Inhibitor screening, IC50 determination, and mechanism of inhibition.

Twenty compounds known to act as potent inhibitors of mammalian NTPDases were tested for their ability to inhibit the PhaHV1 NTPDase-catalyzed conversion of ATP to ADP and AMP. Briefly, 2.4 μg of vNTPDase and 400 μM ATP were incubated for 2 h at 37°C with 10 μM each compound (dissolved in 10% DMSO). Inhibitor-free controls (enzyme plus substrate only) were included in triplicate as blanks, and inhibition values were determined relative to blank conversion values. Reaction samples were diluted 1 in 4 in reaction buffer and analyzed using CE and MG assays. CE conditions were as described above, with 14 to 16 min of separation at −10 kV, and the results of the MG phosphate assays were measured on a PHERAstar plate reader (BMG Labtech) at 623 nm (74).

Concentration-inhibition curves were performed by incubating 2.4 μg of vNTPDase with 400 μM ATP for 2 h at 37°C with inhibitor at a concentration of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 mM in reaction buffer with 10% DMSO in triplicate. No-enzyme (0.01 μM inhibitor and 400 μM ATP only), no-inhibitor, and buffer-only samples were included as reaction controls. Phosphate concentrations were measured using MG phosphate assays, as described above (74). Percent residual protein activity values were calculated relative to the activity for the inhibitor-free controls.

The mechanism of inhibition was determined by testing the inhibitors at 0, 0.1, and 0.2 μM (in 10% DMSO) with substrate (ATP) at 0.1, 0.2, 0.4, 0.8, or 1 mM and 2.4 μg of vNTPDase protein (37°C, 2 h, in reaction buffer). The phosphate released was measured by the MG assay, and the type of inhibition was evaluated graphically from a Hanes-Woolf plot using GraphPad Prism (version 6.0) software (74).