Mechanism-based targeting of bacterial topoisomerases

细菌性病原体会导致毁灭性的疾病，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，它捕获由DNA促旋酶与DNA形成的通常短暂的共价中间体，以产生具有细胞毒性的局部DNA损伤。除了作为2A型拓扑异构酶的促旋酶之外，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）。与DNA促旋酶一样，Topo I形成瞬时共价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。靶向拓扑异构酶I和其他拓扑异构酶的药物的发现和优化受到缺乏用于诱导拓扑异构酶-DNA加合物的基于机制的测定的限制。我们最近开发了一种测定法，RADAR测定法（DNA加合物反应的快速测定法），该测定法定量在用拓扑异构酶毒物处理的细胞中形成的作为特征DNA损伤的共价拓扑异构酶-DNA加合物。我们假设，RADAR检测将是有用的，用于确定新的药物，通过这种机制的功能，并在目前使用的拓扑异构酶毒药的效力进行定量。本申请的目的是验证用于筛选化合物库的毒性细菌拓扑异构酶的药物的RADAR测定。为了实现这一目标，我们将在三个目标中应对当前的挑战。我们将（1）证明M.结核Topo I在分枝杆菌中是有毒的，并且可以通过RADAR测定来检测;（2）调整和优化RADAR测定用于高通量筛选;以及（3）通过筛选TB Alliance TB Active Collection文库来验证RADAR测定用于药物发现。影响：RADAR分析的验证将使其能够应用于发现新的基于机制的药物来治疗结核病和广泛的其他传染病。

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.