Tackling antibiotic resistance through blocking of signalling pathways

随着我们接近“后抗生素时代”，我们比以往任何时候都更需要新的治疗方法来解决抗菌素耐药性问题。目前正在进行临床试验的大多数潜在候选药物都是基于现有的药物类别或作用机制。这项工作提出了一种新的方法：开发一种穿透细胞的“抗抗性肽”，它可以靶向细菌信号，与现有的抗生素结合使用。主要靶点bce是枯草芽孢杆菌中的一种组氨酸激酶，它构成了芽孢杆菌在抗生素杆菌肽存在下作出反应的信号通路的一部分。bce作为二聚体存在，本项目的目的是产生一种肽拮抗剂，破坏二聚体，阻断沿该途径的信号传导，从而使枯草芽孢杆菌易受杆菌肽的影响。现有的肽库筛选方法将在这种情况下应用，任何命中将被合成，纯化和它们与目标的相互作用进行表征。这项工作的目标是一个很好理解的信号通路，在一个模式生物提供概念验证，转化研究的目的。因此，如果这种新方法成功，它可以应用于类似的组氨酸激酶，这些组氨酸激酶构成了更多临床相关病原体中发现的信号通路的一部分。多肽的最佳定位是靶向蛋白质之间的相互作用，如bce二聚化。组氨酸激酶的保守性导致在使用小分子方法时存在特异性问题，同时也无法用于较大的生物制剂。因此，“抗耐药肽”的开发有可能延长现有抗生素的寿命，使新抗生素的应用更加严格，并有助于抗生素管理工作。资助由生物技术和生物科学研究理事会（BBSRC）提供。BBSRC支持对生物系统的开发利用的研究，特别是对健康的综合理解。他们认识到，这样的研究将在老龄化社会中发挥关键作用，使人们更长寿。如上所述，一个已建立的、基于大肠杆菌的细胞内蛋白片段互补试验将用于筛选半合理设计的针对bce的肽拮抗剂文库。这将包括单步选择和竞争性液体培养，直到获得单一的获胜序列。hit采用固相多肽合成法合成，高效液相色谱法纯化。它们与靶标的相互作用将在体外使用圆二色性（CD）、等温滴定量热法（ITC）、交联和SDS-PAGE分析以及分析尺寸排除色谱（aSEC）等分析技术进行表征。最有希望的结合剂的功效将在芽孢杆菌中使用抗生素敏感性和基于发光的信号分析来评估。最后，将在必要时修改命中序列以赋予细胞外显率，以研究潜在的传递方法。

As we approach the 'post-antibiotic era', the need for new treatments to tackle antimicrobial resistance is greater than ever. The majority of potential candidates currently undergoing clinical trials are based on existing drug classes or mechanisms of action. This work proposes to investigate a novel approach: the development of a cell penetrating, 'anti-resistance peptide' that targets bacterial signalling that could be used in conjunction with an existing antibiotic. The primary target, BceS, is a histidine kinase from the bacterium Bacillus subtilis that forms part of the signalling pathway that enables Bacillus to respond in the presence of the antibiotic bacitracin. BceS exists as a dimer and the aim of this project is to generate a peptide antagonist that disrupts dimerisation, blocks signalling along this pathway and consequently renders B. subtilis vulnerable to bacitracin. An existing peptide library screening methodology will be applied in this context and any hits will be synthesised, purified and their interaction with the target characterised.This work targets a well understood signalling pathway in a model organism with the aim of providing proof-of-concept, translational research. Therefore, if this novel approach is successful, it could be applied to similar histidine kinases that form part of signalling pathways found in more clinically relevant pathogens. Peptides are best placed to target protein-protein interactions such as BceS dimerisation. The conserved nature of histidine kinases leads to problems regarding specificity when using a small molecule approach, whilst also being inaccessible for larger biologics. The development of 'anti-resistance peptides' therefore has the potential to extend the life span of existing antibiotics, enable more stringent application of new antibiotics and aid antibiotic stewardship efforts. Funding has been provided by the Biotechnology and Biological Sciences Research Council (BBSRC). The BBSRC supports research into the exploitation of biological systems, particularly with regards to an integrated understanding of health. They recognise that research such as this will play a key part in staying healthy for longer in an aging society.As stated above, an established, intracellular, E. coli-based protein-fragment complementation assay will be used to screen semi-rationally designed libraries for peptide antagonists targeting BceS. This will involve both single step selection and competitive liquid culture until a single winning sequence is obtained. Hits will be synthesised by solid phase peptide synthesis and purified by HPLC. Their interaction with the target will be characterised in vitro using analytical techniques such as circular dichroism (CD), isothermal titration calorimetry (ITC), crosslinking and SDS-PAGE analysis, and analytical size exclusion chromatography (aSEC). The efficacy of the most promising binders will then be assessed in Bacillus using antibiotic susceptibility and luminescence-based signalling assays. Finally, the hit sequences will be modified where necessary to impart cell penetrance, with a view to investigate potential delivery methods.