Mechanism-based targeting of bacterial topoisomerases

细菌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，它能捕获DNA旋转酶与DNA形成的通常短暂的共价中间体，造成局部DNA损伤，具有细胞毒性。除旋转酶是一种2A型拓扑异构酶外，大多数细菌还含有一种结构独特的1A型拓扑异构酶--拓扑异构酶1(Topo I)。与DNA旋转酶一样，Topo I形成一个瞬间的共价DNA加合物，原则上应该是一个很好的药物靶点，但目前还没有针对Topo I的药物。由于缺乏诱导拓扑异构酶-DNA加合物的机制，限制了针对Topo I和其他拓扑异构酶的药物的发现和优化。我们最近开发了一种分析方法，RADAR分析(DNA加合物反应的快速分析)，它可以定量检测在拓扑异构酶毒物处理的细胞中形成的作为标志性DNA损伤的共价拓扑异构酶-DNA加合物。我们假设，雷达分析将有助于识别通过这种机制发挥作用的新药，并用于量化当前使用的拓扑异构酶毒物的效力。这项应用的目标是验证雷达试验用于筛选毒害细菌拓扑异构酶的药物的化合物文库。为了实现这一目标，我们将从三个方面应对当前的挑战。我们将(1)证明结核分枝杆菌Topo I形成的DNA加合物对分枝杆菌有毒性，并可通过雷达检测来检测；(2)调整和优化雷达检测以进行高通量筛选；以及(3)通过筛选结核联盟TB活性集合库来验证雷达检测用于药物发现的有效性。影响：雷达检测的验证将使其能够应用于发现治疗结核病和广泛其他传染病的新的基于机制的药物。

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.