Identifying the Target of a Potent and Selective Inhibitor of Babesia Blood Stages.

项目摘要 扁虱传播的疾病，包括人类巴贝斯虫病，正在上升。由巴贝斯虫属的原生动物引起， 这种感染对脾功能不全或免疫功能低下的人可能会危及生命。基于最新的 CDC的数据显示，2011至2018年间，美国的年度报告病例数量几乎翻了一番，其中一些 正在经历10倍以上增长的国家。免疫功能低下的巴贝斯虫病的治疗选择 个体数量非常有限，有很大的副作用，而且往往无法完全清除寄生虫。这使得 开发新的治疗方法势在必行。我们最近开发了小分子抑制剂C10，它已经 对感染人类的多种巴贝斯虫具有低毒和良好的活性。分子 这种抑制剂的靶点显然是可麻醉的、必不可少的，可能是新的，但目前尚不清楚。使用 结合遗传和生化方法，拟议的实验将识别和验证 这种抑制剂的寄生靶标。在AIM1中，我们将使用µMap确定哪些寄生虫蛋白与C10紧密结合， 一种敏感度和特异度显著提高的光催化化学蛋白质组学平台 过多的方法。在一项平行的工作中，我们选择了多个独立的寄生虫品系， 对C10的抗性。由于编码药物靶点的基因突变是最常见的耐药机制， AIM2将使用全基因组重测序来识别抗病品系中新获得的突变基因，但 在易受影响的父母中不存在，以识别额外的目标候选集合。最后，我们将验证目标 来自AIM1和AIM2的候选基因，通过确认目标的工程突变，如基因敲除或 过度表达，改变对C10的易感性。如果可行，我们还将测试C10是否与C10结合并抑制其活性 重组表达的靶标。在这些实验中将使用C13，几乎相同但不活跃 C10的异构体，以确保靶标特异性。

Project Summary Tick-borne diseases, including human babesiosis, are on the rise. Caused by protozoa in the genus Babesia, this infection can be life-threatening in asplenic or immunocompromised individuals. Based on the most recent CDC data, the number of annual reported cases in the US nearly doubled between 2011 and 2018, with some states experiencing a greater than 10-fold increase. Treatment options for babesiosis in immunocompromised individuals are very limited, have substantial side effects, and often fail to fully clear the parasites. This makes the development of new treatments imperative. We recently developed the small molecule inhibitor C10 that has low toxicity and excellent activity against multiple species of Babesia parasites that infect humans. The molecular target of this inhibitor is clearly druggable, essential, and likely novel but is currently unknown. Using a combination of genetic and biochemical approaches, the proposed experiments will identify and validate the parasite target of this inhibitor. In AIM1 we will identify what parasite proteins that bind tightly to C10 using µMap, a newly developed photocatalytic chemo-proteomics platform with greatly enhanced sensitivity and specificity over current methods. In a parallel effort, we have selected multiple independent parasite lines with partial resistance to C10. Since mutation in genes encoding a drug target is the most common resistance mechanism, AIM2 will use whole-genome resequencing to identify genes with newly acquired mutations in resistant lines but absent from the susceptible parent to identify an additional set of target candidates. Finally, we will validate target candidates from AIM1 and AIM2 by confirming that engineered mutations of the target, such as knockdown or overexpression, alters susceptibility to C10. If feasible, we will also test if C10 binds to and inhibits the activity of the recombinantly expressed target. Throughout these experiments will use C13, a nearly identical but inactive isomer of C10, to ensure target specificity.