A complementary pair of rapid molecular screening assays for RecA activities
A complementary pair of rapid molecular screening assays for RecA activities
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DOI：
10.1016/j.ab.2007.04.021
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发表时间：
2007-08-15
影响因子：
2.9
通讯作者：
Singleton, Scott F.
中科院分区：
文献类型：
--
作者：
Lee, Andrew M.;Wigle, Tim J.;Singleton, Scott F.
细菌RecA蛋白与病原体抗生素耐药性的演变有关，这是一个全球性的问题。可以选择性调节RecA活性的小分子的发现可以用来梳理它在抗生素耐药基因的从头发展和传播中的作用。为了发现可以阻止活性RecA-DNA丝的组装或其随后的ATP依赖性运动活动的小分子配体的目标，我们报告了一对适用于识别ReCA活性抑制剂的快速和稳健的筛选测定的设计和初步验证。一种测定是基于用于监测ATP酶活性的已建立的方法，第二种是使用荧光偏振的RecA-DNA丝组装的新测定。总之，测定结果揭示了互补的试剂组，其可以选择性地仅抑制RecA-DNA细丝的ATP驱动的运动活性，或者完全阻止活性ReCA-DNA细丝的组装。筛选试验可以很容易地配置用于未来的自动化高通量筛选项目，以发现有效的抑制剂，这些抑制剂可以开发成抗生素化疗的新型佐剂，减缓抗生素耐药基因的发展和传播，并增加抗生素治疗指数。(C)2007年爱思唯尔公司All rights reserved.
The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA's activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either the assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhibitors of ReCA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either suppress selectively only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active ReCA-DNA filaments altogether. The screening assays can be readily configured for use in future automated high-throughput screening projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index. (C) 2007 Elsevier Inc. All rights reserved.