Mechanism of Action and Initial, In Vitro SAR of an Inhibitor of the Shigella flexneri Virulence Regulator VirF.
Mechanism of Action and Initial, In Vitro SAR of an Inhibitor of the Shigella flexneri Virulence Regulator VirF.
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DOI：
10.1371/journal.pone.0137410
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Garcia GA
中科院分区：
文献类型：
--
作者：
Emanuele AA;Garcia GA
志贺菌是急性腹泻病的主要病原体之一，每年在全世界夺走100多万人的生命。有多种细菌基因控制志贺菌的致病性，但virF基因可能是最重要的。该基因位于志贺菌的主要致病岛上，编码VirF，一种AraC家族转录激活因子，负责启动志贺菌的致病循环。我们以前已经表明，它是可能的，通过小分子抑制VirF的福氏志贺菌的毒力减弱。在这项研究中，我们探讨了我们的VirF小分子抑制剂的作用机制。为了使这些研究，我们已经开发了一个同源的和有效的表达和纯化系统的VirF，并优化了两种不同的体外VirF-DNA结合试验。我们已经确定，我们的HTS命中化合物之一抑制VirF与DNA的结合，计算的Ki与我们的转录激活和毒力筛选中看到的有效剂量相似。这与作为该命中化合物的作用机制的DNA结合抑制一致。我们还筛选了该化合物的15种商业来源的类似物，并从约100倍的活性范围推断出初始SAR。我们的其他四种HTS命中化合物不抑制DNA结合，但它们确实阻断VirF活性。这表明可以开发具有不同分子机制的VirF抑制剂。采用不同的作用机制来追求命中可能是增强活性并避免可能对任何一种药物产生耐药性的有力方法。
Shigella spp. are among the main causative agents of acute diarrheal illness and claim more than 1 million lives per year worldwide. There are multiple bacterial genes that control the pathogenesis of Shigella, but the virF gene may be the most important. This gene, located on the primary pathogenicity island of Shigella, encodes VirF, an AraC-family transcriptional activator that is responsible for initiating the pathogenesis cycle in Shigella. We have previously shown that it is possible to attenuate the virulence of Shigella flexneri via small molecule inhibition of VirF. In this study, we probed the mechanism of action of our small molecule inhibitors of VirF. To enable these studies, we have developed a homologous and efficient expression and purification system for VirF and have optimized two different in vitro VirF-DNA binding assays. We have determined that one of our HTS hit compounds inhibits VirF binding to DNA with a calculated K i similar to the effective doses seen in our transcriptional activation and virulence screens. This is consistent with inhibition of DNA binding as the mechanism of action of this hit compound. We have also screened 15 commercially sourced analogs of this compound and deduced an initial SAR from the approximately 100-fold range in activities. Our four other HTS hit compounds do not inhibit DNA binding and yet they do block VirF activity. This suggests that multiple agents with different molecular mechanisms of inhibition of VirF could be developed. Pursuing hits with different mechanisms of action could be a powerful approach to enhance activity and to circumvent resistance that could develop to any one of these agents.