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

项目摘要 蜱传播疾病，包括人类巴贝虫病，正在上升。由巴氏杆菌属的原生动物引起， 这种感染在无脾或免疫功能低下的个体中可能危及生命。基于最近的 CDC数据显示，2011年至2018年，美国年度报告病例数几乎翻了一番，其中一些病例 增长超过10倍的国家。免疫功能低下患者巴贝虫病的治疗选择 个体非常有限，具有相当大的副作用，并且常常不能完全清除寄生虫。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， 低毒性和对感染人类的多种巴氏疟原虫的优异活性。分子 该抑制剂靶点显然是可药物化的、必需的且可能是新颖的，但目前是未知的。使用 结合遗传和生物化学的方法，拟议的实验将确定和验证 这种抑制剂的寄生虫目标。在AIM1中，我们将使用µ Map确定哪些寄生虫蛋白与C10紧密结合， 新开发的光催化化学蛋白质组学平台，具有极大增强的灵敏度和特异性 超过目前的方法。在一个平行的努力，我们已经选择了多个独立的寄生线与部分 抗C10。由于编码药物靶标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鉴定抗性品系中新获得突变的基因， 以识别另外一组目标候选物。最后，我们将验证目标 通过确认靶的工程化突变，如敲低或缺失， 过表达，改变对C10的易感性。如果可行，我们还将检测C10是否结合并抑制 重组表达的靶标。在这些实验中，将使用C13，一种几乎相同但无活性的 C10异构体，以确保靶特异性。

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.