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，作为赖氨酸乙酰化标记的表观遗传“阅读器”，存在于多种核蛋白中，并在染色质生物学中发挥多样化的生物学功能。溴结构域的失常与人类疾病如癌症的发病机制密切相关，因此溴结构域已成为药物发现中的治疗靶点。鉴于溴结构域高度的结构相似性，开发溴结构域抑制剂的关键步骤之一是评估其选择性，以避免脱靶效应。尽管已经鉴定出众多溴结构域抑制剂，但评估活细胞中内源溴结构域的选择性抑制剂的新方法仍属必需。在本研究中，我们报道了一种光亲和探针——光溴杂环化合物（photo-BS），它能够实现对活细胞中溴结构域抑制剂的广泛谱系分析。photo-BS能够在体外和活细胞中对重组溴结构域和内源溴结构域包含蛋白（BCPs）进行光诱导交联。photo-BS诱导的溴结构域标记可以被相应的溴结构域抑制剂选择性地竞争。蛋白质组学分析揭示了photo-BS从活细胞中捕获了42种已知BCP中的28种。对两种溴结构域抑制剂——溴杂环化合物和GSK6853的评估，导致了已知以及先前未表征的溴结构域靶点的识别。总体而言，我们建立了一个化学蛋白质组学平台，用于全面评估溴结构域抑制剂在活细胞中对其内源BCPs选择性的影响。