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. 2020 Jan 15;11(5):706–712. doi: 10.1021/acsmedchemlett.9b00560

Structural and in Vivo Characterization of Tubastatin A, a Widely Used Histone Deacetylase 6 Inhibitor

Sida Shen ‡, Michal Svoboda §, Guangming Zhang ∥, Maria A Cavasin ⊥,#, Lucia Motlova §, Timothy A McKinsey ⊥,#, James H Eubanks ∥, Cyril Bařinka §, Alan P Kozikowski †,*
PMCID: PMC7236036  PMID: 32435374

Abstract

graphic file with name ml9b00560_0006.jpg

Tubastatin A, a tetrahydro-γ-carboline-capped selective HDAC6 inhibitor (HDAC6i), was rationally designed 10 years ago, and has become the best investigated HDAC6i to date. It shows efficacy in various neurological disease animal models, as HDAC6 plays a crucial regulatory role in axonal transport deficits, protein aggregation, as well as oxidative stress. In this work, we provide new insights into this HDAC6i by investigating the molecular basis of its interactions with HDAC6 through X-ray crystallography, determining its functional capability to elevate the levels of acetylated α-tubulin in vitro and in vivo, correlating PK/PD profiles to determine effective doses in plasma and brain, and finally assessing its therapeutic potential toward psychiatric diseases through use of the SmartCube screening platform.

Keywords: HDAC selectivity, zinc-binding group, phenylhydroxamate, brain penetration, neurological disorders

Histone deacetylases (HDACs) are a class of enzymes responsible for removing acetyl groups from the lysine side chains of proteins. A total of 18 known mammalian HDACs have been identified that are divided into Zn2+-dependent (Class I, IIa, IIb, IV) and NAD+-dependent (Class III) families.1 The NAD+-dependent sirtuins (SIRTs) are for the most part unaffected by HDAC inhibitors.2 Compared to other isoforms, HDAC6, which belongs to the Class IIb, is structurally and functionally unique. It contains two tandem deacetylase domains (CD1 and CD2), the C-terminal zinc finger domain with ubiquitin-binding properties (ZnF-UBP), and the N-terminal microtubule-binding domain.3,4 Moreover, HDAC6 is largely localized within the cytosol and is responsible for regulating the acetylation of specific cytosolic nonhistone proteins, including α-tubulin, HSP-90, peroxiredoxin, cortactin, and HSF-1.4 α-Tubulin was recognized as the first physiological substrate of HDAC6, and acetylation at lysine 40 of α-tubulin regulated by HDAC6 is involved in the microtubule-mediated intracellular trafficking and signaling in neuronal cells.5,6 It has been reported that SIRT2, a NAD+-dependent deacetylase, can also regulate the acetylation status of tubulin.7 However, several later studies have demonstrated that it is HDAC6 that acts as the primary tubulin deacetylase in vitro and in vivo.4,8,9 In the past decade, a number of pharmacological studies demonstrated that selective inhibition of HDAC6 is an effective approach to ameliorate symptoms of neurodegenerative diseases associated with defective axonal transport or impaired brain-derived neurotrophic factor (BDNF) trafficking, such as Alzheimer’s disease (AD),10 Charcot–Marie–Tooth Disease (CMT),11−13 amyotrophic lateral sclerosis (ALS),14,15 and Rett syndrome (RTT).16,17 Therefore, HDAC6 has emerged as an attractive target for discovering potential therapies for a variety of neurological disorders.

HDAC inhibitors share a common pharmacophore template consisting of a zinc-binding group (ZBG) that coordinates the zinc ion in the active site, a linker that occupies the hydrophobic tunnel, and a capping group that interacts with amino acid residues present in the rim region. In 2010, we built a HDAC6 homology model based on a HDAC8 crystal structure and found that the rim region of the HDAC6 pocket is much wider (17.5 Å) than that of HDAC1 (12.5 Å). An ensuing modeling study led to the eventual identification of a tetrahydro-γ-carboline-capped selective HDAC6 inhibitor (HDAC6i), named tubastatin A (TubA), bearing a phenylhydroxamate as a linker-ZBG.18 The discovery of TubA has sparked an extensive interest in the development of novel selective HDAC6is in the past decade.19 Recent findings also suggest that TubA is a potent HDAC10 inhibitor, as determined from two ligand displacement assays.20 This report stands in contrast to our earlier studies that indicated only modest potency against HDAC10.18,21 These differences are difficult to rationalize at present and may warrant further studies. One explanation might stem from differences in the assay types used, namely, probe displacement assays vs enzymatic assays, respectively.

