4-aminoquinolines block heme iron reactivity and interfere with artemisinin action

Abstract

Artemisinin-based combination therapies (ACTs) remain the mainstay of treatment for Plasmodium falciparum malaria, despite reports of ACT treatment failure. ACTs consist of an artemisinin and a longer-lived partner drug, which is often a quinoline. Given that heme is central to the mechanism of action of artemisinins and some quinolines, we hypothesized that these antimalarials would exhibit strong drug-drug interactions. Previous studies using standard 48 h or 72 h assays identified additive to mildly antagonistic interactions between artemisinins and quinolines. Here, we sought to re-evaluate these interactions using a pulsing assay that better mimics the short in vivo half-life of artemisinins. We found that chloroquine (CQ), piperaquine (PPQ), and amodiaquine substantially antagonize dihydroartemisinin (DHA), the active metabolite of artemisinins. CQ-DHA antagonism was notably exacerbated in CQ-resistant parasites, resulting in a superantagonistic phenotype in isobolograms. Further, we found that CQ co-treatment conferred artemisinin resistance to Kelch 13 wild type parasites in the ring stage survival assay. Using a small molecule probe to measure chemically reactive heme in live parasites, we determined that quinolines block artemisinin activation by rendering cytosolic heme inert. Finally, we probed beyond traditional ACTs, evaluating interactions of the proposed triple ACT, DHA-PPQ-Mefloquine, as well as OZ439-quinoline combinations, which were all found to be antagonistic. Collectively, these in vitro data suggest that peroxide-quinolines may have liabilities as combination therapies.