Target Validation for I-BET151-Induced Differentiation in the African Trypanosome

项目摘要/摘要 非洲锥虫是一种原生动物寄生虫，可导致多种哺乳动物患上严重且往往致命的疾病。 在撒哈拉以南非洲，包括人类(昏睡病)。像许多其他寄生虫一样，非洲的 锥虫在其哺乳动物宿主和宿主体内的生命周期中经历了一系列不同的阶段 昆虫媒介，泽蝇，它们在新陈代谢、形态和毒力上有很大的不同。确实有 我们目前对协调这种转变的分子机制的认识中存在几个关键差距 在这两个阶段之间。我们的长期目标是解开生命周期阶段调控的分子基础 非洲锥虫并利用这一知识操纵分化过程用于治疗 目的。在最近的实验中，我们发现了一种名为i-BET151的小分子，它最初是 被设计为人类溴域抑制剂，对人体内细长的血流形态有深远的影响 通过将这一毒力阶段转变为原环状形式，这种形式普遍存在于Tse-Tse的中肠-- 谢飞向量。重要的是，这种I-BET151诱导的分化削弱了寄生虫的防御机制，如 作为抗原变异，从而使其容易被宿主免疫系统攻击，这可被利用来 治疗目的。事实上，用i-BET151处理过的寄生虫感染小鼠，结果令人印象深刻 与未经治疗的锥虫感染小鼠的存活率进行比较。在这些原则证明研究之前 可以转化为一种新的化疗，我们需要首先了解和验证I- 非洲锥虫中的BET151，作为I-BET151的靶标(S)，这些都是负责强表型的 在锥虫体内的基因还没有被确认。在这项建议中，我们试图澄清I-BET151的目标(S)在 非洲锥虫使用三种互补的化学蛋白质组策略。在第一种方法中，我们将 通过在锥虫细胞裂解物的珠基质上亲和捕获来丰富I-BET151相互作用的蛋白质，随后 通过质谱学进行蛋白质鉴定。以更具生理相关性的方式捕获I-BET151目标 在第二种方法中，我们将在锥虫细胞中使用原位光交联法，并识别修改过的 蛋白质的质谱学分析。在第三种方法中，I-BET151的泛素化和蛋白酶体降解- 相互作用的蛋白质将被双功能小分子诱导，这种小分子被称为蛋白水解靶向 嵌合体，或PROTAC，其中I-BET151连接到E3泛素连接酶招募配体。总而言之， 我们提出的研究将通过识别与锥虫相关的蛋白质和复合体来推动锥虫生物学的发展 寄生虫分化。这些研究也将促进相关生命周期阶段调控的研究。 在世界范围内造成毁灭性死亡和发病率的寄生虫。最后，拟议中的实验将 药物诱导分化治疗的基础，它有可能成为一种新的变革性策略 控制和消灭致病寄生虫。

PROJECT SUMMARY/ABSTRACT African trypanosomes are protozoan parasites that cause severe and often fatal disease in a variety of mammals in Sub-Saharan Africa, including humans (sleeping sickness). Like many other parasites, the African trypanosome progresses through a series of distinct stages during its life cycle in its mammalian host and its insect vector, the tse-tse fly, which dramatically differ in their metabolism, morphology, and virulence. There are several critical gaps in our current knowledge of the molecular mechanisms that orchestrate the transitions between these stages. Our long-term goal is to unravel the molecular basis of life-cycle stage regulation in the African trypanosome and to harness this knowledge to manipulate the differentiation process for therapeutic purposes. In recent experiments, we discovered that a small molecule called I-BET151, which was originally designed as a human bromodomain inhibitor, has a profound effect on the long slender bloodstream form in the mammal by transforming this virulent stage into the procyclic form, which is prevalent in the midgut of the tse- tse fly vector. Importantly, this I-BET151-induced differentiation cripples the parasite’s defense mechanism such as antigenic variation and, thus, renders it vulnerable by the host immune system, which can be exploited for therapeutic purposes. Indeed, infection of mice with I-BET151-treated parasites resulted in an impressive survival rate in comparison to mice infected with untreated trypanosomes. Before these proof-of-principle studies can be translated into a new chemotherapy, we need to first understand and validate the mode of action of I- BET151 in the African trypanosome, as the target(s) of I-BET151 that are responsible for the strong phenotype in trypanosomes have not yet been identified. In this proposal, we seek to elucidate the target(s) of I-BET151 in the African trypanosome using three complementary chemoproteomic strategies. In the first approach, we will enrich I-BET151-interacting proteins by affinity capture on a bead matrix from trypanosome cell lysate, followed by protein identification using mass spectrometry. To capture I-BET151 targets in a more physiologically relevant condition, we will use in situ photo-crosslinking in trypanosome cells in the second approach and identify modified proteins by mass spectrometry. In a third approach, ubiquitination and proteasomal degradation of I-BET151- interacting proteins will be induced using bifunctional small molecules known as a PROteolysis TArgeting Chimeras, or PROTACs, in which I-BET151 is conjugated to an E3 ubiquitin ligase-recruiting ligand. Collectively, our proposed research will advance trypanosome biology by identifying proteins and complexes involved in parasite differentiation. These studies will also stimulate research of life-cycle stage regulation in related parasites that cause devastating mortality and morbidity worldwide. Finally, the proposed experiments will lay the foundation for drug-induced differentiation therapy, which has the potential as a novel transformative strategy to control and kill pathogenic parasites.