Recent crystallographic studies of Danio rerio HDAC6 (drHDAC6) complexes with phenylhydroxamate-based selective HDAC6is revealed an unusual monodentate Zn2+ coordination geometry in the active site. At the same time, a bidentate Zn2+ coordination is observed in the majority of HDAC6 complexes with hydroxamate-based HDACis comprising a flexible aliphatic linker.22−24 Furthermore, the phenylhydroxamate moiety usually nestles in the hydrophobic tunnel established by F583 and F643, engaging in a double π-stacking arrangement. Clearly, the combination of the rigid phenylhydroxamate moiety and a bulky, hydrophobic capping group engaging the L1-loop pocket of HDAC6 (comprising residues H463, P464, F583, and L712) is critical for stabilization of the monodentate coordination mode that is slightly less energetically favorable (0.5 kcal/mol) compared to the bidentate-binding mode.25

To understand the molecular basis of interactions between TubA and HDAC6, we solved a crystal structure of the drHDAC6-CD2/TubA complex to an ultrahigh resolution limit of 1.1 Å. As cocrystallization experiments have not yielded any diffraction quality crystals, the complex was prepared by soaking 4 mM TubA solution into ligand-free HDAC6 crystals for 1 h. The Fo–Fc difference electron density map representing the active-site-bound TubA was of excellent quality and allowed for the unambiguous fitting of the inhibitor in the final stages of the refinement (Figure 1A). The hydroxamate moiety coordinates the active-site Zn2+ ion in a monodentate fashion similar to previously reported structures of related phenylhydroxamates (Figures 1B and S1 and Table S1).22,24−28 Likewise, the phenyl ring of the linker is wedged between the side chains of F583, F643, and H614 with distances of 3.7, 4.6, and 5.0 Å between the respective ring centers. The indole moiety of the cap group is positioned against a hydrophobic patch delineated by amino acids of the L1-loop pocket—side chains of H463, P464, F583, and L712 (Figure 1C). On the other hand, the methylpiperidine moiety of the cap is located in a solvent-rich environment (five surrounding water molecules), and its direct interactions with the enzyme are limited to van der Waals contacts with the side chains of F643 and L712 (Figure 1C).

Figure 1.

Figure 1

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Crystal structure of the drHDAC6-CD2/TubA complex. (A) Detailed view of the residues delineating the active site and the substrate tunnel. The Fo–Fc map (green mesh) is contoured at 4.0 σ, and the inhibitor and selected HDAC6 residues are shown in stick representation with atoms colored gray/yellow (carbon), red (oxygen), and blue (nitrogen). The active-site zinc ion and water molecules are shown as orange and red spheres, respectively. (B) Detailed view of monodentate coordination around the active-site zinc ion. The inhibitor and selected HDAC6 residues are shown in stick representation with the same coloring schemes as in Figure 1A. (C) Interactions between the capping moiety and the “L1-loop pocket” formed by the side chains of H463, P464, F583, and L712 (colored blue). The surface of HDAC6 is shown in a semitransparent surface representation.

In the original article, TubA exhibited neuroprotection in a dose-dependent manner (1.0–10 μM) in a homocysteic acid (HCA)-induced neuron model of oxidative stress, via elevating the acetylated status of peroxiredoxin, another physiological HDAC6 substrate, thus qualifying TubA as the first neuroprotective hydroxamic-acid-based HDACi without neurotoxicity.18 This initial finding inspired the further investigation of TubA in various animal models of neurological disorders. A summary in Table S3 indicates that administration of TubA for weeks in a dose range from 25 to 100 mg/kg by intraperitoneal (IP) or subcutaneous (SC) injection exhibited efficacy and tolerability in distinct animal models of neurological disorders, autoimmune diseases, and cardiac and pulmonary diseases. Recent findings reveal that TubA’s brain penetration is limited (B/P ratio = 0.15) due to a high efflux ratio of the drug,21 and treatment of TubA at the dose of 10 mg/kg (IP) did not enhance the level of acetylated α-tubulin (Ac-α-tubulin) in the brains of wild-type mice.29 Intriguingly, TubA administration (25 mg/kg, IP) exhibited efficacy in the models of Alzheimer’s disease (AD),30,31 Parkinson’s disease (PD),32 and stroke,33 thus demonstrating its blood–brain barrier (BBB) permeability under these conditions. Therefore, to better understand the required amount of TubA in vivo, we carried out PK/PD studies to measure the effective dose.

