Salisbury Cleo M, Cravatt Benjamin F
The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
J Am Chem Soc. 2008 Feb 20;130(7):2184-94. doi: 10.1021/ja074138u. Epub 2008 Jan 25.
Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive "clickable" probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.
组蛋白去乙酰化酶（HDACs）是表观基因组的关键酶调节剂，也是抗癌治疗的有前景的靶点。最近，我们开发了一种光反应性“可点击”探针SAHA-BPyne，用于在天然生物系统中报告HDAC活性和复合物形成。在此，我们研究了SAHA-BPyne以及相关的基于潜在活性的HDAC探针的选择性、灵敏度和抑制特性。虽然我们鉴定出了几种强效HDAC抑制剂探针，并在天然蛋白质组制剂中标记了HDAC复合物成分，但SAHA-BPyne在分析活细胞中的HDAC活性方面明显更具优势。有趣的是，SAHA-BPyne作为基于原位活性的探针性能增强，不能仅仅归因于其与HDAC结合的效力，这意味着该分子的其他特征是在生物系统中进行有效活性位点导向标记的关键。最后，我们通过比较用细胞毒性剂小白菊内酯处理的癌细胞中HDAC1的活性和表达，证明了HDAC原位分析的价值。这些结果强调了基于活性的蛋白质分析在研究HDAC功能方面的实用性，并可能为未来基于点击化学的光反应性探针用于原位分析其他酶活性的开发提供见解。
