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

Abstract

Epigenetic reader proteins, such as bromo-domains, are often associated with diseases such as cancer and inflammation. 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 non-selective towards other class I bromodomains PCAF, GCN5, and CECR2. Here, we use a BZ1 analog, BZ1-THQ, to design a small molecule NanoBRET tracer, MS-1, for assessing inhibitor functional activity through live-cell target engagement against the BPTF bromodomain. Further, we investigate the versatility of MS-1 against PCAF, GCN5, and CECR2. We observe that MS-1 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 EC50 values of commercially available and in-house inhibitors to demonstrate tracer versatility for future target engagement studies and inhibitor development. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/689777v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@149371forg.highwire.dtl.DTLVardef@1c1e652org.highwire.dtl.DTLVardef@f81fbaorg.highwire.dtl.DTLVardef@1eb18f1_HPS_FORMAT_FIGEXP M_FIG C_FIG