HDAC6 ( IC50 = 15 nM ); HDAC8 ( IC50 = 854 nM ); HDAC1 ( IC50 = 16400 nM )
The primary target of Tubastatin A (TubA, AG-CR1-3900) is histone deacetylase 6 (HDAC6), with an IC50 of 15 nM for recombinant human HDAC6 catalytic domain. It exhibits high selectivity for HDAC6, as IC50 values against other HDAC isoforms (HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11) are all >10 μM [1]
A secondary off-target of Tubastatin A (TubA, AG-CR1-3900) is metallocarboxypeptidase-like protein 2 (MBLAC2), with an IC50 of ~2.3 μM for recombinant human MBLAC2 enzymatic activity. It shows no significant inhibition against MBLAC1 (a homolog of MBLAC2) at concentrations up to 10 μM [7,9]

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].

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1. Neuroprotective activity in primary cortical neurons: - Primary cortical neurons isolated from E18 rat embryos were cultured for 7 days, then subjected to oxygen-glucose deprivation (OGD, 3 hours: glucose-free medium, 95% N2/5% CO2). Tubastatin A (TubA, AG-CR1-3900) (100 nM, 500 nM, 1 μM) was added 1 hour before OGD. After 24 hours of reoxygenation, MTT assay showed that cell viability increased from 40% (OGD control) to 75% in the 1 μM TubA group. LDH release (a marker of cell death) was reduced by 50% in the 1 μM group. Western blot analysis revealed a 2.5-fold increase in acetyl-α-tubulin levels, a 3.6-fold increase in Bcl-2 (anti-apoptotic protein) expression, and a 40% decrease in cleaved caspase-3 (apoptotic marker) compared to OGD controls [1]
2. Regulation of actin dynamics in cancer cells: - MDA-MB-231 breast cancer cells were treated with Tubastatin A (TubA, AG-CR1-3900) (0.5 μM, 1 μM, 2 μM) for 24 hours. Transwell migration assays showed that 1 μM TubA reduced the number of migrating cells by 40%. Western blot detected a 2.8-fold increase in acetyl-cortactin (a substrate of HDAC6) and a 35% decrease in phosphorylated NEDD9 (a regulator of actin dynamics). Immunofluorescence microscopy (confocal) revealed reduced cortactin accumulation at the cell leading edge, with DAPI staining for nuclei [2]
3. Modulation of T-regulatory (Treg) cell function: - CD4+CD25+ Treg cells were isolated from C57BL/6 mouse spleens using magnetic beads and treated with Tubastatin A (TubA, AG-CR1-3900) (500 nM, 1 μM, 2 μM) for 48 hours. Immunofluorescence showed that 2 μM TubA decreased nuclear Foxp3 (a Treg master transcription factor) fluorescence intensity by 30%. ELISA analysis of cell supernatants demonstrated a 30% reduction in IL-10 and a 25% reduction in TGF-β (suppressive cytokines). Co-culture of Tregs with CFSE-labeled CD4+CD25- T cells showed that 2 μM TubA reduced Treg-mediated suppression of T-cell proliferation (inhibition rate from 60% to 30%) [3]
4. Enhancement of misfolded protein clearance in chronic kidney disease models: - HK-2 cells (human proximal tubular epithelial cells) were treated with high glucose (30 mM D-glucose) plus Tubastatin A (TubA, AG-CR1-3900) (0.5 μM, 1 μM) for 72 hours. Western blot showed a 2.2-fold increase in the LC3-II/LC3-I ratio (autophagy marker) and a 45% decrease in ubiquitinated protein accumulation in the 1 μM group. Immunofluorescence indicated that 1 μM TubA increased nuclear localization of TFEB (a transcription factor regulating autophagy) from 20% to 60% of cells. CCK-8 assay showed cell viability increased from 65% (high glucose control) to 85% [4]
5. Interaction with dysferlin and promotion of myotube repair: - C2C12 myoblasts were differentiated into myotubes (5 days in 2% horse serum) and treated with Tubastatin A (TubA, AG-CR1-3900) (0.5 μM, 1 μM) for 24 hours. Laser-induced myotube membrane damage and FM4-64 staining (for repair sites) showed that 1 μM TubA increased repair rate from 30% to 80% at 30 minutes post-damage. Co-immunoprecipitation (co-IP) with anti-dysferlin antibody revealed a 2.0-fold increase in HDAC6 binding to dysferlin. Western blot detected a 3.0-fold increase in acetyl-α-tubulin [5]
6. Inhibition of off-target MBLAC2: - Recombinant human MBLAC2 enzyme was incubated with a fluorescent substrate (GSH-AMC) and Tubastatin A (TubA, AG-CR1-3900) (0.1 μM-10 μM). Fluorescence measurement (405 nm emission) showed 50% enzyme inhibition at 2.3 μM. LC-MS analysis of MBLAC2 substrate (ceramide-1-phosphate) in HEK293T cells treated with 2 μM TubA showed a 2.0-fold increase in substrate levels, confirming in-cell MBLAC2 inhibition [7,9]

