Abstract
During the preclinical development of small molecule inhibitors, compounds or compound libraries are typically first screened using purified target enzymes in vitro to select candidates with high potency. In the later stages of the development, however, functional cell-based assays may provide biologically more relevant data. In this chapter, we describe a detailed protocol for determining the potency of inhibitors targeting human histone deacetylase 6 in complex cellular environments. Cells are first treated with a dilution series of tested compounds, cell lysates separated by SDS-PAGE, and electrotransferred to a blotting membrane. The inhibitor potency is then determined indirectly by quantifying the levels of acetylated tubulin as a surrogate readout.
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Acknowledgments
This work was funded by the CAS (RVO: 86652036), the Czech Science Foundation (21-31806), and 5R01CA249248-02 from the NIH.
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Mikesova, J., Ondrakova, M., Jelinkova, I., Ptacek, J., Novakova, Z., Barinka, C. (2023). Determining Potency of Inhibitors Targeting Histone Deacetylase 6 by Quantification of Acetylated Tubulin in Cells. In: Krämer, O.H. (eds) HDAC/HAT Function Assessment and Inhibitor Development. Methods in Molecular Biology, vol 2589. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2788-4_29
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DOI: https://doi.org/10.1007/978-1-0716-2788-4_29
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