Sivelestat

Porcine pancreatic elastase (Elastin Products Company, Owensville, MO) was dissolved in Tris-HCl (pH 8.0) to give a concentration of 75 μg/mL. N-succinyl-Ala-Ala-Ala-p-nitroanilide (Sigma-Aldrich, St. Louis, MO) was dissolved in Tris-HCl (pH 8.0) to give a 2 mM substrate solution. 1 μL of varying doses of compound 1, symplostatin 5, sivelestat (Sigma-Aldrich) or solvent control (DMSO), 5 μL of elastase solution and 79 μL of Tris-HCl (pH 8.0) were pre-incubated at room temperature for 15 min in a 96-well plate. At the end of the incubation, 15 μL of substrate solution was added to each well by using multichannel pipette, and the reaction was monitored by recording the absorbance at 405 nm every 30 s for 30 min on SpectraMax M5 (Molecular Devices, Sunnyvale, CA).
The inhibitory activity against human neutrophil elastase was also determined using the same procedure with minor modifications. The assay was carried by using 100 μg/mL human neutrophil elastase (Elastin Products Company) and 2 mM N-(OMe-succinyl)-Ala-Ala-Pro-Val-p-nitroanilide (Sigma-Aldrich), both prepared in 0.1 M Tris-0.5 M NaCl (pH 7.5).
Enzyme activity in each well was calculated based on the initial slope of the reaction curve, expressed as a percentage of the initial slope of the uninhibited reaction. IC₅₀ calculations were done by GraphPad Prism 6 based on triplicate experiments.