Development of novel chemical probes to image penicillin binding proteins in Staphylococcus aureus

The shortage of new antibiotics has given rise to unprecedented levels of antimicrobial resistance and demands research into the complex mechanisms utilised by bacteria to adapt, grow and evolve as a species. One of these mechanisms is the dynamicity that bacteria possess to grow and divide whilst maintaining a flexible but rigid structure. The key determinant for these attributes is the unique polymer, peptidoglycan, a major component of eubacterial cell walls responsible for cellular shape and size. Penicillin binding proteins (PBPs) are a group of enzymes that mediate the construction of peptidoglycan and are therefore crucial for cellular viability. They are the target of some of the most clinically effective antibiotics, β-lactams, which operate by inhibiting PBP function, causing cell apoptosis. Despite the function of PBPs being known, certain strains of bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA) express PBPs which resist such antibiotics. The reasons behind this are not understood. In addition, the precise roles of PBPs in peptidoglycan assembly and mechanisms which follow their inhibition remain elusive. To help elucidate this, a series of chemical probes were developed to bind to PBPs for imaging in fluorescence microscopy.

Novel cephalosporin scaffolds demonstrated good antibacterial activity against Staphylococcus aureus and were subsequently ligated to various fluorescent dyes via click chemistry. Each of the five functionalised chemical probes retained antibacterial activity and successfully imaged PBPs in Staphylococcus aureus using widefield microscopy. The chemical probes displayed specificity for PBPs which was hypothesised to be a result of the size or charge exhibited by each compound. Chemical probe Ceph-1 Cy5 55 imaged PBPs in stochastic optical reconstruction microscopy and gave the highest resolution fluorescent images of PBPs in any organism to date. A potential PBP2a binder was also synthesised for biological testing against MRSA and subsequent ligation to fluorophores. A moenomycin based fluorescent probe to bind to a subset of PBPs and transglycosylase enzymes in Staphylococcus aureus was prepared and demonstrated localised fluorescence in widefield microscopy. A novel oxetanyl dipeptide scaffold was synthesised but did not induce any antibacterial activity. Finally, the chemical methodology for a series oxetanyl phosphonates as potential β-lactamase inhibitors was established.