Cell experiment:

Undifferentiated RN46A-B14 cells, a line of immortalized rat raphe neuronal precursors, are grown. They are treated with 2.5 μM ACY-738, ACY-775, tubastatin A, 0.6 μM TSA or vehicle (0.1% DMSO) for 4 h. Samples are processed using histone extraction kit and quantified using protein assay.

Animal experiment:

Mice are tested for immobility in the TST. At 30 min or 2 h after i.p. injection of ACY-738 (5, 50 mg/kg), ACY-775 (5, 50 mg/kg), and citalopram (0.5, 2, 20 mg/kg), a combination of the previous, or vehicle, mice are attached to the test rig and time immobile over 6 min is recorded. For open-field activity mice are injected with ACY-738 or ACY-775 at 5, 10, or 50 mg/kg or vehicle and allowed to explore. Activity is recorded[2].

ACY-775 is a potent and selective inhibitor of the of histone deacetylase 6 (HDAC6) with an IC50 of 7.5 nM.

In vehicle-treated cells, α-tubulin is mainly presented in the deacetylated form, while histone 3 is clearly acetylated. Upon treatment with ACY-775, a clear enhancement of the acetylation of α-tubulin is visible, while histone acetylation remains unaltered. Acetylation of α-tubulin is visualized by immunofluorescence and the intensity in the neurites of the neurons is quantified and normalized to the length of the fluorescent signal. In vehicle-treated DRG neurons, acetylated α-tubulin is already present. Upon treatment with ACY-775 the signal intensity of acetylated α-tubulin increases significantly. Significant increase in motility of mitochondria and also the total number of mitochondria within the neurites are observed compare with vehicle-treated DRG neurons. A significantly higher number of retrogradely transport mitochondria is observed in DRG neurons treated with ACY-775 compare with vehicle-treated cells[1].

Biodistribution profiles of ACY-738, ACY-775, and tubastatin A are examined after acute dosing at 5 or 50 mg/kg over 2 h. At t=30 min after acute 50 mg/kg injection, respective plasma levels of ACY-738 and ACY-775 are 515 ng/mL (1.9 μM) and 1359 ng/mL (4.1 μM). Elimination from plasma is rapid, with plasmatic half-life of 12 min and concentration below 10 ng/mL after 2 h. Nevertheless, areas under concentration time curves for brain and plasm calculated over 2 h for both ACY-738 and ACY-775 lead to ratios >1. When ACY-738 (5 mg/kg) or ACY-775 (50 mg/kg) are administered repeatedly in wild-type mice at 24 h, 4 h, and 30 min before killing, significant increases in α-tubulin acetylation are observed in all tested brain regions[2].

[1]. Veronick Benoy, et al. Development of Improved HDAC6 Inhibitors as Pharmacological Therapy for Axonal Charcot-Marie-Tooth Disease. Neurotherapeutics. 2017 Apr; 14(2): 417-428. [2]. Jeanine Jochems et al. Antidepressant-Like Properties of Novel HDAC6-Selective Inhibitors with Improved Brain Bioavailability. Neuropsychopharmacology. 2014 Jan; 39(2): 389-400.