| Assay Method Information | |
| | Cellular Target Engagement Assay |
| Description: | NanoBRET target engagement was performed against the catalytic domain 2 (CD2) of HDAC6, with minor modifications of kit manufacturer protocol (Promega). Expression of exogenous NanoLuc-HDAC6(CD2) fusion and in HEK293T was achieved following transient transfection with FuGENE HD Transfection Reagent (Promega). The intracellular target engagement assay on HDAC6(CD2) was performed in a 384-well plate format with 8,000 cells per well and a tracer concentration of 0.125 μM or 0.600 μM for HDAC6(CD2). Compounds (dissolved in 100% DMSO) or DMSO (vehicle) were added either manually by diluting them in culture medium at 8× final assay concentration and then adding 5 μL to assay plate or in an automated way by adding 160 nL compound to 5 μL OptiMEM without phenol red (Gibco) using the Echo650 (Labcyte). Tracer solution was added to the cells before seeding 35 μL cell/tracer mixture in the assay plate. The final reaction volume was 40 μL, final DMSO concentration was 1.4% (manual compound addition) or 1.25% (automated compound addition). Assay plates were incubated at 37° C. in a humidified atmosphere containing 5% C02 for 2 hours. The NanoBRET Nano-glo Substrate/Inhibitor was prepared by diluting NanoBRET Nano-Glo Substrate (1:332) and Extracellular Inhibitor (1:1000) in assay medium (Promega). The NanoBRET TE Nano-glo Substrate/Inhibitor was added to cells and measurement of NanoBRET donor and acceptor signal (460-80 and 647-75, respectively) was performed at room temperature with either the EnVision Xcite (PerkinElmer) or the CLARIOstar (BMG Labtech) plate reader 1-2 minutes after NanoLuc substrate addition. BRET ratios were calculated from acceptor/donor signal ratio (mBRET=acceptor/donor*1000) and normalized for each plate. Percentage inhibition was calculated by setting the mBRET obtained with cells without tracer to 100%, while the mBRET obtained with uninhibited cells with tracer was set to 0%. IC50-values were calculated from the percentage inhibition with the log(inhibitor) vs. response—Variable slope (four parameters) nonlinear regression in GraphPad Prism software. |
| Affinity data for this assay | |
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