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

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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.