Malachite green assay for measuring NTPDase activity (CD39 and NTPDases2, –3 and 8)

Effects of compounds on ATP hydrolysis by CD39, NTPDase2, −3 or −8, and by human cancer cell membrane preparations were evaluated using the malachite green assay.54 The substrate concentration of ATP was 50 µM for CD39 and the cancer cell membrane preparations, and 100 µM for NTPDase2, −3 or −8. The assay was performed in clear half area plates in a total volume of 50 µL of 10 mM HEPES containing 2 mM CaCl₂ and 1 mM MgCl₂ (pH 7.4) in the presence of a maximal concentration of 2% DMSO. The mixtures of inhibitor and enzyme were preincubated at 37°C for 5 min. Subsequently, the enzymatic dephosphorylation of ATP was started by addition of the substrate. The samples were incubated at 37°C for 15 min, then the reaction was stopped by adding the malachite green detection reagents (20 µL of malachite green solution (0.6 mM) and 30 µL of ammonium molybdate solution (20 mM) in sulfuric acid (1.5 M)). Absorption of the samples at 600 nm was measured using the BMG PHERAstar FS plate reader (BMG Labtech GmbH, Ortenberg, Germany) after 20 min of incubation at 25°C. The corrected absorption was calculated by subtracting the absorption of the negative control samples, which were incubated with denatured enzyme (90°C, 15 min).

The kinase inhibitors were initially investigated at 10 µM concentration at human CD39, natively expressed in high density in human umbilical cord membrane preparations at a protein concentration of 50.0 ng per well. Further studies were performed using CD39 and the respective NTPDase proteins expressed in COS-7 cell membranes (ca. 100 ng of protein depending on enzymatic activity, adjusted to ensure 10%–20% of substrate conversion).33 47 The respective enzyme was incubated with or without 50 µM ceritinib and 50 or 100 µM ATP (K_m (CD39)=17 µM; K_m (NTPDase2)=70 µM; K_m (NTPDase3)=75 µM; K_m (NTPDase8)=46 µM).48 Full concentration-inhibition curves were determined with inhibitor concentrations ranging from 0.1 to 300 µM. For the inhibition type experiments, 57.0 ng of recombinant human CD39 was incubated with 0, 5, 10, 15 and 20 µM of ceritinib and increasing substrate (ATP) concentrations of 10, 25, 50, 100 or 150 µM. All experiments were performed in three independent iterations (n=3), and the obtained data were processed and plotted with GraphPad Prism V.8 software (San Diego, USA).