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].

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1. Neuroprotection in rat middle cerebral artery occlusion (MCAO) model: - Male SD rats (250-300 g) underwent MCAO (90-minute occlusion via intraluminal suture). Tubastatin A (TubA, AG-CR1-3900) was dissolved in 10% DMSO + 90% normal saline and administered via intraperitoneal (i.p.) injection: 10 mg/kg 1 hour before occlusion, and 20 mg/kg 24 hours after occlusion. Vehicle controls received equal volumes of solvent. At 24 hours post-reperfusion, Bederson neurofunctional scores (0-4, higher = worse) decreased from 3.0 (vehicle) to 1.2 (TubA group). TTC staining showed cerebral infarct volume reduced by 40%-55% in the TubA group. Western blot of brain tissues revealed a 2.8-fold increase in acetyl-α-tubulin and a 50% decrease in cleaved caspase-3 [1]
2. Modulation of Treg function in mouse melanoma model: - Female C57BL/6 mice (6-8 weeks old) were subcutaneously injected with 5×10⁵ B16 melanoma cells. Tubastatin A (TubA, AG-CR1-3900) (5 mg/kg, dissolved in 10% DMSO + 90% saline) was administered i.p. daily for 7 days, starting on the day of tumor inoculation. Vehicle controls received solvent. Tumor volume (calculated as length × width² / 2) was 40% larger in the TubA group than in controls at day 14. Splenic Tregs isolated at day 14 showed a 2.0-fold increase in acetyl-α-tubulin and a 35% decrease in nuclear Foxp3 (immunofluorescence) compared to controls [3]

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.

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1. Recombinant HDAC6 activity assay: - Recombinant human HDAC6 catalytic domain was mixed with fluorogenic substrate Boc-Lys(Ac)-AMC in reaction buffer (50 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM DTT). Tubastatin A (TubA, AG-CR1-3900) was added at concentrations ranging from 0.1 nM to 10 μM, and the mixture was incubated at 37°C for 60 minutes. Trypsin-containing developer solution was added to cleave deacetylated substrate, releasing fluorescent AMC. Fluorescence intensity was measured at 360 nm (excitation) and 460 nm (emission). IC50 was calculated by nonlinear regression of percentage activity (vs. vehicle) against log drug concentration, resulting in an IC50 of 15 nM for HDAC6. For selectivity testing, the same protocol was used with recombinant HDAC1-5,7-11; IC50 >10 μM for all [1]
2. Recombinant MBLAC2 activity assay: - Recombinant human MBLAC2 was incubated with fluorescent substrate GSH-AMC in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA). Tubastatin A (TubA, AG-CR1-3900) (0.1 μM-10 μM) was added, and the mixture was incubated at 37°C for 30 minutes. Fluorescence was measured at 360 nm (excitation) and 405 nm (emission). IC50 was determined by dose-response curves, showing 50% inhibition at 2.3 μM. To test MBLAC1 selectivity, recombinant MBLAC1 was used with the same substrate; no inhibition was observed at 10 μM TubA [7,9]

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.

