New approaches to characterise and help optimize antibiotics that target penicillin-bind-proteins (PBPs) and more effectively cross the G-ve cell wall to tackle antimicrobial resistance (AMR)

Outer membrane (OM) penetration is an important factor which can make it difficult to develop new antibiotics to treat Enterobacteriaceae and Pseudomonas aeruginosa infections. Deeper insights into the structure-activity relationships that improve penetration will enable the development of the next generation of agents to treat these serious infections.

As β-lactams remain the most important class of therapeutic agents we have chosen to focus our research on gaining a deeper understanding of their OM penetration and efflux propensities. Beyond their therapeutic importance, β-lactam inhibitors target periplasmic bacterial targets and therefore the additional challenge of also crossing the inner membrane doesn’t need to be dealt with.

Initially, this project will develop and validate novel (mass spectrometry based) penetration assays to extensively profile the penetration of a set of reference β-lactam compounds against isogenic and clinically relevant strains of E. coli. Once the assays are validated, they will be used to profile three different sets of mechanistically distinct and specially designed and synthesized monobactams, lactivicins and boronates against the same set of strains.

In parallel we will establish novel in vitro assays using synthetic cells walls, using the microfluidics platform available at Cambridge with Dr J Cama. This will involve the generation of increasingly complex vesicles with defined porin composition and within the core of the vesicle defined target proteins. This system once developed will provide a state of the art platform to better characterise compounds that can penetrate G-ve bacterial cell walls.

Finally we will also use state of the art high resolution single cell Raman Spectroscopic analysis with Prof S. Evans, Leeds, to further characterise compound permeation into cells.

Results from these studies will enable an improved view of the properties that lead to greater OM penetration of β-lactam inhibitors in Gram negative bacteria for these important classes of therapeutics. To achieve all of the above goals we have assembled a multidisciplinary consortium of experts in penicillin binding protein enzymology and microbiology (Warwick), β-lactam inhibitor synthesis (Oxford), microfluidics (Cambridge), and biophysics (Leeds). Together, this new consortium will share new insights into how to improve OM penetration in Enterobacteriaceae and Pseudomonas aeruginosa pathogens.