Purification of P. c; chabaudi aminopeptidase, and antimalarial effects of the aminopeptidase inhibitor bestatin

The malarial parasite utilises amino acids drawn from the host haemoglobin for it's intraerythrocytic development. The endopeptidases described to date will only produce peptides from host haemoglobin. In order to obtain free aminoacids for incorporation in malarial proteins the malarial parasite requires exopeptidases (i.e. aminopeptidases and carboxypeptidases). Aminopeptidase activity was described in human malarial parasites Plasmodium falciparum rodent malarial parasites Plasmodium chabaudi chabaudi and Plasmodium berghei. These enzymes were shown to be inhibited by the aminopeptidase inhibitor bestatin. In light of these findings, the antimalarial effects of bestatin were investigated both in vitro and in vivo. Bestatin inhibited the in vitro growth of P. c. chabaudi, of chloroquine sensitive P. falciparum clone FCH5.C2 and chloroquine resistant P. falciparum clone K 1/Thailand. P. chabaudi infection is naturally lethal to male mice whereas female mice develop protective immunity against a similar infection. In vivo investigation of the effects of bestatin showed that this aminopeptidase inhibitor could induce self-healing of P. c. chabaudi infection in male CD1 mice. The aminopeptidase was purified by a combination of sizeexclusion HPLC and affinity chromatography and, alternatively, by a combination of size-exclusion HPLC and anion exchange chromatography. The aminopeptidase activity appears to be composed of four different aminopeptidases with an apparent monomeric molecular weight range of 60 to 70 kDa. Comparative studies of the inhibition of the P. c. chabaudi and porcine kidney aminopeptidase by bestatin indicate that the active site of the malarial aminopeptidase could differ to the mammalian aminopeptidase. Nucleotide probes/primers to clone this aminopeptidase(s) have been designed from the sequences of bestatin and metal ions binding site of several leucine aminopeptidases. This thesis shows that the malarial aminopeptidase is a potential chemotherapeutic target with bestatin and/or bestatin analogues as potential antimalarial drugs. The difference between the binding site of bestatin on mammalian and malarial aminopeptidase could be exploited for the design of drugs that would be specific to the malarial parasite enzyme.