Biochemical and Behavioral Characterization of IN14, a New Inhibitor of HDACs with Antidepressant-Like Properties

Abstract

Evidence suggests that histone deacetylases (HDACs) inhibitors could be used as an effective treatment for some psychiatric and neurological conditions such as depression, anxiety and age-related cognitive decline. However, non-specific HDAC inhibiting compounds have a clear disadvantage regarding their efficacy and safety, thus the need to develop more selective ones. The present study evaluated the toxicity, the capacity to inhibit HDAC activity and antidepressant-like activity of three recently described class I HDAC inhibitors IN01, IN04 and IN14, using A.salina toxicity test, in vitro fluorometric HDAC activity assay and forced-swimming test, respectively. Our data show that IN14 possesses a better profile than the other two. Therefore, the pro-cognitive and antidepressant effects of IN14 were evaluated. In the forced-swimming test model of depression, intraperitoneal administration of IN14 (100 mg/Kg/day) for five days decreased immobility, a putative marker of behavioral despair, significantly more than tricyclic antidepressant desipramine, while also increasing climbing behavior, a putative marker of motivational behavior. On the other hand, IN14 left the retention latency in the elevated T-maze unaltered. These results suggest that novel HDAC class I inhibitor IN14 may represent a promising new antidepressant with low toxicity and encourages further studies on this compound.

Keywords: antidepressants; chromatin remodeling; class I HDAC; drug design; elevated T maze; epigenetics; forced-swimming test; toxicity.

Figure 1

(A) Chemical structures of compounds IN01 (left), IN04 (center) and IN14 (right). (B) Yield and purity of the new histone deacetylases (HDAC) inhibitors synthetized.

Figure 2

Percentage of mortality of A. salina exposed to HDAC inhibitors. Acute toxicity (LC₅₀) of IN01, IN04, IN14 and sodium phenylbutyrate (PB) on A. salina in seawater medium. The concentrations of the compounds (IN01, IN04 and IN14) and the HDAC inhibitor, phenylbutyrate (PB), are expressed in log10 concentration. Each point represents the mean ± SEM of 30 nauplii.

Figure 3

IN14 inhibits HDAC activity in HeLa Nuclear Extract. Data are expressed as mean ± SEM. Experiments were performed by technical triplicate. HDAC activity (arbitrary fluorescent units [AFU]); PB: phenylbutyrate; TSA: Trichostatin A (positive control); IN01-IN04-IN14: synthesized compounds. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Figure 4

Detection of IN14 by LC-MS. (A) IN14 chromatogram and electrospray ionization (ESI) (+) mass spectrum. Extracted ion chromatograms (EIC) of m/z = 236 in samples of cerebrospinal fluid (B), plasma (C), and urine (D). Mice (n = 5) were treated with IN14 for one day (red line) and five days (black line), fluid extraction and LC-MS analysis are described in the Materials and Methods section.

Figure 5

Performance of mice treated with vehicle, IN14, phenylbutyrate (PB) or Desipramine hydrochloride (DMI) during forced swimming test. Bars indicate the incidence of swimming (swim), climbing (climb) and immobility (immobile) during a 5-min test. Data are expressed as mean ± SEM (n = 8). * p  <  0.05; *** p  <  0.001 vs. vehicle.

Figure 6

Pre-training intraperitoneal administration of phenylbutyrate (PB) or IN14 does not affect memory performance. (A) Elevated T-maze learning curves of intact mice during the training session and retention latencies. * p < 0.05, *** p < 0.001 vs. the first acquisition latency (AL1), n = 9. (B) Escape latency from the open arm in the elevated T-maze (ETM) test. No significant differences were observed among different groups. ns: p = 0.5250, EL: escape latency; RL: retention latencies.