A path to tackle liver-stage parasites Malaria parasites are evolutionarily prepared to resist drug attack. Resistance is emerging to even the latest frontline combination therapies, which target the blood stages of the Plasmodium parasite. As an alternative strategy, Antonova-Koch et al. investigated the possibilities of drugs against liver-stage parasites (see the Perspective by Phillips and Goldberg). To do so, they devised a luciferase-reporter drug screen for the rodent parasite Plasmodium berghei. Three rounds of increasingly stringent screening were used. From this regime, several chemotypes that inhibit Plasmodium mitochondrial electron transport were identified. Excitingly, several new scaffolds, with as-yet-unknown modes of action but solely targeting the parasites' liver stages, emerged as promising drug leads for further development. Science, this issue p. eaat9446; see also p. 1112 Screening of more than half a million compounds for their ability to inhibit liver-stage Plasmodium development yields thousands of candidates. INTRODUCTION Malaria remains a devastating disease, affecting 216 million people annually, with 445,000 deaths occurring primarily in children under 5 years old. Malaria treatment relies primarily on drugs that target the disease-causing asexual blood stages (ABS) of Plasmodium parasites, the organisms responsible for human malaria. Whereas travelers may rely on short-term daily chemoprotective drugs, those living in endemic regions require long-term malaria protection such as insecticide-treated nets (ITNs) and vector control. However, ITNs do not fully shield individuals from malaria, may lose potency with time, and can be bulky and difficult to use. Another concern is that mosquitos may become resistant to the active insecticides that are used in ITNs and vector control. RATIONALE As the possibility of malaria elimination becomes more tangible, the ideal antimalarial medicine profile should include chemoprotection. Chemoprotective medicines typically work against the exoerythrocytic parasite forms that invade and develop in the liver and are responsible for the earliest asymptomatic stage of the infection. Such medicines could be formulated to provide long-acting prophylaxis, safeguarding individuals that are living near or traveling to areas that have been cleared of parasites. Long-acting chemoprotection in endemic regions could also greatly reduce circulating parasite numbers and potentially replace a vaccine in an elimination campaign. Although millions of compounds have been screened for activity against parasite ABS, and some have been subsequently tested for potential prophylactic activity, large-scale searches that begin with prophylactic activity have not been performed because of the complexity of the assay: This assay requires the production of infected laboratory-reared mosquitoes and hand-dissection of the sporozoite-infected salivary glands from mosquito thoraxes. RESULTS To discover leads for next-generation chemoprotective antimalarial drugs, we used luciferase-expressing Plasmodium spp. parasites, dissected from more than a million mosquitoes over a 2-year period, to test more than 500,000 compounds for their ability to