Chemical inhibition of a CBASS anti-bacteriophage defense

Multidrug-resistant bacteria necessitate innovative antibacterial strategies. Bacteriophages (phages) offer a promising alternative; however, bacterial immune defenses limit their effectiveness. Small-molecule inhibitors of these defenses may facilitate mechanistic studies and serve as adjuvants to enhance phage therapy. Here, we identify inhibitors targeting the bacterial cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector nuclease Cap5. Cap5 is hypothesized to degrade genomic DNA in virally infected cells, leading to cell death through abortive infection. Guided by the crystal structure of the Cap5 SAVED domain bound to its activating ligand, we performed structure-guided virtual screening to identify candidate inhibitors. Biochemical assays revealed ∼16% of the top docking hits inhibited Cap5. Cellular assays revealed one compound could enter E. coli cells and inhibit Cap5 activity. Our integrated approach-combining structure-based virtual screening with biochemical validation-provides a framework for discovering small-molecule inhibitors of bacterial immune defenses to advance adjunctive therapies and deepen our understanding of phage-bacteria interactions.