The discovery of new antibiotics effective against Gram-negative bacteria is a major challenge due to the permeability barrier of the 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 multidrug resistant 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 rigorously explored to understand whether derivatives have separable membrane permeabilising activity and antibacterial properties.
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.
In this interdisciplinary project the student will work at the interface of computational chemistry, medicinal chemistry and microbiology.
Year 1: The project will involve initial training and utilisation of molecular modelling and computational techniques to study the interaction of bisbiguanides with Gram-negative membranes and design probes for synthesis. To serve as probes and for SAR determination, fluorophores with different sensitivities to the polar environment will be added onto existing scaffolds.
Year 2-3: The student will use a range of molecular biology techniques and a new bacterial membrane impedance assay to explore the interaction and partitioning of probes with bacterial membranes from a range of species and with known resistance modifications. This will allow us to develop a new understanding of the Gram-negative membrane barrier and chemical features to overcome them which can be applied to enhance activity of known antibacterial scaffolds with poor penetration.
Year 3-4: Optimisation of probes to separate the bacterial and mammalian membrane disrupting activity. These modifications, based on our improved understanding of mechanism of binding and disruption, may allow the development of more refined membrane probes or candidate antimicrobial agents for further development.
