CanB, a Druggable Cellular Target in Mycobacterium tuberculosis .

Treatment against tuberculosis can lead to the selection of drug-resistant strains. To tackle this serious threat, new targets from 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, , and enzymological approaches. CanB is one of the three β-carbonic anhydrases that catalyze the reversible reaction of CO hydration to form HCO and H. To this end, we precisely demonstrated that CanB is essential for the survival of the pathogen by constructing conditional mutants. In addition, to search for CanB inhibitors, conditional 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 of wild-type strain and 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 screening proved to have a high affinity as CanB ligands endowed with antitubercular activity.