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1. Primary cortical neuron OGD and neuroprotection assay: - E18 rat cortical neurons were plated in 96-well plates (for MTT/LDH) or 6-well plates (for western blot) and cultured in neurobasal medium with B27 supplement for 7 days. For OGD, medium was replaced with glucose-free DMEM, and cells were placed in a 95% N2/5% CO2 incubator for 3 hours. Tubastatin A (TubA, AG-CR1-3900) (100 nM-1 μM) was added 1 hour before OGD. After OGD, medium was replaced with normal neurobasal medium for 24 hours. MTT reagent (5 mg/mL) was added (10 μL/well) for 4 hours; formazan was dissolved in DMSO, and absorbance was read at 570 nm. LDH release was measured using a colorimetric kit (absorbance at 490 nm). For western blot, cells were lysed in RIPA buffer with protease inhibitors; 20 μg protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against acetyl-α-tubulin, Bcl-2, and cleaved caspase-3 [1]
2. MDA-MB-231 cell migration and actin dynamics assay: - MDA-MB-231 cells were seeded in 6-well plates (for western blot) or Transwell inserts (for migration). For migration assays, 5×10⁴ cells in serum-free medium were added to upper inserts, and medium with 10% FBS was added to lower chambers. Tubastatin A (TubA, AG-CR1-3900) (0.5 μM-2 μM) was added to both chambers. After 24 hours, cells on the lower surface were fixed with methanol, stained with crystal violet, and counted. For western blot, cells were treated with TubA for 24 hours, lysed, and probed with antibodies against acetyl-cortactin and phosphorylated NEDD9. For immunofluorescence, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, stained with anti-cortactin antibody and DAPI, and imaged by confocal microscopy [2]
3. Mouse Treg cell isolation and function assay: - C57BL/6 mouse spleens were homogenized, and CD4+CD25+ Tregs were isolated using magnetic bead sorting. Tregs were plated in 24-well plates (1×10⁵ cells/well) and treated with Tubastatin A (TubA, AG-CR1-3900) (500 nM-2 μM) for 48 hours. For Foxp3 immunofluorescence, cells were fixed, permeabilized, stained with anti-Foxp3 antibody and DAPI, and imaged. For ELISA, cell supernatants were collected to measure IL-10 and TGF-β. For suppression assays, Tregs were co-cultured with CFSE-labeled CD4+CD25- T cells (1:2 ratio) and anti-CD3/CD28 beads; CFSE dilution (T-cell proliferation) was analyzed by flow cytometry [3]
4. HK-2 cell high glucose and autophagy assay: - HK-2 cells were seeded in 6-well plates and cultured in DMEM with 10% FBS. Medium was replaced with DMEM containing 30 mM D-glucose (high glucose) plus Tubastatin A (TubA, AG-CR1-3900) (0.5 μM-1 μM) for 72 hours. For western blot, cells were lysed and probed with antibodies against LC3 and ubiquitin. For TFEB immunofluorescence, cells were fixed, stained with anti-TFEB antibody and DAPI, and the percentage of nuclear TFEB-positive cells was counted. Cell viability was measured by CCK-8 assay (absorbance at 450 nm) [4]
5. C2C12 myotube repair and co-IP assay: - C2C12 cells were seeded in 6-well plates (for western blot/co-IP) or glass coverslips (for repair assay) and differentiated into myotubes with 2% horse serum for 5 days. Tubastatin A (TubA, AG-CR1-3900) (0.5 μM-1 μM) was added for 24 hours. For repair assays, myotubes were stained with FM4-64 (membrane dye), and a laser was used to induce membrane damage; repair was monitored by confocal microscopy (FM4-64 uptake). For co-IP, cell lysates were incubated with anti-dysferlin antibody and protein A/G beads; precipitated proteins were analyzed by western blot with anti-HDAC6 antibody. Acetyl-α-tubulin was detected by western blot [5]

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.

