Tackling antibiotic resistance through blocking of signalling pathways

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.