Enzyme inhibition assays
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Isolated recombinant human protein was used for the HDAC1, 2, 4, 5, 6, 7, 8, 9, 10 and 11 assays, and HDAC3/NcoR2 complex was used for the HDAC3 assay. Substrate for HDAC1, 2, 3, 6, 10 and 11 assays was a fluorogenic peptide from p53 residues 379 ~ 382 (RHKKAc); substrate for HDAC8 was fluorogenic diacyl peptide based on residues 379 ~ 382 of p53 (RHKAcKAc). Acetyl-Lys (trifluoroacetyl)-AMC substrate was used for HDAC4, 5, 7 and 9 assays. Tubastatin A was dissolved in DMSO and tested in 10-dose IC50 mode with 3-fold serial dilution starting at 30 μM. Control Compound Trichostatin A was tested in a 10-dose IC50 with 3-fold serial dilution starting at 5 μM. IC50 values were extracted by curve-fitting the dose/response slopes.
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Applications
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Adoptively transferred B6/Rag1-/- mice which were treated with Tubastatin A HCl recovered body weights and normal stool formation. In addition, the histologic examination showed reduced mononuclear cell infiltration, preservation of goblet cells, as well as intact mucosal surfaces.
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References:
[1]. Kyle V. Butler, Jay Kalin, Camille Brochier, et al. Rational Design and Simple Chemistry Yield a Superior, Neuroprotective HDAC6 Inhibitor, Tubastatin A [J]. J. Am. Chem. Soc., 2010, 132 (31), pp 10842–10846.
[2]. Kappeler KV, Zhang J, Dinh TN, et al. Histone deacetylase 6 associates with ribosomes and regulates de novo protein translation during arsenite stress [J]. Toxicol Sci. 2012 May;127(1):246-255.
[3]. de Zoeten EF, Wang L, Butler K, Beier UH, Akimova T, Sai H, Bradner JE, Mazitschek R, Kozikowski AP, Matthias P, Hancock WW. 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.
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