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”，存在于具有不同生物学功能的多种核蛋白中，并在染色质生物学领域扮演重要角色。溴结构域的功能失调与人类疾病（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。鉴于溴结构域的高度结构相似性，在溴结构域抑制剂的开发过程中，评估其选择性以避免脱靶效应成为关键步骤。尽管已鉴定出众多溴结构域抑制剂，但评估活细胞中内源性溴结构域抑制剂选择性的新方法仍亟待开发。在本研究中，我们报道了一种光亲和探针——光溴素（photo-BS）的开发，该探针能够对活细胞中的溴结构域抑制剂进行广泛谱系分析。photo-BS能够在体外及活细胞中诱导重组溴结构域与内源性溴结构域蛋白（BCPs）之间的光交联。通过光溴素诱导的溴结构域标记可被相应的溴结构域抑制剂选择性竞争。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——溴素和GSK6853的评估，不仅识别了已知靶点，还发现了之前未表征的溴结构域靶点。综上所述，我们建立了一个化学蛋白质组学平台，用于全面评估活细胞中溴结构域抑制剂对其内源性BCPs的选择性。