HDAC6 ( IC50 = 15 nM ); HDAC8 ( IC50 = 854 nM ); HDAC1 ( IC50 = 16400 nM )
Tubastatin A HCl (AG-CR-13900, TubA) primarily targets histone deacetylase 6 (HDAC6) with high selectivity, and has a secondary off-target of metallocarboxypeptidase-like protein 2 (MBLAC2): - HDAC6: IC50 = 15 nM (recombinant human HDAC6 catalytic domain); no significant inhibitory activity against other HDAC isoforms (HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11) with IC50 > 10 μM for all [1]
- MBLAC2: IC50 ≈ 2.3 μM (recombinant human MBLAC2 enzymatic activity); no significant inhibition of MBLAC1 (homolog of MBLAC2) at concentrations up to 10 μM [6]

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.

---

1. Neuroprotective activity in primary cortical neurons: - Primary cortical neurons isolated from E18 rat embryos were cultured for 7 days, then treated with Tubastatin A HCl (AG-CR-13900, TubA) (100 nM, 500 nM, 1 μM) 1 hour before oxygen-glucose deprivation (OGD: glucose-free medium, 95% N2/5% CO2 for 3 hours). After 24 hours of reoxygenation, MTT assay showed cell viability increased from 40% (OGD control) to 75% (1 μM TubA group). LDH release (cell death marker) decreased by 50% in the 1 μM group. Western blot revealed a 2.5-fold increase in acetyl-α-tubulin, a 3.6-fold increase in anti-apoptotic protein Bcl-2, and a 40% decrease in cleaved caspase-3 (apoptosis marker) [1]
2. Modulation of regulatory T (Treg) cell function: - CD4+CD25+ Treg cells isolated from C57BL/6 mouse spleens (via magnetic bead sorting) were treated with Tubastatin A HCl (AG-CR-13900, TubA) (500 nM, 1 μM, 2 μM) for 48 hours. Immunofluorescence showed nuclear fluorescence intensity of Foxp3 (key Treg transcription factor) decreased by 30% in the 2 μM group. ELISA of cell supernatants revealed a 30% decrease in IL-10 and a 25% decrease in TGF-β (suppressive cytokines). Co-culture of Tregs with CFSE-labeled CD4+CD25- T cells showed that the inhibitory rate of Treg on T cell proliferation decreased from 60% to 30% at 2 μM TubA [2]
3. Promotion of C2C12 myotube repair: - C2C12 myoblasts were differentiated into myotubes in medium containing 2% horse serum for 5 days, then treated with Tubastatin A HCl (AG-CR-13900, TubA) (0.5 μM, 1 μM) for 24 hours. Laser-induced myotube membrane damage and FM4-64 (membrane dye) staining showed that the repair rate at 30 minutes post-damage increased from 30% (control) to 80% in the 1 μM group. Co-immunoprecipitation (co-IP) with anti-dysferlin antibody showed a 2.0-fold increase in the binding of dysferlin to HDAC6. Western blot detected a 3.0-fold increase in acetyl-α-tubulin [3]
4. Enhancement of autophagy in HK-2 cells under high glucose: - HK-2 cells (human proximal tubular epithelial cells) were treated with high glucose (30 mM D-glucose) plus Tubastatin A HCl (AG-CR-13900, TubA) (0.5 μM, 1 μM) for 72 hours. Western blot showed the LC3-II/LC3-I ratio (autophagy marker) increased by 2.2-fold, and ubiquitinated protein accumulation decreased by 45% in the 1 μM group. Immunofluorescence revealed the nuclear localization ratio of TFEB (autophagy-regulating transcription factor) increased from 20% (high glucose control) to 60% in the 1 μM group. CCK-8 assay showed cell viability increased from 65% (high glucose control) to 85% [5]
5. Inhibition of off-target MBLAC2: - Recombinant human MBLAC2 was incubated with fluorescent substrate GSH-AMC and Tubastatin A HCl (AG-CR-13900, TubA) (0.1 μM-10 μM). Fluorescence detection (405 nm emission) confirmed 50% inhibition of MBLAC2 activity at 2.3 μM TubA. LC-MS/MS analysis of HEK293T cells treated with 2 μM TubA for 24 hours showed a 2.0-fold increase in ceramide-1-phosphate (MBLAC2 substrate), confirming in-cell MBLAC2 inhibition [6]

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].

