Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite’s Chloroquine Resistance Transporter

Abstract

Mutations in the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite’s digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite’s hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite’s survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite’s hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite’s sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.

Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic

“…PfCRT’s pleiotropic drug-transport properties are thought to impact other antimalarials by altering their local concentration at their site of action 159 . Drugs might also be able to bind PfCRT isoforms, resulting in impaired function and parasite hyper-susceptibility 175,176 .…”

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

“…At the cellular level, PfCRT is a multi-pass transporter embedded in the intra-erythrocytic parasite’s digestive vacuole (DV) membrane, with enigmatic functions that may include transport of ions and/or peptides [18–21]. In the absence of PfCRT structural information, mutational approaches have guided studies into the effect of specific PfCRT mutations on drug transport and parasite growth [16,22–24]. …”

The transportome of the malaria parasite

“…PfCRT, PfMDR1, and PfUGT) impart significant fitness costs to the parasite (Rosenthal, ; Lim et al ., ) to the extent that for PfCRT and PfMDR1, the withdrawal of the relevant antimalarial from a malarious region tends to result in the re‐emergence of parasites carrying the wild‐type allele (reviewed by Rosenthal, ) or the emergence of additional polymorphisms in the mutant allele that re‐sensitise the parasite to the drug and for which the main purpose may be to reinstate fitness (Summers et al ., ; Pelleau et al ., ). The propensity of the essential transporters to be subject to conflicting selection forces could be exploited to delay or combat resistance through the rational design of drug combinations (Yuan et al ., ; Summers et al ., ; Lukens et al ., ; Richards et al ., ; Martin et al ., ).…”

“…PfCRT, PfMDR1, and PfUGT) impart significant fitness costs to the parasite (Rosenthal, 2013;Lim et al, 2016) to the extent that for PfCRT and PfMDR1, the withdrawal of the relevant antimalarial from a malarious region tends to result in the re-emergence of parasites carrying the wild-type allele (reviewed by Rosenthal, 2013) or the emergence of additional polymorphisms in the mutant allele that re-sensitise the parasite to the drug and for which the main purpose may be to reinstate fitness (Summers et al, 2012;Pelleau et al, 2015). The propensity of the essential transporters to be subject to conflicting selection forces could be exploited to delay or combat resistance through the rational design of drug combinations (Yuan et al, 2011;Summers et al, 2012;Lukens et al, 2014;Richards et al, 2016;Martin et al, 2018). Elucidating the mechanisms by which polymorphisms in the 26 transporters confer, or contribute to, antimalarial drug resistance will require significant investment as it will entail the expression and characterisation of Plasmodium transporters in heterologous systems as well as the development of live parasite assays (and genetically modified parasite lines) to assess the impact of the polymorphisms on transport functions in situ.…”

“…Consistent with their importance to all aspects of Plasmodium biology, transporters are increasingly being identified as mediators of antimalarial drug resistance as well as drug targets themselves (e.g. Lim et al, 2016;Richards et al, 2016;Cowell et al, 2018). In this review, several large data sets as well as the findings from many smaller-scale studies of Plasmodium transporters -including investigations of gene essentiality, protein subcellular localisation, predicted or observed function, and associations with drug resistance -have been collated and analysed to expand and deepen our understanding of the parasite's transportome.…”