IDENTIFICATION OF THE ANTIMALARIAL TARGET OF PEPSTATIN ESTERS

胃酶抑素酯抗疟靶点的鉴定

• 批准号: 9285725
• 负责人: Daniel E. Goldberg
• 金额: $ 38.13万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9285725
• 关键词: Alleles; Antibodies; Antimalarials; Aspartic Endopeptidases; Binding; Binding Proteins; Biochemical; Biology; Chemicals; Country; Data; Development; Disease; Drug resistance; Ensure; Esters; Family; Genetic; Immunoprecipitation; Malaria; Modification; Mutation; Parasite resistance; Parasites; Pepstatins; Plasmodium falciparum; Preparation; Prodrugs; Protease Inhibitor; Proteins; Resistance; Rodent Model; Role; Specificity; Testing; base; cellular targeting; chemotherapy; drug development; inhaled nitric oxide; inhibitor/antagonist; insight; interest; killings; microbial; mutant; nanomolar; new therapeutic target; novel; novel therapeutics; overexpression; public health relevance

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.

描述（由申请人提供）：疟疾控制方面的进展受到耐药寄生虫传播的威胁。非常需要新的抗疟化疗，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酶抑素的微生物制备中的活性成分是一种作为前药的酯化衍生物。胃酶抑素酯对培养物中的恶性疟原虫表现出纳摩尔级效力，己酯的 IC50 为 19 nM。这表明胃酶抑素酯的靶标虽然尚未阐明，但是可药物化的。识别细胞 针对这些化合物的目标，我们艰难地选择了抗性突变寄生虫。这些突变体在一种功能未知的小蛋白质上发生了改变，我们称之为“TITLE”。我们建议调查 TITLE 是否是胃酶抑素酯的直接目标。我们将获得 TITLE 在胃酶抑素酯抗性中的作用的遗传学证据，将进行化学生物学研究以鉴定直接靶标，并将找到 TITLE 和直接靶标（如果与 TITLE 不同）的相互作用蛋白。具体来说： 1) 我们将进行等位基因替换研究，以确定 TITLE 对于耐药性很重要。 TITLE 可以充当 PSE 目标、目标的修饰符或化合物的修饰符。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。我们将评估野生型和突变型寄生虫中 PSE 的积累和修饰。 2) 我们将使用生物素化、可光交联的胃酶抑素进行下拉研究。这将使我们能够确认 TITLE 与抑制剂的细胞相互作用，或者在 TITLE 不是直接结合蛋白的情况下识别真正的靶标。我们将利用酯化胃酶抑素与非酯化胃酶抑素的差异结合来确保特异性。 3) 我们将使用抗标签和抗 TITLE 抗体来标记并下拉 TITLE。如果 TITLE 不是直接目标，则目标 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化胃酶抑素下拉，但清洗不那么严格，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确定 PSE 的目标，将产生对 PSE 机制的见解，并为我们提供抗疟药物开发的有前景的新途径。