Antidepressant-like properties of novel HDAC6-selective inhibitors with improved brain bioavailability

Abstract

HDAC inhibitors have been reported to produce antidepressant and pro-cognitive effects in animal models, however, poor brain bioavailability or lack of isoform selectivity of current probes has limited our understanding of their mode of action. We report the characterization of novel pyrimidine hydroxyl amide small molecule inhibitors of HDAC6, brain bioavailable upon systemic administration. We show that two compounds in this family, ACY-738 and ACY-775, inhibit HDAC6 with low nanomolar potency and a selectivity of 60- to 1500-fold over class I HDACs. In contrast to tubastatin A, a reference HDAC6 inhibitor with similar potency and peripheral activity, but more limited brain bioavailability, ACY-738 and ACY-775 induce dramatic increases in α-tubulin acetylation in brain and stimulate mouse exploratory behaviors in novel, but not familiar environments. Interestingly, despite a lack of detectable effect on histone acetylation, we show that ACY-738 and ACY-775 share the antidepressant-like properties of other HDAC inhibitors, such as SAHA and MS-275, in the tail suspension test and social defeat paradigm. These effects of ACY-738 and ACY-775 are directly attributable to the inhibition of HDAC6 expressed centrally, as they are fully abrogated in mice with a neural-specific loss of function of HDAC6. Furthermore, administered in combination, a behaviorally inactive dose of ACY-738 markedly potentiates the anti-immobility activity of a subactive dose of the selective serotonin reuptake inhibitor citalopram. Our results validate new isoform-selective probes for in vivo pharmacological studies of HDAC6 in the CNS and reinforce the viability of this HDAC isoform as a potential target for antidepressant development.

Figure 1

Selectivity, potency, and pharmacokinetic properties of HDAC6 inhibitors used in this study. Top row (a–c): dose-dependent enzymatic inhibition of recombinant HDAC1, HDAC2, HDAC3, and HDAC6 incubated with (a) tubastatin A, (b) ACY-738, or (c) ACY-775. Middle row: (d) heat map summary of IC50 values for the novel inhibitors ACY-738 and ACY-775 and reference compounds SAHA, MS-275, and ACY-1215 (n=2 per condition). Brighter reds denote higher inhibitory potency of compounds on corresponding HDAC isoform, black background indicates lack of activity. (e) Ratios of brain concentration to plasma concentration over time after acute 5 mg/kg i.p. injection. Bottom rows (f): distribution of HDAC2, HDAC3, HDAC6, and TPH2 mRNA as seen after in situ hybridization on sagittal views of the mouse brain. Average expression intensities range from low (blue) to high (red). Pictures courtesy of the Allen Mouse Brain Atlas (Allen Institute for Brain Science; http://www.brain-map.org/).

