Resminostat hydrochloride (RAS2410 hydrochloride) is an effective inhibitor of HDAC1/HDAC3/HDAC6 (IC50: 42.5/50.1/71.8 nM), respectively, and shows less potent activities against HDAC8 (IC50: 877 nM).

HDAC3:50.1 nM, HDAC1:42.5 nM, HDAC6:71.8 nM, HDAC8:877 nM

Resminostat hydrochloride (5 μM) induces histone acetylation in myeloma cells. Resminostat hydrochloride has a substrate competitive binding mode (Ki: 27 nM). Resminostat hydrochloride (5 μM) induces histone hyperacetylation in myeloma cells. Resminostat inhibits cell growth, induces apoptosis and inhibits MM cell proliferation. Resminostat (5 μM) also modulates expression of bcl-2 family proteins and inhibits Akt pathway signalling downstream of Akt. Resminostat inhibits cell growth in head and neck squamous cell carcinoma cell lines (IC50 for SCC25: 0.775 μM; CAL27: 1.572 μM; and FaDu: 0.899 μM). Resminostat (1.25/2.5 μM) has a synergistic effect with irradiation on HNSCC cell lines. Resminostat in combination with cisplatin induces a downregulation of survivin. However, Resminostat shows no effect on Mcl-1 and p-AKT expression. Resminostat reduces viability of HCC cells with the co-therapy of AZD-2014, (IC50s: 0.07-0.89 μM).

Forty microliter enzyme buffer (15 mM Tris HCl pH 8.1, 0.25 mM EDTA, 250 mM NaCl, 10% v:v glycerol) containing HDAC1, 3, 6 or 8 activity, 29 μL enzyme buffer and 1 μL resminostat [HCl] at different concentrations are added to a 96-well microtitre plate and the reaction started by the addition of 30 μL substrate peptide Ac-NH-GGK(Ac)-AMC (HDAC1, 3 and 6 assays, final concentrations 6 μM for HDAC1, 10 μM for HDAC6 and 25 μM for HDAC3/DAD) or Ac-RHK(Ac)K(Ac)-AMC (HDAC8 assay, final concentration 50 μM). After incubation for 180 min (HDAC1, HDAC6, HDAC8) or 120 min (HDAC3) at 30°C, the reaction is terminated by the addition of 25 μL stop solution (50 mM Tris HCl pH 8, 100 mM NaCl, 0.5 mg/mL trypsin and 2 μM trichostatin A [TSA]). After incubation at room temperature for further 40 min, fluorescence is measured using a Wallac Victor2 1420 multilabel counter (extinction 355 nm, emission 460 nm) for quantification of AMC generated by tryptic cleavage of the deacetylated peptide. For the calculation of the 50% inhibitory concentration (IC50) values the fluorescence in wells without test compound (1% DMSO, negative control) is set as 100% enzymatic activity and the fluorescence in wells with 2 μM TSA (positive control) are set at 0% enzymatic activity (background fluorescence substracted).

Resminostat is dissolved in DMSO.A CCK-8 cell proliferation assay is used to investigate the antiproliferative effect of resminostat on HNSCC cells. Cells are seeded into 96-well plates at a density of 3 × 105/well. After 24 hours of growth, the cells are treated with resminostat and cisplatin, either alone or in combination and incubated for 72 hours. Untreated cells maintained in RPMI and equal concentrations of dimethylsulfoxide served as control. After 72 hours, cell proliferation is measured by CCK-8.