Bisbiguanide analogues as probes for assessing membrane barrier function in multidrug resistant Gram-negative bacteria.

Aim:
The key aim of this project is to develop a number of bisbiguanide-based membrane active probes to investigate the barrier properties of the Gram-negative membrane. The overarching aim is to identify chemical features of small molecules that enable them to penetrate and accumulate inside Gram-negative bacteria.
Remit:
This project aligns with the BBSRC "Bioscience for Health" theme and addresses BBSRC priority "Combatting Antimicrobial Resistance" (specifically "underpinning the development of novel antimicrobials and alternatives to antimicrobials"). This project focuses on mechanistic understanding of "effects and interactions of drugs with cells", "understanding the mechanisms of drug resistance, prevention and combating drug resistance" and inlcludes "research that underpins the development of strategies to mitigate the effects of antimicrobial resistance", "technology development for replacing animal use in drug testing". As such the project is well aligned with BBSRC remit.
Background, Rational and Importance:
The discovery of new antibiotics effective against Gram-negative bacteria is a major challenge due to the permeability barrier of two-membrane cell envelope of Gram-negative bacteria and limited chemical diversity of compound libraries to probe this barrier. The expansion of the chemical space of antibacterial tool compounds and identification of compounds that can penetrate the Gram-negative membrane barrier is urgent to understand the chemical features of compounds that are able to overcome the Gram-negative membrane barrier. The industrial applicants have previously been involved in screening of the Prestwick Chemical Library, commissioned by ANTRUK, and identified a small limited number of compounds which either showed direct antimicrobial effects against MDR Gram-negative pathogens or which were able to synergise with specific antibiotics to potentiate their activity against resistant isolates (Hind et al 2019). Two related compounds, chlorhexidine and alexidine, showed high levels of potentiation against all of the strains tested. These bisbiguanides are well characterised antiseptic agents used in a wide range of clinical settings, but the scaffold has not been rigourously explored to understand whether derivatives have separable membrane permeabilising activity and antibacterial properites.
Bisbiguanides offer an excellent chemical scaffold for probe development due to their ability to interact with Gram-negative membranes. We will use these probes to improve the understanding of the molecular basis for low-permeability barriers of the problematic Gram-negative pathogens and define the physicochemical properties that enable uptake of various compounds into bacterial cells using probes synthesied as part of this PhD project. Coupled with other methods for studying membrane perturbation, including whole genome sequencing of ESKAPE mutants generated by serial passage (where resistance emerges), and strain libraries with known modifications in membrane structure/function, will provide a powerful new basis for understanding uptake, and the ability of compounds to penetrate or disrupt bacterial membranes.
The study will also look at the properties of membrane active compounds like bisbiguanides which restrict their potential use for systemic therapy; specifically their eukaryotic cell toxicity. The project will extend the studies exploring interaction of biguanides with the bacterial membrane to include an assessment of their activity in mammalian membranes, to further extend our understanding of their structure activity relationship.