Transporters involved in resistance to antimalarial drugs

Abstract

The ability to treat and control Plasmodium falciparum infection through chemotherapy has been compromised by the advent and spread of resistance to antimalarial drugs. Research in this area has identified the P. falciparum chloroquine resistance transporter (PfCRT) and the multidrug resistance-1 (PfMDR1) transporter as key determinants of decreased in vitro susceptibility to several principal antimalarial drugs. Transfection-based in vitro studies are consistent with clinical findings of an association between mutations in the pfcrt gene and failure of chloroquine treatment, and between amplification of the pfmdr1 gene and failure of mefloquine treatment. Many countries are now switching to artemisinin-based combination therapies. These incorporate partner drugs of which some have an in vitro efficacy that can be modulated by changes in pfcrt or pfmdr1. Here, we summarize investigations of these and other recently identified P. falciparum transporters in the context of antimalarial mode of action and mechanisms of resistance.

Figure 1

Predicted structure and representative haplotypes of P. falciparum chloroquine resistance transporter. (a) PfCRT is predicted to have ten transmembrane domains, with its N and C termini located on the cytoplasmic side of the digestive vacuole membrane (adapted, with permission, from Ref. [20]). Mutations identified in pfcrt cDNA sequences from CQR lines (black circles), the crucial K76T mutation common to all CQR strains (red) and the S163R mutation identified in amantadine- and halofantrine-resistant parasites (yellow circle) [24] are indicated. (b) Representative pfcrt haplotypes.

Figure 2

Mechanistic models of PfCRT-mediated chloroquine resistance. (a) Chloroquine-sensitive parasites. In sensitive parasites expressing wild-type PfCRT, the weak base chloroquine (CQ) concentrates (broken arrow) in the digestive vacuole. The acidic environment of the digestive vacuole promotes diprotonation of chloroquine, trapping it within the organelle. This charged form is thought to bind hematin, preventing its detoxification into hemozoin. pH estimates for the cytoplasm and digestive vacuole are taken from Refs [17] and [39], respectively. (b) Energy-coupled drug efflux. In this model, chloroquine resistance is due to carrier-mediated drug efflux out of the parasite digestive vacuole. Glucose addition to the parasite culture medium, which results in reduced chloroquine accumulation in resistant parasites, is accompanied by production of ATP by the parasite [36,38]. Chloroquine-resistant parasites show an energy-dependent trans-stimulation effect characteristic of carrier-mediated transport [37,38]. Mutant PfCRT has been proposed to function as the drug effluxer (filled arrow) [48]. (c) Facilitated diffusion of charged drug. This model (also known as ‘charged drug leak’ [20]) considers the possible effects of mutations in PfCRT on transport function. Mutations in PfCRT alter the substrate specificity of the transporter and facilitate the diffusion (broken arrow) of diprotonated chloroquine from the digestive vacuole down an electrochemical gradient maintained by a digestive vacuole proton pump. Reversal agents, such as verapamil, are monobasic and have been proposed to interact with mutant PfCRT, blocking the passive efflux of drug in resistant parasites [49]. (d) Indirect effects of altered digestive vacuole pH. CQR parasites have been reported to have a more acidic digestive vacuole than have sensitive parasites [39]. This model postulates that alterations in the pH of the digestive vacuole, caused directly or indirectly by changes in the transport properties of mutant PfCRT, results in reduced accumulation of drug at its site of action. The increased rate of hematin aggregation and hemozoin formation at acidic pH has been proposed to reduce the amount of target available for chloroquine binding [17]. The excess unbound drug could alter the equilibrium of passive drug accumulation (broken arrows) or could be transported out of the digestive vacuole by mutant PfCRT (filled arrow) [44,77].

Figure 3

Predicted structure and genetic polymorphisms in P. falciparum multidrug resistance-1. (a) PfMDR1 has two homologous halves, each with six predicted transmembrane domains and a nucleotide-binding pocket [56]. The nucleotide-binding domains (NBD1 and NBD2; orange boxes) are each formed by large cytoplasmic domains. Polymorphic amino acids found in the K1 allele (N86Y) and the 7G8 allele (Y184F, S1034C, N1042D and D1246Y) are indicated. The D1246Y mutation is located in the predicted NBD2. (b) Representative haplotypes, including the one most commonly associated with amplification of pfmdr1 copy number.