Positional isomers of Indolyl-benzodiazepines display dissimilar binding and recruitment of BET transcriptional regulators to targeted genomic loci.

BET proteins contain two tandem bromodomains (BD1 and BD2) that bind histone acetyl-lysine residues that can be targeted with small molecule inhibitors such as IBET-762, which bears a triazolo benzodiazepine core. Here, we report the consequences of substituting the pendant chlorobenzene moiety of IBET-762. Substitution with larger ring structures diminishes bromodomain binding, and even subtle changes on the phenyl ring significantly impact affinity. Structural analysis, molecular docking, and molecular dynamics simulations of four indolyl-benzodiazepine derivatives indicate that ligand selectivity arises from interaction with His433 in BD2. Their ability to engage BET proteins in cells was tested by incorporating them into heterobifunctional synthetic genome readers and regulators (SynGRs). The relative activity of each SynGR corresponded to the affinity of the tethered BET ligand for the BD2 domain. The development and structure-activity relationship analysis of these BET ligands provides a blueprint for the construction of increasingly selective BET inhibitors and proximity-inducing molecules.