A modular platform for in cellulo target engagement profiling of chromatin reader inhibitors.

The dysregulation of acylation-reading bromodomain proteins is implicated in many diseases, making them important therapeutic targets. However, drug development is hindered by a lack of high-throughput cellular assays to measure target engagement and off-target binding in living cells. To address this gap, we developed a panel of cell lines that express synthetic bromodomain constructs, termed Acyl-eCRs. We demonstrate that Acyl-eCRs recapitulate the same response to bromodomain inhibitors and PROTACs as endogenous proteins, allowing for quantitative assessment of drug effects. We introduce two complementary flow cytometry-based assays to evaluate inhibitor-target engagement: a competitive binding assay leveraging PROTAC-induced degradation, and a nuclear retention assay measuring displacement of bromodomains from chromatin. Our approach circumvents laborious protein purification and in vitro characterization, providing a scalable, physiologically relevant method for assessing inhibitor potency and specificity. This platform provides a scalable strategy for quantitative target-engagement profiling of chromatin reader inhibitors in living cells.