IV and PO plasma PK parameters of TubA were measured in CD1 mice and are shown in Table 1. The half-lives through two administrations are less than 1 h with a high plasma clearance rate (IV), resulting in low AUC values that may be related to the compound’s instability in mouse plasma (t1/2 = 30 min).34TubA is relatively stable in mouse liver microsome and hepatocyte (t1/2 > 60 min),34 while its high efflux ratio (9.83) significantly impairs its absorption in the GI tract,21 resulting in low oral bioavailability (∼6%). IP injection is thus a preferred administration route that circumvents efflux in the GI tract. Additionally, TubA has a high Vss value (4.14) indicating broad distribution.

Table 1. Mouse Plasma PK Studies of TubAa.

PK parameters IV (3 mg/kg) PO (30 mg/kg)
Cl (mL/min/kg) 222 n/a
t1/2 (h) 0.35 0.86
C0 (ng/mL) 1245 135
C1h (ng/mL) 29.7 41.9
AUC (h*ng/mL) 227 134
Vss (L/kg) 4.14 n/a
F% n/a 5.9
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a

PK parameters were profiled by Pharmaron, Inc. (Irvine, CA). TubA was tested in a formulation composed of 5% DMSO in “10% HP-β-CD in saline” (pH adjusted with 0.5 M HCl). Data are presented as the mean from three male CD1 mice.

As Ac-α-tubulin has been considered as an effective biomarker to reflect the functional effect of HDAC6is in the clinic,35 we carried out a PD study with TubA (10 mg/kg, IP) to measure the change of Ac-α-tubulin in murine heart. We found that Ac-α-tubulin reached its maximum level at 1 h after administration and subsequently decreased in a time-dependent manner (Figure 2). As TubA exhibits good liver metabolic stability and therefore a first-pass effect may not significantly influence its absorption, we used the IV PK data in Table 1 to predict the TubA concentration in plasma upon IP injection (10 mg/kg). The calculated total concentrations at the initial point (C0) and 1 h time point (C1h) are around 3735 and 89 ng/mL (Table 1), which are equal to 11.1 and 0.27 μM, respectively. Protein-binding data in mouse plasma (66.05%, performed by Pharmaron) allowed us to determine the concentration of unbound TubA at the initial point (3.77 μM) and 1 h (0.09 μM). With an EC50 value for the increase of intercellular Ac-α-tubulin levels of 0.145 μM (measured in N2a cells; Figure 3), TubA’s initial unbound concentration is thus 26-fold greater than its EC50, causing a significant increase of Ac-α-tubulin at the dose of 10 mg/kg. However, the unbound concentration had fallen below the EC50 value at 1 h. Notably, only a trace amount of TubA was detected in the PK studies after 2 h due to its short half-life. However, the Ac-α-tubulin level remained elevated up to 8 h, indicating that the initial drug exposure plays a critical role in triggering α-tubulin acetylation.

Figure 2.

Figure 2

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PD studies of TubA in murine heart. Male C57BL/6 mice were treated with TubA (10 mg/kg, IP) and sacrificed at 1, 4, 8, and 24 h time points after administration (n = 3–4 per group). Tissue punches from mouse left ventricles were collected, and the levels of Ac-α-tubulin were measured as a biomarker to reflect the effect of TubA. Data are expressed as the mean and standard error of the resulting normalized densitometric ratios of Ac-α-tubulin to total tubulin. * denotes statistical significance compared to vehicle at p < 0.05.

Figure 3.

Figure 3

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Structures of 1, 2, and TubA and effects on intracellular tubulin acetylation levels. α-Tubulin acetylation was evaluated by Reaction Biology Corp. (Malvern, PA) in N2a cells. Compounds were tested in a 10-dose EC50 mode in singlet with 3-fold serial dilution starting at 100 μM.

