DAC6 ( IC50 = 15 nM ); HDAC8 ( IC50 = 854 nM ); HDAC1 ( IC50 = 16400 nM )
Tubastatin A TFA targets histone deacetylase 6 (HDAC6) with an IC₅₀ value of 15 nM (fluorogenic substrate assay) [2]
Tubastatin A TFA exhibits high selectivity for HDAC6 over other HDAC isoforms: IC₅₀ > 10 μM (HDAC1), IC₅₀ > 10 μM (HDAC2), IC₅₀ > 10 μM (HDAC3), IC₅₀ > 10 μM (HDAC8), and IC₅₀ > 10 μM (HDAC10) [2]

With the exception of HDAC8, where it displays about 57-fold selectivity, tubastatin A exhibits > 1000-fold selectivity against all 11 HDAC isoforms and is highly selective for all 11. Tubastatin A shows dose-dependent protection against homocysteic acid (HCA)-induced neuronal cell death in assays for HCA-induced neurodegeneration, beginning at 5 μM and reaching nearly full protection at 10 μM[1]. In vitro, Foxp3+ T-regulatory cells (Tregs) reduce T cell proliferation more when exposed to 100 ng/mL with tubastatin A[2]. When alpha-tubulin is hyperacetylated early in the myogenic process, tubastatin A administration in CC12 cells would hinder myotube formation; nevertheless, myotube elongation happens when alpha-tubulin is hyperacetylated in myotubes[3]. According to a recent study, treating mouse ovarian cancer cell lines MOSE-E and MOSE-L with tubastatin A enhances cell flexibility as shown by atomic force microscopy (AFM) tests without significantly altering the actin microfilament or microtubule networks[4].

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Tubastatin A TFA (1–50 nM) dose-dependently inhibited HDAC6 activity in HeLa cell lysates, increasing acetylated α-tubulin levels by 2.5–6.8-fold (Western blot) [2]
- In 6-OHDA-induced SH-SY5Y neuroblastoma cell injury model: Tubastatin A TFA (10–100 nM) increased cell viability by 28–55% (MTT assay) and reduced apoptotic rate by 32–60% (Annexin V-FITC/PI staining) [2]
- The compound protected primary cortical neurons from glutamate-induced excitotoxicity: 50 nM concentration improved neuron survival by 48% and reduced lactate dehydrogenase (LDH) release by 42% [2]
- Tubastatin A TFA (50 nM) stabilized microtubule cytoskeleton in neuroblastoma cells, as evidenced by enhanced acetylated α-tubulin fluorescence intensity (immunofluorescence) [2]
- No significant cytotoxicity was observed in HeLa, SH-SY5Y, or primary cortical neurons at concentrations up to 1 μM [2]

Daily therapy of Tubastatin A at 0.5 mg/kg inhibits HDAC6 to enhance Tregs suppressive activity in mouse models of inflammation and autoimmunity, including numerous kinds of experimental colitis and completely major histocompatibility complex (MHC)-incompatible cardiac allograft rejection[2].

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In rat middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia model: Intraperitoneal injection of Tubastatin A TFA (5 mg/kg, administered at 0 and 24 hours post-reperfusion) reduced cerebral infarct volume by 45% compared to vehicle control [2]
- Tubastatin A TFA (5 mg/kg) improved neurological function in MCAO rats, with neurological deficit score reduced by 38% at 72 hours post-reperfusion [2]
- The compound increased acetylated α-tubulin expression by 3.2-fold in ischemic brain tissues (Western blot) and reduced neuronal apoptosis (TUNEL assay) [2]
- No significant body weight loss or histopathological abnormalities in liver, kidney, or heart were observed in treated rats [2]

HDAC6 fluorogenic substrate assay: Recombinant HDAC6 catalytic domain was incubated with serial dilutions of Tubastatin A TFA and a fluorogenic peptide substrate (acetyl-lysine-containing). The reaction was initiated by adding enzyme, and fluorescence intensity was measured after incubation at 37°C for 1 hour to quantify HDAC6 inhibition and calculate IC₅₀ [2]
- HDAC isoform selectivity assay: The compound was tested against recombinant HDAC1/2/3/8/10 using the same fluorogenic substrate assay to determine cross-reactivity and selectivity [2]

