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ABSTRACT Multidrug-resistant (MDR) bacteria pose a significant public health challenge, underscoring the urgent need for innovative antibacterial strategies. Bacteriophages (phages), viruses that specifically target bacteria, offer a promising alternative; however, bacterial immune defenses often limit their effectiveness. Developing small-molecule inhibitors of these defenses can facilitate mechanistic studies and serve as adjuvants to enhance phage therapy. Here, we identify novel inhibitors targeting the bacterial cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector Cap5. Cap5 is an HNH endonuclease activated by a cyclic nucleotide 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 that approximately 16% of the top docking hits exhibited inhibitory activity. Further cellular assays demonstrated that one potent compound could enterE. colicells and inhibit Cap5 activity. Our integrated approach—combining structure-based virtual screening with biochemical validation—provides a robust framework for discovering small-molecule inhibitors of bacterial immune defenses to advance adjunctive therapies and deepen our understanding of phage-bacteria interactions.
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This content will become publicly available on February 21, 2026
Chemical inhibition of a bacterial immune system
Abstract The rise of antibiotic resistance motivates a revived interest in phage therapy. However, bacteria possess dozens of anti-bacteriophage immune systems that confer resistance to therapeutic phages. Chemical inhibitors of these anti-phage immune systems could be employed as adjuvants to overcome resistance in phage-based therapies. Here, we report that anti-phage systems can be selectively inhibited by small molecules, thereby sensitizing phage-resistant bacteria to phages. We discovered a class of chemical inhibitors that inhibit the type II Thoeris anti-phage immune system. These inhibitors block the biosynthesis of a histidine-ADPR intracellular ‘alarm’ signal by ThsB and prevent ThsA from arresting phage replication. These inhibitors promiscuously inhibit type II Thoeris systems from diverse bacteria—including antibiotic-resistant pathogens. Chemical inhibition of the Thoeris defense improved the efficacy of a model phage therapy against a phage-resistant strain ofP. aeruginosain a mouse infection, suggesting a therapeutic potential. Furthermore, these inhibitors may be employed as chemical tools to dissect the importance of the Thoeris system for phage defense in natural microbial communities.
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- Award ID(s):
- 2143636
- PAR ID:
- 10646148
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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