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Characterisation and evaluation of novel potential target (tubulin) for antimalarial chemotherapy

Low, Chee Kin Andrew (2004) Characterisation and evaluation of novel potential target (tubulin) for antimalarial chemotherapy. PhD thesis, Murdoch University.

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Malaria has long affected the world both socially and economically. Annually, there are 1.5-2.7 million deaths and 300-500 million clinical infections (WHO, 1998). Several antimalarial agents (such as chloroquine, quinine, pyrimethamine, cycloguanil, sulphadoxine and others) have lost their effectiveness against this disease through drug resistance being developed by the malarial parasites (The- Wellcome-Trust, 1999). Although there is no hard-core evidence of drug resistance shown on the new antimalarial compounds (artemisinin and artesunate), induced resistant studies in animal models have demonstrated that the malarial parasites have capabilities to develop resistance to these compounds (Ittarat et al., 2003; Meshnick, 1998; Meshnick, 2002; Walker et al., 2000). Furthermore, a useful vaccine has yet to be developed due to the complicated life cycle of the malarial parasites (The- Wellcome-Trust, 1999). As such, the re-emergence of this deadly infectious disease has caused an urgent awareness to constantly look for novel targets and compounds.

In this present study, Plasmodium falciparum (clone 3D7) was cultured in vitro in human red blood cells for extraction of total RNA which was later reverse transcribed into cDNA. The alphaI-, alphaII- and beta-tubulin genes of the parasite were then successfully amplified and cloned into a bacterial protein expression vector, pGEX- 6P-1. The tubulin genes were then sequenced and analysed by comparison with previously published homologues. It was found that the sequenced gene of alpha-Itubulin was different at twelve bases, of which only six of these had resulted in changes in amino acid residues. alphaII- and beta-tubulin genes demonstrated 100% sequence similarity with the published sequences of clone 3D7, but differences were observed between this clone and other strains (strains NF54 and 7G8) of beta-tubulin. Nevertheless, the differences were minor in alphaI- and beta-tubulins and there was greater than 99% homology. Subsequently, all three Plasmodium recombinant tubulin proteins were separately expressed and purified. Insoluble aggregates (inclusion bodies) of these recombinant tubulins were also refolded and have been tested positive for their structural characteristics in Western blot analysis.

Both soluble and refolded recombinant tubulins of malaria were examined in a drugtubulin interaction study using sulfhydryl reactivity and fluorescence quenching techniques. Known tubulin inhibitors (colchicine, tubulozole-c and vinblastine) and novel synthetic compounds (CCWA-110, 239 and 443) were used as the drug compounds to determine the dynamics and kinetics of the interactions. In addition, mammalian tubulin was also used to determine the potential toxicity effects of these compounds. Similarities were observed with other published reports in the binding of colchicine with the recombinant tubulins, hence confirming proposed binding sites of this compound on the Plasmodium recombinant tubulins. Two synthetic compounds (CCWA-239 and 443) that have previously tested positive against P. falciparum in vitro were found to bind effectively with all three tubulin monomers, while displaying low binding interactions with the mammalian tubulin, thus indicating that these compounds have potential antimalarial activity. Therefore, this study has satisfied and fulfilled all the aims and hypotheses that have previously been stated.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Biomedical Sciences
Supervisor(s): Reynoldson, Jim
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