CanB, a druggable cellular target in mycobacterium tuberculosis

Treatment against tuberculosis can lead to the selectionof drug-resistant Mycobacterium tuberculosis strains. To tackle thisserious threat, new targets from M. tuberculosis are needed to develop novel effective drugs. In this work, we aimedto provide a possible workflow to validate new targets and inhibitorsby combining genetic, in silico, and enzymologicalapproaches. CanB is one of the three M. tuberculosis & beta;-carbonic anhydrases that catalyze the reversible reactionof CO2 hydration to form HCO3 (-) and H+. To this end, we precisely demonstrated that CanBis essential for the survival of the pathogen in vitro by constructing conditional mutants. In addition, to search forCanB inhibitors, conditional canB mutants were alsoconstructed using the Pip-ON system. By molecular docking and minimuminhibitory concentration assays, we selected three molecules thatinhibit 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-killingassay. We further studied the interactions of these molecules withCanB using enzymatic assays and differential scanning fluorimetrythermal shift analysis. In conclusion, the compounds identified bythe in silico screening proved to have a high affinityas CanB ligands endowed with antitubercular activity.