Tubastatin A (TubA, AG-CR1-3900) 1252003-15-8_HDAC_DNA Damage_Signaling Pathways_Products

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.

Physicochemical Properties

Molecular Formula	C20H21N3O2
Molecular Weight	335.4
Exact Mass	371.14
Elemental Analysis	C, 71.62; H, 6.31; N, 12.53; O, 9.54
CAS #	1252003-15-8
Related CAS #	1252003-15-8; 1239034-70-8 (TFA) ; 1252003-15-8 1310693-92-5 (HCl);
PubChem CID	49850262
Appearance	White solid powder
LogP	3.125
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	3
Heavy Atom Count	25
Complexity	478
Defined Atom Stereocenter Count	0
SMILES	O=C(NO)C1=CC=C(CN2C3=C(CN(C)CC3)C4=C2C=CC=C4)C=C1
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; 1252003-15-8; Tubastatin-A; Tubastatin A (free base); Tubastatin A BASE; 2XTSOX1NF8; N-hydroxy-4-((2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)methyl)benzamide; Tubastatin A(free base); 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)

Biological Activity

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.

Additional Infomation

Tubastatin A is a pyridoindole that is 1,2,3,4-tetrahydro-5H-pyrido[4,3-b]indole which is substituted on the tetrahydropyridine nitrogen by a methyl group and on the indole nitrogen by a p-[N-(hydroxy)aminocarbonyl]benzyl group. It is a histone deacetylase 6 (HDAC6) inhibitor that is selective against all the other isozymes (1000-fold) except HDAC8 (57-fold). It has a role as an EC 3.5.1.98 (histone deacetylase) inhibitor. It is a pyridoindole, a hydroxamic acid and a tertiary amino compound.

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.

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.

PeptideDB

Tubastatin A (TubA, AG-CR1-3900) 1252003-15-8

CAS No.: 1252003-15-8

Physicochemical Properties

Biological Activity

Solubility Data