The project aims at a novel means of getting antibiotics into bacteria. It exploits the active uptake of an antibiotic-boosting catalyst into the bacterial cell. The catalyst is designed to convert a masked prodrug, which has itself no biological activity to its active form once it has reached its bacterial target. The targeting group is a compound utilised by bacteria for the uptake of essential iron. The idea is to link the catalyst to the targeting group for active accumulation by the bacterial cell. Human cells will remain unaffected since they do not import the bacterial targeting group. In addition, the chosen catalyst is a synthetic compound that allows for an activation reaction that has no equivalent in biology. Hence, the prodrugs cannot be activated in the cells of the human host. Side effects would be reduced and resistance mechanisms avoided.
Two prodrug forms of the fluoroquinolone antimicrobial ciprofloxacin were investigated and activity tests confirmed the masking of their antibacterial activity. Both were found suitable for further studies. Subsequently, a selection of synthetic catalysts was tested and found not to have antimicrobial activity in their own right at physiologically relevant concentrations. Upon addition of the prodrugs to the catalyst-containing growth medium, the antimicrobial activity of the fluoroquinolone was unmasked, confirming that the catalytic activation of the prodrug could be achieved in aqueous media and at physiological pH.
In the next step it was confirmed that the targeting siderophore group was required to allow the synthetic catalysts to enter bacterial cells. In addition, the antimicrobial activty of a series of siderophore-drug conjugates was assessed using E. coli and S. aureus strains under both iron-deficient and iron-sufficient conditions. Standard screening protocols are now established that allow time-dependent bacterial cell growth to be monitored and evaluated in an automated way and these will be used by chemists in the research group in the future. In addition, an E. coli mutant that is unable to transport iron siderophores into the cell was obtained to serve as a negative control. The insights obtained have allowed us to start a new collaboration with Prof. Eszter Boros at Stony Brook University in New York, who is now following the uptake of our siderophore conjugates into E. coli by means of radioactive Ga-67.
The results obtained from the antimicrobial activity screens and the radiolabelling revealed key structural requirements for the targeted delivery of cargo, such as catalysts or drugs, into the bacterial cell. Difficulties with the synthesis of the siderophore-catalyst conjugates could not be overcome for the planned intracellular catalytic tests to be completed within the timescale of the project. The studies are still ongoing and they are greatly facilitated by the protocols that were developed with CFH support.