| Assay Method Information | |
| | Enzymatic Activity Assay |
| Description: | For each test compound, 100× concentrated DMSO solutions at 8 doses were prepared and then diluted in assay buffer (25 mM Tris-HCl, pH 8, 130 mM NaCl, 0.05% Tween-20, 10% Glycerol) to obtain 5× concentrated solutions in relation to the final concentrations (typical final concentration range 6.4-200000 nM or 0.18-50000 nM, final DMSO content 1%). Then 10 μL solution of each test compound concentration were placed on a 96-well plate in triplicate and 15 μL of 3.33× concentrated enzyme solution in the assay buffer containing 3.33× concentrated BSA (final BSA concentration 2 mg/mL for HDAC4, HDAC5 and HDAC9 or 1 mg/mL for other isoforms) and in the case of HDAC6-3.33× concentrated TCEP (final TCEP concentration 200 μM) were added to each well. After a period of preincubation (incubation times and temperatures vary for different isoforms and are shown in table 1) 25 μL of solution containing the substrate were added. As substrate, FLUOR DE LYS deacetylase substrate (Enzo Life Sciences, cat: BML-KI104, FdL), FLUOR DE LYS -Green substrate (Enzo Life Sciences, cat: BML-KI572, FdL_G) or Boc-Lys(Tfa)-AMC (Bachem, cat: 4060676.005, Tfal) 2× concentrated solution in assay buffer were used. Following a reaction period (reaction times and temperatures vary for different isoforms and are reported in Table 1), 50 μL of the development solution consisting of concentrate FLUOR DE LYS developer I (Enzo Life Sciences, ca: BML-KI105), diluted 200 times in buffer (50 mM Tris-HCl, pH 8, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) plus 2 μM TSA was added and, after 25 minutes at room temperature in the dark, using the Victor 1420 Multilabel Counter Perkin Elmer Wallac instrument, the fluorescence reading was carried out. Enzymatic activity on recombinant human HDAC6 and HDAC1 was evaluated (Table 2) for each synthesized compound. All compounds tested resulted virtually inactive (IC50 > 30 μM) on HDAC1. |
| Affinity data for this assay | |
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