Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells

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 in vitro 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（bromodomains）是识别赖氨酸乙酰化标记的表观遗传“阅读器”，广泛存在于多种核蛋白中，在染色质生物学领域发挥多样生物学功能。溴结构域功能异常与癌症等人类疾病的发病机制密切相关，因此已成为药物研发的重要治疗靶点。由于溴结构域间结构相似性较高，开发溴结构域抑制剂的关键环节之一是评估其选择性以规避脱靶效应。尽管目前已鉴定出大量溴结构域抑制剂，但仍需开发新方法以在活细胞内评估抑制剂对内源性溴结构域的选择性。 本研究报道了一种光亲和探针——光溴孢菌素（photo-bromosporine，photo-BS）的开发，该探针可在活细胞中对溴结构域抑制剂进行广谱靶点谱分析。实验证实，photo-BS可在体外及活细胞内实现重组溴结构域与内源性含溴结构域蛋白（bromodomain-containing proteins, BCPs）的光诱导交联。其介导的溴结构域标记可被相应溴结构域抑制剂特异性竞争性阻断。蛋白质组学分析显示，photo-BS可从活细胞中捕获42种已知含溴结构域蛋白中的28种。针对溴孢菌素与GSK6853两种溴结构域抑制剂的评估实验，不仅鉴定出了已知的溴结构域靶点，还发现了此前未被表征的新型靶点。 综上，本研究构建了一套化学蛋白质组学平台，可在活细胞内全面评估溴结构域抑制剂对内源性含溴结构域蛋白的选择性。