Home Epigenetics HDAC inhibitor - Apoptosis related activator - Autophagy activator Tubastatin A For research use only.

Tubastatin A

Tubastatin A is a potent and selective HDAC6 inhibitor with IC50 of 15 nM in a cell-free assay. It is selective against all the other isozymes (1000-fold) except HDAC8 (57-fold). Tubastatin A promotes autophagy and increases apoptosis.

Tubastatin A Chemical Structure

Tubastatin A Chemical Structure

CAS No. 1252003-15-8

Purity & Quality Control

Tubastatin A Related Products

Signaling Pathway

HDAC Signaling Pathways

HDAC Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
Sf9 Function assay 2 hrs Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 (1 to 1215 residues) expressed in sf9 cells preincubated with enzyme followed by fluorogenic Arg-His-Lys-Lys(Ac)-AMC substrate addition measured after 2 hrs by fluorescence assay, IC50 = 0.0035 μM. 27541357
Sf9 Function assay Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 (1 to 1215 residues) expressed in sf9 cells using RHK-K(Ac)-AMC as substrate by fluorescence assay, IC50 = 0.011 μM. 27541357
Sf9 Function assay 30 mins Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 expressed in baculovirus infected sf9 cells using fluorogenic HDAC substrate 3 after 30 mins by fluorescence assay, IC50 = 0.013 μM. 29549837
Sf9 Function assay 90 mins Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 expressed in Sf9 cells using RHKK(Ac) as substrate after 90 mins by fluorimetric method, IC50 = 0.0137 μM. 28038324
Sf9 Function assay 2 hrs Inhibition of human recombinant HDAC6 expressed in Sf9 cells incubated for 2 hrs using RHKK-Ac fluorogenic substrate, IC50 = 0.015 μM. 23009203
Sf9 Function assay Inhibition of human recombinant HDAC6 expressed in baculovirus/sf9 cells using RHKKAc as substrate, IC50 = 0.015 μM. 23905680
Sf9 Function assay 90 mins Inhibition of human recombinant N-terminal GST-tagged full length HDAC6 expressed in insect SF9 cells using fluorogenic ZMAL as substrate after 90 mins by fluorescence-based assay, IC50 = 0.0304 μM. 30092367
HeLaS3 Function assay 15 mins Inhibition of HDAC6 in human HeLaS3 cells preincubated for 15 mins followed by HDAC-Glo substrate addition measured after 30 to 45 mins by ELISA, IC50 = 0.031 μM. 28337317
insect cells Function assay 4 hrs Inhibition of human recombinant HDAC6 expressed in baculovirus infected insect cells using BATCP as substrate after 4 hrs by UHPLC-ESI-MS/MS analysis, IC50 = 0.0349 μM. 27650925
SHSY5Y Function assay 8 hrs Inhibition of HDAC6 in human SHSY5Y cells using BATCP as substrate after 8 hrs by UHPLC-ESI-MS/MS analysis, IC50 = 0.0943 μM. 27650925
SHSY5Y Function assay 8 hrs Inhibition of HDAC in human SHSY5Y cells using MAL as substrate after 8 hrs by UHPLC-ESI-MS/MS analysis, IC50 = 0.1221 μM. 27650925
Sf9 Function assay 4 hrs Inhibition of full length human recombinant C-terminal FLAG/His-tagged HDAC1 expressed in baculovirus infected sf9 cells using fluorogenic HDAC substrate 3 after 4 hrs fluorescence assay, IC50 = 0.718 μM. 29549837
Sf9 Function assay Inhibition of human recombinant HDAC8 expressed in baculovirus/sf9 cells using RHKAcKAc as substrate, IC50 = 0.854 μM. 23905680
Sf9 Function assay 1 hr Inhibition of full length human recombinant C-terminal FLAG/His-tagged HDAC1 expressed in baculovirus infected sf9 cells using fluorogenic HDAC substrate 3 after 1 hr fluorescence assay, IC50 = 0.967 μM. 29549837
SHSY5Y Function assay 8 hrs Inhibition of HDAC1 in human SHSY5Y cells using MOCPAC as substrate after 8 hrs by UHPLC-ESI-MS/MS analysis, IC50 = 1.1097 μM. 27650925
Sf9 Function assay 30 mins Inhibition of full length human recombinant C-terminal FLAG/His-tagged HDAC1 expressed in baculovirus infected sf9 cells using fluorogenic HDAC substrate 3 after 30 mins by fluorescence assay, IC50 = 1.54 μM. 29549837
Sf9 Function assay 90 mins Inhibition of human recombinant C-terminal His/FLAG-tagged full length HDAC1 expressed in insect SF9 cells using fluorogenic ZMAL as substrate after 90 mins by fluorescence-based assay, IC50 = 1.91 μM. 30092367
HCT116 Antiproliferative assay 72 hrs Antiproliferative activity against human HCT116 cells after 72 hrs by MTT assay, IC50 = 2 μM. 27541357
HCT116 Antiproliferative assay Antiproliferative activity against human HCT116 cells, IC50 = 2 μM. 29945795
Sf9 Function assay 30 mins Inhibition of full length human recombinant C-terminal His-tagged HDAC3/N-terminal GST-tagged NCOR2 (95 to 489 residues) expressed in baculovirus infected sf9 cells using fluorogenic HDAC substrate 3 after 30 mins by fluorescence assay, IC50 = 2.26 μM. 29549837
HeLa Function assay 6 hrs Inhibition of HDAC6 in human HeLa cells assessed as reduction in K40 hyperacetylation of alpha-tubulin incubated for 6 hrs by immunofluorescence assay, IC50 = 2.5 μM. 25454270
HL60 Antiproliferative assay 48 hrs Antiproliferative activity against human HL60 cells after 48 hrs in presence of JAK2 inhibitor CYT-387 by CCK-8 assay, IC50 = 2.54 μM. 29940115
K562 Antiproliferative assay 48 hrs Antiproliferative activity against human K562 cells after 48 hrs in presence of JAK2 inhibitor CYT-387 by CCK-8 assay, IC50 = 2.54 μM. 29940115
HEL Antiproliferative assay 48 hrs Antiproliferative activity against human HEL cells after 48 hrs in presence of JAK2 inhibitor CYT-387 by CCK-8 assay, IC50 = 2.54 μM. 29940115
HeLaS3 Function assay 15 mins Inhibition of HDAC1 in human HeLaS3 cells preincubated for 15 mins followed by HDAC-Glo substrate addition measured after 30 to 45 mins by ELISA, IC50 = 2.7 μM. 28337317
HeLaS3 Function assay 15 mins Inhibition of HDAC3 in human HeLaS3 cells preincubated for 15 mins followed by HDAC-Glo substrate addition measured after 30 to 45 mins by ELISA, IC50 = 2.9 μM. 28337317
Jurkat Cytotoxicity assay 72 hrs Cytotoxicity against human Jurkat cells assessed as growth inhibition after 72 hrs by MTS assay, IC50 = 3.38 μM. 24304348
MCF7 Antiproliferative assay 72 hrs Antiproliferative activity against human MCF7 cells after 72 hrs by MTT assay, IC50 = 3.7 μM. 27541357
MCF7 Antiproliferative assay Antiproliferative activity against human MCF7 cells, IC50 = 3.7 μM. 29945795
HL60 Antiproliferative assay 48 hrs Antiproliferative activity against human HL60 cells after 48 hrs by CCK-8 assay, IC50 = 3.75 μM. 29940115
K562 Antiproliferative assay 48 hrs Antiproliferative activity against human K562 cells after 48 hrs by CCK-8 assay, IC50 = 3.75 μM. 29940115
HEL Antiproliferative assay 48 hrs Antiproliferative activity against human HEL cells after 48 hrs by CCK-8 assay, IC50 = 3.75 μM. 29940115
HeLaS3 Function assay 15 mins Inhibition of HDAC2 in human HeLaS3 cells preincubated for 15 mins followed by HDAC-Glo substrate addition measured after 30 to 45 mins by ELISA, IC50 = 3.9 μM. 28337317
CAL27 Antiproliferative assay 72 hrs Antiproliferative activity against human CAL27 cells measured after 72 hrs by MTT assay, IC50 = 4.6 μM. 28581289
PC3 Antiproliferative assay 72 hrs Antiproliferative activity against human PC3 cells after 72 hrs by MTT assay, IC50 = 8.6 μM. 27541357
PC3 Antiproliferative assay Antiproliferative activity against human PC3 cells, IC50 = 8.6 μM. 29945795
Sf9 Function assay 30 mins Inhibition of full length human recombinant C-terminal His-tagged HDAC2 expressed in baculovirus infected sf9 cells using fluorogenic HDAC substrate 3 after 30 mins by fluorescence assay, IC50 = 9.97 μM. 29549837
MDA-MB-231 Antiproliferative assay 72 hrs Antiproliferative activity against human MDA-MB-231 cells after 72 hrs by MTT assay, IC50 = 10.4 μM. 27541357
