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

I-BET151 诱导非洲锥虫分化的目标验证

• 批准号: 10218818
• 负责人: Erik Debler
• 金额: $ 26.17万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10218818
• 关键词: Affinity; Affinity Chromatography; Africa South of the Sahara; African; African Trypanosomiasis; Antigenic Variation; Binding; Biological Models; Biology; Blood Circulation; Bromodomain; CRISPR/Cas technology; Cell Membrane Permeability; Cells; Chagas Disease; Complement; Crosslinker; Defense Mechanisms; Development; Diazomethane; Differentiation Therapy; Disease; Environment; Evaluation; Foundations; Gene Expression Regulation; Genes; Genetic; Genomics; Goals; Human; Immobilization; Immune system; In Situ; In Vitro; Infection; Insect Vectors; Knock-out; Knowledge; Leishmania; Leishmaniasis; Life Cycle Stages; Ligands; Link; Longitudinal Studies; Maintenance; Mammals; Mass Spectrum Analysis; Metabolism; Methodology; Midgut; Molecular; Molecular and Cellular Biology; Morbidity - disease rate; Morphology; Mus; Organism; Parasites; Parasitic Diseases; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Phenotype; Physiological; Plant Resins; Process; Protac; Proteins; Proteomics; RNA Interference; Recombinant Proteins; Regulation; Research; Series; Survival Rate; Testing; Therapeutic; Translating; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Tsetse Flies; Ubiquitination; Validation; Virulence; Virulent; base; cancer cell; chemoproteomics; chemotherapy; crosslink; design; experimental study; global health; in vivo; inhibitor/antagonist; knock-down; mortality; novel; protein complex; protein degradation; protein protein interaction; recruit; small molecule; ubiquitin-protein ligase; ultraviolet irradiation; vector

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.

项目概要/摘要 非洲锥虫是原生动物寄生虫，可在多种哺乳动物中引起严重且往往致命的疾病 撒哈拉以南非洲地区，包括人类（昏睡病）。与许多其他寄生虫一样，非洲寄生虫 锥虫在其哺乳动物宿主及其生命周期中经历了一系列不同的阶段 昆虫媒介采采蝇，它们的新陈代谢、形态和毒力方面存在显着差异。有 我们目前对协调转变的分子机制的了解存在几个关键差距 在这些阶段之间。我们的长期目标是揭示生命周期阶段调控的分子基础 非洲锥虫并利用这些知识来操纵分化过程以进行治疗 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 设计为人类溴结构域抑制剂，对细长的血流形式具有深远的影响 哺乳动物通过将这种有毒阶段转化为原循环形式，这种形式普遍存在于tse的中肠中 谢飞矢量。重要的是，这种 I-BET151 诱导的分化削弱了寄生虫的防御机制，例如 作为抗原变异，因此使其容易受到宿主免疫系统的攻击，从而可被利用 治疗目的。事实上，用 I-BET151 处理的寄生虫感染小鼠产生了令人印象深刻的结果 与感染未经处理的锥虫的小鼠相比，存活率。在进行这些原理验证研究之前 能否转化为新的化疗方案，我们首先需要了解并验证I-的作用方式 非洲锥虫中的 BET151，作为 I-BET151 的靶标，导致强表型 锥虫中尚未被鉴定。在本提案中，我们试图阐明 I-BET151 的目标 使用三种互补的化学蛋白质组学策略来研究非洲锥虫。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富集 I-BET151 相互作用蛋白，然后 使用质谱法进行蛋白质鉴定。以更具生理相关性的方式捕获 I-BET151 目标 在这种情况下，我们将在第二种方法中在锥虫细胞中使用原位光交联并鉴定修饰的 通过质谱分析蛋白质。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使用称为蛋白质水解靶向的双功能小分子来诱导 嵌合体或 PROTAC，其中 I-BET151 与 E3 泛素连接酶招募配体缀合。总的来说， 我们提出的研究将通过鉴定参与锥虫的蛋白质和复合物来推进锥虫生物学 寄生虫分化。这些研究也将促进相关领域生命周期阶段调控的研究。 导致全世界毁灭性死亡和发病的寄生虫。最后，所提出的实验将奠定 药物诱导分化治疗的基础，有潜力成为一种新的变革策略 控制和杀死致病寄生虫。