Development of a sensitive luminescent probe to uncover new BRD4 inhibitors in living cells

Bromodomain-containing protein 4 (BRD4) is a reader of acetylated histones that regulates the invasion, metastasis and proliferation of cancer. Inhibition of BRD4 activity is an emerging strategy for anticancer. In this study, an iridium based-luminescent probe for screening BRD4 inhibitors in cellulo was developed. The K293 probe was designed by linking a BRD4 nuclear translocation inhibitor as a "binding unit" to an iridium complex as a luminescent "signal unit". K293 selectively binds to BRD4 in living cancer cells. Candidate compounds compete with K293 for BRD4, resulting in a decrease in luminescence signal. The long luminescence lifetime of K293 allows its luminescence signal to be distinguished from interfering fluorescence using time-resolved techniques in order to improve sensitivity. Hence, even potential BRD4 inhibitors that are undetectable by conventional fluorescence methods can be identified. As a proof-of-concept, Azelastine, an FDA-approved histamine 1 (H1) antagonist was identified to be a potential BRD4 inhibitor using the K293 probe. Azelastine binds to BRD4 protein in the cytoplasm to prevent its translocation into the nucleus, and suppresses cancer cell growth and migration. The results demonstrated that the probe could be useful for the future drug repurposing.