Bz-Nle-Lys-Arg-Arg-AMC (Synonyms: Benzoyl-Nle-Lys-Arg-Arg-AMC, Benzoyl-Nle-Lys-Arg-Arg-7-amino-4-methylcoumarin, Bz-Nle-Lys-Arg-Arg-AMC)

Preparation Method

FRET assay was performed to evaluate NS2B-NS3 protease activity and its inhibition by flavonoids. NS2B-NS3 protease in buffer solution (20 mM HEPES, 20 % glycerol and 1 mM CHAPS, pH 9.0) was pre-incubated at 37 °C for 30 min, and the reaction was initiated by adding 100 μM of the substrate Bz-Nle-Lys-Arg-Arg-AMC. The fluorescence was monitored per 15 s in 10 min with a microplate spectrophotometer at 360 nm and 450 nm for excitation and emission, respectively.

The inhibition assay was performed in 100 μL of buffer containing 20 mM HEPES (pH 9.0), 20 % glycerol, 1 mM CHAPS, 0.1 μM NS2B-NS3 protease and 100 μM substrate with varying concentrations of inhibitors. NS2B-NS3 protease was pre-incubated with inhibitors at 37°C for 30 min, and the reaction was initiated by adding 100 μM of the substrate Bz-Nle-Lys-ArgArg-AMC. After 10 min, the fluorescence was monitored with a microplate spectrophotometer at 360 nm and 450 nm for excitation and emission, respectively. Aprotinin (1 μM) was used as a positive control.

Reaction Conditions

100 µM for 10 min

References:

[1]: Zou M, Liu H, Li J, Yao X, Chen Y, Ke C, Liu S. Structure-activity relationship of flavonoid bifunctional inhibitors against Zika virus infection. Biochemical pharmacology. 2020 Jul 1;177:113962.