Structure and Drug Resistance of the Plasmodium falciparum Transporter PfCRT

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide(1). Clinical resistance to the chemically-related current first-line combination drug piperaquine (PPQ) has now emerged regionally, thwarting its efficacy(2). Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum chloroquine resistance transporter PfCRT, a 49 kDa member of the drug/metabolite transporter (DMT) superfamily that traverses the membrane of the parasite’s acidic digestive vacuole (DV)(3–9). Here we present the 3.2 Å structure of the PfCRT isoform from CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy (cryo-EM) and fragment antigen-binding (Fab) technology. Mutations contributing to CQ and PPQ resistance localize primarily to moderately-conserved sites on distinct helices lining a central negatively-charged cavity, implicating this as the principal site of interaction with positively-charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, with these drugs being mutually competitive. This isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism driving CQ resistance(5), but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America respectively(6,9), reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest distinct mechanistic features mediating CQ and PPQ resistance in PfCRT variants. These data provide the first atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.