Although TubA exhibits limited BBB permeability, it showed efficacy in several animal models of CNS disorders. Therefore, we investigated its functional ability to increase the levels of Ac-α-tubulin in the brain cortex and compared it to two brain-penetrant HDAC6is. In our prior work, pyrazole-capped compound 1 and 6-fluoroindoline-capped compound 2 were identified as selective HDAC6is with favorable physicochemical properties and permeability coefficients in the MDCK-MDR1 cell assay, suggesting good brain uptake.21 A 10-dose EC50 mode α-tubulin acetylation assay in N2a cells validated that 1, 2, and TubA are capable of increasing the levels of Ac-α-tubulin in a dose-dependent manner and in a good correlation with their HDAC6 potency.

Brain/plasma PK studies demonstrated that compounds 1 and 2 exhibited good brain exposure in mice at the time point of 8 min (Figure 4). Although the B/P ratios increase at the 60 min time point, the brain levels at equilibrium are very low, possibly due to drug efflux.

Figure 4.

Figure 4

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Brain/plasma PK studies in mouse and α-tubulin acetylation in the mouse brain cortex. PK studies were performed by Pharmaron. Drugs were administered to two male CD1 mice via IV injection (3 mg/kg). Brain and plasma samples were collected at 8 and 60 min time points. Data are presented as the mean ± standard error. α-Tubulin acetylation in the mouse cortex: normalized densitometric ratios of Ac-α-tubulin to total tubulin in cortex 90 min following the second administration of an indicated drug. Compounds were administered to adult male C57BL/6 mice at the dose of 20 mg/kg by two successive IP injections separated by 4 h. Mice were sacrificed 1.5 h following the second injection, and cortical homogenates were isolated and assayed sequentially by Western blotting (n = 3–4 per group). The 100% value reflects the observed baseline ratio in cortical samples from vehicle-treated subjects. Data are expressed as the mean and standard deviation of the resulting ratiometric values. * and ** denote statistical significance compared to vehicle at p < 0.05 and p < 0.01, respectively.

As anticipated, TubA exhibited a low brain uptake (<100 ng/g) consistent with its high efflux ratio. Animal studies using TubA demonstrated that the impaired levels of Ac-α-tubulin in the brain of CNS disorder models (e.g., AD and PD) were reversed at the daily dose of 25 mg/kg through IP injection (Table 1).30−33 Based on the brain PK data in Figure 4 and the brain-homogenate-binding result (90.6%, determined by Pharmaron), we calculated that the free drug exposure in the wild-type mouse brain at 8 min after 25 mg/kg administration is just around 0.1 μM, which is lower than its EC50 in the N2a neuronal cell line. It is evident that the BBB exhibits breakdown and disruption in mouse models of AD and other neurodegenerative disorders,36 causing normally poorly BBB-penetrable HDAC6is, such as TubA (25 mg/kg, IP) and ACY-1215 (25 mg/kg, IP),30 to become more brain accessible, thus allowing them to reach effective doses in the brain in these neurodegenerative models.

Compounds 1, 2, and TubA were administrated to mice at doses of 20 mg/kg by IP injection twice a day to overcome their short half-lives in brain tissue. The levels of Ac-α-tubulin in the mouse brain cortex were measured to compare the functional activity of these three compounds. Results shown in Figure 4 demonstrate that all studied compounds significantly increased Ac-α-tubulin levels, while the rather minor differences that were observed are, however, inconsistent with their PK profiles. As compound 1 has lower cellular potency compared to 2 and TubA (Figure 3), a higher dose might be required to reach an effective drug concentration. Compound 2 and TubA showed similar ability to increase Ac-α-tubulin levels. Compound 2 is more brain-penetrant but is also more lipophilic, which may lead to higher brain tissue-binding, resulting in lower concentration of the free, unbound drug.37 Additionally, Ac-α-tubulin accounts for approximately 75% of the total brain tubulin, so any putative increase in Ac-α-tubulin levels would be marginal and difficult to quantify accurately.21 To the contrary, lower levels of Ac-α-tubulin are detected in the brains of neurodegenerative disorder models (e.g., AD) that improve the dynamic range of this assay, allowing for more accurate quantification upon inhibitor treatment.10,33,38

