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

In vitro activity: Tubastatin A is largely 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]. Alpha-tubulin hyperacetylation early in the myogenic process would impair myotube formation in CC12 cells treated with Tubastatin A; however, myotube elongation happens when alpha-tubulin is hyperacetylated in myotubes[3]. According to a recent study, treatment with tubastatin A increases cell elasticity as measured by atomic force microscopy (AFM) tests in mouse ovarian cancer cell lines MOSE-E and MOSE-L[4] without significantly altering the actin microfilament or microtubule networks.

In mouse models of inflammation and autoimmunity, such as multiple forms of experimental colitis and fully major histocompatibility complex (MHC)-incompatible cardiac allograft rejection, daily treatment with Tubastatin A at 0.5 mg/kg inhibits HDAC6 to promote Tregs suppressive activity[2].

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.

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.

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 every day. 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. The mice's continued weight loss and the consistency of their feces are observed. When the study comes to an end, paraffin sections of colons stained with hematoxylin and eosin or Alcian Blue are either immunoperoxidase stained for Foxp3+ Treg infiltration or graded histologically.

[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.

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]

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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]

C21H22N2O2

334.411585330963

334.168

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

1239034-70-8

68380216

Typically exists as solid at room temperature

1.2±0.1 g/cm3

522.8±50.0 °C at 760 mmHg

270.0±30.1 °C

0.0±1.4 mmHg at 25°C

1.624

4.08

0

3

4

25

475

0

O(C)C(C1C=CC(=CC=1)CN1C2C=CC=CC=2C2CN(C)CCC1=2)=O

RMAYYLGDIMEEOS-UHFFFAOYSA-N

InChI=1S/C21H22N2O2/c1-22-12-11-20-18(14-22)17-5-3-4-6-19(17)23(20)13-15-7-9-16(10-8-15)21(24)25-2/h3-10H,11-14H2,1-2H3

methyl 4-[(2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5-yl)methyl]benzoate

1239034-70-8; Methyl 4-((2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)methyl)benzoate; Benzoic acid, 4-[(1,2,3,4-tetrahydro-2-methyl-5H-pyrido[4,3-b]indol-5-yl)methyl]-, methyl ester; SCHEMBL12804583; RMAYYLGDIMEEOS-UHFFFAOYSA-N;

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.)