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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

基本信息

批准号：
10334561
负责人：
Erik Debler
金额：
$ 19.77万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10334561
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 knock-down mortality novel protein complex protein degradation protein protein interaction recruit small molecule ubiquitin-protein ligase ultraviolet irradiation vector

项目摘要

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.

项目总结/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发生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，非洲锥虫在其哺乳动物宿主中的生命周期中经历一系列不同的阶段，昆虫媒介，tse-tse蝇，它们在代谢、形态和毒力方面有很大的不同。有我们目前对协调这些转变的分子机制的认识中存在几个关键的空白在这些阶段之间。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，并利用这些知识来操纵分化过程，目的在最近的实验中，我们发现了一种名为I-BET 151的小分子，它最初是作为一种人布罗莫结构域抑制剂，对人的细长血流形式有深远的影响，哺乳动物通过将该毒性阶段转化为前循环形式，这在TSE的中肠中普遍存在， tse飞行矢量。重要的是，这种I-BET 151诱导的分化削弱了寄生虫的防御机制，作为抗原变异，因此，使其易受宿主免疫系统的攻击，这可以被利用，治疗目的。事实上，用I-BET 151处理的寄生虫感染小鼠导致了令人印象深刻的结果。存活率与感染未处理锥虫的小鼠相比。在这些原理验证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的作用模式，非洲锥虫中的BET 151，作为I-BET 151的靶点，其负责强表型在锥虫体内还没有发现。在该提案中，我们寻求阐明I-BET 151在以下中的靶点：非洲锥虫使用三个互补的化学蛋白质组学策略。在第一种方法中，我们将通过在来自锥虫细胞裂解物的珠基质上进行亲和捕获来富集I-BET 151相互作用蛋白，通过质谱法进行蛋白质鉴定。为了以更生理相关的方式捕获I-BET 151靶标，条件下，我们将在锥虫细胞中使用原位光交联的第二种方法，并确定修饰蛋白质质谱在第三种方法中，I-BET 151的泛素化和蛋白酶体降解是一种新的方法。将使用称为PROteolysis Targeting的双功能小分子诱导相互作用蛋白质嵌合体或PROTAC，其中I-BET 151与E3泛素连接酶募集配体缀合。总的来说，我们提出的研究将通过鉴定与锥虫生物学有关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生命周期阶段调控的研究。寄生虫在世界范围内造成毁灭性的死亡率和发病率。最后，拟议的实验将奠定药物诱导分化治疗的基础，这有可能成为一种新的变革策略控制和杀死致病寄生虫。