Identification of essential kinases in Leishmania

摘要 有20多种利什曼原虫属的病原是利什曼病的病原体。 每种疾病都有不同的症状，从轻微的皮损到严重的皮损 内脏利什曼病，如果不治疗是致命的。利什曼病是通过咬一口静脉血传播的。 沙蝇。在沙蝇体内，寄生虫作为前鞭毛体生长，然后传播给哺乳动物。 在喂食沙蝇媒介后寄主。在哺乳动物体内，细胞被巨噬细胞吞噬。 吞噬小体的酸性环境与寄主温度的升高相结合诱导 分化为无鞭毛体阶段。无鞭毛体不仅存活在巨噬细胞内，而且还会增殖。 利什曼病主要被描述为一种贫困疾病，因为它感染的人主要是贫困人口。 世界各地。目前还没有治疗利什曼病的疫苗，用于治疗利什曼病的药物也有 有毒或过于昂贵的资源是贫乏疾病的受害者，也对一些人产生抗药性 毒品开始成为该领域的一个问题。由于这一点，需要新药来治疗 利什曼病。由于缺乏常规工具，治疗利什曼原虫的药物开发进一步复杂化 例如RNAi和条件性基因敲除。作为一类，蛋白激酶提供了一类优秀的新的 潜在的毒品目标。它们的ATP结合位点虽然在进化上是保守的，但仍然包含足够的多样性 蛋白激酶之间的相互作用，使小分子抑制剂能够被分离到特定的蛋白激酶。完毕 37种药物已被FDA批准用于靶向蛋白激酶。绝大多数的蛋白激酶 耐受ATP结合位点后面的一个大的氨基酸的突变。这种氨基酸被称为 当守门人的残留物变成较小的氨基酸，如甘氨酸或丙氨酸时，会打开 它可以与一类含有大链的ATP类似物结合，这种结合位点被称为碰碰式激酶 抑制剂(BKI)。BKI将选择性地与突变蛋白相互作用，因为其他蛋白激酶都含有 将BKI从ATP结合位点排除在外的更大的守门人残基。这些突变的蛋白质是指 到模拟敏感(AS)。我们建议为一些蛋白激酶产生AS等位基因 利什曼原虫，根据相关病原体布鲁氏锥虫中已知的基本激酶清单，然后 用AS等位基因替换野生型内源基因。然后我们将确定一个可以绑定AS的BKI- 使用体内生长试验或体外结合试验的等位基因。表达AS蛋白激酶的菌株 然后分化成无鞭毛体并感染巨噬细胞。然后我们将确定 通过将BKI添加到感染的巨噬细胞并寻找生长，蛋白激酶的重要性 无鞭毛体的叛逃者。在这个项目完成后，我们将证明所需的技术 审问利什马尼亚的亲属，寻找新的毒品目标。

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.