Tackling antibiotic resistance through blocking of signalling pathways

通过阻断信号通路应对抗生素耐药性

• 批准号: 2749192
• 金额: --
• 依托单位: University of Bath
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2749192
• 关键词: Tackling; antibiotic; resistance; through; blocking

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.

随着我们接近“后抗生素时代”，对新疗法的需求比以往任何时候都更大。目前正在进行临床试验的大多数潜在候选药物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：开发一种细胞穿透性的“抗耐药肽”，其靶向细菌信号传导，可与现有的抗生素结合使用。主要靶标BceS是来自枯草芽孢杆菌的组氨酸激酶，其形成使芽孢杆菌能够在抗生素杆菌肽存在下作出反应的信号通路的一部分。BceS以二聚体形式存在，该项目的目的是产生一种肽拮抗剂，其破坏二聚化，阻断沿着该途径的信号传导，从而产生B。枯草杆菌对杆菌肽敏感。现有的肽库筛选方法将应用于此背景下，任何命中将被合成，纯化和他们的相互作用与目标characterized.This工作的目标是一个很好理解的信号通路在一个模式生物的目的，提供概念验证，转化研究。因此，如果这种新的方法是成功的，它可以应用于类似的组氨酸激酶，形成在临床上更相关的病原体中发现的信号通路的一部分。肽最适合靶向蛋白质-蛋白质相互作用，如BceS二聚化。组氨酸激酶的保守性质导致当使用小分子方法时关于特异性的问题，同时对于较大的生物制剂也不可接近。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。资金由生物技术和生物科学研究理事会提供。BBSRC支持对生物系统开发的研究，特别是关于健康的综合理解。他们认识到，像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。基于大肠杆菌的蛋白片段互补分析将用于筛选针对BceS的肽拮抗剂的半合理设计的文库。这将涉及单步选择和竞争性液体培养，直到获得单个获胜序列。命中将通过固相肽合成法合成，并通过HPLC纯化。将使用分析技术（如圆二色谱（CD）、等温滴定量热法（ITC）、交联和SDS-PAGE分析以及分析型尺寸排阻色谱法（aSEC））在体外表征其与靶标的相互作用。最有前途的粘合剂的效力，然后将在芽孢杆菌中使用抗生素敏感性和发光为基础的信号分析进行评估。最后，在必要时将修改命中序列以赋予细胞转染率，以研究潜在的递送方法。