Identification of essential kinases in Leishmania

利什曼原虫必需激酶的鉴定

• 批准号: 9974477
• 负责人: BRYAN C JENSEN
• 金额: $ 27.9万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-09 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9974477
• 关键词: African Trypanosomiasis; Alanine; Alleles; Amino Acids; Back; Binding; Binding Sites; Biological Assay; Bite; CRISPR/Cas technology; Case Study; Cell Cycle; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Chagas Disease; Cutaneous; Cytokinesis; Defect; Development; Disease; Double-Stranded RNA; Drug Targeting; Drug resistance; Drug usage; Environment; Essential Genes; Event; Flow Cytometry; Gatekeeping; Generations; Genes; Glycine; Growth; Human; Hydrophobicity; In Vitro; Insecta; Left; Leishmania; Leishmania donovani; Leishmania infantum; Leishmaniasis; Lesion; Malignant Neoplasms; Mammalian Cell; Medical; Microscopy; Mining; Mitosis; Mutate; Mutation; Organism; Parasites; Pathogenicity; Phagolysosome; Phagosomes; Pharmaceutical Preparations; Phenotype; Phlebotominae; Phosphotransferases; Poverty; Proliferating; Protein Analysis; Protein Inhibition; Protein Kinase; Proteins; RNA Interference; RNA interference screen; Resources; S Phase; Side; Site; Symptoms; System; Technology; Temperature; Testing; Trypanosoma brucei brucei; Trypanosoma cruzi; Vaccines; Valine; Visceral Leishmaniasis; Work; World Health Organization; analog; base; cell growth; conditional knockout; drug development; experimental study; feeding; genetic manipulation; human pathogen; in vivo; inhibitor/antagonist; kinase inhibitor; knock-down; knockout gene; macrophage; mutant; neglect; new therapeutic target; novel therapeutics; pathogen; small molecule inhibitor; tool; transmission process; vector

ABSTRACT There are over 20 pathogenic species of the genus Leishmania that are the causative agent for leishmaniasis. With each species causing a disease with different symptoms ranging from mild to severe cutaneous lesions to visceral leishmaniasis that is fatal if left untreated. Leishmania is spread by the bite of the phlebotomine sandfly. Inside the sandfly the parasite grows as promastigotes that are then transmitted to the mammalian host upon feeding of the sandfly vector. Inside the mammalian host the cells are engulfed by macrophages. The acidic environment of the phagosome combined with the increased temperature of the host induce differentiation to the amastigote stage. Amastigotes not only survive within the macrophage but proliferate. Leishmaniasis is mainly described as a disease of poverty since it infects people in mainly impoverished regions of the world. There are no vaccines available for Leishmania and the drugs used to treat it are either toxic or are too expensive for the resource poor victims of the disease, also drug resistance to some of the drugs is starting to be a problem in the field. Due to this, new drugs are needed for the treatment of leishmaniasis. Drug development against Leishmania is further complicated by the lack of conventional tools such as RNAi and condition gene knockouts. As a class, protein kinases provide an excellent class of new potential drug targets. Their ATP-binding site, while evolutionarily conserved, still contain enough diversity between protein kinases such that small molecule inhibitors can be isolated to a specific protein kinase. Over 37 drugs have been approved for use by the FDA targeting protein kinase. A vast majority of protein kinases tolerate mutation of a bulky amino acid at the back of the ATP-binding site. This amino acid referred to as the gatekeeper residue when changed to a smaller amino acid such as glycine or alanine opens up the back of the ATP-binding site such that it can bind a class of ATP-analogs containing large chains called bumped kinase inhibitors (BKIs). BKIs will selectively interact with the mutant protein since the other protein kinases all contain larger gatekeeper residues that exclude the BKI from the ATP binding site. These mutant proteins are referred to as being analog sensitive (AS). We propose generating AS-alleles for a number protein kinase in Leishmania, based on a list of known essential kinases in the related pathogen Trypanosoma brucei, and then replacing the wild-type endogenous genes with the AS-alleles. We will then identify a BKI that can bind the AS- allele using either an in vivo growth assay or an in vitro binding assay. Strains expressing AS-protein kinases will then be differentiated into amastigotes and infected into macrophages. We will then determine the essentiality of the protein kinase by adding the BKI to the infected macrophages and looking for growth defected of the amastigotes. Upon completion of this project we will have proven the technologies required to interrogate the kinome of Leishmania for new drug targets.

抽象的 利什曼原虫属的20多种致病性物种是利什曼病的致病药物。 每种物种都会引起患有不同症状的疾病，从轻度到重度皮肤病变到 如果未治疗，内脏利什曼病是致命的。利什曼原虫被静脉鼻子斑传播 蛉。在沙蝇内，寄生虫随后传播到哺乳动物时生长 饲养沙蝇载体后的主机。在哺乳动物宿主内部，细胞被巨噬细胞吞没。 吞噬体的酸性环境结合宿主的温度升高 区分与amastigote阶段。混合物不仅在巨噬细胞中生存，而且在增殖中生存。 利什曼病主要被描述为一种贫困疾病，因为它主要感染了贫困的人 世界地区。 Leishmania没有可用的疫苗，用于治疗的药物是 有毒或对于这种疾病的资源贫困受害者来说太昂贵了，也对某些疾病的抗药性 药物开始成为该领域的问题。因此，需要新药来治疗 利什曼病。由于缺乏常规工具，针对利什曼尼亚的药物开发更加复杂 例如RNAi和条件基因敲除。作为一堂课，蛋白质激酶提供了一类出色的新课程 潜在的药物靶标。他们的ATP结合站点虽然在进化上保存，但仍然包含足够的多样性 在蛋白激酶之间，可以将小分子抑制剂分离为特定的蛋白激酶。超过 37种药物已被FDA靶向蛋白激酶使用。绝大多数蛋白激酶 在ATP结合位点的背面耐受笨重的氨基酸突变。这个氨基酸称为 当更改为较小的氨基酸（例如甘氨酸或丙氨酸）时，守门人残留物会打开后面 ATP结合位点，以便它可以结合一类ATP-Analogs，这些ATP - Analogs包含称为颠簸激酶的大链 抑制剂（BKIS）。 BKIS将选择性地与突变蛋白相互作用，因为其他蛋白激酶都包含 将BKI排除在ATP结合位点中的较大的网务残基。这些突变蛋白被引用 作为模拟敏感（AS）。我们建议在数量蛋白激酶中生成AS-learleS 利什玛尼亚，基于相关病原体锥虫瘤的已知必需激酶，然后 用AS-Challe代替野生型内源基因。然后，我们将确定一个可以绑定AS-的BKI 使用体内生长测定法或体外结合测定法。表达蛋白激酶的菌株 然后将分化为amastigotes，并将其感染成巨噬细胞。然后，我们将确定 通过将BKI添加到感染的巨噬细胞中并寻找生长，蛋白激酶的重要性 amastigotes缺陷。该项目完成后，我们将证明所需的技术 询问利什曼尼亚的Kinome针对新药靶标的。