Inhibitory activities

ACY-1215 was 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 were 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 was equal to the Michaelis constant (Km), as determined by a titration curve. FTS or MAZ-1675 was diluted in assay buffer to 6-fold the final concentration with 0.3 μM sequencing grade trypsin. The substrate/trypsin mix was added to the enzyme/compound mix and the plate was shaken for 60 seconds and then placed into a SpectraMax M5 microtiter plate reader. The enzymatic reaction was 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 was calculated. HDAC11, sirtuin1, and sirtuin2 assays were performed by Cerep.

Cell lines

MM cells; PBMCs.

Preparation method

Soluble in DMSO > 10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

0.16, 0.8, 4, 20 nM; MM cells: 24 h, PBMCs: 48 h.

Applications

ACY-1215 induces less cytotoxicity in PHA-stimulated or unstimulated PBMCs. In purified CD4+ T cells, ACY-1215 induces toxicity with IC50 value of 2.5 μM. In MM cell lines, ACY-1215 dose-dependently decreases viability with IC50 value of 2-8 μM and induces significant cytotoxicity. In MM.1S cells, ACY-1215 dose-dependently reduces DNA synthesis of MM cells adherent to BMSCs and also inhibits growth induced by IL-6 and IGF-1.

Animal models

Male SCID mice inoculated subcutaneously with MM.1S cells.

Dosage form

50 mg/kg; 5 days a week for 3 weeks; administrated orally.

Preparation method

Dissolved in 10% DMSO in 5% dextrose in water

Applications

In human MM xenograft mouse model, ACY-1215 (50 mg/kg) significantly delays tumor growth. Treatment mice with ACY-1215 plus bortezomib significantly prolonged overall survival (OS) and induces a significant accumulation of polyubiquitinated proteins. Treatment with ACY-1215 plus bortezomib is well tolerated and have no significant influence on the body weight. In female SCID-beige mice inoculated intravenously with MM.1S-LucNeo cells, treatment with ACY-1215 (75 mg/kg) and bortezomib (1.5 mg/kg) significantly inhibits tumor growth and prolongs OS.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Santo L, Hideshima T, Kung AL, et al. Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma. Blood, 2012, 119(11): 2579-2589.