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 áI-, áII- and â-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 áItubulin
was different at twelve bases, of which only six of these had resulted in
changes in amino acid residues. áII- and â-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 & 7G8) of â-tubulin.
Nevertheless, the differences were minor in áI- and â-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.
| Identifer | oai:union.ndltd.org:ADTP/221667 |
| Date | January 2004 |
| Creators | low@wehi.edu.au, CK Andrew Low |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright CK Andrew Low |
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