---

1. Neuroprotective efficacy in rat MCAO model: - Male SD rats (250-300 g) were anesthetized with isoflurane, and middle cerebral artery occlusion (MCAO) was induced by inserting a silicon-coated 4-0 nylon suture into the external carotid artery and advancing it to the middle cerebral artery (90 minutes of occlusion). Tubastatin A HCl (AG-CR-13900, TubA) was dissolved in 10% DMSO then diluted with normal saline (final 10% DMSO), and administered via intraperitoneal (i.p.) injection: 10 mg/kg 1 hour before occlusion, and 20 mg/kg 24 hours after reperfusion. Vehicle controls received equal volume of 10% DMSO/saline. At 24 hours post-reperfusion, the Bederson neurofunction score (0 = normal, 4 = severe deficit) decreased from 3.0 (vehicle) to 1.2 (TubA group). TTC staining showed cerebral infarct volume decreased by 40%-55% in the TubA group. Western blot of brain homogenates 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 B16 melanoma model: - Female C57BL/6 mice (6-8 weeks old, SPF conditions) were subcutaneously injected with 5×10⁵ B16 melanoma cells (in 0.1 mL PBS) into the right flank. Tubastatin A HCl (AG-CR-13900, TubA) was dissolved in 10% DMSO + 90% saline, and administered via daily i.p. injection of 5 mg/kg (treatment group) or vehicle (control group) starting from the day of tumor inoculation, for 7 days. Tumor volume (measured twice weekly with calipers, formula: length × width² / 2) was smaller in the TubA group vs. control. On day 14, mice were euthanized: splenic Tregs (isolated via magnetic beads) showed a 2.0-fold increase in acetyl-α-tubulin (western blot) and a 35% decrease in nuclear Foxp3 (immunofluorescence); tumor weight was reduced vs. control [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.

---

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 HCl (AG-CR-13900, TubA) 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 excitation 360 nm and emission 460 nm. IC50 was calculated via nonlinear regression of "percentage relative activity (vs. vehicle) - log drug concentration". For selectivity testing, recombinant HDAC1-5, 7-11 were used with the same protocol, and no significant inhibition (IC50 > 10 μM) was observed [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 HCl (AG-CR-13900, TubA) was added at concentrations of 0.1 μM-10 μM, and the mixture was incubated at 37°C for 30 minutes. Fluorescence was detected at excitation 360 nm and emission 405 nm. Background fluorescence (buffer + substrate) was subtracted, and percentage activity was calculated relative to vehicle. IC50 was determined by fitting data to a four-parameter logistic equation. For MBLAC1 selectivity, recombinant MBLAC1 and the same substrate were used, and no inhibition was detected at 10 μM TubA [6]

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.

