Functional properties and arrangement of apicomplexan tubulin and the action of anti-tubulin compounds as chemotherapeutic agents

Human malaria and bovine babesiosis are caused by apicomplexan parasites Plasmodium falciparum and Babesia bovis respectively. These two parasites show very similar pathogenesis which is significantly influenced by their apical complex. Members of the apical complex; rhoptry, microneme, apical polar rings and microtubules are intricately involved in the parasite’s biology and are essential for viability and pathogenicity. Apicomplexan microtubules have previously been suggested as plausible drug targets based on several factors. In this study, we have attempted to asses i) the functional role of P. falciparum and B. bovis microtubular system in the parasite’s biology and ii) the feasibility of antimicrotubule drugs as potential antiparasitic agents. Parasite microtubular structures were visualised during different stages of the lifecycle using confocal microscopy. These temporal data have led to better understanding of the potential role played by parasite microtubules in nuclear and apicoplast segregation, maintenance of cell integrity and invasion. The antimicrotubule drugs used on P. falciparum were predicted by in silico docking to bind to well-defined domains of the tubulin dimer. This docking data provides a structure-activity relationship for the binding of antimicrotubule ligands to specific sites and the consequent phenotypic effects as observed by immunofluorescence. The in silico data is also supported by in vitro combination assays displaying antagonism between competing ligands binding to the same site. Growth inhibition studies performed with antimicrotubule drugs leads to the hypothesis that the action of these drugs on their own are too slow to be clinically useful as antimalarials. Their action is recognisable only in the second cycle of parasite division following addition of the drug. However the slow, lingering activity of these drugs could be complementary for fast acting, artemisinin derivatives having short half life. Resistance against current chemotherapeutic agents, residue issues and absence of a globally effective vaccine necessitates the need for increased research efforts on novel effective drugs like microtubule inhibitory agents. Thesis submitted in partial fulfillment of the requirements of the degree of Doctor of Philosophy of the Monash University, Australia and Indian Institute of Technology Bombay, India

Curcumin uptake by parasitised (IE) and uninfected erythrocytes (E) during first and second cycle of <i>P. falciparum</i> growth.

<p>Error bars represent standard error of the mean (n = 3).</p

Sensitivity assays with curcumin in combination with colchicine, paclitaxel and vinblastine and all the drugs individually.

<p>Panels A and B represent growth patterns of parasites subjected to curcumin and colchine individually and in combination, for 48 hours and for 96 hours respectively. Panels C and D represent same scenario for curcumin and paclitaxel treated parasites while panels E and F represent curcumin and vinblastine treated parasites. Concentrations of individual drugs used in the combinations are included for each data point. Error bars represent standard error of the mean (n = 4).</p

Proportion of total parasite cells observed with spindle and subpellicular microtubules in control and treated parasite population.

<p>Proportion of total parasite cells observed with spindle and subpellicular microtubules in control and treated parasite population.</p

Controls: Treatment of <i>P. falciparum</i> with antimitotic drugs.

<p>Microtubule stabilizing and destabilizing compounds exert contrasting effects on their target. Panel A shows <i>P. falciparum</i> without any drug treatment after 24 hours. Panel B shows parasites 24 hours after treated with 500 nM paclitaxel. Images in panel C represents parasites treated with 100 nM vinblastine, after 24 hours. Molar concentration of the drugs was chosen on the basis of published IC₅₀.</p

Comparative growth patterns of cultures containing curcumin pre-treated and non pre-treated host erythrocytes.

<p>For pre-treatment, erythrocytes were incubated with 5 µM curcumin for 48 h and then mixed with parasite-infected untreated erythrocytes to a final parasitemia of 0.5% and final hematocrit of 3%. In no pre-treatment cultures, erythrocytes were not given any pre-treatment with curcumin. No drug control parasite culture with no drug. Error bars represent standard error of the mean (n = 4).</p

Predictive binding of curcumin, colchicine, paclitaxel and vinblastine to <i>P. falciparum</i> tubulin dimer.

<p>Figures showing bound poses of curcumin diketo form at the interface of tubulin dimer. <i>P. falciparum</i> tubulin is represented as dimer of alpha (yellow) and beta (green) subunit. Panel A shows all the predicted bound poses, mostly at the interface of the dimer. Panel B shows the most probable binding pose according to Autodock (Rank 1) with the curcumin diketo form at the interface of alpha and beta tubulin monomers. Panel C shows binding sites of colchicine (purple), paclitaxel (red) and vinblastine (brown) on parasite tubulin dimer.</p

Combination indices of curcumin with colchicine, paclitaxel and vinblastine over a course of 96 hours.

<p>ND* not determined: ΣFIC90 for curcumin-colchicine combination could not be determined because the maximum growth inhibition of treated parasites, even after 96 hours, was 35%.</p

Morphological changes induced in <i>P. falciparum</i> by curcumin treatment.

<p>Panel A and B represent the morphology of schizonts and late trophozoites in untreated cultures. Panel C and D represent the morphological changes in schizonts and late trophozoites in cultures treated for 96 hours with 5 µM curcumin.</p