Kinetic and docking studies of phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX and XII evidence a new binding mode within the enzyme active site.

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, coumarins (acting as prodrugs) and polyamines. A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) in a different manner, is reported here. Kinetic measurements allowed us to identify hydroxy-/methoxy-substituted benzoic acids as well as di-/tri-methoxy benzenes as submicromolar-low micromolar inhibitors of the four CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II allowed us to understand the inhibition mechanism. This new class of inhibitors binds differently compared to all other classes of inhibitors known to date: they were found between the phenol-binding site and the coumarin-binding site, filling thus the middle of the enzyme cavity. They exploit different interactions with amino acid residues and water molecules from the CA active site compared to other classes of inhibitors, offering the possibility to design CAIs with an interesting inhibition profile compared to the clinically used sulfonamides/sulfamates.