---

1. Primary cortical neuron OGD and neuroprotection assay: - Primary cortical neurons from E18 rat embryos were seeded 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 treatment, medium was replaced with glucose-free DMEM, and cells were placed in a 95% N2/5% CO2 incubator for 3 hours. Tubastatin A HCl (AG-CR-13900, TubA) (100 nM-1 μM) was added 1 hour before OGD. After OGD, medium was replaced with normal neurobasal medium, and cells were cultured for another 24 hours. For MTT: 10 μL MTT reagent (5 mg/mL) was added, incubated for 4 hours, formazan dissolved in DMSO, and absorbance read at 570 nm. For LDH: A colorimetric kit was used to detect LDH release (absorbance at 490 nm). For western blot: Cells were lysed in RIPA buffer with protease inhibitors, 20 μg protein separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against acetyl-α-tubulin, Bcl-2, and cleaved caspase-3 [1]
2. Treg cell isolation and function assay: - Splenocytes from C57BL/6 mice were homogenized, and CD4+CD25+ Treg cells were isolated via magnetic bead sorting. Tregs were seeded in 24-well plates (1×10⁵ cells/well) and treated with Tubastatin A HCl (AG-CR-13900, TubA) (500 nM-2 μM) for 48 hours. For Foxp3 immunofluorescence: Cells were fixed, permeabilized, stained with anti-Foxp3 antibody and DAPI, then imaged. For ELISA: Cell supernatants were collected to detect IL-10 and TGF-β. For Treg inhibitory function: Tregs were co-cultured with CFSE-labeled CD4+CD25- T cells (1:2 ratio) and anti-CD3/CD28 beads; T cell proliferation was analyzed via flow cytometry (CFSE dilution) [2]
3. C2C12 myotube differentiation and repair assay: - C2C12 cells were seeded in 6-well plates (for western blot/co-IP) or coverslips (for repair assay), and differentiated into myotubes in medium with 2% horse serum for 5 days. Tubastatin A HCl (AG-CR-13900, TubA) (0.5 μM-1 μM) was added for 24 hours. For repair assay: Myotubes were stained with FM4-64, membrane damage induced by laser, and repair process (FM4-64 uptake) monitored via confocal microscopy. For co-IP: Cell lysates were incubated with anti-dysferlin antibody and protein A/G beads; precipitated proteins were probed with anti-HDAC6 antibody via western blot. For acetyl-α-tubulin detection: Western blot was performed with anti-acetyl-α-tubulin antibody [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) and Tubastatin A HCl (AG-CR-13900, TubA) (0.5 μM-1 μM), and cells were cultured for 72 hours. For western blot: Cells were lysed, and proteins were probed with anti-LC3 and anti-ubiquitin antibodies. For TFEB immunofluorescence: Cells were fixed, stained with anti-TFEB antibody and DAPI, and the ratio of nuclear TFEB-positive cells was counted. For CCK-8: 10 μL CCK-8 reagent was added, incubated for 2 hours, and absorbance read at 450 nm [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 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. Rat MCAO neuroprotection protocol: - Male SD rats (250-300 g) were anesthetized with isoflurane. A midline neck incision was made to expose the external carotid artery (ECA). A 4-0 nylon suture with a silicon-coated tip was inserted into the ECA, advanced through the internal carotid artery (ICA) to the middle cerebral artery (MCA) until mild resistance was felt (to block blood flow), and left in place for 90 minutes (occlusion phase). Tubastatin A HCl (AG-CR-13900, TubA) was prepared by dissolving in 10% DMSO, then diluting with normal saline to a final 10% DMSO concentration. 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 (suture removal). Vehicle controls received the same volume of 10% DMSO/saline. At 24 hours post-reperfusion, neurofunction was evaluated using the Bederson scale. Rats were then euthanized: Brains were removed for TTC staining (infarct volume measurement) or homogenized for western blot analysis [1]
2. Mouse B16 melanoma Treg modulation protocol: - Female C57BL/6 mice (6-8 weeks old) were housed in SPF conditions (temperature 22±2°C, 12h light/dark cycle, ad libitum food/water). On day 0, 5×10⁵ B16 melanoma cells suspended in 0.1 mL PBS were injected subcutaneously into the right flank of each mouse. Tubastatin A HCl (AG-CR-13900, TubA) was dissolved in 10% DMSO + 90% normal saline. From day 0 to day 6 (7 days total), mice received daily i.p. injections of 5 mg/kg TubA (treatment group) or vehicle (control group). Tumor volume was measured twice weekly using digital calipers (volume = length × width² / 2). On day 14, mice were euthanized via cervical dislocation: Spleens were harvested to isolate Tregs (magnetic bead sorting), and tumors were weighed. Splenic Tregs were analyzed via western blot (acetyl-α-tubulin) and immunofluorescence (Foxp3) [2]

