Abstract by Mikkel Stampe Madsen

Bacterial resistance to a broad range of antibiotics is an increasingly serious problem. If antimicrobial resistance development remains unaddressed, deaths caused by multidrug-resistant bacteria are projected to surge to 10 million people in 2050. Bacteria develop resistance through various mechanisms, one of which involves upregulating the expression of multi-component efflux pumps. These pumps exploit a proton gradient to actively expel toxic compounds from the bacterial cytosol, thereby reducing the efficacy of antimicrobial agents.

To tackle this challenge, four distinct series of efflux pump inhibitor conjugates were synthesised, employing different shuttle approaches to facilitate internalisation of efflux pump inhibitors. Once inside the bacteria, the efflux pump inhibitor can block the efflux of other antibiotics, and thus enhance the activity of other antibiotics. The conjugates were prepared by synthesising mono-functionalised shuttles and conjugating them to one of two distinct, but chemically similar inhibitors. The four shuttles comprise: (i) aminoglycosides, (ii) bacteria-penetrating peptidomimetics, (iii) siderophores, and (iv) antimicrobial peptides. Compounds of each class were synthesised, and evaluated in vitro against P. aeruginosa, an antibiotic-resilient Gram-negative bacteria, known to have a high level of efflux pumps.

The majority of the intended target molecules, including representative conjugates from each class, were successfully synthesised. The number of compounds synthesised varied across the four distinct classes, with some classes yielding a greater diversity of compounds than others did due to differences in the complexity of their synthesis routes.

Microbiological evaluation of all synthesised compounds revealed that conjugates employing tobramycin as the shuttle exhibited significantly more efficient internalisation in P. aeruginosa compared to those employing other shuttles classes. Adding as little as 2 µg/mL of the tobramycin-based potentiator significantly enhanced the activity of the following antibiotics ciprofloxacin, rifampicin and minocycline, reducing their MICs to less than 1/8 of their initial values. Ciprofloxacin was potentiated to an extent so that its MIC was below the clinical breakpoint. Furthermore, rifampicin and minocycline tested in combination with the tobramycin-inhibitor conjugates, displayed activity in concentrations below 0.5 µg/mL despite normally being inactive against P. aeruginosa. While tobramycin is the most efficient shuttle, some antimicrobial peptides conjugates also showed activity, albeit to a lesser extent.