Despite the rising threat of antibiotic resistance, recent decades have seen a decline in antibiotic development. Discovery and development of compounds that inhibit the growth of Gram-negative bacteria is especially important. Target-based antibiotic drug discovery describes the identification of appropriate targets and establishment of bespoke assays for inhibitors of those targets. The bacterial Sec-machinery, a major system of protein translocation across and into the cytoplasmic membrane, is essential across bacteria, differs from the human counterpart and contributes to the virulence and antibiotic resistance of many pathogens, namely export of most β-lactamases. It therefore represents a promising target for development of antibiotic drugs, both as a standalone target and in terms of potentiating β-lactam antibiotics. This work describes the design of a whole-cell split-luciferase-based assay to monitor inhibition of the Sec-machinery. After validation with a model Sec substrate and known inhibitors of the machinery, the assay was scaled up for use in a local screen of 5000 diverse synthetic compounds, giving an average Z’-factor (robustness measure) of 0.71 and primary hit rate of 0.22%. Compared to a commercially available Sec inhibitor, hits have greater inhibitory activity against protein translocation in the Gram-negative bacterium Escherichia coli but negligeable effects on bacterial growth. A secondary assay strategy to complement the screen system and characterise hits is proposed. The split-luciferase-based assay was also adapted for analysing the role of Sec in export of metallo-β-lactamases IMP-1 and NDM-1, yielding data consistent with traditional assays for β-lactamase activity. Both enzymes are dependent on signal recognition particle for efficient translocation, in contrast to predictions based on signal sequence. Overall, this work further establishes the bacterial Sec-machinery as an antibiotic target, demonstrates how to design a successful strategy for Sec inhibitor discovery and provides insights into the types of Sec inhibitor most likely to be useful antibiotic leads.
The bacterial Sec-machinery as an antibiotic target
Salter, T. (Author). 21 Mar 2023
Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)