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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：
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
项目类别：
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

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/文摘