Unprecedented carbonic anhydrase inhibition mechanism: Targeting histidine 64 side chain through a halogen bond.

2,2'-Thio-bis(4,6-dichlorophenol), namely bithionol, is a small molecule endowed with a multifaceted bioactivity. Its peculiar polychlorinated phenolic structure makes it a suitable candidate to explore its potentialities in establishing interaction patterns with enzymes of MedChem interest, such as the human carbonic anhydrase (hCA) metalloenzymes. Herein, bithionol was tested on a panel of specific hCAs through the stopped-flow technique, showing a promising micromolar inhibitory activity for the hCA II isoform. X-ray crystallographic studies revealed an unprecedented halogen-bond interaction between one chlorine of bithionol and the N3(ε) atom of the hCA II catalytically active histidine residue, His64. Then, quantum mechanics calculations based on the fragment molecular orbital method allowed us to estimate the strength of this bond (~2.9 kcal/mol) and highlighted the contribution of a rich hydrophobic interaction network within the isoenzyme. Interestingly, the compound inactivity against the hCA III isoform, characterized by His64Lys and Leu198Phe mutations, supported the key role played by halogen bonding in the enzyme affinity. This finding might pave the way for the development of a new class of hCA inhibitors characterized by such chemical features, with the halogen bond being a key ligand-receptor interaction.