Tackling antibiotic resistance through blocking of signalling pathways

随着我们接近“后抗生素时代”，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。这项工作建议研究一种新的方法：开发一种穿透细胞的“抗耐药肽”，它针对细菌信号，可以与现有的抗生素一起使用。主要靶标BceS是来自枯草杆菌的组氨酸激酶，它构成了使芽孢杆菌能够在抗生素杆菌肽存在的情况下做出反应的信号通路的一部分。BceS以二聚体的形式存在，该项目的目标是产生一种多肽拮抗剂，它可以扰乱二聚化，阻断这一途径上的信号，从而使枯草杆菌对杆菌肽敏感。现有的多肽库筛选方法将在此背景下应用，任何命中都将被合成、纯化并表征它们与靶标的相互作用。这项工作的目标是在模型生物体中众所周知的信号通路，目的是提供概念验证和翻译研究。因此，如果这一新方法成功，它可以应用于类似的组氨酸激酶，这些酶构成了在更多临床相关病原体中发现的信号通路的一部分。多肽最适合于靶向蛋白质-蛋白质相互作用，如BceS二聚化。组氨酸激酶的保守性导致了在使用小分子方法时的特异性问题，同时对于较大的生物制品也是不可获得的。因此，“抗药性多肽”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。资金由生物技术和生物科学研究理事会(BBSRC)提供。BBSRC支持对生物系统开发的研究，特别是在对健康的综合理解方面。他们认识到，这样的研究将在老龄化社会中保持更长时间的健康方面发挥关键作用。如上所述，将使用建立的细胞内、基于大肠杆菌的蛋白质片段互补分析来筛选针对BceS的肽拮抗剂的半理性设计的文库。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。采用固相合成法合成HITS，并用高效液相色谱法进行纯化。利用圆二色谱(CD)、等温滴定量热法(ITC)、交联和十二烷基硫酸钠-PAGE(SDS-PAGE)分析以及分析尺寸排阻色谱(ASEC)等分析技术，将在体外表征它们与目标的相互作用。然后，将使用抗生素敏感性和基于发光的信号分析来评估最有希望的粘合剂在芽孢杆菌中的有效性。最后，将在必要时修改命中序列以传递细胞穿透性，以期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.