Date Published: July 21, 2016
Publisher: Public Library of Science
Author(s): Sashika N. Richards, Megan N. Nash, Eileen S. Baker, Michael W. Webster, Adele M. Lehane, Sarah H. Shafik, Rowena E. Martin, Akhil B. Vaidya.
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.
Originally identified as the protein responsible for conferring resistance to the ‘wonder-drug’ chloroquine (CQ) [1, 2], the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT) has become a key player in the malaria parasite’s steadily expanding resistance to drugs [3–5]. The isoforms of PfCRT that confer CQ resistance (PfCRTCQR) render the parasite less susceptible to many other compounds [6–10], but also simultaneously induce hypersensitivity to a subset of structurally-diverse molecules [6, 10–18]. This phenomenon, whereby resistance to one drug causes hypersensitivity to another, is known as ‘inverse susceptibility’ or ‘collateral sensitivity’ and has been observed in a wide range of pathogens and cancer cells [19–22]. The growing awareness of the propensity of drug-resistant pathogens to exhibit hypersensitivity to one or more other drugs has sparked interest in the potential for exploiting this Achilles’ heel to combat existing drug resistance and to delay the emergence of resistance to new drugs . However, it is not known how PfCRTCQR isoforms induce hypersensitivity to certain drugs and, more generally, much remains to be understood about the molecular mechanisms that underpin collateral sensitivity in pathogens and cancer cells [20–22].
Our work provides mechanistic explanations for the patterns of inverse susceptibility induced by PfCRT. First, we confirmed our previous observation  of there being a positive correlation between the capacity of a given PfCRT isoform for mediating CQ transport and the magnitude of CQ resistance achieved by the respective parasite (S7A Fig) and extended this relationship to include, with the notable exception of the QN-hypersensitive lines, a positive correlation between the quinine or quinidine transport activity of PfCRT and the parasite’s in vitro responses to these drugs (Fig 5A and S7B Fig). Moreover, we showed that in most cases, the isoforms of PfCRTK1 that possessed CQ transport activity also transported quinine and quinidine and vice versa. Together, these findings confirm a common role for PfCRT in reducing the accumulation of CQ, quinine, and quinidine within the DV and explain the tendency of CQ-resistant parasites to exhibit decreased susceptibilities to quinine and quinidine. However, our results resolve this phenomenon further by providing a fundamental insight into PfCRT-induced drug phenotypes: whether a given isoform of PfCRT alters the parasite’s susceptibility to a drug, and to what extent and in which direction, depends on the kinetics of the drug’s transport via PfCRT.