Ricolinostat (Rocilinostat) is an orally bioavailable, specific inhibitor of histone deacetylase 6 (HDAC6) with potential antineoplastic activity.

HDAC6:4.7 nM, HDAC8:100 nM, HDAC1:58 nM, HDAC3:51 nM, HDAC2:48 nM

在浆细胞瘤模型和弥散性MM模型中,ACY-1215易被肿瘤组织吸收并且不会在组织中积累,能够抑制肿瘤生长.

在极低剂量时,ACY-1215能够显著诱导α-tubulin乙酰化,同时在较高剂量时,能够诱导组蛋白H3和H4组蛋白赖氨酸的乙酰化。在HDAC4, HDAC5, HDAC7, HDAC9, HDAC11, Sirtuin1,和 Sirtuin2,对HDAC8中,ACY-1215,ACY-1215(IC50=0.1 μM) 有轻微的活性。

ACY-1215 is dissolved and subsequently 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 the final concentration. HDAC enzymes are diluted to 1.5-fold of the final concentration in assay buffer and pre-incubated with ACY-1215 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[1].

ACY-1215 is dissolved in DMSO (10 mM) and stored, and then in diluted with appropriate culture medium before use[1]. The effect of ACY-1215 with or without Bortezomib on the viability of MM cell lines, patient MM cells, and PBMCs is assessed by measuring MTT dye absorbance. PBMCs from healthy donors are isolated and stimulated with 2.5 μg/mL of phytohemagglutinin (PHA) for 48 hours in the presence of increasing concentrations of ACY-1215. DNA synthesis is measured by tritiated thymidine uptake. CD4+ T cells are purified from human blood with the Rosette Sep negative-selection kit. Cells are stimulated by CD3/CD28 Dynabeads for 7 days in the presence of compounds. Cell viability is assessed using alamarBlue. MM cells (2-3×104 cells/well) are incubated in 96-well culture plates with medium and different concentrations of ACY-1215, Bortezomib, and/or recombinant IL-6 (10 ng/mL) or insulin-like growth factor-1 (IGF-1; 50 ng/mL) for 24 hours at 37°C, and tritiated thymidine incorporation is measured[1].