Hdac6

Manufactured by BPS Biosciences

Sourced in United States

HDAC6 is a recombinant protein produced by BPS Biosciences. It is a histone deacetylase enzyme that catalyzes the removal of acetyl groups from lysine residues on histone and non-histone proteins.

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Human recombinant HDAC6 Recombinant HDAC6

12 protocols using hdac6

HDAC Enzyme Inhibition Assay

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The HDAC assay kit was purchased from BPS Bioscience (San Diego, CA). The assays were carried out in black NUNC 96-well plates. Each well contained a volume of 50 μl including buffer (BPS Bioscience, catalogue no. 50031), the mixture of proteins (HDAC-1 (0.033 mg/ml, BPS Bioscience, catalogue no. 50051) or HDAC-6 (0.025 mg/ml, BPS Bioscience, catalogue no. 50006) and competing proteins), inhibitor (at the IC50), BSA (1 mg/mL, Sigma-Aldrich), and HDAC substrate 3 (20 μM, BPS Bioscience, catalogue no. 50037). Upon addition of substrate, the plate was incubated at 37 °C for 30 min. HDAC assay developer (50 μL, BPS Bioscience, catalogue no. 50030) was then added to each well and the plate incubated for 15 min at room temperature. The fluorescence was recorded at excitation and emission wavelengths of 360 and 460 nm, respectively. Blank wells containing no inhibitor or protein were subtracted from all wells. The control wells, containing no inhibitor, were arbitrarily set as 100% activity. The assays were performed in triplicate, and each assay contained each inhibitor/competing protein combination was conducted three times. The data were normalized to values measured for uninhibited enzyme. Assays were reported as mean ± standard deviation.

Chen Y, & Cohen S.M. (2015). Investigating the Selectivity of Metalloenzyme Inhibitors in the Presence of Competing Metalloproteins. ChemMedChem, 10(10), 1733-1738.

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HDAC Inhibition Assay Protocol

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HDAC-1 and −6 were purchased from BPS Bioscience (BPS Bioscience catalog #50051 and 50006, San Diego, CA, USA) and the assay was carried out as instructed by manufacturer. The assays were carried out in black 96-well plates (Costar). Each well contained a volume of 50 μL including buffer (25 mM Tris/HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 0.1 mg/mL BSA, pH 8.0), HDAC (3.8 ng/well of HDAC-1, 50 ng/well of HDAC-6, BPS Bioscience, catalogue no. 50051 and no. 50006, respectively), inhibitor (various concentrations, 1 mM – 5 μM), and HDAC substrate 3 (20 μM, BPS Bioscience, catalogue no. 50037). Prior to adding substrate, the plate was preincubated for 5 min. Upon addition of substrate, the plate was incubated at 37 °C for 30 min. HDAC assay developer (50 μL, BPS Bioscience, catalogue no. 50030) was added to each well and the plate incubated for 15 min at room temperature. The fluorescence was recorded at excitation and emission wavelengths of 360 and 460 nm, respectively. The negative control wells, containing no inhibitor, were arbitrarily set as 100% activity. The positive control wells, containing 200 μM SAHA, were arbitrarily set as 0% activity. HDAC activity was defined as the ratio of fluorescence in the inhibitor wells relative to the negative control wells, expressed as a percentage. The assays were performed in triplicate.

Chen A.Y., Thomas P.W., Stewart A.C., Bergstrom A., Cheng Z., Miller C., Bethel C.R., Marshall S.H., Credille C.V., Riley C.L., Page R.C., Bonomo R.A., Crowder M.W., Tierney D.L., Fast W, & Cohen S.M. (2017). Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1. Journal of medicinal chemistry, 60(17), 7267-7283.

