M170:Federici:High-Throughput LanthaScreen® Cellular Assays for Interrogating Post-Translational Modifications of p53 and Histone H3

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Mark Federici, Thomas Machleidt, Spencer Hermanson, Coby Carlson and Kun Bi
Discovery and ADMET Systems • Life Technologies • 501 Charmany Drive • Madison, Wisconsin 53719 • USA

Post-translational modifications such as phosphorylation, acetylation, methylation and ubquitination play important roles in regulating the structure and functions of histones and transcription factors such as p53, which in turn regulate essential gene expression. Dysregulation of these post-translational modifications has been linked to cancer and metabolic diseases. In particular, histone deacetylases (HDACs) have been pursued as valuable targets for various therapeutic interventions. However, detection of these enzymatic activities in a cell-based format has remained intractable using HTS-compatible technologies. To enable the drug discovery for these post-translational modification enzymes, we have developed and validated high-throughput compatible LanthaScreen® cellular assays for the analysis of histone and/or p53-specific acetylation, methylation and phosphorylation in cell backgrounds of interest. The cell cycle-dependent acetylation of histone H3 at Lys9 and the phosphorylation of histone H3 at Ser10 can be detected with terbium-labeled anti-Histone H3 acetyl-lys 9 and anti-phospho-Ser10 specific antibodies, respectively. In addition, trimethylation of Histone H3 at Lys4 can be measured with terbium-labeled anti-trimethyl lys4 antibody. We have also applied this technology and developed cellular assays for measuring DNA-damage induced phosphorylation at Ser15 and acetylation at Lys382 of p53. These assays were further validated with both small molecule inhibitor and siRNA against specific HDAC family members. Our results suggest that the deacetylation of Histone H3 Lys9 is mediated by type I/II HDACs, whereas the deacetylation of p53 at Lys382 is mediated synergistically by both SIRT1 and type I/II HDAC activities. Together, these assays enable cellular interrogation of multiple enzymatic activities responsible for post-translational modifications of challenging targets such as histones and p53.

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