3.2. In Vitro HDACs Inhibitory Assay

All the HDAC enzymes were brought from BPS Bioscience. Ten μL of enzyme solution (HDAC1, HDAC6 or HDAC8) was mixed with different concentrations of tested compound (50 μL). The mixture was incubated at 37 °C for 5 min, followed by adding 40 μL fluorogenic substrate (Boc-Lys(acetyl)-AMC for HDAC1 and HDAC6, Boc-Lys(triflouroacetyl)-AMC for HDAC8). After incubation at 37 °C for 30 min, the mixture was quenched by addition of 100 μL of developer containing trypsin and Trichostatin A. Over another incubation at 37 °C for 20 min, fluorescence intensity was measured using a microplate reader at excitation and emission wavelengths of 390 nm and 460 nm, respectively. The inhibition rates were calculated from the fluorescence intensity readout of tested wells relative to those of control wells, and the IC₅₀ values were calculated using Prism non-linear curve fitting method.