HDAC Inhibition Assays

Assays were performed in 96-well plates (Fischer Scientific, cat. #: 3686) in HEPES buffer (final volume: 25 µL/well). Asuha-containing peptide inhibitors (5-fold dilution series, or 50 µM and 5 µM concentration for HDAC4) were incubated with enzyme [HDAC1 (0.5‒4 nM), HDAC2 (0.5‒3 nM), HDAC3/NCoR2 (1 nM), HDAC8 (0.2‒0.5 nM), HDAC4 (0.01 nM) or HDAC6 (0.5‒1 nM)] and substrate [HDACs 1‒3 and 6: Ac-Leu-Gly-Lys(Ac)-AMC (20 µM), HDACs 4 and 8: Ac-Leu-Gly-Lys(Tfa)-AMC (20 µM and 100 µM, respectively)] for 30 min at 37 °C (AMC: 7-amino-4-methylcoumarin). A solution of trypsin (25 µL, 0.4 mg/mL; final concentration of 0.2 mg/mL) was subsequently added, and the assay was allowed to develop for 15 min at room temperature. Then, fluorescence was recorded and analyzed to afford residual enzymatic activity relative to control wells and, assuming a standard fast-on/fast-off mechanism, IC₅₀ values were obtained by fitting the resulting data to the dose–response equation with variable Hill slope (Eq. ¹). Inhibition K_i values were obtained by using the Cheng-Prusoff equation (Eq. ²) and Michaelis-Menten constants as reported (HDAC1: K_M = 6 µM, HDAC2: K_M = 3 µM, HDAC3: K_M = 6 µM, HDAC6: K_M = 16 µM, HDAC8: K_M = 190 µM)⁵⁷. Assays were performed twice with two internal replicates, and data was analyzed using GraphPad Prism 7 or 8.