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HDAC Inhibition Assay Protocol

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The assays were carried out by Shanghai ChemPartner Co., Ltd (Shanghai, China), according to our previous method [23 (link),24 (link)]. Briefly, different concentrations of compounds were incubated with recombinant human HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8 (BPS Biosciences, San Diego, CA, USA) at room temperature for 15 min, which was followed by adding Ac-peptide-AMC substrates to initiate the reaction in Tris-based assay buffer. Reaction mixtures were incubated at room temperature for 60 min in HDAC1, HDAC2, HDAC3 and HDAC6 assays, and were incubated for 240 min in HDAC8 assay. Then, the stop solution containing trypsin was added. The coupled reaction was incubated for another 90 min at 37 °C. Fluorescent AMC released from substrate was measured in SynergyMx (BioTek, Winooski, VT, US) using filter sets as excitation = 355 nm and emission = 460 nm. IC₅₀ values were calculated by GraphPad Prism software (7.0 version., GraphPad Software, San Diego, CA, USA).

Zhang B., Ruan Z.W., Luo D., Zhu Y., Ding T., Sui Q, & Lei X. (2020). Unexpected Enhancement of HDACs Inhibition by MeS Substitution at C-2 Position of Fluoro Largazole. Marine Drugs, 18(7), 344.

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Acetylation and Deacetylation Assay

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ACY-738 was synthesized and kindly provided by Acetylon Pharmaceuticals, Inc. (Boston, MA). We purchased the recombinant proteins p300 (Enzo Life Sciences Inc., Farmingdale, NY), HDAC6 (BPS Biosciences, San Diego, CA), and tubulin (Cytoskeleton Inc., Denver, CO). We also purchased phosphatase inhibitor mixtures II and III, nicotinamide, trichostatin A, DNase, acetyl-CoA, dextran sulfate (6,500–10,000 Da), thioflavin S, Coomassie Blue, and β-mercaptoethanol from Sigma-Aldrich. In addition, we purchased DTT (Fisher), protease inhibitor mixture, and isopropyl 1-thio-d-galactopyranoside from EMD Millipore (Billerica, MA).

Carlomagno Y., Chung D.E., Yue M., Castanedes-Casey M., Madden B.J., Dunmore J., Tong J., DeTure M., Dickson D.W., Petrucelli L, & Cook C. (2017). An acetylation–phosphorylation switch that regulates tau aggregation propensity and function. The Journal of Biological Chemistry, 292(37), 15277-15286.

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HDAC Inhibition Assay Protocol

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The assays were carried out by Shanghai ChemPartner Co., Ltd. (Shanghai, China). Briefly, different concentrations of compounds were incubated with recombinant human HDAC1, HDAC2, HDAC3, HDAC6, and HDAC8 (BPS Biosciences, San Diego, CA, USA) at room temperature for 15 min, which was followed by adding Ac-peptide-AMC substrates to initiate the reaction in Tris-based assay buffer. Reaction mixtures were incubated at room temperature for 60 min in the HDAC1, HDAC2, HDAC3, and HDAC6 assays, and were incubated for 240 min in the HDAC8 assay. Then a stop solution containing trypsin was added. The coupled reaction was incubated for another 90 min at 37 °C. Fluorescent AMC released from substrate was measured in SynergyMx (BioTek, Winooski, VT, USA) using filter sets as excitation = 355 nm and emission = 460 nm. IC₅₀ values were calculated by GraphPad Prism version 4.00 Windows (GraphPad Software, San Diego, CA, USA).

Zhang B., Shan G., Zheng Y., Yu X., Ruan Z.W., Li Y, & Lei X. (2019). Synthesis and Preliminary Biological Evaluation of Two Fluoroolefin Analogs of Largazole Inspired by the Structural Similarity of the Side Chain Unit in Psammaplin A. Marine Drugs, 17(6), 333.