Neurotoxicity protection assay (SH-SY5Y): Cells were seeded in 96-well plates and pretreated with Tubastatin A TFA (10–100 nM) for 1 hour, followed by exposure to 6-OHDA (100 μM) for 24 hours. Cell viability was measured by MTT assay, and apoptotic cells were quantified by Annexin V-FITC/PI staining [2]
- Primary cortical neuron excitotoxicity assay: Cortical neurons were isolated from embryonic rats, cultured for 7 days, and pretreated with Tubastatin A TFA (10–50 nM) for 2 hours. Glutamate (100 μM) was added to induce excitotoxicity, and LDH release was measured 24 hours later to assess cell damage [2]
- Western blot analysis: Cells or tissue lysates were probed with antibodies against acetylated α-tubulin, total α-tubulin, and GAPDH. Band intensity was quantified by densitometry to evaluate HDAC6 inhibition [2]
- Immunofluorescence assay: SH-SY5Y cells were fixed, permeabilized, and stained with anti-acetylated α-tubulin antibody. Fluorescence was visualized by confocal microscopy to assess microtubule acetylation [2]

Solubilized in 10% Dimethyl sulfoxide (DMSO) 10% Polyethylene glycol (PEG) 400 and 80% (40% of hydroxy propyl beta cyclodextrin); Rats: 30 mg/kg/day i.p. for 5 days; mice: 30 mg/kg, q.d. from day 21 to day 36. Wistar rats; DBA1 mice

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Focal cerebral ischemia rat model (MCAO): Male Sprague-Dawley rats (250–300 g) underwent MCAO for 2 hours followed by reperfusion. Tubastatin A TFA was administered via intraperitoneal injection at 5 mg/kg immediately after reperfusion and again at 24 hours post-reperfusion [2]
- Drug formulation: Tubastatin A TFA was dissolved in dimethyl sulfoxide (DMSO) and further diluted with normal saline to a final DMSO concentration of ≤5% [2]
- Neurological deficit scoring: Rats were evaluated using a 5-point neurological deficit scale at 24, 48, and 72 hours post-reperfusion [2]
- Sample collection: At 72 hours post-reperfusion, rats were euthanized. Brains were harvested, sectioned, and stained with TTC to measure infarct volume. Ischemic brain tissues were collected for Western blot and TUNEL assay [2]

In vitro toxicity: CC₅₀ > 1 μM in SH-SY5Y cells, HeLa cells and primary cortical neurons [2] - Acute in vivo toxicity: No death or obvious behavioral abnormalities (sleepiness, ataxia) were observed in rats after intraperitoneal injection of up to 20 mg/kg of Tubastatin A TFA [2] - Subchronic toxicity (7 days, rats): Tubastatin A TFA (5 mg/kg, intraperitoneal injection, twice) did not cause significant changes in body weight, hematological parameters or liver and kidney function indicators (ALT, AST, creatinine) [2] - Plasma protein binding: 88% (rat plasma, ultrafiltration) [2]

[1]. Histone deacetylase 6 and heat shock protein 90 control the functions of Foxp3(+) T-regulatory cells. Mol Cell Biol. 2011 May;31(10):2066-78.

[2]. Rational Design and Simple Chemistry Yield a Superior, Neuroprotective HDAC6 Inhibitor, Tubastatin A J. Am. Chem. Soc., 2010, 132 (31), pp 10842-10846.

[3]. Dysferlin interacts with histone deacetylase 6 and increases alpha-tubulin acetylation. PLoS One. 2011;6(12):e28563.

[4]. Actin filaments play a primary role for structural integrity and viscoelastic response in cells. Integr Biol (Camb). 2012 May;4(5):540-9.

[5]. HDAC6 Inhibition Promotes Transcription Factor EB Activation and Is Protective in Experimental Kidney Disease. Front Pharmacol. 2018 Feb 1;9:34.

[6]. Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target. Nat Chem Biol. 2022 Apr 28.

Tubastatin A is a pyridoindole compound, chemically named 1,2,3,4-tetrahydro-5H-pyrido[4,3-b]indole, in which the tetrahydropyridine nitrogen atom is substituted with a methyl group, and the indole nitrogen atom is substituted with a p-[N-(hydroxy)aminocarbonyl]benzyl group. It is a histone deacetylase 6 (HDAC6) inhibitor with selectivity for all other isoenzymes except HDAC8 (1000-fold higher selectivity, compared to 57-fold selectivity for HDAC8). It is an EC 3.5.1.98 (histone deacetylase) inhibitor. It is a pyridoindole, hydroxamic acid, and tertiary amine compound.

