biostudies-literatureCui HNational Center for Advancing Translational SciencesNCATS NIH HHSCenter for Regenerative Medicine, Mayo Clinic MinnesotaNCRR NIH HHSSt Jude Cancer CenterUniversity of MinnesotaAmerican Lebanese Syrian Associated CharitiesNCI NIH HHSNational Institute of General Medical SciencesNIGMS NIH HHSMinnesota Masonic Charities2342-2360https://www.ebi.ac.uk/biostudies/studies/S-EPMC9683399biostudies-literatureUnknown65(3)Chemical probes for epigenetic proteins are essential tools for dissecting the molecular mechanisms for gene regulation and therapeutic development. The bromodomain and extra-terminal (BET) proteins are master transcriptional regulators. Despite promising therapeutic targets, selective small molecule inhibitors for a single bromodomain remain an unmet goal due to their high sequence similarity. Here, we address this challenge via a structure-activity relationship study using 1,4,5-trisubstituted imidazoles against the BRD4 N-terminal bromodomain (D1). Leading compounds <b>26</b> and <b>30</b> have 15 and 18 nM affinity against BRD4 D1 and over 500-fold selectivity against BRD2 D1 and BRD4 D2 via ITC. Broader BET selectivity was confirmed by fluorescence anisotropy, thermal shift, and CETSA. Despite BRD4 engagement, BRD4 D1 inhibition was unable to reduce c-Myc expression at low concentration in multiple myeloma cells. Conversely, for inflammation, IL-8 and chemokine downregulation were observed. These results provide new design rules for selective inhibitors of an individual BET bromodomain.Journal of medicinal chemistryA Structure-based Design Approach for Generating High Affinity BRD4 D1-Selective Chemical Probes.PMC9683399UL1 TR002494P30 CA021765GM140837-01P30 GM124165T32 GM008700S10 RR029205R35 GM118047R35 GM140837GM118047T32 GM132029CA21765Zahid HAihara HLee REPomerantz WCKJohnson JAKhanal SDivakaran AHarki DAScholtz CRGee CTLee ALEllingson MOShah VHHoell ZJCui HGriffith ECShi KfalseA Structure-based Design Approach for Generating High Affinity BRD4 D1-Selective Chemical Probes.Chemical probes for epigenetic proteins are essential tools for dissecting the molecular mechanisms for gene regulation and therapeutic development. The bromodomain and extra-terminal (BET) proteins are master transcriptional regulators. Despite promising therapeutic targets, selective small molecule inhibitors for a single bromodomain remain an unmet goal due to their high sequence similarity. Here, we address this challenge via a structure-activity relationship study using 1,4,5-trisubstituted imidazoles against the BRD4 N-terminal bromodomain (D1). Leading compounds <b>26</b> and <b>30</b> have 15 and 18 nM affinity against BRD4 D1 and over 500-fold selectivity against BRD2 D1 and BRD4 D2 via ITC. Broader BET selectivity was confirmed by fluorescence anisotropy, thermal shift, and CETSA. Despite BRD4 engagement, BRD4 D1 inhibition was unable to reduce c-Myc expression at low concentration in multiple myeloma cells. Conversely, for inflammation, IL-8 and chemokine downregulation were observed. These results provide new design rules for selective inhibitors of an individual BET bromodomain.2022-01-01T00:00:00Z2022 Feb2026-05-03T10:39:54.176Z2025-04-19T06:04:40.662ZS-EPMC96833993500706110.1021/acs.jmedchem.1c01779