MDA-MB-231 Antiproliferative assay Antiproliferative activity against human MDA-MB-231 cells, IC50 = 10.4 μM. 29945795
Cal27CisR Antiproliferative assay 72 hrs Antiproliferative activity against human Cal27CisR cells measured after 72 hrs by MTT assay, IC50 = 10.8 μM. 28581289
LNCAP Cytotoxicity assay 72 hrs Cytotoxicity against androgen-dependent human LNCAP cells assessed as growth inhibition after 72 hrs by MTS assay, IC50 = 10.88 μM. 24304348
Cal27CisR Function assay 18 hrs Inhibition of HDAC in human Cal27CisR cells using Boc-Lys(epsilon-Ac)-AMC as substrate preincubated for 18 hrs followed by substrate addition measured after 3 hrs by fluorescence assay, IC50 = 12.1 μM. 28581289
KB Cytotoxicity assay 72 hrs Cytotoxicity against human KB cells after 72 hrs by MTS assay, IC50 = 14.81 μM. 25899338
THLE2 Cytotoxicity assay 72 hrs Cytotoxicity against human THLE2 cells after 72 hrs by vialight cell proliferation assay, LC50 = 15.1 μM. 29549837
CAL27 Function assay 18 hrs Inhibition of HDAC in human CAL27 cells using Boc-Lys(epsilon-Ac)-AMC as substrate preincubated for 18 hrs followed by substrate addition measured after 3 hrs by fluorescence assay, IC50 = 16.1 μM. 28581289
Sf9 Function assay 2 hrs Inhibition of human recombinant HDAC1 expressed in Sf9 cells incubated for 2 hrs using RHKK-Ac fluorogenic substrate, IC50 = 16.4 μM. 23009203
Sf9 Function assay Inhibition of human recombinant HDAC1 expressed in baculovirus/sf9 cells using RHKKAc as substrate, IC50 = 16.4 μM. 23905680
B16 Growth inhibition assay 48 hrs Growth inhibition of mouse B16 cells incubated for 48 hrs by MTT assay, GI50 = 40.5 μM. 23009203
KB Function assay 14 uM 24 hrs Inhibition of HDAC1 in human KB cells assessed as increase in histone H4 acetylation at 14 uM after 24 hrs by Western blotting analysis 25899338
KMS-12-BM Function assay 15 uM up to 48 hrs Inhibition of HDAC6 in human KMS-12-BM cells assessed as increase in acetylated tubulin level at 15 uM up to 48 hrs by immunoblot method 27541357
MOLM14 Function assay 15 uM up to 48 hrs Inhibition of HDAC6 in human MOLM14 cells assessed as increase in acetylated tubulin level at 15 uM up to 48 hrs by immunoblot method 27541357
U937 Function assay 2 uM 18 hrs Inhibition of HDAC6 in human U937 cells assessed as increase in alpha-tubulin acetylation at Lys-40 residue at 2 uM after 18 hrs by Western blot method 28337317
LNCAP Function assay 24 hrs Inhibition of HDAC6 in human LNCAP cells assessed as inhibition of DHT-induced alpha-tubulin deacetylation by measuring increase in alpha-tubulin acetylation measured after 24 hrs relative to control 27717544
human Function assay 24 hrs Antagonist activity at AR in human LNCAP cells assessed as suppression of DHT-induced AR protein level measured after 24 hrs relative to control 27717544
DAOY qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for DAOY cells 29435139
SJ-GBM2 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for SJ-GBM2 cells 29435139
A673 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for A673 cells 29435139
SK-N-MC qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for SK-N-MC cells 29435139
BT-37 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for BT-37 cells 29435139
NB-EBc1 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for NB-EBc1 cells 29435139
U-2 OS qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for U-2 OS cells 29435139
Saos-2 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for Saos-2 cells 29435139
SK-N-SH qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for SK-N-SH cells 29435139
NB1643 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for NB1643 cells 29435139
LAN-5 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for LAN-5 cells 29435139
Rh18 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for Rh18 cells 29435139
OHS-50 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for OHS-50 cells 29435139
RD qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for RD cells 29435139
Rh30 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for Rh30 cells 29435139
Rh41 qHTS assay qHTS of pediatric cancer cell lines to identify multiple opportunities for drug repurposing: Primary screen for Rh41 cells 29435139
HEL Cell cycle assay 1 to 5 uM 48 hrs Cell cycle arrest in human HEL cells assessed as accumulation at G1 phase at 1 to 5 uM after 48 hrs propidium iodide staining based flow cytometry 29940115
HeLa Function assay 2 uM 12 hrs Inhibition of HDAC6 in human HeLa cells assessed as increase in acetyl-tubulin level at 2 uM after 12 hrs by Western blot analysis 29533873
HEK293 Function assay 10 uM 24 hrs Inhibition of HDAC1 in HEK293 cells assessed as increase in histone H3 acetylation at 10 uM after 24 hrs by Western blot method 28523102
MV4-11 Function assay 200 nM 24 hrs Inhibition of HDAC6 in human MV4-11 cells assessed as accumulation of acetylated alpha-tubulin at 200 nM after 24 hrs by Western blot analysis 29738953
HCT116 Cell cycle assay 5 uM 48 hrs Cell cycle arrest in human HCT116 cells assessed as accumulation at sub-G1 phase at 5 uM after 48 hrs by propidium iodide staining-based flow cytometric method 28038324
PC12 Neuroprotective assay 10 uM 24 hrs Neuroprotective activity against H2O2-induced toxicity in rat PC12 cells assessed as cell viability at 10 uM pretreated for 24 hrs followed by H2O2 challenge and measured after 12 hrs by MTT assay relative to control 30385227
PC12 Neuroprotective assay 5 uM 24 hrs Neuroprotective activity against 6-OHDA-induced toxicity in rat PC12 cells assessed as increase in cell viability at 5 uM pretreated for 24 hrs followed by 6-OHDA challenge and measured after 12 hrs by MTT assay 30385227
PC12 Neuroprotective assay 10 uM 24 hrs Neuroprotective activity against 6-OHDA-induced toxicity in rat PC12 cells assessed as increase in cell viability at 10 uM pretreated for 24 hrs followed by 6-OHDA challenge and measured after 12 hrs by MTT assay 30385227
PC12 Cytoprotective assay 24 hrs Cytoprotective activity against H2O2-induced damage in rat PC12 cells assessed as decrease in ROS accumulation preincubated for 24 hrs followed by H2O2 challenge measured after 12 hrs by DCFH-DA dye-based fluorescence analysis 30385227
PC12 Cytoprotective assay 24 hrs Cytoprotective activity against H2O2-induced damage in rat PC12 cells assessed as decrease in ROS accumulation preincubated for 24 hrs followed by H2O2 challenge measured after 12 hrs by DCFH-DA dye-based inverted fluorescence microscopic analysis 30385227
PC12 Neuroprotective assay 5 to 10 uM 24 hrs Neuroprotective activity against 6-OHDA-induced toxicity in rat PC12 cells assessed as increase in cell viability at 5 to 10 uM pretreated for 24 hrs followed by 6-OHDA challenge and measured after 12 hrs coincubated with ebselen by MTT assay 30385227
PC12 Antioxidant assay 5 uM 24 hrs Antioxidant activity against H2O2-induced oxidative stress in rat PC12 cells assessed as decrease in ROS accumulation at 5 uM preincubated for 24 hrs followed by H2O2 challenge and measured after 12 hrs by DCFH-DA dye-based fluorescence analysis 30385227
PC12 Antioxidant assay 5 uM 24 hrs Antioxidant activity against H2O2-induced oxidative stress in rat PC12 cells assessed as decrease in ROS accumulation at 5 uM preincubated for 24 hrs followed by H2O2 challenge and measured after 12 hrs coincubated with ebselen by DCFH-DA dye-based fluore 30385227
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Biological Activity

