Biochemistry fluorescence assay of NEP inhibitors

Biochemistry fluorescence assay of NEP inhibitors were performed by Molecular Devices Flexstation III (Molecular Devices) which can provided the fluorescence detection²². NEP can catalyze the substrate into fluorescent production. So the fluorescence intensity can indicate the inhibition activity of compounds. When the compounds with inhibition activity put into the system, the enzyme produce less production and the fluorescence would weaken. Recombinant human NEP expressed and purified from HEK 293 cells and positive NEP inhibitor of DL-Thiorphan was supplied by Sigma-Aldrich. Substrate of DNS-Gly-(pNO₂)Phe-β-Ala (DGNPA) for NEP were chemically synthesized by the Beijing Scilight Biotechnology Ltd., Co. (Beijing, China). TCM NEP inhibitor of 3,5,3′-triiodothyronine was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

The sample divided into six groups. Group 1 was considered 100% hydrolyze group, with substrate and enzyme. Group 2 added substrate without the enzyme, and considering as control group. Group 3 was received the substrate with enzyme and DL-Thiorphan in 50 μM as the positive control. The control group was used to verify the experiment system. Group 4, 5, 6 was given the substrate with enzyme and 3,5,3′-triiodothyronine at the doses of 50, 100, and 200 nM.

NEP (final concentration 1 µg/mL) was incubated for 15 min at 37 °C in a total volume of 100 µL of 50 mM Tris/HCl buffer, pH 7.4 (final concentration 1 μM) and increasing concentrations of 3,5,3′-triiodothyronine^23,24. The reaction was initiated by the addition of the DGNPA to a final concentration of 37 µM, and was stopped after 30 min at 37 °C by adding 100 µL of DMSO. The 200 µL mixture was measured by fluorescence (λ_ex = 342 nm, λ_em = 525 nm). All the measurements were done in black 96-well plates. Mixtures of 0% hydrolysis consisted of the buffer and DGNPA only, while mixtures of 100% hydrolysis were the group without inhibitor²⁵. The inhibition ratio of NEP was computed by reference to 100% hydrolysis and the inhibition activity values of 3,5,3′-triiodothyronine were determined accordingly.