Calcium dysregulation is the critical initiator in high dose chloroquine-induced programmed cell death of Plasmodium falciparum

Understanding the mechanism of action of novel antimalarial drugs is a key goal in eradicating malaria. Previously we have explored a possible alternative cell death mechanism that is elicited by micromolar amounts of the antimalarial chloroquine (CQ) against Plasmodium falciparum. The phenotypes assessed were calcium redistribution, mitochondrial depolarization and parasite digestive vacuole membrane permeabilization. Identifying the source or initiator of calcium release in the parasite will reveal a potential new therapeutic target. From timepoint studies we revealed the simultaneous loss of mitochondrial integrity along with intracellular calcium dysregulation, followed by downstream membrane rupture. Using a series of calcium efflux inhibitors and antioxidants, mitochondrial calcium (Ca2+mt) was implicated in initiating this cascade of parasite cell death phenotypes. Having established the mechanism with a high dose concentration of CQ, we expanded the search for similar phenotypic outcomes in a high-content screening of the Medicines for Malaria Venture (MMV) Pathogen Box. Three “hit” compounds were subjected to further characterization assays and the top performing compound (MMV085071) was identified. Ten analogues based on the top hit were assessed with dose-response assays and one lead analogue (MMV1545561) was selected as the top candidate based on its efficacy. Both parent and lead compound were investigated for their ability to redistribute Ca2+mt. While the effects were statistically significant, the lack of complete reversal of compound-induced calcium dysregulation and mitochondrial collapse by inhibitors suggested that Ca2+mt’s role is more limited as compared to that of chloroquine. Furthermore, it is likely that the novel screening compounds are directed at multiple parasite targets and due to their highly efficacious nature, further follow-up work to ascertain their mechanism of action is warranted. P. falciparum 3D7 strain parasites were synchronized with sorbitol and MACS sorting to enrich those in schizont stage. The enriched schizonts were seeded at 1.5-2% parasitemia at 1.25% haematocrit. Further synchronisation by sorbitol was performed 12 hours post seeding to ensure all parasites were synchronised at the ring stage. Drug treatments starts when parasites reach mid-to-late trophozoite stage. The infected RBCs were given fresh MCM and incubated for 4 hours, with 3uM MMV 085071, MMV 1545561, CQ or the same volume of DMSO resuspended in MCM medium.

阐明新型抗疟药物的作用机制，是根除疟疾的核心目标之一。此前我们针对恶性疟原虫（Plasmodium falciparum），探究了由微摩尔级浓度抗疟药物氯喹（chloroquine, CQ）诱导的一种潜在替代性细胞死亡机制。本次研究评估的表型包括钙重分布、线粒体去极化以及疟原虫消化泡膜通透化。明确疟原虫内钙释放的源头或起始因子，有望发现全新的治疗靶点。通过时间点实验，我们发现线粒体完整性的丧失与胞内钙稳态失调同时发生，随后出现下游膜破裂事件。通过一系列钙外流抑制剂与抗氧化剂的实验，我们证实线粒体钙（mitochondrial calcium, Ca²⁺mt）参与启动了这一系列疟原虫细胞死亡表型的级联反应。在利用高剂量氯喹明确该机制后，我们通过疟疾药品事业会（Medicines for Malaria Venture, MMV）病原体库（Pathogen Box）的高内涵筛选，拓展寻找具有相似表型效应的化合物。共筛选得到3个命中化合物，随后对其开展进一步的表征实验，并确定活性最优的化合物为MMV085071。针对该最优命中化合物设计合成了10个衍生物，通过剂量反应实验评估其活性后，基于药效筛选出活性最佳的先导衍生物MMV1545561。我们分别对原始命中化合物与先导化合物进行了关于线粒体钙重分布能力的探究。尽管实验结果具有统计学显著性，但抑制剂无法完全逆转化合物诱导的钙稳态失调与线粒体崩解，这表明相较于氯喹，线粒体钙在该类化合物引发的细胞死亡过程中作用更为有限。此外，本次筛选得到的新型化合物可能靶向疟原虫的多个靶点，且其药效优异，因此有必要开展后续研究以明确其具体作用机制。采用山梨醇同步化与磁性活化细胞分选（magnetic-activated cell sorting, MACS）技术对恶性疟原虫3D7株（P. falciparum 3D7）进行处理，以富集裂殖体阶段的疟原虫。将富集得到的裂殖体以1.5%~2%的虫血症率（parasitemia）接种于1.25%血细胞比容（haematocrit）的培养体系中。接种12小时后，再次通过山梨醇进行同步化处理，以确保所有疟原虫均同步发育至环状体阶段。待疟原虫发育至中晚期滋养体阶段时，开始施加药物处理。将感染红细胞更换为新鲜的MCM培养基，分别加入终浓度3μM的MMV085071、MMV1545561、氯喹，或等体积的二甲基亚砜（dimethyl sulfoxide, DMSO）对照，于MCM培养基中孵育4小时。