Figure 2

Effects of HDAC6 inhibitors on α-tubulin acetylation at lysine 40 (K40) and histone H3 acetylation at lysine 9 (H3K9) (a) in neuronal cell culture and (b, c) in the CNS in vivo after acute and subchronic administration. (a) In RN46A-B14 serotonergic cell line, treatment for 4 h with the non-selective HDAC inhibitor TSA (0.6 μM) or the selective HDAC6 inhibitors tubastatin A (TubA, 2.5 μM), ACY-738 (2.5 μM), and ACY-775 (2.5 μM) all increased α-tubulin acetylation at K40. In contrast, only TSA significantly increased H3K9 acetylation in RN46A-B14 cells. Results are shown as percent change normalized to vehicle treatment condition. The horizontal hard line depicts average and dotted lines depict SEM of vehicle-treated wells. Representative western blot images are presented. None of the treatments significantly altered total α-tubulin levels (n=3 per condition, middle row) or total levels of histone H3 protein (data not shown). (b) In vivo, a single i.p. administration of ACY-738 (5 mg/kg) and ACY-775 (50 mg/kg) increased K40 acetylated α-tubulin levels measured at 30 min, 1 h, and 4 h time-points in mouse whole-brain lysates. In contrast, no significant changes were detected at any time-point after administration of tubastatin A (10 mg/kg), as expected based on the limited CNS bioavailability of this compound. Representative western blot images are presented. (c) Increases in α-tubulin K40 acetylation induced by administration of ACY-738 (5 mg/kg) and ACY-775 (50 mg/kg) are occluded in neuron-specific HDAC6 KO mice that have hyperacetylated α-tubulin at baseline. Drugs (5 mg/kg) were administered subchronically (at 24 h, 4 h, and 30 min before killing) to HDAC6 KO and WT littermates. Changes in tubulin acetylation were measured in tissue lysates from cortex (ctx), hippocampus (Hpc), dorsal raphe nucleus (DRN), and cerebellum (cb). Tubulin acetylation levels in drug-treated mice are expressed as percent change from vehicle-treated mice of corresponding genotype. Horizontal lines depict average tubulin acetylation±SEM in vehicle-treated mice. Representative western blot images of the changes induced by each drug treatment, in the hippocampus (left column) and dorsal raphe (right column) of WT and HDAC6 KO mice. Note the markedly enhanced baseline levels of α-tubulin K40 acetylation in HDAC6 KO compared with WT mice treated with vehicle. Also note the lack of increase in KO treated with ACY-738 and ACY-775 in contrast to WT. (d) Histone H3 lysine 9 (H3K9) acetylation measured by ChIP is increased at BDNF promoter 4 and cFOS promoters after subchronic treatment with a high-dose sodium butyrate (1.2 g/kg). (e) Lack of effect of subchronic treatment with a behaviorally active dose of ACY-738 (5 mg/kg) on histone H3 lysine 9 (H3K9) acetylation at BDNF promoter 4 and cFOS promoters. *P<0.05, **P<0.01, ***P<0.001 vs vehicle (n=2–3 per condition).

Figure 3

Effects of HDAC6-selective inhibition in anxiety tests. (a–c) Activity was assessed in open-field test 1 h prior and 2 h after acute systemic administration of ACY-738, ACY-775, or tubastatin A. (a) ACY-738 and (b) ACY-775 increased exploration when administered at 50 mg/kg but not lower doses (arrow indicates drug administration). (c) Beam breaks accumulated over 2 h post treatment. Note the lack of effect of tubastatin A (10 mg/kg) and blockade of the effect of ACY-738 and ACY-775 in mice with neural cell-specific conditional HDAC6 KO. (n=5–8 per condition). (d) Acute administration of ACY-738 or ACY-775 produces anxiolytic-like effects in marble-burying test (n=10–18 per condition) and (e) in NIH test (n=8–11 per condition), but (f) has no effect in elevated plus maze (n=5 per condition). Data expressed as mean values±SEM. * P<0.05, ** P<0.01, ***P<0.001 vs vehicle.

Figure 4

HDAC6 inhibitors ACY-738 and ACY-775 have antidepressant-like properties. (a) In the TST acute administration of ACY-738 and ACY-775 produce anti-immobility effects in NIH Swiss mice resembling those of the reference antidepressant citalopram. Effects are seen 30 min after acute 50 mg/kg i.p. injection, but not for lower dose (5 mg/kg). Combination of subactive doses of HDAC6-selective inhibitors (5 mg/kg) and citalopram (0.5 mg/kg) produces robust potentiation of anti-immobility activity (n=8–29 per condition). (b) In C57BL/6J mice, the anti-immobility effect of ACY-738 is blocked by a neural cell-selective KO of HDAC6, whereas citalopram effect is potentiated by HDAC6 KO. No difference was observed between HDAC6 WT and KO when treated with vehicle (n=6–15 per condition). (c) Timeline showing treatment period, social defeat and social interaction (SI) testing. Chronic treatment with HDAC6-selective inhibitors prevents development of avoidance after social defeat as observed in (d) an increased average interaction time and (e) a decrease in time spent in the corners of the test arena, as compared with vehicle-treated mice that underwent social defeat. (f) In addition, treatment with SSRI or HDAC6 inhibitors results in a greater percentage of resilient mice compared with vehicle treatment. (g) No change in total distance traveled during the test period was observed (n=11–15 per condition). Data expressed as mean values±SEM. ⁺P<0.05 vs vehicle, ^#P<0.01 vs vehicle, *P<0.01 vs undefeated control.