Exploring the substrate specificity of the Plasmodium falciparum 'chloroquine resistance transporter'

Abstract

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. The native substrates of PfCRT were investigated in both the Xenopus oocyte expression system and isogenic parasite lines expressing different pfcrt isoforms, using complementary physiological, biochemical, and metabolomic approaches. Host-derived peptides of 4-11 amino acid residues, varying in both charge and composition, were identified as the substrates of PfCRT in vitro and in situ, and the protein was shown not to mediate the non-specific transport of ions and other metabolites. Drug-resistance-conferring mutations were found to reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. The transport of peptides and peptide mimics via PfCRT is saturable and inhibited by verapamil. The results presented in this Thesis indicate that PfCRT transports peptides from the lumen of the parasite's digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The long-awaited resolution of PfCRT's substrate-specificity and physiological role will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials. It is becoming increasingly apparent that certain mutations in PfCRT alter the parasite's susceptibility to a range of pharmacons. Here, the interactions of PfCRT with a structurally diverse group of compounds - methylene blue, quinacrine, amantadine, and piperaquine - were investigated. Parasite susceptibility to methylene blue, quinacrine, and amantadine against CQ-resistant and CQ-sensitive P. falciparum was measured. Parasites expressing CQ-resistance-conferring isoforms of PfCRT exhibit reduced susceptibility to methylene blue and quinacrine but increased susceptibility to amantadine. The ability of wild-type and mutant isoforms of PfCRT to transport methylene blue, quinacrine, amantadine, and piperaquine when expressed at the surface of Xenopus oocytes was also assessed. Mutant isoforms of PfCRT mediated transport of methylene blue, quinacrine, amantadine, and piperaquine, whereas wild-type PfCRT did not. Furthermore, transport of these four drugs via mutant PfCRT was saturable and inhibited by verapamil. In this Thesis, possible mechanistic explanations for PfCRT-induced drug resistance to methylene blue, quinacrine, and piperaquine and PfCRT-induced drug hypersensitivity to amantadine are described. First, methylene blue, quinacrine, and piperaquine, which normally accumulate inside the digestive vacuole and therewithin exert their antimalarial effects, are transported out of this compartment via mutant isoforms of PfCRT. The second scenario shows that although amantadine also sequesters within the digestive vacuole, the parasite's hypersensitivity to this drug arises from the CQ-resistance-conferring isoforms of PfCRT mediating the transport of amantadine from the digestive vacuole into the cytosol, where it can better access its antimalarial target. These insights provide a foundation for understanding clinically-relevant observations of drug resistance and inverse drug susceptibilities in the malaria parasite.