Department of Chemistry , The University of Hong Kong , Pokfulam Road , Hong Kong , China.
Center of Biomedical Analysis , Tsinghua University , Beijing 100084 , China.
J Am Chem Soc. 2019 Jul 24;141(29):11497-11505. doi: 10.1021/jacs.9b02738. Epub 2019 Jul 9.
Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.
溴结构域是组蛋白赖氨酸乙酰化修饰的“读取器”，存在于具有不同生物学功能的多种核蛋白中，在染色质生物学中发挥作用。溴结构域功能异常与人类疾病（如癌症）的发病机制有关。因此，溴结构域已成为药物发现的治疗靶点。鉴于溴结构域具有高度的结构相似性，开发溴结构域抑制剂的关键步骤是评估其选择性，以避免脱靶效应。虽然已经鉴定出许多溴结构域抑制剂，但仍需要新的方法来评估它们对活细胞内内源性溴结构域的抑制剂选择性。在这里，我们报告了一种光亲和探针 photo-bromosporine（photo-BS）的开发，该探针可用于在活细胞中广泛分析溴结构域抑制剂。photo-BS 允许在重组溴结构域和内源性含有溴结构域的蛋白质（BCPs）中诱导光诱导交联，无论是在细胞内还是在细胞外。相应的溴结构域抑制剂可选择性竞争 photo-BS 诱导的溴结构域标记。蛋白质组学分析显示，photo-BS 从活细胞中捕获了 28 个已知的 BCP 中的 28 个。对两种溴结构域抑制剂 bromosporine 和 GSK6853 的评估导致鉴定出已知的以及以前未表征的溴结构域靶标。总之，我们建立了一个化学蛋白质组学平台，可全面评估溴结构域抑制剂对活细胞内内源性 BCP 的选择性。
