Secretory human carbonic anhydrase VI (CA VI) has emerged as a potential
drug target due to its role in pathological states, such as excess
acidity-caused dental caries and injuries of gastric epithelium.
Currently, there are no available CA VI-selective inhibitors or
crystallographic structures of inhibitors bound to CA VI. The present
study focuses on the site-directed CA II mutant mimicking the active
site of CA VI for inhibitor screening. The interactions between CA
VI-mimic and a series of benzenesulfonamides were evaluated by
fluorescent thermal shift assay, stopped-flow CO₂ hydration
assay, isothermal titration calorimetry, and X-ray crystallography.
Kinetic parameters showed that A65T, N67Q, F130Y, V134Q, L203T mutations
did not influence catalytic properties of CA II, but inhibitor
affinities resembled CA VI, exhibiting up to 0.16 nM intrinsic affinity
for CA VI-mimic. Structurally, binding site of CA VI-mimic was found to
be similar to CA VI. The ligand interactions with mutated side chains
observed in three crystallographic structures allowed to rationalize
observed variation of binding modes and experimental binding affinities
to CA VI. This integrative set of kinetic, thermodynamic, and structural
data revealed CA VI-mimic as a useful model to design CA VI-specific
inhibitors which could be beneficial for novel therapeutic applications.