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1. Rat MCAO model for neuroprotection: - Male SD rats (250-300 g) were anesthetized with isoflurane. A 4-0 nylon suture with a silicon-coated tip was inserted into the external carotid artery and advanced into the middle cerebral artery to induce occlusion for 90 minutes. Tubastatin A (TubA, AG-CR1-3900) was prepared by dissolving in 10% DMSO followed by dilution with normal saline (final 10% DMSO). The drug was administered via i.p. injection: 10 mg/kg at 1 hour before occlusion, and 20 mg/kg at 24 hours after reperfusion. Vehicle controls received the same volume of 10% DMSO/saline. At 24 hours post-reperfusion, rats were evaluated for neurofunction using the Bederson scale (0 = normal, 4 = severe deficit). Rats were then euthanized, and brains were removed for TTC staining (to measure infarct volume) or western blot analysis of brain homogenates [1]
2. Mouse B16 melanoma model for Treg modulation: - Female C57BL/6 mice (6-8 weeks old) were housed under SPF conditions. B16 melanoma cells (5×10⁵ cells in 0.1 mL PBS) were injected subcutaneously into the right flank. Tubastatin A (TubA, AG-CR1-3900) was dissolved in 10% DMSO + 90% saline. Starting on the day of tumor inoculation, mice received daily i.p. injections of 5 mg/kg TubA (treatment group) or solvent (control group) for 7 days. Tumor volume was measured twice weekly with calipers (volume = length × width² / 2). On day 14, mice were euthanized; spleens were collected to isolate Tregs (magnetic beads), and tumors were weighed. Splenic Tregs were analyzed by western blot (acetyl-α-tubulin) and immunofluorescence (Foxp3) [3]

1. Mouse Plasma Pharmacokinetics: - Female ICR mice (20-25 g) were administered a single intraperitoneal injection of Tubastatin A (TubA, AG-CR1-3900) at a dose of 20 mg/kg. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. Plasma was separated by centrifugation (3000×g, 10 min, 4℃). Drug concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (mobile phase: acetonitrile/aqueous solution containing 0.1% formic acid). Pharmacokinetic parameters were calculated using a non-compartmental model: maximum plasma concentration (Cmax) = 8.5 μM (0.5 h), terminal half-life (t₁/₂) = 3.2 h, area under the concentration-time curve (AUC₀₋∞) = 28.6 μM·h [1]
2. Tissue distribution in mice: - Mice were sacrificed 1 hour after intraperitoneal injection of 20 mg/kg Tubastatin A (TubA, AG-CR1-3900). Tissues (brain, liver, kidney, lung, spleen) were collected, homogenized in PBS (1:3 w/v), and extracted with acetonitrile. Drug concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS): liver = 12.5 μM, kidney = 9.8 μM, lung = 7.2 μM, spleen = 5.5 μM, brain = 2.1 μM. The brain tissue/plasma concentration ratio was 0.25, indicating that its blood-brain barrier penetration was limited [1].
3. Plasma protein binding rate: Tubastatin A (TubA, AG-CR1-3900) was added to human plasma to make its final concentrations of 1 μM and 10 μM, respectively. The samples were incubated at 37°C for 30 minutes, and then ultrafiltered at a centrifugation force of 3000×g (molecular weight cutoff 30 kDa) for 15 minutes. The drug concentration in the ultrafiltrate (free drug) and plasma (total drug) was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The plasma protein binding rate at both concentrations was >95% [1].

1. Acute toxicity in mice: Female ICR mice (20-25 g) were randomly divided into four groups (n=6 per group) and intraperitoneally injected with 0 mg/kg (solvent control), 50 mg/kg, 100 mg/kg, or 200 mg/kg of Tubastatin A (TubA, AG-CR1-3900), respectively. Mortality and clinical symptoms (lethargy, diarrhea) were monitored for 7 consecutive days. No deaths were observed in the 50 mg/kg group; 1 out of 6 mice died in the 100 mg/kg group; and 4 out of 6 mice died in the 200 mg/kg group. At the 100 mg/kg dose, a transient decrease in body weight (8% of initial body weight) was observed on day 3, which was fully recovered by day 5. Serum biochemical indicators (ALT, AST, creatinine, BUN) on day 7 showed no significant changes compared with the control group in the 50 mg/kg and 100 mg/kg groups [1]
2. Chronic toxicity in rats: - Male SD rats (250-300 g) were divided into 3 groups (n=8 per group) and injected intraperitoneally daily with Tubastatin A (TubA, AG-CR1-3900) at doses of 0 mg/kg (excipient), 10 mg/kg or 20 mg/kg for 28 days. Body weight was measured weekly; no significant differences were observed between the groups. Blood samples were collected on day 28 for routine blood tests (white blood cell count, red blood cell count, platelet count) and serum biochemical tests (ALT, AST, BUN, creatinine); no abnormal values were detected. Major organs (brain, liver, kidney, spleen, heart) were removed, fixed with formalin and stained with HE; no pathological damage was observed [1]. 3. MBLAC2-related off-target toxicity: - Female C57BL/6 mice (20–25 g) were administered a single intraperitoneal injection of 20 mg/kg of Tubastatin A (TubA, AG-CR1-3900) or its carrier (n=4 per group). Twenty-four hours after administration, the liver was removed, and ceramide-1-phosphate (MBLAC2 substrate) levels were measured by LC-MS/MS. The TubA group showed a 2.0-fold increase in hepatic ceramide-1-phosphate levels, but serum ALT (a marker of liver injury) remained within the normal range. No histological changes were observed in liver sections [7,9].

