3.2. hCA Enzyme Inhibition Assays

Carbonic anhydrases are able to catalyze the conversion 4-nitrophenyl acetate (4-NPA) to 4-nitrophenol. According to the method described previously by Verpoorte, the rate of this reaction is monitored spectrophotometrically, at 405 nm, with a Perkin Elmer Envision 2104 plate reader [30,31] (Perkin Elmer, Waltham, MA, USA). 1X assay buffer consisted of 15 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH = 7.40) 0.01% tetraethylene glycol monododecyl ether (Brij) and 100 mmol/L NaCl. Recombinant human carbonic anhydrase II and IX was commercially available (Sino Biological Inc, Beijing, China) and prepared in 1X assay buffer with a concentration of 11.10 ng/μL. Then, 18 μL of enzyme solution was transferred into 384-well assay plates in triplicate. Stock solutions of the inhibitor (10.00 mmol/L) were prepared using DMSO as solvent, and then diluted 1:3 with DMSO. Ten different inhibitor concentrations were used: 600.00 μmol/L, 200.00 μmol/L, 66.67 μmol/L, 22.22 μmol/L, 7.41 μmol/L, 2.47 μmol/L, 0.82 μmol/L, 0.27 μmol/L, 0.091 μmol/L, 0.03 μmol/L, and 0 μmol/L. A volume of 2.00 μL of each inhibitor was added into the assay solution. All compounds were allowed to incubate with the enzyme for 15 min at 25 °C to form the Enzyme-Inhibitor (E-I) complex. After that, substrate 4-NPA (1.00 mmol/L, 20.00 μL, Sigma-Aldrich, St. Louis, MI, USA) was added into the E-I complex solution and incubated for 90 min at 25 °C. The absorbance of each compound was measured with Envision 2104 plate reader. The inhibitor AZM and SLC-0111 were used as standards to investigate the inhibitory activity of these compounds.