Description Tubastatin A is a potent and selective HDAC6 inhibitor with IC50 of 15 nM in a cell-free assay. It is selective against all the other isozymes (1000-fold) except HDAC8 (57-fold). Tubastatin A promotes autophagy and increases apoptosis.
Targets
HDAC6 [1]
(Cell-free assay)
15 nM
In vitro
In vitro Tubastatin A is selective at all isozymes except HDAC8 and maintains over 1000-fold selectivity against all isoforms excluding HDAC8, where it has approximately 57-fold selectivity. Tubastatin A preferentially induces α-tubulin hyperacetylation at 2.5 μM. Slight induction of histone hyperacetylation is seen for Tubastatin A at 10 μM. Tubastatin A displays dose-dependent protection against homocysteic acid-induced neuronal cell death starting at 5 μM with near complete protection at 10 μM. [1] Tubastatin A (10 μM) induces an increase in acetylated-α-tubulin levels and the restoration of primary cilia expression in the cholangiocarcinoma cell lines (18-fold); and the restoration of primary cilia correlated with downregulated Hedgehog (Hh) and MAPK signaling pathways, as well as decreased cell proliferation rates (in average by 50%) and invasion (by 40%). [2] Tubastatin A shows significant inhibition of TNF-α and IL-6 in LPS stimulated human THP-1 macrophages with an IC50 of 272 nM and 712 nM. Tubastatin A inhibits nitric oxide (NO) secretion in murine Raw 264.7 macrophages dose depenndently with an IC50 of 4.2 μM. [3]
Kinase Assay HDAC enzymatic assays
Tubastatin A is dissolved and diluted in 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) to 6-fold of the final concentration. HDAC enzymes are diluted to 1.5-fold of the final concentration in assay buffer and pre-incubated with Tubastatin A for 10 minutes before the addition of the substrate. The amount of FTS (HDAC1, HDAC2, HDAC3, and HDAC6) or MAZ-1675 (HDAC4, HDAC5, HDAC7, HDAC8, and HDAC9) used for each enzyme is equal to the Michaelis constant (Km), as determined by a titration curve. FTS or MAZ-1675 is diluted in assay buffer to 6-fold the final concentration with 0.3 μM sequencing grade trypsin. The substrate/trypsin mix is added to the enzyme/compound mix and the plate is shaken for 60 seconds and then placed into a SpectraMax M5 microtiter plate reader. The enzymatic reaction is monitored for release of 7-amino-4-methoxy-coumarin over 30 minutes, after deacetylation of the lysine side chain in the peptide substrate, and the linear rate of the reaction is calculated.
Cell Research Cell lines Human cholangiocarcinoma cell lines HuCCT-1
Concentrations ~10 μM
Incubation Time 21 days
Method

