CanB, a Druggable Cellular Target in Mycobacterium tuberculosis

Treatment against tuberculosis can lead to the selection of drug-resistant Mycobacterium tuberculosis strains. To tackle this serious threat, new targets from M. tuberculosis are needed to develop novel effective drugs. In this work, we aimed to provide a possible workflow to validate new targets and inhibitors by combining genetic, in silico, and enzymological approaches. CanB is one of the three M. tuberculosis β-carbonic anhydrases that catalyze the reversible reaction of CO₂ hydration to form HCO₃^- and H⁺. To this end, we precisely demonstrated that CanB is essential for the survival of the pathogen in vitro by constructing conditional mutants. In addition, to search for CanB inhibitors, conditional canB mutants were also constructed using the Pip-ON system. By molecular docking and minimum inhibitory concentration assays, we selected three molecules that inhibit the growth in vitro of M. tuberculosis wild-type strain and canB conditional mutants, thus implementing a target-to-drug approach. The lead compound also showed a bactericidal activity by the time-killing assay. We further studied the interactions of these molecules with CanB using enzymatic assays and differential scanning fluorimetry thermal shift analysis. In conclusion, the compounds identified by the in silico screening proved to have a high affinity as CanB ligands endowed with antitubercular activity.