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

Abstract

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.