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.

细菌病原体引起毁灭性疾病，必须提高我们治疗细菌感染的能力。当前使用中最有效的药物之一是氟喹诺酮抗生素，该抗生素会捕获DNA Gyse与DNA形成的正常瞬时共价中间体，以产生细胞毒性的局部DNA损伤。除Gyrase是2A型拓扑异构酶外，大多数细菌还含有结构上不同的1A型拓扑异构酶，拓扑异构酶1（Topo I）。像DNA回旋酶一样，TOPO I形成了瞬态共价DNA加合物，原则上应该是一个极好的药物靶标，但目前没有药物针对的是TopoI I.靶向Topo I和其他topo i和其他topoisomerases的药物的发现和优化受到基于机制的基于机制的测定的限制，无法诱导topoisomerase-DNA加入。我们最近开发了一种测定法，雷达测定法（DNA加合反应的快速测定），该测定法量化了用拓扑异构酶毒药处理的细胞中形成的共价拓扑异构酶-DNA加合物。我们假设雷达测定法对于鉴定通过该机制起作用的新药物和量化拓扑异构酶毒药的效力将很有用。该应用的目的是验证雷达分析的筛查化合物文库的毒药中毒性细菌拓扑异构酶的药物。为了实现这一目标，我们将解决三个目标的当前挑战。我们将（1）表明，结核分枝杆菌I形成的DNA加合物在分枝杆菌中有毒，可以通过雷达测定法检测。（2）适应并优化雷达测定法以进行高吞吐量筛选；（3）通过筛选结核病联盟结核病收集库来验证雷达测定法以发现药物发现。影响：对雷达测定法的验证将使其应用于发现新机制的药物以治疗结核病和多种其他传染病。