Solving the Artemisinin Dormancy Conundrum

描述(申请人提供)：青蒿素及其衍生物(ART)是疟疾的一线治疗药物。抗逆转录病毒疗法可迅速清除寄生虫血症并缓解症状(1)。然而，ART单一疗法与高复发率(2-3)相关，需要将ART与不同化学类别的抗疟药(ART联合疗法或ACT)相结合。尽管ACT具有突出的活性，但已有恶性疟原虫感染复发的报告(4-6例)。导致复发的机制尚不清楚。ART介导的复发与休眠的环期寄生虫的聚集和寄生虫细胞周期的中断有关。本研究的目的是阐明ART诱导休眠的分子机制。ART是一种倍半萜三氧六环内酯，含有一种内过氧桥，是自由基的来源。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引发的氧化应激激活了一些寄生虫中的eIF2？(真核起始因子2？)激酶，导致蛋白质合成的全局抑制和休眠状态。我们记录了经ART处理的伯氏疟原虫休眠环期eIF2高水平的磷酸化，并表明Salubrine(SAL)显著延长了休眠时间。SAL是eIF2？-P磷酸酶(7-8)的特异性抑制剂。我们建议确定由ART诱导的氧化应激激活的eIF2？激酶。在疟原虫基因组中有三种eIF2蛋白：eIK1、eIK2和PK4(9)。为了找出哪一个对ART压力做出反应，我们将分析ART对P.berghei的激酶敲除的影响。我们预计相关突变体不会产生休眠环。我们已经获得了eIK2(-)寄生虫的克隆(8)，并将测试它们的复发情况。PbeIK2(-)血液阶段将被注射到小鼠和接受ART治疗的动物身上。寄生虫清除后，我们将检查感染是否复发。我们将生成eIK1基因敲除克隆并如上所述对其进行测试。然而，由于PK4对红细胞期(10-11)的发育是必需的，所以不能产生PK4(-)突变体。在亚致死剂量的ART治疗前后，通过对PK4转录的分析，将获得PK4在ART介导的休眠中可能发挥作用的证据。我们将在含有小分子化学物质的数据库中搜索“休眠激酶”的抑制剂。该激酶的鉴定应该有助于未来开发防止休眠和克服ART治疗失败的药物。

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.