IDENTIFICATION OF THE ANTIMALARIAL TARGET OF PEPSTATIN ESTERS

描述(由申请人提供)：疟疾控制方面的进展受到抗药性寄生虫传播的威胁。迫切需要新的抗疟疾化疗，更根本的是，需要新的化疗靶点。我们发现，天冬氨酸蛋白酶抑制剂胃抑素在微生物制剂中的活性成分是一种作为前体药物的酯化衍生物。胃抑素酯在培养中显示出抗恶性疟原虫的纳摩尔效力，对己酯的IC50为19 NM。这表明，胃抑素酯的靶点，虽然尚未阐明，但可以下药。识别蜂窝电话 这些化合物的靶标，我们有困难地选择抗药性突变寄生虫。突变体中有一种功能未知的小蛋白发生了变化，我们称之为标题。我们建议调查标题是否是胃抑素酯的直接靶标。我们将获得标题在胃抑素酯耐药中的作用的Geneti证据，将进行化学生物学研究以确定直接靶标，并将找到标题和直接靶标的相互作用蛋白，如果不同于标题的话。具体地说：1)我们将进行等位基因替换研究，以确定标题对抗性是重要的。标题可以作为PSE目标，作为目标的修饰物或化合物的修饰物。我们将使用野生型和突变型标题等位基因进行过度表达研究，以深入了解标题的作用。我们将评估PSE在野生型和突变型寄生虫中的积累和修饰。2)我们将使用生物素化的、可光交叉连接的胃抑素版本进行下拉研究。这将使我们能够确认TITLE与抑制剂的细胞相互作用，或者如果TITLE不是直接结合蛋白，则识别真正的靶点。我们将利用酯化和非酯化胃抑素的不同结合来确保特异性。3)我们将使用抗标签和抗标题抗体对标题进行标记和下拉。如果标题不是直接目标，则目标2中确定的目标将被类似地标记和删除。如上所述，目标也将被衍生的胃抑素拉下，但洗涤不那么严格，以保持与其他蛋白质的相互作用。我们将对下拉列表进行MS-MS分析，并通过相互免疫沉淀确认相互作用的蛋白质。该实验计划包括一种新颖的并行下拉方法。我们预计这些研究将使我们能够建立PSES的靶点，将对PSE机制产生深刻的见解，并将为我们提供一条有希望的抗疟疾药物开发的新途径。

DESCRIPTION (provided by applicant): Advances in the control of malaria are threatened by the spread of drug-resistant parasites. There is a great need for new antimalarial chemotherapy and more fundamentally, for new chemotherapeutic targets. We have discovered that the active principle in microbial preparation of the aspartic protease inhibitor pepstatin is an esterified derivative that acts as a prodrug. Pepstatin esters show nanomolar potency against Plasmodium falciparum in culture, with an IC50 for the hexyl ester of 19 nM. This suggests that the target of pepstatin esters, though yet to be elucidated, is druggable. To identify the cellular target of these compounds we have, with difficulty, selected resistant mutant parasites. The mutants have alterations in a small protein of unknown function that we call TITLE. We propose to investigate whether or not TITLE is the direct target of pepstatin esters. We will obtain geneti evidence for the role of TITLE in resistance to pepstatin esters, will perform chemical biology studies to identify the direct target and will find interacting proteins for TITLE and for the diret target if that is different from TITLE. Specifically: 1) We will perform allelic replacement studie to establish that TITLE is important for resistance. TITLE could be acting as the PSE target, as a modifier of the target or as a modifier of the compound. We will perform overexpression studies using wild-type and mutant TITLE alleles to gain insight into TITLE's role. We will assess PSE accumulation and modification in wild-type and mutant parasites. 2) We will perform pull-down studies using a biotinylated, photo-cross linkable version of pepstatin. This will enable us to confirm cellular interaction of TITLE with inhibitor, or to identify the real target if TITLE isnot the direct binding protein. We will take advantage of differential binding of esterified vs. non-esterified pepstatin to ensure specificity. 3) We will tag and pull down TITLE using anti-tag and anti-TITLE antibodies. If TITLE is not the direct target, the target identified in aim 2 will be similarly tagged and pulled down. The target will also be pulled down with derivatized pepstatin as above but washed less stringently to preserve interactions with other proteins. We will perform MS-MS analysis on the pull-downs and will confirm interacting proteins by reciprocal immunoprecipitation. The experimental plan comprises a novel parallel pull-down approach. We anticipate that these studies will allow us to establish the target of PSEs, will yield insights ino PSE mechanism and will provide us with a promising new avenue for antimalarial drug development.