As regulators of transcription, epigenetic proteins that interpret post-translational modifications to N-terminal histone tails are essential for maintaining cellular homeostasis. When dysregulated, “reader” proteins become drivers of disease. In the case of bromodomains, which recognize N-ε-acetylated lysine, selective inhibition of individual bromodomain-and-extra-terminal (BET)-family bromodomains has proven challenging. We describe the >55-fold N-terminal-BET bromodomain selectivity of 1,4,5-trisubstitutedimidazole dual kinase−bromodomain inhibitors. Selectivity for the BRD4 N-terminal bromodomain (BRD4(1)) over its second bromodomain (BRD4(2)) arises from the displacement of ordered waters and the conformational flexibility of lysine-141 in BRD4(1). Cellular efficacy was demonstrated via reduction of c-Myc expression, inhibition of NF-κB signaling, and suppression of IL-8 production through potential synergistic inhibition of BRD4(1) and p38α. These dual inhibitors provide a new scaffold for domain-selective inhibition of BRD4, the aberrant function of which plays a key role in cancer and inflammatory signaling.

In memory of FranÅoisD iederich who inspired ag eneration of scientists for tackling problems in molecular recognition Abstract: Bromodomain and extra-terminal (BET) family proteins,BRD2-4 and T, are important drug targets;however, the biological functions of each bromodomain remain illdefined. Chemical probes that selectively inhibit asingle BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here,w e develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays,w es how that our lead compound is 9-33 fold selective for BRD4 D1 over the other BET bromodomains.X -rayc rystallography supports ar ole for the selectivity based on reorganization of an onconserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers,B RD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation mayshed light on differential BET bromodomain functions.

Inhibitor discovery for protein−protein interactions has proven difficult due to the large protein surface areas and dynamic interfaces involved. This is particularly the case when targeting transcription-factor−protein interactions. To address this challenge, structural biology approaches for ligand discovery using X-ray crystallography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy have had a significant impact on advancing small molecule inhibitors into the clinic, including the U.S. Food and Drug Administration approved drug, Venetoclax. Inspired by the protein-observed NMR approach using 1 H− 15 N-HSQC NMR which detects chemical shift perturbations of 15 N-labeled amides, we have applied a complementary protein-observed 19 F NMR approach using 19 F-labeled side-chains that are enriched at protein−proteininteraction interfaces. This protein-observed 19 F NMR assay is abbreviated PrOF NMR to distinguish the experiment from the more commonly employed ligand-observed 19 F NMR methods. In this Account, we describe our efforts using PrOF NMR as a ligand discovery tool, particularly for fragment-based ligand discovery (FBLD). We metabolically label the aromatic amino acids on proteins due to the enrichment of aromatic residues at protein interfaces. We choose the 19 F nucleus due to its high signal sensitivity and the hyperresponsiveness of 19 F to changes in chemical environment. Simultaneous labeling with two different types of fluorinated aromatic amino acids for PrOF NMR has also been achieved. We first describe the technical aspects of considering the application of PrOF NMR for characterizing native protein−protein interactions and for ligand screening. Several test cases are further described with a focus on a transcription factor coactivator interaction with the KIX domain of CBP/p300 and two epigenetic regulatory domains, the bromodomains of BRD4 and BPTF. Through these case studies, we highlight medicinal chemistry applications in FBLD, selectivity screens, structure−activity relationship (SAR) studies, and ligand deconstruction approaches. These studies have led to the discovery of some of the first inhibitors for BPTF and a novel inhibitor class for the N-terminal bromodomain of BRD4. The speed, ease of interpretation, and relatively low concentration of protein needed for NMR-based binding experiments affords a rapid, structural biology-based method to discover and characterize both native and new ligands for bromodomains, and it may find utility in the study of additional epigenetic proteins and transcription-factor−protein interactions.

The Bromodomain and Extra Terminal (BET) family of proteins recognize post-translational N-ε-acetylated lysine modifications, regulating transcription as "reader" proteins. Bromodomain inhibitors are interesting targets for the development of potential cancer, inflammation, and heart disease treatments. Several dual kinase-bromodomain inhibitors have been identified by screening kinase inhibitor libraries against BET proteins. Although potentially useful from a polypharmacology standpoint, multitarget binding complicates deciphering molecular mechanisms. This report describes a systematic approach to mitigating kinase activity in a dual kinase-bromodomain inhibitor based on a 1,2,3-triazole-pyrimidine core. By modifying the triazole substituent and altering the pyrimidine core, this structure−activity relationship study enhanced BET activity while reducing the p38α kinase activity >90,000-fold. A BRD4-D1 cocrystal structure indicates that the 1,2,3triazole is acting as a N-ε-acetylated lysine mimic. A BRD4 sensitive cell line, MM.1S, was used to demonstrate activity in cells, which is further supported by reduced c-Myc expression.

Single-particle analysis of biosensors that use charge transfer as the means for analyte-dependent signaling with semiconductor nanoparticles, or quantum dots, was examined. Single-particle analysis of biosensors that use energy transfer show analyte-dependent switching of nanoparticle emission from off to on. The charge-transfer-based biosensors reported here show constant emission, where the analyte (maltose) increases the emission intensity. By monitoring the same nanoparticles under various conditions, a single charge-transfer-based biosensor construct (one maltose binding protein, one protein attachment position for the reductant, one type of nanoparticle) showed a dynamic range for analyte (maltose) detection spanning from 100 pM to 10 μM while the emission intensities increase from 25 to 175% at the single-particle level. Since these biosensors were immobilized, the correlation between the detected maltose concentration and the maltose-dependent emission intensity increase could be examined. Minimal correlation between maltose detection limits and emission increases was observed, suggesting a variety of reductant-nanoparticle surface interactions that control maltose-dependent emission intensity responses. Despite the heterogeneous responses, monitoring biosensor emission intensity over 5 min provided a quantifiable method to monitor maltose concentration. Immobilizing and tracking these biosensors with heterogeneous responses, however, expanded the analyte-dependent emission intensity and the analyte dynamic range obtained from a single construct. Given the wide dynamic range and constant emission of charge-transfer-based biosensors, applying these single molecule techniques could provide ultrasensitive, real-time detection of small molecules in living cells.