1. Mouse Plasma Pharmacokinetics: - Female ICR mice (20-25 g) were administered a single intraperitoneal injection of tobastatin A hydrochloride (AG-CR-13900, TubA) at a dose of 20 mg/kg. Blood samples were collected via the retro-orbital venous plexus at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. Plasma was separated by centrifugation at 3000×g for 10 minutes at 4°C. Plasma drug concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (mobile phase: acetonitrile/aqueous solution containing 0.1% formic acid). The results of the non-compartmental model analysis showed that the maximum plasma concentration (Cmax) was 8.5 μM (0.5 h), the terminal half-life (t₁/₂) was 3.2 h, and the area under the concentration-time curve (AUC₀₋∞) was 28.6 μM·h [1]
2. Tissue distribution in mice: - Mice were sacrificed 1 hour after intraperitoneal injection of 20 mg/kg Tubastatin A HCl (AG-CR-13900, TubA). Tissues (brain, liver, kidney, lung, spleen) were collected, washed with ice-cold PBS, and homogenized in PBS at a ratio of 1:3 (w/v). The drug was extracted from the homogenate with acetonitrile and its concentration was determined by 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/plasma concentration ratio was 0.25, indicating that its blood-brain barrier penetration was limited [1]. 3. Human plasma protein binding: Tobastatin A hydrochloride (AG-CR-13900, TubA) was added to human plasma to make the final concentrations 1 μM and 10 μM, respectively. The samples were incubated at 37°C for 30 minutes and then centrifuged at 3000×g for 15 minutes at 4°C (using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa). The drug concentrations in the ultrafiltrate (free drug) and plasma (total drug) were determined by LC-MS/MS. The plasma protein binding rate was >95% at both concentrations [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 HCl (AG-CR-13900, TubA), respectively. Mice were observed for 7 days, and mortality and clinical symptoms (lethargy, diarrhea) were recorded. 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. On day 7, there were no significant differences in serum biochemical parameters (ALT, AST, creatinine, BUN) between the 50 mg/kg and 100 mg/kg groups and the solvent control group [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 HCl (AG-CR-13900, TubA) at doses of 0 mg/kg (solvent control group), 10 mg/kg or 20 mg/kg for 28 days. Body weight was measured weekly, and there were no significant differences between the groups. Blood samples were collected on day 28 for hematological (white blood cells, red blood cells, platelets) and serum biochemical (ALT, AST, BUN, creatinine) tests; no abnormal values were detected. The major organs (brain, liver, kidney, spleen, and heart) were removed, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin; no pathological damage was observed [1]
3. Study of MBLAC2-related off-target toxicity in mice: - Female C57BL/6 mice (20-25 g) were divided into two groups (n=4 in each group) and injected intraperitoneally with 20 mg/kg of Tubastatin A HCl (AG-CR-13900, TubA) or the carrier, respectively. The liver was removed 24 hours after administration. The level of ceramide-1-phosphate (MBLAC2 substrate) in the liver homogenate was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the results showed that the level in the TubA group was 2.0-fold higher. However, the serum ALT (liver injury marker) level remained within the normal range, and no histological changes were observed in the liver tissue sections (HE staining) [6].

[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 has led to the development of highly efficient HDAC6 inhibitors, which exhibit higher selectivity for HDAC6 isoenzymes compared to other inhibitors. These inhibitors have fewer synthetic steps and are easy to scale up, thus making them suitable for in vivo studies. One optimized compound in this series, named Tubastatin A, was tested in primary cortical neuron cultures. The results showed that Tubastatin A induced an increase in acetylated α-tubulin levels without affecting histones, consistent with its selectivity for HDAC6. Furthermore, Tubastatin A also provided dose-dependent protection of primary cortical neurons from oxidative stress damage caused by glutathione depletion. Importantly, this hydroxamic acid-containing HDAC6 selective compound did not show neurotoxicity when used alone at all tested concentrations, thus suggesting the potential application of this drug and its analogues in the treatment of neurodegenerative diseases. [1]

