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

项目总结/摘要 抗生素的过度使用和滥用给细菌带来了进化压力， 或绕过药物靶点或以其他方式产生耐药性，使大部分 我们现有的药物和杀虫剂无效。新型抗生素提供了暂时的 缓解由于快速发展的阻力，虽然几家制药公司已经 退出这一研究领域。细菌生物膜进一步使许多细菌的治疗复杂化。 细菌感染这些细胞聚集体导致各种健康状况， 已知会在大多数医疗器械的表面定居。此外，它们还庇护着大量的 持续存在的细胞-“休眠”细胞，其不生长并且耐受大多数抗生素。 不幸的是，大多数现有的治疗靶向代谢过程，这些代谢过程在这些细胞中暂停。 短暂的细菌亚群。总之，我们正面临着一场完美的抵抗风暴， 这可能会导致数百万人死亡，并破坏我们目前的医学方法， 除非我们找到一个彻底的解决方案。 为此，我们已经确定了一个潜在的抗生素目标-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，并且与以下因素有关： 抗性和耐受机制，包括水平基因转移、诱变和细胞 组织逮捕SOS基因的转录被阻遏蛋白酶莱克萨抑制， 在与丝状蛋白RecA* 相互作用时切割以暴露SOS启动子区。一 先前的高通量筛选鉴定了莱克萨裂解的有效抑制剂。我们提出了一个 研究改进这种抑制剂，更好地了解其作用和效果。使用初步 构效关系（SAR）研究为指导，我们设计了一个22-25个类似物库 进行更深入的SAR活动，包括专门设计用于克服潜在危险的模拟物。 外排挑战。此外，我们提出了具有共价陷阱的肽片段来模拟 莱克萨蛋白酶的天然底物，并不可逆地抑制其功能。利用我们最有效的 抑制剂，我们将研究莱克萨抑制的下游生物学效应，包括 获得性抗生素耐药性和生物膜形成。我们还计划使用光亲和探针， 识别蛋白质内的抑制剂结合位点和方向。独特的跨学科 这一建议的方法将阐明这些抑制剂的机制，并将奠定 为解决抵抗和宽容危机的新战略奠定了基础。

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have afforded temporary relief due to quick development of resistance, although several pharmaceutical companies have withdrawn from this area of research.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.