Disulfide bond formation is an essential step in the folding of many proteins that enter the secretory pathway. Disulfide bonds between cysteine residues act as covalent braces that impart stability, activity and protection to proteins from degradation. Most organisms encode machinery to catalyse this important process, known as oxidative protein folding. In bacteria the DiSulfide Bond (Dsb) enzymes catalyse the formation of disulfide bonds, with DsbA being the best studied thiol redox catalyst.
We have developed small molecule inhibitors of Escherichia coli DsbA (EcDsbA) from several chemical classes. Using a pipeline of cell-based assays specific for DsbA activity (in vitro and in vivo) that can be utilised from high-throughput screening and hit discovery, to early preclinical lead candidate validation, we showed that DsbA inhibitors attenuate virulence in different Gram‐negative pathogens by directly inhibiting diverse DsbA homologues. While inhibitors reduced virulence but not growth under standard laboratory conditions, pathogenic bacteria grown under pathophysiological in vitro conditions showed slowed growth in the presence of DsbA inhibitors, phenocopying isogenic dsbA null mutants. Passaging experiments with Salmonella enterica serovar Typhimurium under conditions that rapidly induce resistance to ciprofloxacin did not result in detectable DsbA inhibitor resistance. Furthermore, no drug-specific adaptations were detected in inhibitor‐treated S. Typhimurium, while pathogen virulence remained susceptible to DsbA inhibition. Finally, DsbA inhibitors from different classes showed synergistic effects and some restored clinical susceptibility to carbapenem antibiotics in KPC-producing uropathogenic E. coli isolates by disrupting functional carbapenemase biogenesis in cells.
Our work demonstrates that DsbA inhibitors hold broad potential as novel agents with multifaceted antimicrobial activity against critical-priority Gram-negative pathogens: from evolutionarily robust pathoblockers and antibiotic potentiators for last-line drugs, to likely stand-alone antibiotics for niche-specific infections. This presentation will summarise the evidence from recent published and unpublished studies that showcase the diverse potential of DsbA inhibitors as antimicrobials for further development.