Boc-Gly-Arg-Arg-AMC

Protocol for protease assay [1]:

1. Preparing an enzymatic reaction mixture with a total volume of 100µL containing standard buffer solution (Tris HCl buffer pH 8.5), a constant amount (0.57 µmol/L) of DENV2 NS2B-NS3 solution and the candidate inhibitor compound (200 mg/L in DMSO).
2. The mixtures were incubated at 37℃ and shaken at 200 rpm for 10 min, and then 10 mM of peptide substrate Boc-Gly-Arg-Arg-AMC was added to initiate the reaction and incubation with shaking at 200 rpm was carried out for another 60 min.
3. The assays were in triplicate and corrected by subtracting the absorbance of their respective blank.
4. The selected compounds were used to generate an IC₅₀ value in the range (6.25-200 mg/L) of sample concentration.
5. The IC₅₀ values were determined graphically from inhibition curves (log inhibitor concentration vs. percentage of inhibition) using graphpad module.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1]. Osman H, Idris NH, et,al. 3,5-Bis(arylidene)-4-piperidones as potential dengue protease inhibitors. Acta Pharm Sin B. 2017 Jul;7(4):479-484. doi: 10.1016/j.apsb.2017.04.009. Epub 2017 May 4. PMID: 28752033; PMCID: PMC5518655.