Tubastatin A (TubA, AG-CR1-3900)

Alias: Tubastatin A hydrochloride; Tubastatin A HCl; TSA HCl; Tubastatin A; TubA, AG-CR1-3900

Cat No.:V0275 Purity: ≥98%

COA Product Data Instructions for use SDS

Tubastatin A (TubA, AG-CR1-3900) Chemical Structure CAS No.: 1252003-15-8
Product category: HDAC

This product is for research use only, not for human use. We do not sell to patients.

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Nature 2021, 597(7874):119-125.

Cell 2020,183:1202-1218.e25.

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Cell 2020,183:1202-1218.e25.

Science Adv 2022:8,eabm9427.

Nature 2021,597(7874):119-125.

Cell 2020,183:1202-1218.e25.

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InvivoChem's Tubastatin A (TubA, AG-CR1-3900) has been cited by 1 publication

[1] J Enzyme Inhib Med Chem. 2023 Dec;38(1):2201408.

Purity & Quality Control Documentation

Current batch：

Purity: ≥98%

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Instructions

Product Description
Bioactivity & Protocols
Physicochemical Properties
Solubility Data
Calculators
Biological Data

Product Description

Tubastatin A (TubA, AG-CR13900), a tubacin derivative, is a potent and selective HDAC6 (Histone deacetylase 6) inhibitor (IC50 = 15 nM in a cell-free assay) with potential anticancer and anti-inflammatory activity. It showed that HDAC6 has the highest selectivity among the HDAC isoforms(excluding HDAC8 for which the IC50 is 0.9 μM), more than 1,000-fold.

Biological Activity I Assay Protocols (From Reference)

Targets

HDAC6 ( IC50 = 15 nM ); HDAC8 ( IC50 = 854 nM ); HDAC1 ( IC50 = 16400 nM )

ln Vitro

In vitro activity: is inherently selective for each of the 11 HDAC isoforms and retains over 1000-fold selectivity against all isoforms, with the exception of HDAC8, where selectivity is only about 57 layers. Tubastatin A initiates dose-dependent protection against homocysteic acid (HCA)-induced neuronal cell death as early as 5 μM and achieves near-complete protection at 10 μM in assays for HCA-induced neurodegeneration[1]. Tubastatin A suppresses T cell proliferation in vitro at 100 ng/mL by increasing Foxp³⁺ T-regulatory cells (Tregs)[2]. When alpha-tubulin is hyperacetylated early in the myogenic process, Tubastatin A treatment in CC12 cells would impair myotube formation; however, 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 increases cell elasticity as shown by atomic force microscopy (AFM) tests without significantly altering the actin microfilament or microtubule networks[4].

ln Vivo

Regular administration of Tubastatin A at 0.5 mg/kg suppresses HDAC6 to enhance Tregs suppressive activity in murine models of inflammation and autoimmunity, encompassing various types of experimental colitis and cardiac allograft rejection that is completely MHC-incompatible[2].

Enzyme Assay

In the assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, and 20 μM tris(2-carboxyethyl)phosphine), tubastatin A is dissolved and diluted to six times the final concentration levels. Prior to adding the substrate, HDAC enzymes are diluted in assay buffer to 1.5 times the final concentration and pre-incubated for 10 minutes with tubastatin A. The enzymes' respective amounts of FTS (HDAC1, HDAC2, HDAC3, and HDAC6) or MAZ-1675 (HDAC4, HDAC5, HDAC7, HDAC8, and HDAC9) are calculated using a titration curve to determine the Michaelis constant (K_m). Trypsin of sequencing grade 0.3 μM is used to dilute FTS or MAZ-1675 in assay buffer to a final concentration six times. The plate is put into a SpectraMax M5 microtiter plate reader after the substrate/trypsin mix has been added to the enzyme/compound mix and shaken for 60 seconds. Following the peptide substrate's lysine side chain's deacetylation, the enzymatic reaction is watched for the release of 7-amino-4-methoxy-coumarin over a 30-minute period. The reaction's linear rate is then computed.

Cell Assay

Primary cortical neuron cultures are prepared, as previously mentioned, from the cerebral cortex of fetal Sprague-Dawley rats (embryonic day 17). 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. pH 7.5 solutions that are 100 times concentrated are used to dilute HCA. Tubastatin A is administered to neurons at the specified concentrations in addition to HCA. After a day, the MTT assay is used to determine viability.