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In Vitro HDAC Inhibition Assay

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The in vitro HDACs inhibitory assay of all compounds was performed by Sundia MediTech Company, Ltd. (Shanghai, China). It was conducted by fluorescent assay. Briefly, all compounds were serially diluted to certain concentrations. Then, the HDAC1 enzymes (Active Motif, 31504), HDAC2 (BPS bioscience, 50002), HDAC5 (BPS bioscience, 50005), HDAC6 (BPS bioscience, 50006), HDAC8 (Active Motif, 31566), HDAC11 (BPS bioscience, 50011), and compounds solution were dissolved in 1×Assay buffer, which was then added into a 384-well microplate. It was mixed briefly with gentle centrifuging and the plate was incubated at r.t. Substrate peptide and Trypsin mixture were added to each well, shaken for 1 minute, and the 384-plate was placed in a BioTek Synergy plate reader for data collection.

Wang S., Han S., Cheng W., Miao R., Li S., Tian X, & Kan Q. (2022). Design, Synthesis, and Biological Evaluation of 2-Anilino-4-Triazolpyrimidine Derivatives as CDK4/HDACs Inhibitors. Drug Design, Development and Therapy, 16, 1083-1097.

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HDAC Enzyme Inhibition Assay Protocol

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Recombinant human HDAC1 and HDAC6 were purchased from BPS Bioscience. Recombinant human HDAC8 was expressed and purified as previously described [24 (link)]. Recombinant smHDAC8 enzyme was produced in E. coli cells and purified by a method previously described by us [24 (link)]. Inhibition assays of smHDAC8 and human HDACs were performed as already described in previous publications [24 (link),56 (link)]. Briefly, the commercial Fluor de Lys kit (BML-KI178) was used for testing inhibition of smHDAC8 and hsHDAC8. Test compounds, Fluor de Lys-HDAC8 substrate (50 µM) and enzyme were incubated for 90 min at 37 °C with subsequent addition of 50 μL Developer II (BML-KI176) and further incubation for 45 min at 30 °C. Fluorescence was measured in a plate reader (BMG Polarstar) with excitation at λ = 390 nm and emission at λ = 460 nm. Inhibition tests of human HDAC1 and 6 were conducted using ZMAL (Cbz-(Ac)Lys-AMC) as substrate and trypsin as a developer. After incubation of the compounds, ZMAL (10.5 µM) and enzyme for 90 min at 37 °C, 60 μL of trypsin was added and further incubated for 20 min at 37 °C. Trichostatin A (2 µM) was used in both assays to stop the reaction. Fluorescence was measured similarly as mentioned above. IC₅₀ values were determined with OriginPro (version 9.0.0, Northampton, MA, USA). IC₅₀ Values in Table 1 are given as mean ± S.E.

Simoben C.V., Robaa D., Chakrabarti A., Schmidtkunz K., Marek M., Lancelot J., Kannan S., Melesina J., Shaik T.B., Pierce R.J., Romier C., Jung M, & Sippl W. (2018). A Novel Class of Schistosoma mansoni Histone Deacetylase 8 (HDAC8) Inhibitors Identified by Structure-Based Virtual Screening and In Vitro Testing. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(3), 566.

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High-Throughput Screening of Epigenetic Modulators

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Four cell lines (human colon cancer cell line HCT116, human embryonic kidney cell line HEK293, human liver cancer cell line HepG2, and human B lymphoma cell line SU-DHL-6), fetal bovine serum (FBS), Eagle's Minimum Essential Medium (EMEM), and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from American Type Cell Collection (ATCC, Manassas, VA, USA). H9-derived human neural stem cells, defined FBS, trypsin-EDTA, fibronectin, basal fibroblast growth factor (bFGF), epidermal growth factor (EGF), KnockOut™ Dulbecco's Modified Eagle Medium (DMEM)/F12, and penicillin-streptomycin, StemPro^® Neural Supplement were acquired from Life Technologies, Carlsbad, CA, USA. Chemicals and epigenetic compound libraries were purchased from Cayman Chemicals (Ann Arbor, MI), Santa Cruz Biotechnology (Dallas, TX, USA), SelleckChem (Houston, TX, USA), and Sigma-Aldrich (St. Louise, MO, USA). HDAC-Glo I/II and CellTiter-Glo reagents were purchased from Promega, Madison, WI. Fluorogenic assay kits for HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and HDAC10 were acquired from BPS Bioscience (San Diego, CA). White solid bottom 1536-well assay plates were purchased from Greiner Bio-One (Monroe, NC).

