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

点击翻译按钮获取中文摘要

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.