Small molecules targeting the eubacterial β-sliding clamp discovered by combined and screening approaches.

Antibiotic resistance stands as the foremost post-pandemic threat to public health. The urgent need for new, effective antibacterial treatments is evident. Protein-protein interactions (PPIs), owing to their pivotal role in microbial physiology, emerge as novel and attractive targets. Particularly promising is the α-subunit/β-sliding clamp interaction, crucial for the replicative competence of bacterial DNA polymerase III holoenzyme. Through pharmacophore-based virtual screening, we identified 4,000 candidate small molecule inhibitors targeting the β-clamp binding pocket. Subsequently, these candidates underwent evaluation using the BRET assay in yeast cells. Following this, three hits and 28 analogues were validated via Protein Thermal Shift and competitive ELISA assays. Among them, thiazolo[4,5-]-pyrimidinedione and benzanilide derivatives exhibited micromolar potency in displacing the β-clamp protein partner and inhibiting DNA replication. This screening campaign unveiled new chemical classes of α/β-clamp PPI disruptors capable of inhibiting DNA polymerase III activity, which lend themselves for further optimisation to improve their antibacterial efficacy.