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Tubastatin A HCl, the hydrochloride salt of Tubastatin A (also known as TubA, AG-CR-13900), is a tubacin analog that acts as a potent and specific inhibitor of histone deacetylase 6 (HDAC6) with potential antitumor, neuroprotective and anti-inflammatory activities. Its selectivity (>1,000-fold) for inhibiting HDAC6 is greater than that of other HDAC isoforms, with the exception of HDAC8, whose IC50 is 0.9 μM.
Physicochemical Properties
| Molecular Formula | C20H21N3O2.HCL |
| Molecular Weight | 371.86 |
| Exact Mass | 371.14 |
| Elemental Analysis | C, 64.60; H, 5.96; Cl, 9.53; N, 11.30; O, 8.60 |
| CAS # | 1310693-92-5 |
| Related CAS # | 1310693-92-5 (HCl); 1239034-70-8 (TFA) ; 1252003-15-8 |
| PubChem CID | 57336514 |
| Appearance | White to yellow solid powder |
| LogP | 3.927 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 3 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 26 |
| Complexity | 478 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C(C1=CC=C(C=C1)CN2C3=C(C4=C2C=CC=C4)CN(C)CC3)NO.[H]Cl |
| InChi Key | LJTSJTWIMOGKRJ-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C20H21N3O2.ClH/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;/h2-9,25H,10-13H2,1H3,(H,21,24);1H |
| Chemical Name | N-hydroxy-4-[(2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5-yl)methyl]benzamide;hydrochloride |
| Synonyms | AG-CR-13900; TubA; Tubastatin A hydrochloride; Tubastatin A hydrochloride; Tubastatin A HCl; 1310693-92-5; Tubastatin A (Hydrochloride); UHCM2AYJVX; N-hydroxy-4-[(2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5-yl)methyl]benzamide;hydrochloride; N-hydroxy-4-[(1,2,3,4-tetrahydro-2-methyl-5H-pyrido[4,3-b]indol-5-yl)methyl]benzamide hydrochloride; Benzamide, N-hydroxy-4-[(1,2,3,4-tetrahydro-2-methyl-5H-pyrido[4,3-b]indol-5-yl)methyl]-, hydrochloride (1:1); .Tubastatin A HCl; TSA HCl |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | HDAC6 ( IC50 = 15 nM ); HDAC8 ( IC50 = 854 nM ); HDAC1 ( IC50 = 16400 nM ) | |
| ln Vitro |
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| ln Vivo |
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| Enzyme Assay | The Reaction Biology HDAC Spectrum platform is utilized for the execution of enzyme inhibition experiments. Isolated recombinant human protein is utilized in the HDAC1, 2, 4, 5, 6, 7, 8, 9, 10, and 11 assays; the HDAC3/NcoR2 complex is utilized in the HDAC3 test. Fluorogenic peptide derived from p53 residues 379–382 (RHKKAc) serves as the substrate for HDAC1, 2, 3, 6, 10, and 11 assays; fluorogenic diacyl peptide derived from p53 residues 379–382 (RHKAcKAc) serves as the substrate for HDAC8. For the HDAC4, 5, 7, and 9 assays, acetyl-Lys (trifluoroacetyl)-AMC substrate is utilized. After dissolving tubastatin A in DMSO, it is tested in 10-dose IC50 mode using a 3-fold serial dilution regimen that begins at 30 μM. Trichostatin A (TSA), the control compound, is tested in a 10-dose IC50 using a 3-fold serial dilution that begins at 5 μM. Curve-fitting the dose/response slopes yields IC50 values. | |
| Cell Assay | The cerebral cortex of fetal Sprague-Dawley rats (embryonic day 17) is used to cultivate primary cortical neurons. Twenty-four hours after plating, all experiments are started. Glutamate-mediated excitotoxicity cannot harm the cells in these circumstances. Cells are washed with warm PBS before being put in minimum essential medium with 5.5 g/L glucose, 10% fetal calf serum, 2 mM L-glutamine, and 100 μM cystine for cytotoxicity investigations. The glutamate analogue homocysteate (HCA; 5 mM) is added to the media to cause oxidative stress. HCA is prepared by diluting solutions that have been concentrated 100 times and adjusted to pH 7.5. Neurons are treated with Tubastatin A at the indicated concentrations in addition to HCA. The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is used to determine viability after a 24-hour period. | |
| Animal Protocol |
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| References |
[1]. Rational Design and Simple Chemistry Yield a Superior, Neuroprotective HDAC6 Inhibitor, Tubastatin A J. Am. Chem. Soc., 2010, 132 (31), pp 10842-10846. [2]. 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. [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. |
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| Additional Infomation |
Structure-based drug design combined with homology modeling techniques were used to develop potent inhibitors of HDAC6 that display superior selectivity for the HDAC6 isozyme compared to other inhibitors. These inhibitors can be assembled in a few synthetic steps, and thus are readily scaled up for in vivo studies. An optimized compound from this series, designated Tubastatin A, was tested in primary cortical neuron cultures in which it was found to induce elevated levels of acetylated alpha-tubulin, but not histone, consistent with its HDAC6 selectivity. Tubastatin A also conferred dose-dependent protection in primary cortical neuron cultures against glutathione depletion-induced oxidative stress. Importantly, when given alone at all concentrations tested, this hydroxamate-containing HDAC6-selective compound displayed no neuronal toxicity, thus, forecasting the potential application of this agent and its analogues to neurodegenerative conditions.[1] Dysferlin is a multi-C2 domain transmembrane protein involved in a plethora of cellular functions, most notably in skeletal muscle membrane repair, but also in myogenesis, cellular adhesion and intercellular calcium signaling. We previously showed that dysferlin interacts with alpha-tubulin and microtubules in muscle cells. Microtubules are heavily reorganized during myogenesis to sustain growth and elongation of the nascent muscle fiber. Microtubule function is regulated by post-translational modifications, such as acetylation of its alpha-tubulin subunit, which is modulated by the histone deacetylase 6 (HDAC6) enzyme. In this study, we identified HDAC6 as a novel dysferlin-binding partner. Dysferlin prevents HDAC6 from deacetylating alpha-tubulin by physically binding to both the enzyme, via its C2D domain, and to the substrate, alpha-tubulin, via its C2A and C2B domains. We further show that dysferlin expression promotes alpha-tubulin acetylation, as well as increased microtubule resistance to, and recovery from, Nocodazole- and cold-induced depolymerization. By selectively inhibiting HDAC6 using Tubastatin A, we demonstrate that myotube formation was impaired when alpha-tubulin was hyperacetylated early in the myogenic process; however, myotube elongation occurred when alpha-tubulin was hyperacetylated in myotubes. This study suggests a novel role for dysferlin in myogenesis and identifies HDAC6 as a novel dysferlin-interacting protein.[3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.62 mg/mL (7.05 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.62 mg/mL (7.05 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (5.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 4: ≥ 2.08 mg/mL (5.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 5: ≥ 2.08 mg/mL (5.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 6: ≥ 0.52 mg/mL (1.40 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 7: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6892 mL | 13.4459 mL | 26.8918 mL | |
| 5 mM | 0.5378 mL | 2.6892 mL | 5.3784 mL | |
| 10 mM | 0.2689 mL | 1.3446 mL | 2.6892 mL |