Periodic Reporting for period 3 - ANTIBIOCLICKS (BioInspired Clicked Siderophore-Antibiotics)
Reporting period: 2023-09-01 to 2025-02-28
Antibiotic drug resistant bacteria are a major threat to global healthcare, and predicted to cause more deaths than cardiovascular disease by 2050. The World Health Organisation has classed research into novel antibiotic research and development a priority, particularly critical for antibiotic resistant tuberculosis and Gram-negative infections. Antibiotic drug development against these bacterial is however challenging, particularly as it is complicated to design antibiotics that easily cross the bacterial cell envelope to enter the bacterium. One attractive approach to overcome this permeability barrier is to hijack the bacterial machinery used to take up iron from the environment. Bacteria achieve this iron acquisition by synthesizing and secreting molecules named siderophores, which bind and solubilise environmental iron. These siderophore iron complexes are then taken up by specific uptake pumps to deliver into the bacterial cytoplasm where the iron can be liberated and used. A number of natural product antibiotics named sideromycins have been discovered that are composed of an antibiotic warhead attached to such a siderophore molecules. These sideromycins are actively taken up through siderophore uptake systems of the bacteria for intracellular delivery of the antibiotic, thus allowing them to bypass the permeability barrier.
Research in the host lab into natural product production by a soil bacterium discovered a novel such siderophore-antibiotic conjugate. Unlike previous examples of natural sideromycins, here it was discovered that the siderophore-antibiotic conjugate could be chemically synthesised form the individual siderophore and antibiotic in a previously unexploited conjugation reaction. This finding had the important implication that custom siderophore-antibiotic conjugates could be rationally generated by coupling antibiotics of interest to the natural siderophores used by target bacteria, particularly those bacteria that pose the greatest challenge to drug discovery today. Together this would allow for specific delivery of antibiotics into bacteria of interest, and evade the bacterial penetration barrier.
The overall objectives of the Antibioclicks research program are to 1. Characterise the mechanism behind the chemical conjugation reaction identified, 2. Generate custom siderophore-antibiotic/fluorophore conjugates to target E.coli K. pneumoniae, A. baumannii, P. aeruginosa and M. tuberculosis 3. Elucidate the mechanism by which the siderophore-antibiotic conjugates are taken up by bacteria and engage with their target, and finally 4. Translate these in vitro findings to improving the treatment or diagnosis of bacterial infections in mice.
Research in the host lab into natural product production by a soil bacterium discovered a novel such siderophore-antibiotic conjugate. Unlike previous examples of natural sideromycins, here it was discovered that the siderophore-antibiotic conjugate could be chemically synthesised form the individual siderophore and antibiotic in a previously unexploited conjugation reaction. This finding had the important implication that custom siderophore-antibiotic conjugates could be rationally generated by coupling antibiotics of interest to the natural siderophores used by target bacteria, particularly those bacteria that pose the greatest challenge to drug discovery today. Together this would allow for specific delivery of antibiotics into bacteria of interest, and evade the bacterial penetration barrier.
The overall objectives of the Antibioclicks research program are to 1. Characterise the mechanism behind the chemical conjugation reaction identified, 2. Generate custom siderophore-antibiotic/fluorophore conjugates to target E.coli K. pneumoniae, A. baumannii, P. aeruginosa and M. tuberculosis 3. Elucidate the mechanism by which the siderophore-antibiotic conjugates are taken up by bacteria and engage with their target, and finally 4. Translate these in vitro findings to improving the treatment or diagnosis of bacterial infections in mice.
Research thus far has unravelled the chemical mechanism by which the siderophores and antibiotics are conjugated together to form the sideromycins, clearly demonstrating it broader translatability to for the formation of alternative biomimetic conjugates. By understanding this reaction mechanism, alternative synthetic approaches have been identified that now also allow for this conjugation reaction to occur, and this has also revealed that alternative antibiotics can also be coupled to siderophores by this reaction, greatly increasing the scope of this technology. To allow for the synthesis of custom siderophore conjugates, the lab has now chemically synthesised a panel of siderophore molecules, a variety of antibiotics, and a number of fluorophores. By coupling these fluorophores onto the siderophores, their active uptake into bacteria could be visualised and confirmed by fluorescence microscopy. When alternative antibiotics were coupled to the siderophores, these conjugates were found to retain antibiotic activity and able to reach their periplasmic and cytoplasmic targets. Investigation into the mechanism of uptake for a number of the conjugates has also confirmed that these molecules hijack the iron uptake system for their penetration. This invention is now patented and a manuscript describing this work submitted.
The technology uncovered in Antbioclicks allows for the synthesis on previously unexplored configurations of siderophore-antibiotic conjugates, and opens the door to the rational biomimetic synthesis of conjugates with the siderophores of the most important bacterial pathogens. It is planned that the potential of such conjugates are defined in E.coli K. pneumoniae, A. baumannii, P. aeruginosa and M. tuberculosis. This work will validate the mechanism by which these conjugates are taken up by bacteria, and if there is any intercellular processing of the conjugates once inside the bacteria. It is planned that the utility of these molecules will finally be evaluated in vivo.