Animal Protocol

In adoptive transfer and dextran sodium sulfate (DSS) models of colitis, the effects of HDAC6 targeting are assessed in groups of ten mice each. For five days, WT B6 mice's pH-balanced tap water is supplemented with freshly made 4% (wt/vol) DSS (MP Biomedicals). Colitis is evaluated by daily monitoring of body weight, stool consistency, and fecal blood. Mice are treated daily for 7 days with either tubacin or niltubacin (0.5 mg/kg of body weight/day, i.p.). Hemoloccult feces are graded as 0 (absent), 2 (occult), or 4 (gross). Stool consistency is graded as 0 (hard), 2 (soft), or 4 (diarrhea). In order to evaluate the prevention of colitis in a T cell-dependent model, B6/Rag1−/− mice receive an intraperitoneal injection of CD⁴⁺ CD45RBhi T cells (1×10⁶) isolated from WT mice using magnetic beads (>95% cell purity, flow cytometry) along with CD⁴⁺ CD²⁵⁺ Tregs (1.25×10⁵) isolated from HDAC6^−/− or WT mice using magnetic beads (>90% Treg purity, flow cytometry). The mice are then observed every two weeks for signs of colitis. In order to evaluate treatment for established T cell-dependent colitis, CD⁴⁺ CD45RBhi cells (1×10⁶) are intraperitoneally injected into B6/Rag1^−/− mice. After colitis manifests, mice are also given treatment with HDAC6i (tubastatin A) or HSP90i (17-AAG) or CD⁴⁺ CD²⁵⁺ Tregs (5×10⁵ cells), which were isolated from HDAC6^−/− or WT mice as previously described. Weight loss and stool consistency in the mice are continuously observed. Colon paraffin sections stained with hematoxylin and eosin or Alcian Blue are either immunoperoxidase stained for Foxp³⁺ Treg infiltration or graded histologically at the end of the study.

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]. NEDD9 regulates actin dynamics through cortactin deacetylation in an AURKA/HDAC6-dependent manner. Mol Cancer Res. 2014 May;12(5):681-93.

[3]. 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.

[4]. Role of HDAC6 in Transcription Factor EB Mediated Clearance of Misfolded Proteins in Chronic Kidney Disease. University of Toronto.Nov-2017.

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

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

[7]. Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target [published online ahead of print, 2022 Apr 28]. Nat Chem Biol. 2022;10.1038/s41589-022-01015-5.

[8]. Selective Inhibition of Histone Deacetylase 10: Hydrogen Bonding to the Gatekeeper Residue is Implicated. J Med Chem. 2019;62(9):4426-4443.

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

These protocols are for reference only. InvivoChem does not independently validate these methods.

Physicochemical Properties

Molecular Formula	C20H21N3O2
Molecular Weight	335.4
Exact Mass	335.16
Elemental Analysis	C, 71.62; H, 6.31; N, 12.53; O, 9.54
CAS #	1252003-15-8
Related CAS #	1252003-15-8;1310693-92-5 (HCl)
Appearance	White solid powder
SMILES	CN1CCC2=C(C1)C3=CC=CC=C3N2CC4=CC=C(C=C4)C(=O)NO.Cl
InChi Key	GOVYBPLHWIEHEJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H21N3O2/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)
Chemical Name	N-hydroxy-4-[(2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5-yl)methyl]benzamide
Synonyms	Tubastatin A hydrochloride; Tubastatin A HCl; TSA HCl; Tubastatin A; TubA, AG-CR1-3900
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
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility Data

Solubility (In Vitro)

DMSO: 12.5~67 mg/mL (37.3~199.8 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.73 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 12.5 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 2: ≥ 1.25 mg/mL (3.73 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 12.5 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.

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Solubility in Formulation 3: ≥ 1.25 mg/mL (3.73 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

Solubility in Formulation 4: 4% DMSO+30% PEG 300: 5mg/mL

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.9815 mL	14.9076 mL	29.8151 mL
	5 mM	0.5963 mL	2.9815 mL	5.9630 mL
	10 mM	0.2982 mL	1.4908 mL	2.9815 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?

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The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

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Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:

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The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box

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Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4 c12h18n3o4
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Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
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In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)

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Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)

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Calculation results

Working concentration： mg/mL；

Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O，mix and clarify.

Note： (1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.

Biological Data

Comparison of histone and α-tubulin hyperacetylation for TSA, Tubastatin A, and Tubacin. J Am Chem Soc . 2010 Aug 11;132(31):10842-6.

C2C12 or 134/04 myoblasts were seeded in growth media on Day -1, then switched to differentiation media on Day 0. Cells were mock-treated (Mock) or treated with 7.5 µM Tubastatin A beginning on different days post-induction of myogenic differentiation (Day 0, Day 2, Day 4). On Day 5, cells were fixed and stained with an anti-desmin antibody and DAPI. PLoS One . 2011;6(12):e28563.