3.2.3. Enzyme Inhibition

The inhibitory effect of the tested compounds was measured using colorimetric PDE Activity Assay Kit (Abcam, Cambridge, UK). This assay allows the quantification of 5′-nucleotide released in the reaction catalyzed by tested phosphodiesterase, PDE5, using the additional enzyme 5′-nucleotidase to cleave the mentioned product, and next, using modified Malachite Green assay, the phosphate is quantified. The assay was carried in a 96-well plate at a volume of 40 μL in an assay buffer, containing 40 ng recombinant human PDE5A/PDE5 protein (Abcam), 200 μM cGMP, 25 kU 5′-nucleotidase and serially diluted ligand to preserve 4% of DMSO. The reaction was initiated with the enzyme and kept going for 20 min at 37 °C. The reaction was stopped by adding 80 μL of Green Assay reagent. After incubation at room temperature for 30 min (to develop color proportional to phosphate content), the absorbance at 620 nm was measured with The SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). Inhibitors concentrations varied in the range of 2 pM–200 μM. Each experiment was repeated 4–5 times and IC₅₀ values were calculated by global fitting of the sigmoidal dose response equation implemented in Origin 9.0 package (www.originlab.com). For each experiment, control reaction with no enzyme added and without inhibitor was also performed.