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旋合酶与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。除了回转酶是一种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回转酶一样，Topo I形成一种瞬时共价DNA加合物，原则上应该是一个很好的药物靶点，但目前还没有药物靶向Topo I。由于缺乏基于机制的诱导拓扑异构酶-DNA加合物的试验，靶向Topo I和其他拓扑异构酶的药物的发现和优化受到限制。我们最近开发了一种检测，RADAR检测（DNA加合物反应快速检测），该检测量化了共价拓扑异构酶-DNA加合物，这些加合物是在拓扑异构酶中毒处理的细胞中作为特征DNA损伤形成的。我们假设RADAR分析将有助于识别通过这种机制起作用的新药，并用于量化目前使用的拓扑异构酶毒药的效力。本应用程序的目的是验证雷达测定法筛选毒化细菌拓扑异构酶的化合物文库。为实现这一目标，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加合物在分枝杆菌中是有毒的，可以通过RADAR试验检测到；(2)调整和优化RADAR法，以实现高通量筛选；(3)通过筛选TB Alliance TB Active Collection文库，验证RADAR方法在药物发现中的应用。影响：RADAR试验的验证将使其能够应用于发现治疗结核病和各种其他传染病的基于新机制的药物。

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.