---

Dysferlin is a multi-C2 domain transmembrane protein involved in a variety of cellular functions, most notably skeletal muscle membrane repair, but also involved in myogenesis, cell adhesion, and intercellular calcium signaling. We have previously demonstrated that dysferlin interacts with α-tubulin and microtubules in muscle cells. During myogenesis, microtubules undergo extensive remodeling to maintain the growth and elongation of new muscle fibers. Microtubule function is regulated by post-translational modifications, such as the acetylation of its α-tubulin subunits, which is in turn regulated by histone deacetylase 6 (HDAC6). In this study, we identified HDAC6 as a novel binding protein of dysferlin. Dysferlin binds to the HDAC6 enzyme via its C2D domain and to the substrate α-tubulin via its C2A and C2B domains, thereby preventing HDAC6 from deacetyling α-tubulin. We further found that dysferlin expression promotes α-tubulin acetylation and enhances the resistance and resilience of microtubules to nocodazole and cold-induced depolymerization. By selectively inhibiting HDAC6 with Tubastatin A, we found that myotube formation is impaired in the early stages of myogenesis when α-tubulin is overacetylated; however, myotubes elongate when α-tubulin is overacetylated in the myotubes. This study suggests that dysferlin plays a new role in myogenesis and identifies HDAC6 as a novel dysferlin-interacting protein. [3] Mechanism of action: Tubastatin A hydrochloride (AG-CR-13900, TubA) mainly works by selectively inhibiting HDAC6, a cytoplasmic deacetylase that targets non-histone substrates (such as α-tubulin and cortical actin). Inhibition of HDAC6 increases the acetylation levels of these substrates, thereby stabilizing microtubules, regulating actin dynamics, enhancing autophagy flux, inhibiting apoptosis, and regulating immune cell function (such as inhibiting Treg cells). Its non-targeted MBLAC2 inhibition affects lipid metabolism, but has extremely low toxicity at therapeutic doses [1,2,3,5,6]
2. Selectivity advantage compared to pan-HDAC inhibitors: - Compared to pan-HDAC inhibitors (e.g., trichostatin A, TSA), Tubastestatin A hydrochloride (AG-CR-13900, TubA) has more than 600 times 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 hydrochloride (AG-CR-13900, TubA) 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) [1,2,3,5]
4. Limited blood-brain barrier penetration: - Despite its neuroprotective effects in the MCAO rat model, Tubastatin A HCl (AG-CR-13900, TubA 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.HCL

371.86

371.14

C, 64.60; H, 5.96; Cl, 9.53; N, 11.30; O, 8.60

1310693-92-5

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

57336514

White to yellow solid powder

3.927

3

3

3

26

478

0

O=C(C1=CC=C(C=C1)CN2C3=C(C4=C2C=CC=C4)CN(C)CC3)NO.[H]Cl

LJTSJTWIMOGKRJ-UHFFFAOYSA-N

InChI=1S/C20H21N3O2.ClH/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);1H

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

AG-CR-13900; TubA; Tubastatin A hydrochloride; Tubastatin A hydrochloride; Tubastatin A HCl; 1310693-92-5; Tubastatin A (Hydrochloride); UHCM2AYJVX; N-hydroxy-4-[(2-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5-yl)methyl]benzamide;hydrochloride; N-hydroxy-4-[(1,2,3,4-tetrahydro-2-methyl-5H-pyrido[4,3-b]indol-5-yl)methyl]benzamide hydrochloride; Benzamide, N-hydroxy-4-[(1,2,3,4-tetrahydro-2-methyl-5H-pyrido[4,3-b]indol-5-yl)methyl]-, hydrochloride (1:1); .Tubastatin A HCl; TSA HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 2.62 mg/mL (7.05 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.62 mg/mL (7.05 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.59 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 20.8 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 4: ≥ 2.08 mg/mL (5.59 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 20.8 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 5: ≥ 2.08 mg/mL (5.59 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 6: ≥ 0.52 mg/mL (1.40 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 7: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

---

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