Solving the Artemisinin Dormancy Conundrum

解决青蒿素休眠难题

• 批准号: 8770248
• 负责人: Victor Nussenzweig
• 金额: $ 25.43万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-16 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8770248
• 关键词: Animals; Antimalarials; Artemisinins; Blood; Boxing; Cell Cycle; Chemicals; Combined Modality Therapy; Databases; Development; Dose; Erythrocytes; Eukaryotic Initiation Factor-2; Free Radicals; Future; Genetic; Genetic Transcription; Genome; Infection; Knock-out; Lactones; Life Cycle Stages; Malaria; Mediating; Molecular; Mus; Oxidative Stress; Parasitemia; Parasites; Patients; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Plasmodium; Plasmodium falciparum; Protein Biosynthesis; Protein Kinase Inhibitors; Protein Synthesis Inhibition; Publishing; Recrudescences; Reporting; Role; Sesquiterpenes; Source; Staging; Stress; Symptoms; Testing; Translations; Treatment Failure; artemisinine; drug development; inhibitor/antagonist; killings; mutant; phosphatase inhibitor; prevent; protein kinase inhibitor; public health relevance; response

DESCRIPTION (provided by applicant): Artemisinin and its derivatives (ART) are the first-line treatment in malaria. ART clears parasitaemia and resolves symptoms very rapidly (1). ART monotherapy is however associated with high rates of recrudescence (2-3) requiring the combination of ART with an antimalarial of a different chemical class (ART combination therapy or ACT). Despite ACT's outstanding activity, recrudescences of Plasmodium falciparum infections have been reported (4-6).The mechanisms leading to recrudescence are not known. ART-mediated recrudescence is associated with the accumulation of dormant ring stage parasites and disruption of the parasite's cell cycle. The objective of this proposal is to elucidae the molecular mechanism of ART-induced dormancy. ART is a sesquiterpene trioxane lactone containing an endoperoxide bridge that is a source of free radicals. The reductive cleavage of the bridge is essential for ART anti-malarial activity. We propose that the oxidative stress elicited by ART activates an eIF2¿ (eukaryotic initiation factor 2¿) kinase in some of the parasites, leading to a global inhibition of protein synthesis and the dormant state. We document a high level of eIF2¿ phosphorylation in ART-treated, dormant ring stages of P. berghei parasites, and show that dormancy is significantly extended by Salubrinal (Sal). Sal is a specific inhibitor of eIF2¿-P phosphatase (7-8). We propose to identify the eIF2¿ kinase that is activated by the ART-induced oxidative stress. There are three eIF2¿ kinases in the Plasmodium genome: eIK1, eIK2 and PK4(9). To find which one responds to ART stress we will analyze the effect of ART on the kinase knockouts of P. berghei. We expect that the relevant mutants will not generate dormant rings. We have already obtained clones of eIK2 (-) parasites (8) and will test them for recrudescence. The PbeIK2 (-) blood stages will be injected in mice and the animals treated with ART. After the parasites are cleared we will check for recrudescence of the infection. We will generate eIK1 knockout clones and test them as above. HoweverPK4 (-) mutants cannot be generated because PK4 is essential for the development of erythrocytic stages (10-11). Evidence for a possible role of PK4 in ART-mediated dormancy will be obtained by analysis of PK4 transcription prior to and following treatment with sub-lethal doses of ART. We will search databases containing small chemicals for inhibitors of the "dormancy kinase". The identification of the kinase should facilitate future development of drugs that prevent dormancy and overcome ART treatment failures.

描述（由申请人提供）：青蒿素及其衍生物（ART）是疟疾的一线治疗药物。ART清除寄生虫血症并迅速解决症状（1）。然而，ART单药治疗与高复发率相关（2-3），需要ART与不同化学类别的抗疟药联合治疗（ART联合治疗或ACT）。尽管ACT的活动非常出色，但仍有恶性疟原虫感染复发的报道（4-6），导致复发的机制尚不清楚。ART介导的复发与休眠环期寄生虫的积累和寄生虫细胞周期的破坏有关。本研究的目的是阐明ART诱导休眠的分子机制。ART是含有作为自由基来源的内过氧化物桥的倍半萜三氧杂环己烷内酯。桥的还原切割对于ART抗疟疾活性是必不可少的。我们认为ART引起的氧化应激激活了一些寄生虫中的eIF 2（真核起始因子2）激酶，导致蛋白质合成的全面抑制和休眠状态。我们记录了ART处理的伯氏疟原虫休眠环阶段的高水平eIF 2？磷酸化，并表明Salubrinal（Sal）显著延长了休眠期。Sal是eIF 2 <$-P磷酸酶的特异性抑制剂（7-8）。我们建议鉴定由ART诱导的氧化应激激活的eIF 2 <$激酶。疟原虫基因组中有三种eIF 2？激酶：eIK 1、eIK 2和PK 4（9）。为了发现哪一个响应于ART应激，我们将分析ART对伯氏疟原虫的激酶敲除的影响。我们希望相关的突变体不会产生休眠环。我们已经获得了eIK 2（-）寄生虫的克隆（8），并将测试它们的复发。将PbeIK 2（-）血液阶段注射到小鼠和用ART治疗的动物中。在寄生虫被清除后，我们将检查感染的复发。我们将产生eIK 1敲除克隆并如上所述对其进行测试。然而，不能产生PK 4（-）突变体，因为PK 4对于红细胞阶段的发育是必需的（10-11）。通过分析亚致死剂量ART治疗之前和之后的PK 4转录，将获得PK 4在ART介导的休眠中可能发挥作用的证据。我们将搜索包含“休眠激酶”抑制剂的小化学物质的数据库。激酶的鉴定将有助于未来开发防止休眠和克服ART治疗失败的药物。