IDENTIFICATION OF THE ANTIMALARIAL TARGET OF PEPSTATIN ESTERS

胃酶抑素酯抗疟靶点的鉴定

• 批准号: 8852545
• 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/8852545
• 关键词: Alleles; Antibodies; Antimalarials; Aspartic Endopeptidases; Binding; Binding Proteins; Biochemical; Biology; Chemicals; Country; Data; Development; Disease; Drug Targeting; Drug resistance; Ensure; Esters; Family; Genetic; Genetic screening method; Health; Immunoprecipitation; Inhibitory Concentration 50; Malaria; Modification; Mutation; Parasites; Pepstatins; Plasmodium falciparum; Preparation; Prodrugs; Protease Inhibitor; Proteins; Resistance; Rodent Model; Role; Specificity; base; cellular targeting; chemotherapy; drug development; inhibitor/antagonist; insight; interest; killings; microbial; mutant; novel; overexpression; pepstatin

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）：疟疾控制的进展受到耐药寄生虫传播的威胁。迫切需要新的抗疟化疗，更重要的是，迫切需要新的化疗靶点。我们已经发现，在微生物制备的天冬氨酸蛋白酶抑制剂胃抑素的活性原理是作为前药的酯化衍生物。抑菌素酯在培养中对恶性疟原虫表现出纳米级的抑制作用，其己基酯的IC50值为19 nM。这表明，胃抑素酯的靶点，虽然尚未阐明，是可药物的。来识别细胞