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。我们迫切需要新的抗疟化疗，更重要的是，需要新的化疗靶点。我们已经发现，天冬氨酸蛋白酶抑制剂胃酶抑素的微生物制剂中的活性成分是作为前药的酯化衍生物。胃蛋白酶抑制素酯显示出对培养物中的恶性疟原虫的纳摩尔效力，其中己酯的IC 50为19 nM。这表明胃酶抑素酯的靶点虽然尚未阐明，但却是可药用的。为了识别细胞 以这些化合物为靶标，我们很难选择出具有抗性的突变寄生虫。突变体在一个未知功能的小蛋白质上发生了改变，我们称之为TITLE。我们建议调查是否标题是胃蛋白酶抑制剂酯的直接目标。我们将获得TITLE在胃蛋白酶抑制剂酯抗性中作用的遗传学证据，将进行化学生物学研究以鉴定直接靶标，并将发现TITLE和直接靶标（如果与TITLE不同）的相互作用蛋白。具体而言：1）我们将进行等位基因置换研究，以确定TITLE对耐药性很重要。TITLE可以作为PSE靶标，作为靶标的修饰剂或作为化合物的修饰剂。我们将使用野生型和突变型TITLE等位基因进行过表达研究，以深入了解TITLE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胃酶抑素的光交叉修饰形式进行下拉研究。这将使我们能够证实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，则鉴定真实的靶标。我们将利用酯化与非酯化胃蛋白酶抑制剂的差异结合来确保特异性。3)我们将使用抗标签和抗TITLE抗体标记并下拉TITLE。如果TITLE不是直接目标，则在aim 2中识别的目标将被类似地标记并下拉。靶标也将如上所述用衍生的胃酶抑素拉下，但洗涤不太严格以保留与其他蛋白质的相互作用。我们将对下拉进行MS-MS分析，并通过相互免疫沉淀法确认相互作用的蛋白质。实验计划包括一种新的并行下拉方法。我们期望这些研究将使我们能够建立PSE的靶点，将产生对PSE机制的见解，并将为抗疟药物开发提供有希望的新途径。

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.