Alternative Mechanisms for DNA Engagement by BET Bromodomain-Containing Proteins.

Epigenetic reader domains regulate chromatin structure and modulate gene expression through the recognition of post-translational modifications on histones. Recently, reader domains have also been found to harbor double-stranded (ds) DNA-binding activity, which is as functionally critical as histone association. Here, we explore the dsDNA recognition of the N-terminal bromodomain of the bromodomain and extra-terminal (BET) protein, BRD4.

Dihydropyridine Lactam Analogs Targeting BET Bromodomains.

Inhibitors of Bromodomain and Extra Terminal (BET) proteins are investigated for various therapeutic indications, but selectivity for BRD2, BRD3, BRD4, BRDT and their respective tandem bromodomains BD1 and BD2 remains suboptimal. Here we report selectivity-focused structural modifications of previously reported dihydropyridine lactam 6 by changing linker length and linker type of the lactam side chain in efforts to engage the unique arginine 54 (R54) residue in BRDT-BD1 to achieve BRDT-selective affinity. We found that the analogs were highly selective for BET bromodomains, and generally more selective for the first (BD1) and second (BD2) bromodomains of BRD4 rather than for those of BRDT.

Controlling Intramolecular Interactions in the Design of Selective, High-Affinity Ligands for the CREBBP Bromodomain.

CREBBP (CBP/KAT3A) and its paralogue EP300 (KAT3B) are lysine acetyltransferases (KATs) that are essential for human development. They each comprise 10 domains through which they interact with >400 proteins, making them important transcriptional co-activators and key nodes in the human protein-protein interactome. The bromodomains of CREBBP and EP300 enable the binding of acetylated lysine residues from histones and a number of other important proteins, including p53, p73, E2F, and GATA1.

Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors.

We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson's disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision.

Quantifying the Selectivity of Protein-Protein and Small Molecule Interactions with Fluorinated Tandem Bromodomain Reader Proteins.

Multidomain bromodomain-containing proteins regulate gene expression via chromatin binding, interactions with the transcriptional machinery, and by recruiting enzymatic activity. Selective inhibition of members of the bromodomain and extra-terminal (BET) family is important to understand their role in disease and gene regulation, although due to the similar binding sites of BET bromodomains, selective inhibitor discovery has been challenging. To support the bromodomain inhibitor discovery process, here we report the first application of protein-observed fluorine (PrOF) NMR to the tandem bromodomains of BRD4 and BRDT to quantify the selectivity of their interactions with acetylated histones as well as small molecules.

Overriding ortho selectivity by template assisted meta-C-H activation of benzophenones.

An orthogonal selectivity for distal meta-C-H activation of benzophenone is acheived by overriding the inherent proximal ortho-selectivity through a template assisted metalation approach. This strategy has been successfully utilized in Pd-catalyzed regioselective C-C and C-Si bond formation.