Exploiting quorum sensing inhibition of the natural products fimbrolide and elegaphenone in gram-negative bacteria

多重耐药菌株引起的细菌感染对公共卫生构成重大威胁。特别是革兰氏阴性菌株表现出几乎不可逾越的细胞膜，对治疗具有挑战性。因此，研究基本的细胞机制是一项重要的任务，以确定新的治疗方法。在这里，细菌之间的细胞通讯，称为群体感应（QS），在发病机制的调节中起关键作用。在我们之前的工作中，我们研究了发光模式生物Vibrio harveyi中的飞膜苷天然产物，并确定了QS通路的中心调节因子LuxS作为它们的细胞靶点。在本项目的第一部分，我们希望扩大范围，重点研究LuxS与第二代抑制剂对致病性革兰氏阴性鼠伤寒沙门氏菌的抑制作用。针对这一策略，我们采用鼠伤寒沙门氏菌和哈维氏菌LuxS与金膜苷共结晶的方法，阐明其抑制机制，合理设计改进的结合剂。基于这些结果，将合成不同的膜溴化物类似物，并在已建立的LuxS分析中进行测试。此外，将通过制备相应的探针和随后的化学蛋白质组学研究来分析LuxS在整个鼠伤寒沙门氏菌细胞中最有效的化合物的选择性。有了一种有效的特异性LuxS抑制剂，我们不仅将测试其对鼠伤寒沙门氏菌发病机制的影响，还将阐明其机制之谜。虽然AI-2是鼠伤寒沙门氏菌中唯一的综合QS回路，但其对致病性因子的相应调控至今尚不明确。因此，我们的目标是通过新一代LuxS抑制剂的全蛋白质组分析来研究AI-2在鼠伤寒沙门氏菌发病机制中的作用。项目的第二部分是致力于从秀丽丝桃中分离的天然产物秀丽丝桃酮。该化合物以其对革兰氏阳性菌株的抗菌活性而闻名。然而，在试点实验中，我们令人惊讶地发现，它抑制qs依赖的毒力铜绿假单胞菌，一种具有高度临床相关性的破坏性病原体。由于这种生物活性指向一个有前景的治疗靶点，我们建议合成相应的探针，揭示其细胞机制，并进一步剖析其在QS中的潜在作用。此外，在SAR研究的基础上，将合成新的QS抑制剂，并测试其对革兰氏阴性菌的抗菌活性。综上所述，本项目旨在以天然产物QS抑制剂为切入点，进行合成优化，深入机理研究和开发具有抗菌效力的新型抑制剂，重点关注具有挑战性的革兰氏阴性菌株。

Bacterial infections caused by multiresistant strains pose a major threat to public health. Especially gram-negative strains exhibiting an almost insuperable cell membrane are challenging for treatment. Thus, the study of essential cellular mechanisms is an important task in order to identify novel therapeutic approaches. Here, the cellular communication between bacteria, termed quorum sensing (QS), plays a key role in the regulation of pathogenesis. In our previous work, we investigated fimbrolide natural products in the luminescent model organism Vibrio harveyi and identified LuxS, a central regulator of the QS pathway, as their cellular target. In the first part of this project, we would like to expand the scope and focus on inhibition of LuxS with second generation inhibitors in pathogenic gram-negative S. typhimurium. For this strategy, we employ cocrystallization of S. typhimurium and V. harveyi LuxS with fimbrolide to elucidate its inhibition mechanism and rationally design improved binders. Based on these results, diverse fimbrolide analogs will be synthesized and tested in an established LuxS assay. Furthermore, selectivity of the most potent compound for LuxS in whole S. typhimurium cells will be analyzed via preparation of a corresponding probe and subsequent chemical proteomics studies. With a potent and specific LuxS inhibitor at hand, we will not only test its effect on S. typhimurium pathogenesis but also elucidate a mechanistic puzzle. Although AI-2 is the sole comprehensive QS circuit in S. typhimurium, its corresponding regulation of pathogenicity factors remains elusive so far. Therefore, we aim to investigate the role of AI-2 in S. typhimurium pathogenesis via whole proteome analysis with the new generation of LuxS inhibitors. The second part of the project is dedicated to the natural product elegaphenone isolated of Hypericum elegans. The compound is well-known for its antibacterial activity against gram-positive bacterial strains. However, in pilot experiments we surprisingly identified that it inhibits QS-dependent virulence in Pseudomonas aeruginosa, a devastating pathogen of high clinical relevance. As this bioactivity points towards a promising therapeutic target, we here propose to synthesize a corresponding probe, unravel its cellular mechanism and further dissect its potential role in QS. Moreover, based on SAR studies, new QS inhibitors will be synthesized and tested for their antibacterial activity in gram-negative bacteria.In summary, this project aims to utilize natural product QS inhibitors as a starting point for synthetic optimization, in depth mechanistic studies and development of novel inhibitors with antibacterial potency deliberately focusing on challenging gram-negative strains.