HDAC6is rescue phenotypes in distinct models of neurodegenerative diseases through their ability to regulate axonal transport deficits, remove protein aggregates, and rescue oxidative stress. It has been reported that hdac6-deficient mice exhibit hyperactivity, reduced anxiety, and antidepressant-like behavior,39 while pharmacological inhibition of HDAC6 using NCT-14b or ACY-738/775 showed antidepressant effects.29,39 We thus assessed behavioral effects of TubA using a SmartCube screening platform,40 to explore its therapeutic potential toward psychiatric diseases. The behavioral signatures of mice were captured by a digital video camera, analyzed by computer algorithms, and further compared with a database of behavioral signatures obtained using a set of diverse reference drugs clinically used in treatments of psychiatric diseases.41,42 As shown in Figure 5, the behavioral signatures of TubA at 60 mg/kg (IP) after 15 min of pretreatment revealed anxiolytic, antidepressant, antipsychotic, and cognitive effects with minor side-effects.

Figure 5.

Figure 5

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TubA (60 mg/kg, IP) produced a signature of activity suggesting therapeutic potential toward psychiatric diseases. Compound was injected 15 min before testing. SmartCube assessment was performed by PsychoGenics, Inc. (Tarrytown, NY).

In summary, TubA is a widely used HDAC6 inhibitor with significant therapeutic potential in distinct animal models of neurodegenerative disorders, autoimmune diseases, cardiovascular diseases, and other diseases. In this work, we revealed the molecular basis for its selective inhibition of HDAC6 by solving the structure of a drHDAC6-CD2 crystal complex. Furthermore, we determined its functional ability to enhance the levels of Ac-α-tubulin in vitro and in vivo, correlating these with the ADMET profiles of TubA in plasma and brain. Additionally, we explored new therapeutic potential of TubA toward psychiatric diseases in a behavioral screening platform, which represents the first instance of using this technology to evaluate an HDAC6 inhibitor.

Acknowledgments

This article is dedicated to the memory of Professor Mauricio Botta, a long-time friend and colleague who has served as an inspiration to medicinal chemists, both young and old. We thank Petra Baranova for excellent technical assistance and Jiri Pavlicek for help with crystallization experiments. The synchrotron MX data were collected at beamline P13 operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). We thank T.R. Horn for the assistance with pharmacodynamics analysis of Tubastatin A. We also thank Dr. Werner Tueckmantel for proofreading the article and providing comments.

Glossary

Abbreviations

PK

pharmacokinetics

PD

pharmacodynamics

NAD+

nicotinamide adenine dinucleotide

IP

intraperitoneal

SC

subcutaneous

IV

intravenous

PO

pre-os

Cl

clearness rate

AUC

area under curve

Vss

steady state volume of distribution

F%

oral bioavailability

GI

gastrointestinal

N2a

Neuro2a

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00560.

  • Supplementary figure and tables as well as details of crystallographic study and in vivo α-tubulin acetylation experiments (PDF)

Accession Codes

The atomic coordinates and crystallographic structure factors of the drHDAC6-CD2/tubastatin A complex have been deposited in the Protein Data Bank (www.rcsb.org) with accession code 6THV.

Author Present Address

○ Departments of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, and Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois 60208, United States. (S.S.)

Author Contributions

The manuscript was written through contributions from all the authors. All authors have given their approval to the final version of the manuscript.

This research was supported by NIH grants (NS079183, HD093464, and AG058283 to A.P.K. and HL147558, HL116848, HL127240, and DK119594 to T.A.M.) and CIHR grant (PJT-153015 to J.H.E). This work was also in part supported by the American Heart Association (16SFRN31400013 to T.A.M.), the Czech Science Foundation (15-19640S to C.B.), the CAS (RVO: 86652036 to C.B.), and the project “BIOCEV” (CZ.1.05/1.1.00/02.0109 to C.B.) from the ERDF. We acknowledge CMS-Biocev (“Biophysical techniques, Crystallization, Diffraction, Structural mass spectrometry”) supported by MEYS CR (LM2015043).

The authors declare no competing financial interest.

Supplementary Material

ml9b00560_si_001.pdf (725.3KB, pdf)

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