Inhibition of the Bacterial LexA Repressor-Protease to Halt SOS Response-Mediated Resistance and Biofilm Formation

项目摘要/摘要 抗生素的过度使用和误用给细菌带来了进化压力，迫使其改变 或绕过药物靶标或以其他方式产生抗药性，导致很大比例的 我们现有的药物和杀虫剂无效。新型抗生素暂时提供了 由于抗药性的快速发展而缓解，尽管几家制药公司 退出这一研究领域。细菌生物膜使许多疾病的治疗更加复杂 细菌感染。这些细胞集团有助于各种健康状况，并 已知会在大多数医疗设备的表面定居。此外，它们还庇护了大量的 持久细胞--不生长且对大多数抗生素耐受的“休眠”细胞。 不幸的是，大多数现有的疗法针对的是暂停在这些区域的代谢过程。 细菌的瞬时亚群。总而言之，我们正面临一场完美的抵抗风暴 容忍有可能导致数百万人死亡，并瓦解我们目前在 过程，除非我们找到根本的解决方案。 为此，我们已经确定了一个潜在的抗生素靶点-细菌SOS反应。 这种对遗传毒性应激的反应在细菌中是保守的，并与 抗性和耐受机制，包括水平基因转移、突变和细胞 组织逮捕。SOS基因的转录受到抑制蛋白LexA的抑制，该酶 切割与丝状蛋白RecA*相互作用，暴露SOS启动子区域。一个 以前的高通量筛选发现了一种有效的LexA裂解抑制剂。我们提出了一个 研究改善这种抑制物，更好地了解其作用和效果。使用初步的 以构效关系研究为指导，我们设计了一个包含22-25个类似物的文库 更深入的搜救行动，包括专门为克服潜在威胁而设计的模拟 外流挑战。此外，我们还提出了带有共价陷阱的多肽片段来模拟 LexA蛋白酶的天然底物，不可逆转地抑制其功能。使用我们最强大的 抑制剂，我们将研究LexA抑制的下游生物学效应，包括 获得性抗生素耐药性和生物被膜形成。我们还计划使用光亲和探测器来 确定抑制物在蛋白质中的结合位置和方向。独特的跨学科 这一提议的方法将阐明这些抑制剂的机制，并将为 为解决抵抗和容忍危机的新战略奠定基础。

Project Summary/Abstract The overuse and misuse of antibiotics has put evolutionary pressure on bacteria to alter or bypass the targets of drugs or otherwise develop resistance, rendering a large percentage of our available medicines and pesticides ineffective. Novel antibiotics have afforded temporary relief due to quick development of resistance, although several pharmaceutical companies have withdrawn from this area of research. Bacterial biofilms further complicate treatment of many bacterial infections. These cell conglomerates contribute to a variety of health conditions and are known to colonize the surfaces of most medical devices. Moreover, they shelter high numbers of persister cells— “dormant” cells which are non-growing and tolerant of most antibiotics. Unfortunately, most existing therapies target metabolic processes which are suspended in these transient subpopulations of bacteria. Altogether we are facing a perfect storm of resistance and tolerance which threatens to kill millions and unravel our current approach to medicine in the process, unless we find a radical solution. To this end, we have identified a potential antibiotic target—the bacterial SOS response. This response to genotoxic stress is conserved across bacteria and has been connected to resistance and tolerance mechanisms, including horizontal gene transfer, mutagenesis, and cell division arrest. Transcription of SOS genes is suppressed by the repressor-protease LexA, which cleaves upon interaction with filamentous protein RecA* to expose the SOS promoter region. A previous high throughput screen identified a potent inhibitor of LexA cleavage. We propose a study to improve this inhibitor and better understand its action and effects. Using a preliminary structure-activity relationship (SAR) study as a guide, we have designed a library of 22-25 analogs for a more in-depth SAR campaign, including analogs specifically designed to overcome potential efflux challenges. Additionally, we have proposed peptide fragments with covalent traps to mimic the native substrate of the LexA protease and irreversibly inhibit its function. Using our most potent inhibitors, we will investigate the downstream biological effects of LexA inhibition, including acquired antibiotic resistance and biofilm formation. We also plan to use photoaffinity probes to identify the inhibitor binding site and orientation within the protein. The uniquely interdisciplinary approach of this proposal will elucidate the mechanism of these inhibitors and will lay the groundwork for a novel strategy to address the resistance and tolerance crisis.