Anchorage-independent growth is assessed by growing cells in soft agar. About 25,000 cells suspended in 0.4% agar in culture media are layered over a 1% agar layer in a 6-well plate. Media are added twice a week and pictures are taken after 21 days of incubation. The number and size of colonies are analyzed using the Gel-Pro software.
Experimental Result Images Methods Biomarkers Images PMID
Western blot EGFR / p-AKT / AKT / p-ERK / ERK 29665050
Immunofluorescence α-tubulin / Acetylated tubulin HDAC6 23798680
In Vivo
In vivo Tubastatin A reduces the growth of cholangiocarcinoma in vivo. Tubastatin A (10 mg/kg) induces a 6-fold lower mean tumor weights in syngeneic rat orthotopic model of cholangiocarcinoma, and reduction of the ratios of tumor weight to liver weight and body weight (5- and 5.6-fold, respectively), as well as a greater frequency of ciliated cholangiocytes compared with controls (29% vs 1.4%). Tubastatin A significantly decreases the amount of PCNA-positive cells in the treated tumors compared with vehicle controls (34% vs 65%). [2] Tubastat A shows significant inhibition of paw volume at 30 mg/kg i.p. in a Freund's complete adjuvant (FCA) induced animal model of inflammation. Tubastat A (30 mg/kg i.p.) significant attenuates clinical scores (~ 70%), and IL-6 expression in paw tissues of collagen induced arthritis DBA1 mouse. [3]
Animal Research Animal Models Rat cholangiocarcinoma xenografts BDEneu
Dosages 10 mg/kg
Administration i.p. daily

