Collateral hypersensitivity between ZY19489 and piperaquine neutralizes PfCRT-mediated drug efflux and Plasmodium falciparum resistance

New antimalarial drugs are essential to combat the current emergence and spread of Plasmodium falciparum parasite resistance to first-line artemisinin-based combination therapies. Here, we identify a mechanism of parasite resistance to ZY19489, a triaminopyrimidine currently in a Phase IIb clinical trial. Low-grade resistance was mediated by a novel mutation in the P. falciparum chloroquine resistance transporter PfCRT, which caused a major reduction in asexual blood stage parasite growth rates and a substantial fitness cost. Parasites resistant to ZY19489 lost their chloroquine resistance status and became hypersusceptible to the artemisinin-based combination partner drug piperaquine. All three agents were shown to interfere with parasite-mediated catabolism of host hemoglobin. Uptake studies in PfCRT-containing proteoliposomes provide evidence that ZY19489 can block mutant PfCRT-mediated efflux of piperaquine and chloroquine, creating a scenario of an evolutionary trap whereby resistance to ZY19489 blocks PfCRT efflux-mediated resistance and restores susceptibility to piperaquine and chloroquine. Metabolomic studies revealed that ZY19489 significantly reduces intracellular levels of short hemoglobin-derived peptides (a natural substrate of PfCRT) and leads to higher accumulation of pyrimidine deoxynucleotides. Our data present a marker for tracking the evolution of clinical resistance to ZY19489 and a rationale for pairing this with piperaquine to generate a novel resistance-refractory combination.