Mechanism-based targeting of bacterial topoisomerases

基于机制的细菌拓扑异构酶靶向

基本信息

批准号：
9243114
负责人：
Nancy Maizels
金额：
$ 21.5万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-20 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9243114
关键词：
Address Affect Antibiotics Bacteria Bacterial DNA Topoisomerase I Bacterial Infections Biological Assay Cell Death Cells Ciprofloxacin Collection Communicable Diseases DNA DNA Adducts DNA Damage DNA Gyrase DNA Topoisomerases Detection Disease Drug Targeting Epitopes Escherichia coli Etoposide Evaluation Family Fluoroquinolones Funding Genetic Genus Mycobacterium Goals Growth Human Immunoassay Lead Libraries Measures Metabolism Mycobacterium smegmatis Mycobacterium tuberculosis Pharmaceutical Preparations Pharmacotherapy Poison Proteins Recovery Running Technology Testing Topoisomerase Topoisomerase II Topotecan Tuberculosis Validation base cancer cell cell killing chemotherapeutic agent cytotoxic cytotoxicity design drug discovery experimental study high throughput screening improved in vivo inhibitor/antagonist killings new therapeutic target novel therapeutics pathogen response screening success tool tuberculosis treatment

项目摘要

Bacterial pathogens cause devastating diseases, and it is imperative to improve our ability to treat bacterial infections. Among the most potent drugs in current use are the fluoroquinolone antibiotics, which trap the normally transient covalent intermediate formed by DNA gyrase with DNA, to create local DNA damage that is cytotoxic. In addition to gyrase, which is a type 2A topoisomerase, most bacteria also contain a structurally distinct type 1A topoisomerase, Topoisomerase 1 (Topo I). Like DNA gyrase, Topo I forms a transient covalent DNA adduct and should in principle be an excellent drug target, but no drugs currently target Topo I. Discovery and optimization of drugs that target Topo I and other topoisomerases has been limited by the lack of a mechanism-based assay for induction of topoisomerase-DNA adducts. We recently developed an assay, the RADAR assay (Rapid Assay of the DNA Adduct Response), that quantifies the covalent topoisomerase-DNA adducts formed as signature DNA damage in cells treated with topoisomerase poisons. We hypothesize that the RADAR assay will be useful for identifying new drugs that function by this mechanism, and for quantifying the potency of topoisomerase poisons in current use. The goal of this application is to validate the RADAR assay for screening compound libraries for drugs that poison bacterial topoisomerases. To achieve this, we will address current challenges in three aims. We will (1) show that DNA adducts formed by M. tuberculosis Topo I are toxic in mycobacteria and can be detected by the RADAR assay; (2) adapt and optimize the RADAR assay for high throughput screening; and (3) validate the RADAR assay for drug discovery by screening the TB Alliance TB Active Collection library. Impact: Validation of the RADAR assay will enable its application to discovery of new mechanism- based drugs to treat TB and a wide range of other infectious diseases.

细菌病原体会引起毁灭性的疾病，提高我们治疗细菌感染的能力势在必行。目前使用的最有效的药物是氟喹诺酮类抗生素，它捕获 DNA 旋转酶与 DNA 形成的通常短暂的共价中间体，从而产生具有细胞毒性的局部 DNA 损伤。除了旋转酶（2A 型拓扑异构酶）之外，大多数细菌还含有结构不同的 1A 型拓扑异构酶，即拓扑异构酶 1 (Topo I)。与 DNA 旋转酶一样，Topo I 形成短暂的共价 DNA 加合物，原则上应该是一个极好的药物靶标，但目前没有药物靶向 Topo I。针对 Topo I 和其他拓扑异构酶的药物的发现和优化一直受到缺乏基于机制的拓扑异构酶-DNA 加合物诱导测定。我们最近开发了一种测定法，即 RADAR 测定法（DNA 加合物反应快速测定法），该测定法可对拓扑异构酶毒物处理的细胞中作为标志性 DNA 损伤形成的共价拓扑异构酶-DNA 加合物进行定量。我们假设 RADAR 检测将有助于识别通过这种机制发挥作用的新药，并有助于量化当前使用的拓扑异构酶毒物的效力。此应用的目标是验证 RADAR 测定法，用于筛选毒害细菌拓扑异构酶的药物的化合物库。为了实现这一目标，我们将通过三个目标来应对当前的挑战。我们将 (1) 证明结核分枝杆菌 Topo I 形成的 DNA 加合物在分枝杆菌中具有毒性，并且可以通过 RADAR 测定进行检测； (2) 调整和优化 RADAR 测定以进行高通量筛选； (3) 通过筛选 TB Alliance TB Active Collection 库来验证用于药物发现的 RADAR 测定。影响：RADAR 检测的验证将使其能够应用于发现新的基于机制的药物，以治疗结核病和多种其他传染病。