Molecular analysis of membrane transporters implicated in drug resistance

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Thesis (Ph.D.)--Georgetown University, 2008.; Includes bibliographical references. Resistance to cancer chemotherapeutics and antimalarial drugs is a major obstacle to the successful treatment of these diseases. Membrane transporters have been identified as contributing to this drug resistance. The HuMDR1 protein is thought to reduce accumulation of chemotherapeutics by ATP-dependent transport of the toxic compounds out of the cell. Though previously believed to be a major component of clinical cancer drug resistance, HuMDR1 is now accepted as playing only a minor role, secondary to other mechanisms. The Plasmodium falciparum protein homologue, PfMDR1, may be acting in a similar manner within the malarial parasite, affecting the partitioning of antimalarial drugs amongst cellular compartments. However, the evolving picture of quinoline antimalarial drug resistance may point to a mere modulatory role for PfMDR1 in comparison to another membrane protein, PfCRT, which has been proven to be causative of some drug resistance phenotypes but through an unknown mechanism. Since the protein is native to a subcellular organelle within an intracellular parasite, molecular level analysis of PfMDR1 would benefit from heterologous expression in a simpler system. This thesis reports the successful inducible overexpression of PfMDR1 in Pichia pastoris yeast. The tagged protein can be purified by affinity chromatography and functionally reconstituted in proteoliposomes. ATPase assays of many PfMDR1 variants show the protein to have high basal activity, with very little drug-induced responsiveness. These results support a model in which PfMDR1 acts to modulate the drug resistance profiles determined by PfCRT or to compensate for fitness losses incurred by mutation of PfCRT. All current hypotheses for the molecular mechanism by which PfCRT confers quinoline antimalarial drug resistance entail the direct interaction of the drug molecule with the protein, but evidence for these theories is inferential. This thesis reports the labeling of PfCRT with a photoactivatable chloroquine analogue. The probe is shown to be specific and labeling is efficiently competed with other antimalarial drugs, suggesting a single drug binding site is present in the protein. The photolabeling site is mapped to within 11 amino acids, and a model is proposed in which PfCRT transmembrane helices 1, 9 and 10 form a drug binding pocket.
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