Fluoroskan ascent fl

CycLex® protein phosphatase Cdc25A and B fluorometric assay Kits (Medical & Biological Laboratories Co., Ltd, Japan) were used to evaluate enzyme inhibition by naphthoquinone analogs. Cdc25A and B activity was measured in a 96-well half-area microtiter plate using 3-O-methylfluorescein phosphate (OMFP) as a substrate. Five μL (0.1 μg/μL) of purified recombinant Cdc25 (Cdc25A or B) was mixed with 40 μL of assay mixture and incubated with 5 μL of the test compound at various concentrations in a well. Kinetic measurements were obtained every 30 s for 10 min at 25 °C using a Fluoroskan Ascent FL fluorescence microplate reader (Thermo Electron, MA) with excitation and emission wavelengths at 485 and 538 nm, respectively. For all compounds tested, the concentration of inhibitor that caused 50% inhibition of the enzymatic reaction (IC₅₀) was calculated by plotting % inhibition versus logarithm of inhibitor concentration.

Compounds were dissolved in 100% DMSO at 5 mM stock concentrations. The final concentration of DMSO in the reactions was 1%, and this level of DMSO had no effect on HNE activity. The HNE inhibition assay was performed in black flat-bottom 96-well microtiter plates. Briefly, a buffer solution containing 200 mM Tris–HCl, pH 7.5, 0.01% bovine serum albumin, 0.05% Tween-20, and 20 mU/mL HNE (Calbiochem) was added to wells containing different concentrations of each compound. The reactions were initiated by addition of 25 µM elastase substrate (N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin, Calbiochem) in a final reaction volume of 100 µL/well. Kinetic measurements were obtained every 30 s for 10 min at 25 °C using a Fluoroskan Ascent FL fluorescence microplate reader (Thermo Electron, MA) with excitation and emission wavelengths at 355 and 460 nm, respectively. For all compounds tested, the concentration of inhibitor that caused 50% inhibition of the enzymatic reaction (IC₅₀) was calculated by plotting % inhibition versus logarithm of inhibitor concentration (at least six points). The data are presented as the mean values of at least three independent experiments with relative standard deviations of <15%.

All compounds were dissolved in 100% DMSO at 5 mM stock concentrations. The final concentration of DMSO in the reactions was 1%, and this level of DMSO had no effect on enzyme activity. The HNE inhibition assay was performed in black flat-bottom 96-well microtiter plates. Briefly, a buffer solution containing 200 mM Tris–HCl, pH 7.5, 0.01% bovine serum albumin, and 0.05% Tween-20 and 20 mU/mL of HNE (Calbiochem) was added to wells containing different concentrations of each compound. The reaction was initiated by addition of 25 μM elastase substrate (N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin, Calbiochem) in a final reaction volume of 100 μL/well. Kinetic measurements were obtained every 30 s for 10 min at 25 °C using a Fluoroskan Ascent FL fluorescence microplate reader (Thermo Electron, MA) with excitation and emission wavelengths of 355 and 460 nm, respectively. For all compounds tested, the concentration of inhibitor that caused 50% inhibition of the enzymatic reaction (IC₅₀) was calculated by plotting % inhibition versus logarithm of inhibitor concentration (at least six points). The data are presented as the mean values of at least three independent experiments with relative standard deviations of < 15%.