| Assay Method Information | |
| | Carbonic Anhydrase (Esterase) Biochemical Assay |
| Description: | Compounds were tested in a high-throughput 384-well assay format for their ability to inhibit the human carbonic anhydrase (hCA)-mediated hydrolysis of 4-nitrophenyl acetate (4NPA) (Verpoorte et al, JBC, 1967). 10-dose, 3-fold serial dilutions of compounds were prepared at starting concentrations of 10 mM in 100% DMSO. 200 nl of compounds were then spotted in quadruplicates onto clear 384-well microplates (Perkin Elmer cat #6007640) using a Labcyte ECHO acoustic dispenser. Final starting concentration in the assay was 50 μM. DMSO (no compound) and acetazolamide were included on each microplate as negative and positive controls, respectively.A 1.5 μM solution of hCAI (R&D systems cat #2180-CA) or a 1 μM solution of hCAII (Genscript cat #U3256FL150-4/P5GA002) was prepared in assay buffer (25 mM Tris (pH 7.5), 100 mM NaCl, 1% DMSO) and 20 μl were added to compounds using a Biotek Micro Flo. Following a preincubation at room temperature for 15 minutes, 20 μl of a 4 mM solution of 4NPA substrate (Sigma cat #N8130) in assay buffer were added to start the reaction. Microplates were incubated at room temperature for 60 min after which absorbance at 405 nM was read on an Envision plate-reader. IC50 values were defined as the compound concentration that caused a 50% decrease in absorbance signal and were calculated using a sigmoidal dose-response model to generate curve fits. |
| Affinity data for this assay | |
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