Bacterial β-carbonic anhydrases: a six-step prioritization framework for antibacterial target validation

Bacterial β-carbonic anhydrases (β-CAs) are actively studied as potential targets for next-generation antibacterial agents. Despite extensive biochemical and structural characterization, their validation as isoform-specific therapeutic targets remains incomplete. Here, we developed and applied a six-filter validation framework-encompassing genetic essentiality, physiological relevance, biochemical feasibility, structural rationale, cellular target engagement, and therapeutic context-to integrate disparate data into a strategic prioritization map across representative pathogens (Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Streptococcus pneumoniae). Application of this framework reveals that none of the analyzed β-CA isoforms currently qualifies as a ready therapeutic candidate (Group A), with most falling into context-dependent (Group C) or high-risk (Group D) categories due to specific validation gaps. By systematically defining these gaps, the framework provides a prioritized, target-driven roadmap for future experimental design and antibacterial drug discovery targeting bacterial β-carbonic anhydrases.