates (compounds 18-20 and 1, 5 and 13; with analogues of leucine and homoalanine being of choice), whereas aminopeptidase M1 is preferably inhibited by aminophosphonates containing additional hydrophilic group in side chain, with analogue of homotyrosine (compound 24) being the most active. Although Plasmodium falciparum aminopeptidase M17 (EC 3.4.11.1) prefers aminophosphonates bearing hydrophobic aromatic moiety in their side chain (Cunningham et al 2008), the pattern of activity against this enzyme is also quite different because it is not sensitive to compounds 10-13. Thus, our study indicates that the use of libraries of simple inhibitors could be a useful tool in determining specificities of aminopeptidases and their fingerprints.…”

“…Aminophosphonic acids were available from earlier studies (Lejczak et al 1989;Drąg et al 2005;Cunningham et al 2008) or were synthesized according to the modified literature procedure (Soroka 1989). Acetamide (0.2 mol) was dissolved in acetic acid (40 ml) and cooled in an ice bath followed by addition of acetyl chloride (0.1 mol).…”

“…In this paper, we examined the influence of simple a-aminophosphonic acids on activity of novel metalloaminopeptidase isolated from seeds of barley (Oszywa et al 2013) and compared the results with published data concerning porcine and Plasmodium aminopeptidases (Cunningham et al 2008). …”

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

“…Cunningham et al 51 reported the evaluation of a series of α-aminoalkylphosphonate and phosphonopeptide inhibitors, designed against mammalian M17LAP, for their anti-malaria activity. They then correlated the IC 50 s of these compounds with their inhibitory activity against aminopeptidase activity in malaria cell extracts, and against recombinant PfM17LAP.…”