inhibit liver-stage development of malaria (681 compounds showed a half-maximal inhibitory concentration of <1 μM). Cluster analysis identified potent and previously unreported scaffold families, as well as other series previously associated with chemoprophylaxis. These leads were further tested through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity. This work revealed compound classes that are likely to provide symptomatic relief from blood-stage parasitemia in addition to providing protection. Target identification by use of functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unknown mechanisms of action, some which may disrupt the host pathogen signaling. CONCLUSION Our data substantially expands the set of compounds with demonstrated activity against two known targets of chemoprotective drugs, cytochrome bc1 and dihydroorotate dehydrogenase. These present a rich collection of chemical diversity that may be exploited by members of the community seeking to accelerate malaria elimination with chemoprotection and chemoprophylaxis through open-source drug discovery. A Plasmodium vivax liver-stage schizont on a lawn of hepatocytes. The parasite schizont has been stained with antibodies to parasite HSP70 (red) and UIS4 (yellow). Cell (parasite and hepatoma) nuclei are shown in blue. This study identifies compounds that can prevent the development of these liver-stage parasites and may function as chemoprotective drugs for malaria. To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

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下一代化学保护性抗疟药的化学先导物的开源发现

解决肝脏阶段寄生虫的途径 疟疾寄生虫在进化上已准备好抵抗药物攻击。即使是针对疟原虫血液阶段的最新一线联合疗法也出现了耐药性。作为替代策略，Antonova-Koch 等人。研究了药物对抗肝期寄生虫的可能性（参见菲利普斯和戈德堡的观点）。为此，他们设计了一种针对啮齿动物寄生虫伯氏疟原虫的荧光素酶报告药物筛选。使用了三轮越来越严格的筛选。从该方案中，鉴定了几种抑制疟原虫线粒体电子传递的化学型。令人兴奋的是，几种新的支架，其作用方式尚不为人知，但仅针对寄生虫的肝脏阶段，成为进一步开发的有前途的药物线索。科学，这个问题 p。eaat9446; 另见第。1112 筛选超过 50 万种化合物以抑制肝脏阶段疟原虫发育的能力，产生了数千种候选物。简介 疟疾仍然是一种毁灭性疾病，每年影响 2.16 亿人，其中 445,000 人死亡，主要发生在 5 岁以下儿童中。疟疾治疗主要依赖于针对疟原虫的致病无性血阶段 (ABS) 的药物，疟原虫是导致人类疟疾的生物。旅行者可能依赖短期的日常化学保护药物，而生活在流行地区的人则需要长期的疟疾保护，例如驱虫蚊帐 (ITN) 和病媒控制。然而，ITN 并不能完全保护个人免受疟疾的侵害，可能会随着时间的推移而失去效力，并且体积庞大且难以使用。另一个问题是蚊子可能会对用于 ITN 和病媒控制的活性杀虫剂产生抗药性。基本原理 随着消除疟疾的可能性变得越来越明显，理想的抗疟药物应该包括化学保护。化学保护药物通常对侵入肝脏并在肝脏中发育的红细胞外寄生虫形式起作用，并负责感染的最早无症状阶段。可以配制此类药物以提供长效预防，保护居住在已清除寄生虫地区或前往已清除寄生虫地区的个人。流行地区的长效化学保护也可以大大减少循环寄生虫的数量，并有可能在消除运动中取代疫苗。尽管已经筛选了数百万种化合物的抗寄生虫 ABS 活性，并且随后测试了一些化合物的潜在预防活性，但由于检测的复杂性，尚未进行以预防活性开始的大规模搜索：该检测需要生产受感染的实验室饲养的蚊子和从蚊子胸部的子孢子感染唾液腺的手解剖。结果 为了发现下一代化学保护性抗疟药的先导物，我们使用了表达荧光素酶的疟原虫。在两年内从超过一百万只蚊子中解剖出来的寄生虫，以测试超过 500 只，000 种化合物具有抑制疟疾肝脏阶段发展的能力（681 种化合物的半数抑制浓度 <1 μM）。聚类分析确定了以前未报告的有效支架家族，以及以前与化学预防相关的其他系列。通过预测阶段特异性和多物种抗疟活性的多种表型分析，对这些先导物进行了进一步测试。这项工作揭示了化合物类别，除了提供保护外，还可能缓解血液阶段寄生虫血症的症状。通过使用功能测定、体外进化或代谢分析进行的靶标鉴定揭示了 58 种线粒体抑制剂，但也有许多化学型可能具有以前未知的作用机制，其中一些可能会破坏宿主病原体信号传导。结论 我们的数据极大地扩展了一组化合物，这些化合物对化学保护药物的两个已知靶标具有活性，细胞色素 bc1 和二氢乳清酸脱氢酶。这些提供了丰富的化学多样性集合，社区成员可以利用这些多样性，通过开源药物发现，通过化学保护和化学预防来加速消除疟疾。肝细胞草坪上的间日疟原虫肝脏阶段裂殖体。寄生虫裂殖体已被寄生虫 HSP70（红色）和 UIS4（黄色）抗体染色。细胞（寄生虫和肝癌）细胞核以蓝色显示。这项研究确定了可以防止这些肝脏阶段寄生虫发展的化合物，并且可以作为疟疾的化学保护药物。为了发现下一代化学保护性抗疟药的线索，我们测试了超过 500,000 种化合物抑制表达荧光素酶的疟原虫肝脏阶段发育的能力。寄生虫（681 种化合物的半数最大抑制浓度小于 1 微摩尔）。聚类分析确定了以前未报告的有效支架家族以及以前与化学预防相关的其他系列。通过预测阶段特异性和多物种抗疟活性的多种表型分析进行进一步测试，区分可能通过减少无性血阶段寄生虫血症和可能仅预防疟疾的那些来缓解症状的化合物类别。通过使用功能分析、体外进化、