Tubastatin A (Tubastatin A BASE) is an effective and specific HDAC6 inhibitor (IC50: 15 nM, in a cell-free assay). Its selectivity is 1000-fold against all the other isozymes except HDAC8.

HDAC1:16400 nM, HDAC6:15 nM, HDAC8:854 nM

Tubastatin A (5 μM) dose-dependently protected neuronal cells from homocysteine-induced death, which was completely avoided at 10 μM. Tubastatin A (10 μM) slightly induced histone acetylation. When applied to cholangiocarcinoma cell lines, Tubastatin A (10 μM) induced an increase in the level of acetylated α-tubulin and the restoration of primary cilia expression, which correlated with the down-regulation of Hedgehog and MAPK signaling pathways, and a decrease in the rate of cell proliferation (50% on average) and infiltration (40%). Tubastatin A significantly inhibited TNF-α and IL-6 when acting on lipopolysaccharide-stimulated human THP-1 macrophages (IC50: 272/712 nM). When acting on mouse Raw 264.7 macrophages, Tubastatin A dose-dependently inhibited nitric oxide secretion (IC50: 4.2 μM).

Tubastatin A (5 μM) dose-dependently protected neuronal cells from homocysteine-induced death, which was completely avoided at 10 μM. Tubastatin A (10 μM) slightly induced histone acetylation. When applied to cholangiocarcinoma cell lines, Tubastatin A (10 μM) induced an increase in the level of acetylated α-tubulin and the restoration of primary cilia expression, which correlated with the down-regulation of Hedgehog and MAPK signaling pathways, and a decrease in the rate of cell proliferation (50% on average) and infiltration (40%). Tubastatin A significantly inhibited TNF-α and IL-6 when acting on lipopolysaccharide-stimulated human THP-1 macrophages (IC50: 272/712 nM). When acting on mouse Raw 264.7 macrophages, Tubastatin A dose-dependently inhibited nitric oxide secretion (IC50: 4.2 μM).

HDAC enzymatic assays: Tubastatin A is dissolved and diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, and 20 μM tris(2-carboxyethyl)phosphine) to 6-fold of the final concentration. HDAC enzymes are diluted to 1.5-fold of the final concentration in assay buffer and pre-incubated with Tubastatin A for 10 minutes before the addition of the substrate. The amount of FTS (HDAC1, HDAC2, HDAC3, and HDAC6) or MAZ-1675 (HDAC4, HDAC5, HDAC7, HDAC8, and HDAC9) used for each enzyme is equal to the Michaelis constant (Km), as determined by a titration curve. FTS or MAZ-1675 is diluted in assay buffer to 6-fold the final concentration with 0.3 μM sequencing grade trypsin. The substrate/trypsin mix is added to the enzyme/compound mix and the plate is shaken for 60 seconds and then placed into a SpectraMax M5 microtiter plate reader. The enzymatic reaction is monitored for release of 7-amino-4-methoxy-coumarin over 30 minutes, after deacetylation of the lysine side chain in the peptide substrate, and the linear rate of the reaction is calculated.

Anchorage-independent growth is assessed by growing cells in soft agar. About 25,000 cells suspended in 0.4% agar in culture media are layered over a 1% agar layer in a 6-well plate. Media are added twice a week and pictures are taken after 21 days of incubation. The number and size of colonies are analyzed using the Gel-Pro software.(Only for Reference)