Molecular Mechanisms of Drug Resistance in Plasmodium falciparum Malaria

Abstract

Understanding and controlling the spread of antimalarial resistance, particularly to artemisinin and its partner drugs, is a top priority. Plasmodium falciparum parasites resistant to chloroquine, amodiaquine, or piperaquine harbor mutations in the P. falciparum chloroquine resistance transporter (PfCRT), a transporter resident on the digestive vacuole membrane that in its variant forms can transport these weak-base 4-aminoquinoline drugs out of this acidic organelle, thus preventing these drugs from binding heme and inhibiting its detoxification. The structure of PfCRT, solved by cryogenic electron microscopy, shows mutations surrounding an electronegative central drug-binding cavity where they presumably interact with drugs and natural substrates to control transport. P. falciparum susceptibility to heme-binding antimalarials is also modulated by overexpression or mutations in the digestive vacuole membrane-bound ABC transporter PfMDR1 (P. falciparum multidrug resistance 1 transporter). Artemisinin resistance is primarily mediated by mutations in P. falciparum Kelch13 protein (K13), a protein involved in multiple intracellular processes including endocytosis of hemoglobin, which is required for parasite growth and artemisinin activation. Combating drug-resistant malaria urgently requires the development of new antimalarial drugs with novel modes of action.

Keywords: antimalarial drug resistance; artemisinin-based combination therapy; endocytosis; hemoglobin; k13; pfcrt; piperaquine; transport.

Figure 1

Mechanism of action of quinolines and artemisinins and resistance mechanisms mediated by PfCRT, PfMDR1, and K13 in P. falciparum asexual blood stage parasites. (a) During its asexual blood stage, the P. falciparum parasite, surrounded by its PVM, develops within the host red blood cell. (❶) Quinoline-based antimalarials, including chloroquine, amodiaquine, and piperaquine, concentrate from the parasite cytosol (neutral pH of ~7) into the DV (acidic pH of ~5.2). (❷) Once inside the DV, these weak-base drugs are protonated, existing mostly as pH-trapped charged species that are unable to passively diffuse out through the DV membrane. (❸) As additional molecules diffuse into the DV, their protonated forms bind to the high concentrations of toxic free heme by-product that result from the degradation of host hemoglobin, as well as to grooves on the surfaces of growing hemozoin crystals. The combination of pH trapping and heme binding accounts for the >1,000-fold drug accumulation inside the DV. (❹) The DV membrane protein PfCRT is believed to be involved in transporting peptides released from hemoglobin digestion into the parasite cytosol. (❺) In drug-resistant parasites, mutations in PfCRT enable the efflux of protonated drug molecules out of the DV, away from their heme target. (❻) Mutations in the DV membrane transporter PfMDR1 can also influence parasite susceptibility to these compounds and are thought to enable transport of drugs such as halofantrine, lumefantrine, and mefloquine into the DV, away from their primary site of action. (b) (❶) Artemisinin drugs are activated by cleavage of their endoperoxide by iron protoporphyrin IX (Fe²⁺-heme), a product of parasite-digested hemoglobin. (❷) The Fe²⁺-heme-artemisinin carbon-centered radicals alkylate and damage a multitude of parasite proteins, heme, and lipids and inhibit proteasome-mediated protein degradation. K13 mutations, located primarily in the β-propeller kelch domain, confer artemisinin resistance in young rings. (❸) The loss of K13 function provided by mutations has been shown to cause reduced endocytosis of host hemoglobin and (❹) to extend the duration of ring-stage development, perhaps via PK4-mediated eIF2α phosphorylation. These changes result in lowered levels of hemoglobin catabolism and availability of Fe²⁺-heme as the drug activator and lead to reduced activation of artemisinin drugs. (❺) K13 mutations may activate the unfolded protein response, maintain proteasome-mediated degradation of polyubiquitinated proteins in the presence of artemisinins, and (❻) remove drugs and damaged proteins through an increase in PI3K-mediated vesicular trafficking. (❼) K13 may also help regulate mitochondrial physiology and maintain membrane potential during drug-induced ring-stage quiescence. The asterisk signifies the activated form of ART. Abbreviations: ADQ, amodiaquine; ART, artemisinin; CQ, chloroquine; DV, digestive vacuole; eIF2α, eukaryotic initiation factor 2α; Hb, hemoglobin; HF, halofantrine; LMF, lumefantrine; MFQ, mefloquine; PfCRT, P. falciparum chloroquine resistance transporter; PfMDR1, P. falciparum multidrug resistance 1 transporter; PPQ, piperaquine; PVM, parasitophorous vacuole membrane; RBC, red blood cell.

Figure 2

The structure of 7G8 PfCRT solved by cryogenic–electron microscopy showing the ten transmembrane helices (TM1–10) and two juxtamembrane helices (JM1–2) viewed (a) from the side and (b) from the DV into the central drug-binding cavity. Mutations associated with drug resistance are colored for piperaquine (blue), chloroquine (gold ), or amodiaquine (gold and bold). The substituted amino acids are represented as solid-colored sticks when viewed (b) from the DV only. Abbreviations: ADQ-R, amodiaquine resistance; CQ-R, chloroquine resistance; DV, digestive vacuole; PfCRT, P. falciparum chloroquine resistance transporter; PPQ-R, piperaquine resistance.