Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors

Area of Science:

• Biochemistry
• Enzymology
• Medicinal Chemistry

Background:

• Secretory human carbonic anhydrase VI (CA VI) is implicated in dental caries and gastric issues.
• Selective CA VI inhibitors and structural data are currently lacking.
• Targeting CA VI offers potential therapeutic avenues.

Purpose of the Study:

• To develop and validate a carbonic anhydrase II (CA II) mutant as a mimic of CA VI's active site.
• To screen for and characterize benzenesulfonamide inhibitors using the CA VI-mimic.
• To provide structural insights into inhibitor binding for future drug design.

Main Methods:

• Site-directed mutagenesis of CA II to create a CA VI active site mimic.
• Inhibitor screening using fluorescent thermal shift assay and stopped-flow CO2 hydration assay.
• Thermodynamic characterization via isothermal titration calorimetry and structural analysis through X-ray crystallography.

Main Results:

• The CA VI-mimic (mutant CA II) exhibited inhibitor affinities comparable to CA VI, with intrinsic affinities as low as 0.16 nM.
• Mutations (A65T, N67Q, F130Y, V134Q, L203T) did not significantly alter CA II's catalytic properties.
• Crystallographic structures revealed similarities between the CA VI-mimic and CA VI binding sites, explaining ligand interactions and binding affinities.

Conclusions:

• The developed CA VI-mimic is a valuable tool for screening and designing CA VI-specific inhibitors.
• This approach can facilitate the development of novel therapeutics for conditions associated with CA VI.
• Integrative kinetic, thermodynamic, and structural data support the utility of this model system.