2. Academic Validation
  3. Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors

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

  • Sci Rep. 2019 Sep 3;9(1):12710. doi: 10.1038/s41598-019-49094-0.
Justina Kazokaitė 1 2,  Visvaldas Kairys 3,  Joana Smirnovienė 4,  Alexey Smirnov 4,  Elena Manakova 5,  Martti Tolvanen 6,  Seppo Parkkila 7,  Daumantas Matulis 4
Affiliations

Affiliations

  • 1 Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius, LT-10257, Lithuania. [email protected].
  • 2 Division of Biochemistry, the Netherlands Cancer Institute, Amsterdam, the Netherlands. [email protected].
  • 3 Department of Bioinformatics, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius, LT-10257, Lithuania.
  • 4 Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius, LT-10257, Lithuania.
  • 5 Department of Protein-DNA Interactions, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius, LT-10257, Lithuania.
  • 6 Department of Information Technology, University of Turku, FI-20520, Turku, Finland.
  • 7 Tampere University, Faculty of Medicine and Health Technology; Fimlab Ltd, Tampere University Hospital, Arvo Ylpön katu 34, FI-33520, Tampere, Finland.
PMID: 31481705 DOI: 10.1038/s41598-019-49094-0
Abstract

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.

Figures