Multiple Drugs Compete for Transport via the Plasmodium falciparum Chloroquine Resistance Transporter at Distinct but Interdependent Sites

Sebastiano Bellanca,Robert L Summers,Max Meyrath,Anurag Dave,Megan N Nash,Martin Dittmer,Cecilia P Sanchez,Wilfred D Stein,Rowena E Martin,Michael Lanzer

doi:10.1074/jbc.m114.614206

Abstract

Mutations in the "chloroquine resistance transporter" (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.

Highlights

Mutations in the chloroquine resistance transporter (PfCRT) change the susceptibility of Plasmodium falciparum to diverse antimalarial drugs
The uptake of radiolabeled quinine and quinidine was measured in an acidic medium into Xenopus oocytes expressing either PfCRTDd2 or the wild-type protein from the HB3 strain (PfCRTHB3) as well as into water-injected oocytes
These drugs do not appear to be substrates of the wild-type form of the protein (PfCRTHB3). These observations are consistent with the findings of Lehane et al [19, 21] as well as those of Wellems and colleagues [22, 23]. The former studies showed that chloroquine, quinine, quinidine, and verapamil are each able to induce a proton leak from the digestive vacuole of parasites carrying PfCRTDd2 but not from the digestive vacuole of an isogenic line expressing PfCRTHB3

Summary

Background

Mutations in the chloroquine resistance transporter (PfCRT) change the susceptibility of Plasmodium falciparum to diverse antimalarial drugs. Identified as the main determinant of resistance to chloroquine [3], a synthetic 4-aminoquinoline and a mainstay in previous campaigns to eradicate malaria, mutations in PfCRT are known to have wide-ranging effects on the parasite’s sensitivity to an assortment of pharmacons [4]. These include antimalarial drugs that share the quinoline scaffold (e.g. quinine, amodiaquine, and mefloquine) or that possess structural moieties present in quinoline drugs (e.g. lumefantrine and halofantrine) and encompass a diverse range of compounds that have not been deployed as antimalarial treatments (4 –12). The transporter resides at the membrane of the parasite’s digestive vacuole [3] and is thought to efflux drugs out of this organelle, away from their main target, the detoxification

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EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION