Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates.

The 1,2,4-trioxolanes are a new class of synthetic peroxidic antimalarials currently in human clinical trials. The well known reactivity of the 1,2,4-trioxolane ring towards inorganic ferrous iron and ferrous iron heme is proposed to play a role in the antimalarial action of this class of compounds. We have designed structurally relevant fluorescent chemical probes to study the sub-cellular localization of 1,2,4-trioxolanes in cultured Plasmodium falciparum parasites. Microscopy experiments revealed that a probe fluorescently labeled on the adamantane ring accumulated specifically in digestive vacuole-associated neutral lipid bodies within the parasite while an isosteric, but non-peroxidic congener did not. Probes fluorescently labeled on the cyclohexane ring showed no distinct localization pattern. In their sub-cellular localization and peroxidative effects, 1,2,4-trioxolane probes behave much like artemisinin-based probes studied previously. Our results are consistent with a role for adamantane-derived carbon-centered radicals in the antimalarial action of 1,2,4-trioxolanes, as hypothesized previously on the basis of chemical reactivity studies.

eThe Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (؊)-quinine and (؉)-quinidine. The potency of (؉)-isomers is greater than the (؊)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC 50 ratio of (؊)/(؉) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (؊) and (؉) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids.A lkaloids from the Cinchona tree, exemplified by quinine (QN) and quinidine (QD), have proven to be an important source of antimalarial therapies, especially after resistance to chloroquine (CQ) emerged. QN remains a first-line drug in the treatment of severe malaria in many parts of the world, even with increased use of artemisinin-based combination therapies (70). The Cinchona alkaloids (CA) are aryl amino alcohols where the aryl group is a quinoline (quinoline aminoalcohols). They have four chiral centers, two of which, C-8 and C-9, can have different configurations (66) (Fig. 1). Among the pharmacologically active 8,9-erythro isomers, QN, hydroquinine (HQN), and cinchonidine (CD) all present the S configuration around C-8 and the R configuration at C-9 and are levorotatory (rotate plane polarized light in an anticlockwise [Ϫ] direction). The reverse ordering, R at C-8 and S at C-9, occurs in the respective dextrorotatory (rotate plane polarized light in a clockwise [ϩ] direction) diastereomers, QD, hydroquinidine (HQD), and cinchonine (CN). The 8,9-threo diastereomers 9-epiquinine (EQN) and 9-epiquinidine (EQD) are 8S, 9S, and 8R, 9R, respectively.Against Plasmodium falciparum, the CA diastereomer pairs have a well-established in ...