[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.

Tubastatin A is a pyridoindole compound, chemically named 1,2,3,4-tetrahydro-5H-pyrido[4,3-b]indole, in which the tetrahydropyridine nitrogen atom is substituted with a methyl group, and the indole nitrogen atom is substituted with a p-[N-(hydroxy)aminocarbonyl]benzyl group. It is a histone deacetylase 6 (HDAC6) inhibitor, exhibiting 1000-fold selectivity for all isoenzymes except HDAC8, with a 57-fold selectivity for HDAC8. It also functions as an EC 3.5.1.98 (histone deacetylase) inhibitor. It is a pyridoindole, hydroxamic acid, and tertiary amine compound.

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1. Mechanism of Action: Tubastatin A (TubA, AG-CR1-3900) primarily exerts its effect through the selective inhibition of HDAC6. HDAC6 is a cytoplasmic deacetylase that targets non-histone substrates (e.g., α-tubulin, cortical actin). Inhibition of HDAC6 increases the acetylation levels of these substrates, thereby stabilizing microtubules, regulating actin dynamics, enhancing autophagy flux, inhibiting apoptosis, and modulating immune cell function (e.g., inhibiting Treg cells). Its non-targeting MBLAC2 inhibition affects lipid metabolism, but has minimal toxicity at therapeutic doses [1,2,3,4,5,7,9]
2. Selectivity advantage relative to pan-HDAC inhibitors: Compared to pan-HDAC inhibitors (e.g., tubastatin A, TSA), Tubastatin A (TubA, AG-CR1-3900) exhibits more than 600-fold higher selectivity for HDAC6 than other HDAC subtypes. This reduces the toxicities associated with class I HDAC inhibitors (e.g., hematopoietic suppression, gastrointestinal side effects), making it more suitable for long-term treatment of chronic diseases (e.g., neurodegenerative diseases, chronic kidney disease) [1]
3. Potential clinical applications: Based on in vitro and in vivo data, Tubastatin A (TubA, AG-CR1-3900) has the following potential applications: (1) Neurodegenerative diseases/ischemic stroke (exerting neuroprotective effects through microtubule stabilization and anti-apoptosis); (2) Cancer immunotherapy (modulating Treg function to enhance anti-tumor immunity); (3) Chronic kidney disease (promoting TFEB-mediated clearance of misfolded proteins); (4) Myopathy (improving myotubule repair through dysferlin-HDAC6 interaction) [1,3,4,5]
4. Limited blood-brain barrier penetration: Despite its neuroprotective effects in the MCAO rat model, Tubastatin A (TubA, AG-CR1-3900) has limited blood-brain barrier penetration. AG-CR1-3900 has limited blood-brain barrier penetration (brain/plasma ratio = 0.25). Future improvements (e.g., prodrug, nanoparticle delivery) may be needed to increase its brain concentration in central nervous system diseases [1]

C20H21N3O2

335.4

371.14

C, 71.62; H, 6.31; N, 12.53; O, 9.54

1252003-15-8

1252003-15-8; 1239034-70-8 (TFA) ; 1252003-15-8 1310693-92-5 (HCl);

49850262

White solid powder

3.125

2

3

3

25

478

0

O=C(NO)C1=CC=C(CN2C3=C(CN(C)CC3)C4=C2C=CC=C4)C=C1

GOVYBPLHWIEHEJ-UHFFFAOYSA-N

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)

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

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

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)

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.

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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.

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

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