Heme Detoxification in the Malaria Parasite: A Target for Antimalarial Drug Development.
Heme Detoxification in the Malaria Parasite: A Target for Antimalarial Drug Development.
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DOI：
10.1021/acs.accounts.1c00154
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发表时间：
2021-06-01
影响因子：
18.3
通讯作者：
Egan TJ
中科院分区：
文献类型：
--
作者：
de Villiers KA;Egan TJ
上个世纪，疟疾死亡人数减少了 85% 以上。尽管如此，2018 年仍有 405 000 人死亡，其中大部分是由恶性疟原虫感染造成的。在 21 世纪，大部分进步都来自于使用经过杀虫剂处理的蚊帐和青蒿素联合疗法。然而，在过去的十年中，具有延迟青蒿素清除表型的寄生虫在东南亚出现，威胁到进一步的进展。因此，寻找新药刻不容缓。血期疟疾寄生虫的一个重要过程是疟原虫色素的形成，我们认为它仍然是可行的药物靶点。当用显微镜观察寄生虫时，可以很容易地看到这种由血红素组成的晶体材料。然而，它在寄生虫中形成的过程仍不完全清楚。在早期工作中，我们认识到疟原虫色素的形成是一个生物矿化过程。随后，我们研究了仿生条件下在脂质-水界面催化的合成疟原虫色素（β-血红素）结晶的动力学。这导致我们使用基于中性去污剂的高通量筛选 (HTS) 来筛选 β-血红素形成的抑制剂。获得了针对疟疾寄生虫的良好命中率。同时，我们开发了一种基于吡啶的测定法，事实证明，该测定法可以成功测量未转化为 β-血红素的血红素浓度。吡啶测定适用于确定氯喹和其他临床抗疟药对细胞内疟原虫色素形成的影响。这使得首次能够确定恶性疟原虫中可交换血红素和疟原虫色素的剂量依赖性量。这些研究表明，疟原虫色素抑制剂会导致可交换血红素呈剂量依赖性增加，与寄生虫存活率降低相关。电子光谱成像（ESI）显示血红素铁重新定位到寄生虫细胞质中，而电子显微镜提供了疟原虫色素晶体破坏的证据。这种细胞测定随后扩展到 HTS 发现的各种支架中排名最高的命中。有趣的是，这些支架的寄生虫生长 IC50 值下可交换血红素的量显示出很大的变化。发现可交换血红素的量与寄生红细胞中积累的抑制剂的量相关。这表明血红素抑制剂复合物，而不是游离血红素，导致寄生虫死亡。使用含 Br 化合物的 ESI 证实了 Fe 和 Br 的共定位，并且通过共聚焦拉曼显微镜证实了寄生虫中存在复合物，这都支持了这一点。目前的证据表明抑制剂通过表面吸附阻止疟原虫色素的形成。事实上，我们已经成功地引入了与疟原虫色素的分子对接来寻找新的抑制剂。由此可见，游离血红素的增加导致了杀寄生虫血红素-抑制剂复合物的形成。我们报道了几种芳基甲醇抗疟药在非水介质中的血红素药物复合物的晶体结构。它们形成配位复合物，但大多数其他抑制剂都是非共价相互作用，确定其结构仍然是一个重大挑战。我们认为，未来的关键发展将包括改进测量细胞血红素水平的测定方法、更好地预测 β-血红素抑制的计算机方法，以及确定血红素抑制剂复合物的结构和性质的共同努力。
Over the last century, malaria deaths have decreased by more than 85%. Nonetheless, there were 405 000 deaths in 2018, mostly resulting from Plasmodium falciparum infection. In the 21st century, much of the advance has arisen from deployment of insecticide-treated bed nets and artemisinin combination therapy. However, over the last decade parasites with a delayed artemisinin clearance phenotype have appeared in Southeast Asia threatening further gains. The effort to find new drugs is thus urgent. A prominent process in blood stage malaria parasites, which we contend remains a viable drug target, is hemozoin formation. This crystalline material consisting of heme can be readily seen when parasites are viewed microscopically. The process of its formation in the parasite, however, is still not fully understood. In early work, we recognized hemozoin formation as a biomineralization process. We have subsequently investigated the kinetics of synthetic hemozoin (β-hematin) crystallization catalyzed at lipid-aqueous interfaces under biomimetic conditions. This led us to the use of neutral detergent-based high throughput screening (HTS) for inhibitors of β-hematin formation. A good hit rate against malaria parasites was obtained. Simultaneously we developed a pyridine-based assay which proved successful in measuring concentrations of hematin not converted to β-hematin. The pyridine assay was adapted to determine the effects of chloroquine and other clinical antimalarials on hemozoin formation in the cell. This permitted determination of the dose dependent amounts of exchangeable heme and hemozoin in P. falciparum for the first time. These studies have shown that hemozoin inhibitors cause a dose-dependent increase in exchangeable heme, correlated with decreased parasite survival. Electron spectroscopic imaging (ESI) showed a relocation of heme iron into the parasite cytoplasm, while electron microscopy provided evidence of disruption of hemozoin crystals. This cellular assay was subsequently extended to the top-ranked hits from a wide range of scaffolds found by HTS. Intriguingly, the amounts of exchangeable heme at the parasite growth IC50 values of these scaffolds showed substantial variation. The amount of exchangeable heme was found to be correlated with the amount of inhibitor accumulated in the parasitized red blood cell. This suggests that heme-inhibitor complexes, rather than free heme, lead to parasite killing. This was supported by ESI using a Br-containing compound which showed co-localization of Fe and Br, as well as by confocal Raman microscopy which confirmed the presence of a complex in the parasite. Current evidence indicates that inhibitors block hemozoin formation by surface adsorption. Indeed, we have successfully introduced molecular docking with hemozoin to find new inhibitors. It follows that the resulting increase in free heme leads to formation of the parasiticidal heme-inhibitor complex. We have reported crystal structures of heme-drug complexes for several aryl methanol antimalarials in non-aqueous media. These form coordination complexes, but most other inhibitors interact non-covalently and determination of their structures remains a major challenge. It is our view that key future developments will include improved assays to measure cellular heme levels, better in silico approaches for predicting β-hematin inhibition, and a concerted effort to determine the structure and properties of heme-inhibitor complexes.
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影响因子：
3
作者：
Ambele MA;Egan TJ
通讯作者：
Egan TJ
影响因子：
3.9
作者：
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3.8
作者：
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通讯作者：
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