Bacterial DNA replication requires an orchestration of protein-protein and protein-DNA interactions, so as to faithfully replicate DNA prior to cell division. One protein essential to this process, the β2-sliding clamp, interacts with several proteins via a conserved binding site and mechanism. We have been probing this binding site as a target for novel antimicrobials across Gram-negative bacteria.
Six classes of novel compounds selected via a DNA-encoded chemical screening program were found to target the E. coli β2-sliding clamp. Using a combination of X-ray crystallography, biochemical and biophysical assays, these inhibitors were shown to bind into the β2-sliding clamp binding-pocket to prevent in vitro bacterial DNA replication. Furthermore, the inhibitors had antimicrobial activity against various bacteria, and are bactericidal. Our current exploration into their mechanism of action, using single-molecule live-cell fluorescence imaging techniques, suggests the inhibitors cause the collapse of replication forks and eventual cell lysis. Interestingly, the SOS response, a mutagenic repair pathway activated upon DNA damage, is not induced by most of the compounds. Additionally, the inhibitors were ineffective against the human sliding clamp, PCNA, and some were non-toxic to human cells.
Collectively, these results suggest that targeting the bacterial DNA replication machinery is a promising strategy for the pre-clinical development of a novel antimicrobial compound.