Chemical Information & Solubility

Molecular Weight 335.4 Formula

C20H21N3O2
CAS No. 1252003-15-8 SDF Download Tubastatin A SDF
Smiles CN1CCC2=C(C1)C3=CC=CC=C3N2CC4=CC=C(C=C4)C(=O)NO
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 16.7 mg/mL ( (49.79 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : Insoluble

Ethanol : Insoluble


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In vivo
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Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

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Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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Frequently Asked Questions

Question 1:
We are planning to order some tubastatin A but I found out there are two versions of it. One has HCl and one does not. Which one do you recommend for live cell use? Will the HCl containing version significantly change the pH?

Answer:
S8049 and S2627 have same molecular structure. The only difference is S2627 containing HCl and has higher solubility in DMSO (74 mg/mL vs. S8049 9 mg/mL). Since they are the same molecule, the biological function should be similar. I would recommend to use S2627 for cell culture study.

Question 2:
What’s the vehicle do you recommend to dissolve the compound for in vivo experiments?

Answer:
S8049 Tubastatin A can be dissolved in 2% DMSO/30% PEG 300/PBS at 2.5 mg/mL as a clear solution, and it is also a clear solution in 2% DMSO/ corn oil at 2.5 mg/mL. The drug in 2% DMSO/0.5% Tween 80/PBS is a homogeneous suspension at 2.5 mg/mL at first. After stay for a while, the precipitation goes out at the bottom of the tube.

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