Polyamine Inhibition of LSD1, LSD2 and MAO

LSD1 inhibition assay was performed using the peroxidase-coupled reaction method essentially as described previously.^{30,31 (link)} Human LSD1 (2.8 μM) was incubated with serial dilutions of each polyamine in 50 mM HEPES-Na (pH 7.5) buffer containing 400 μM 4-aminoantipyrine, modified Trinder's reagent TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, dihydrate), and 40 μg mL⁻¹ horseradish peroxidase at 25 °C for 10 min. The reaction mixture was subsequently incubated with 83 μM H3(1–20)K4-dimethylated (K4me2) peptide for 30 min. Inhibition assays for LSD2, MAO-A and MAO-B were performed in a similar manner to the LSD1 inhibition assay, using 167 μM H3(1–20)K4me2 peptide as the substrate for 2.7 μM LSD2, and 50 and 150 μM tyramine as the substrate for 1.4 and 2.8 μM MAO-A and MAO-B, respectively. Absorbance at 562 nm of the hydrogen peroxide by-products generated by H3(1–20)K4me2 demethylation or tyramine oxidation was measured with a 96-well microplate reader (Ultrospec Visible Plate Reader II 96; GE Healthcare). Steady-state reaction ranges from 0 to 10 min (LSD1, LSD2 and MAO-A) or 0 to 30 min (MAO-B) were used for fitting analysis with the equation of competitive inhibition via steady-state kinetic analysis to obtain K_i with GraphPad Prism 6 software (version 6.0e).^{40 (link)}