NanoBRET Tracer Development for Class I Bromodomain Target Engagement in Live Cells

Epigenetic reader proteins, such as bromodomains, are often associated with diseases such as cancer and inflammation. Bromodomain and extra-terminal (BET) bromodomain inhibitors have been studied extensively; however, non-BET bromodomains are understudied. Moreover, available high-throughput biological assays to assess inhibitors are limited. One non-BET bromodomain-containing protein, BPTF, has a recently reported inhibitor, BZ1, with an in vitro affinity of 6.3 nM. Additionally, BZ1 is known to be nonselective toward other class I bromodomains PCAF, GCN5, and CECR2. Here, we use a BZ1 analog, BZ1-THQ, to design a small-molecule NanoBRET tracer, <b>MS-1</b>, for assessing inhibitor functional activity through live-cell target engagement against the BPTF bromodomain. Further, we investigate the versatility of <b>MS-1</b> against PCAF, GCN5, and CECR2. We observe that <b>MS-1</b> is a broadly applicable NanoBRET tracer for class I bromodomains, effectively binding BPTF, PCAF, GCN5, and CECR2 in HEK293T cells at low to sub-micromolar concentrations. We report IC₅₀ values of commercially available and in-house inhibitors to demonstrate tracer versatility for future target engagement studies and inhibitor development.