4.4. Urease Activity Inhibition and Kinetic Characterization

In vitro urease inhibition screening assays were conducted to further narrow the virtual screened compounds for further IC₅₀ determination and kinetic characterization. The urease inhibition assay was performed with slight modifications of the Berthelot alkaline phenol-hypochlorite method [35]. Candidate compounds filtered by virtual screening were diluted in a methanol solution. The assay mixture containing 15 μL of ruminal microbial urease and 15 μL of the candidate compounds with 70 μL of urea solution (50 mM, 0.15 g urea diluted in a HEPES buffer (pH 7.5, 50 mM)) was incubated in a water bath at 37 °C for 30 min. The final concentration for the candidate compounds in the assay mixture was 0.5 mM. The ammonia that was produced was measured using 50 μL of a phenol reagent containing 10.0 g/L phenol and 50 mg/L of sodium nitroprusside, and 50 μL of alkali reagent containing 5 g/L sodium hydroxide and 8.4 mL/L of sodium hypochlorite solution, at 37 °C for 30 min. The absorbance of each well was measured at 625 nm by a micro plate spectrophotometer (Thermo Scientific Varioskan Flash G-282, Thermo Scientific, Waltham, MA, USA). The endogenous nitrogen (blank) was measured by adding a 70 μL HEPES buffer (pH 7.5, 50 mM) containing no urea and 20 μL methanol solution in the first 30 min of the reaction mixture. The 100% initial activity was determined by adding a 15 μL methanol solution in the first 30 min of the reaction mixture. The urease inhibition (%) was calculated by the following formula:

where a is the absorbance of inhibitory well, b is the absorbance of 100% initial activity without an inhibitor, and c is the absorbance of the endogenous nitrogen (blank). All the assays were performed in triplicate.

One compound was selected for further determination of the IC₅₀ value and inhibition mode based on the most potent inhibition potency at the concentration of 0.5 mM. Different concentrations of the compound were added to the assay mixture at 500, 250, 125, 62.5, 41.7, 27.8, 18.5, 8.2, and 0 µM final concentrations for determination of the IC₅₀ value. The IC₅₀ value of acetohydroxamic acid was also tested at the same series of concentrations as a reference. The residual activity of the addition of each concentration of the compounds was calculated by the following formula:

The IC₅₀ value was calculated by plotting each concentration of urease inhibitor against the corresponding residual activity of the enzyme using the nonlinear regression curve fitting function of GraphPad Prism, version 8.0.1 (GraphPad Software, San Diego, CA, USA).

The kinetic of the compound was determined by varying the concentration of urea in the presence of different compound 6238-0047 concentrations of 0.25 and 0.5 mM in the assay mixture. The control group only added the solvent of compound 6238-0047. The concentrations for the substrate urea varied between 100, 50, 25, 12.5, 6.25, and 3.125 mM in the assay mixture, and the procedure for measuring urease activity was the same as the urease inhibition assay. Maximal initial velocities were determined from the initial linear portion of the absorbance up to 10 min after the addition of the enzyme.

Kinetic parameters Km and Vmax were calculated by the nonlinear fitting of different substrate concentrations against the velocities of the reaction at each different concentrations of compound 6238-0047 using the GraphPad Prism software as mentioned before.