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Structure-based drug design combined with homology modeling techniques has led to the development of highly efficient HDAC6 inhibitors with higher selectivity for HDAC6 isoenzymes compared to other inhibitors. These inhibitors can be assembled in several synthetic steps, making them easily scaled up for in vivo studies. One of the optimized compounds in this series, named Tubastatin A, was tested in primary cortical neuron cultures. Tubastatin A was found to induce an increase in acetylated α-tubulin levels without affecting histone levels, consistent with its HDAC6 selectivity. Tubastatin A also protected primary cortical neuron cultures from glutathione depletion-induced oxidative stress in a dose-dependent manner. Importantly, this hydroxamic acid-containing HDAC6-selective compound did not show neurotoxicity when used alone at all tested concentrations, thus this compound and its analogues are promising for the treatment of neurodegenerative diseases. [1] Dysferlin is a multi-C2 domain transmembrane protein involved in a variety of cellular functions, most notably skeletal muscle membrane repair, as well as myogenesis, cell adhesion and intercellular calcium signaling. We have previously demonstrated that dysferlin interacts with α-tubulin and microtubules in muscle cells. During myogenesis, microtubules undergo extensive remodeling to maintain the growth and elongation of new muscle fibers. Microtubule function is regulated by post-translational modifications, such as the acetylation of its α-tubulin subunit, a process regulated by histone deacetylase 6 (HDAC6). In this study, we identified HDAC6 as a novel binding partner for dysferlin. Dysferlin binds to HDAC6 via its C2D domain and to the substrate α-tubulin via its C2A and C2B domains, thereby preventing HDAC6 from deacetyling α-tubulin. We further found that dysferlin expression promotes α-tubulin acetylation and enhances the resistance and resilience of microtubules to nocodazole and cold-induced depolymerization. By selectively inhibiting HDAC6 using Tubastatin A, we demonstrated that myotubule formation is impaired when α-tubulin is overacetylated early in the myogenic process; however, myotubule elongation occurs when α-tubulin is overacetylated within the myotubule. This study revealed a novel role of dysferlin in myogenesis and identified HDAC6 as a new dysferlin-interacting protein. [3]

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Tubastatin A TFA is a highly selective and potent HDAC6 inhibitor that targets the catalytic domain of HDAC6 through rational drug design. [2]
- Its neuroprotective mechanism includes inhibiting HDAC6-mediated α-tubulin deacetylation, stabilizing the microtubule cytoskeleton, reducing neuronal apoptosis, and mitigating excitotoxicity. [2]
- This compound does not significantly inhibit class I HDACs (HDAC1-3) or other class II HDACs, thereby minimizing off-target effects associated with pan-HDAC inhibitors. [2]
- It is a valuable tool compound for studying the biology of HDAC6 and has potential therapeutic applications in neuroinflammatory and neurodegenerative diseases (e.g., stroke, Alzheimer's disease). [2]

C20H21N3O2

335.39964

335.163

C, 58.79; H, 4.93; F, 12.68; N, 9.35; O, 14.24

1239262-52-2

Tubastatin A Hydrochloride;1310693-92-5;Tubastatin A;1252003-15-8

50898504

Typically exists as solid at room temperature

1.3±0.1 g/cm3

1.668

2.14

3

8

3

32

561

0

O=C(NO)C1=CC=C(CN2C3=C(CN(C)CC3)C4=C2C=CC=C4)C=C1.O=C(O)C(F)(F)F

AVAOVICSJJIYRZ-UHFFFAOYSA-N

InChI=1S/C20H21N3O2.C2HF3O2/c1-22-11-10-19-17(13-22)16-4-2-3-5-18(16)23(19)12-14-6-8-15(9-7-14)20(24)21-25;3-2(4,5)1(6)7/h2-9,25H,10-13H2,1H3,(H,21,24);(H,6,7)

N-hydroxy-4-[(2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5-yl)methyl]benzamide;2,2,2-trifluoroacetic acid

1239262-52-2; N-Hydroxy-4-((2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)methyl)benzamide 2,2,2-trifluoroacetate; Tubastatin A TFA; Tubastatin TFA salt; Tubastatin A (TFA); N-hydroxy-4-[(2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5-yl)methyl]benzamide;2,2,2-trifluoroacetic acid; Tubastatin A (trifluoroacetate salt); Benzamide, N-hydroxy-4-[(1,2,3,4-tetrahydro-2-methyl-5H-pyrido[4,3-b]indol-5-yl)methyl]-, 2,2,2-trifluoroacetate (1:1);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)