Hsu C.W., Shou D., Huang R., Khuc T., Dai S., Zheng W., Klumpp-Thomas C, & Xia M. (2016). Identification of HDAC inhibitors using a cell-based HDAC I/II assay. Journal of biomolecular screening, 21(6), 643-652.

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HDAC Inhibitor Screening and Analysis

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Dulbecco’s Modified Eagle’s medium (DMEM) with L-glutamine was purchased from GenDEPOT (Barker, TX, USA) and RPMI 1640 medium, fatal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA). Antibodies specific for α-tubulin, Ac-α-tubulin, Histone H3, Ac-Histone H3, PARP, caspase 3, cleaved caspase 8, β-actin HDAC1, and HDAC6 were purchased from Cell Signalling Technology (Boston, MA, USA). Rad52 antibody and goat anti-rabbit IgG horseradish peroxidase conjugate were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Cell Titre 96 Aqueous One Solution cell proliferation assay kit was purchased from Promega (Madison, WI, USA). Amersham ECL select Western blotting detection reagent was purchased from GE Healthcare (Waukesha, WI, USA). HDAC fluorogenic assay kits (HDAC1, HDAC3, HDAC6, and HDAC8) were purchased from BPS Bioscience (San Diego, CA, USA). OxiSelect™ Comet Assay Kit was purchased from Cell Biolabs, Inc (San Diego, CA, USA).

Song Y., Park S.Y., Wu Z., Liu K.H, & Seo Y.H. (2020). Hybrid inhibitors of DNA and HDACs remarkably enhance cytotoxicity in leukaemia cells. Journal of Enzyme Inhibition and Medicinal Chemistry, 35(1), 1069-1079.

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HDAC1 and HDAC6 Inhibition Assay

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The inhibition of human HDAC1 and 6 was determined as previously described [35 (link)]. OptiPlate-96 black microplates (Perkin Elmer Inc., Waltham, MA, USA) were used with an assay volume of 50 µL. A total of 5 µL test compound or control, diluted in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 0.1 mg/mL BSA), was incubated with 35 µL of the fluorogenic substrate ZMAL (Z-Lys(Ac)-AMC) (21.43 µM in assay buffer) and 10 µL of human recombinant HDAC1 (BPS Bioscience Inc., San Diego, CA, USA, Catalog# 50051) or HDAC6 (BPS Bioscience Inc., San Diego, CA, USA, Catalog# 50006) at 37 °C. After an incubation time of 90 min, 50 µL of 0.4 mg/mL trypsin in trypsin buffer (50 mM Tris-HCl, pH 8.0, and 100 mM NaCl) was added, followed by further incubation at 37 °C for 30 min. Fluorescence was measured with an excitation wavelength of 355 nm and an emission wavelength of 460 nm using a Fluoroskan Ascent microplate reader (Thermo Scientific, Waltham, MA, USA). All compounds were tested at least twice and in duplicates, and the 50% inhibitory concentration (IC₅₀) was determined by plotting dose response curves and nonlinear regression with GraphPad Prism (San Diego, CA, USA).

von Bredow L., Schäfer T.M., Hogenkamp J., Tretbar M., Stopper D., Kraft F.B., Schliehe-Diecks J., Schöler A., Borkhardt A., Bhatia S., Held J, & Hansen F.K. (2022). Synthesis, Antiplasmodial, and Antileukemia Activity of Dihydroartemisinin–HDAC Inhibitor Hybrids as Multitarget Drugs. Pharmaceuticals, 15(3), 333.

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