Design and characterization of bivalent BET inhibitors

Tanaka, Minoru; Roberts, Justin M.; Seo, Hyuk‐Soo; Souza, Amanda; Paulk, Joshiawa; Scott, Thomas G.; DeAngelo, Stephen L.; Dhe‐Paganon, Sirano; Bradner, James E.

doi:10.1038/nchembio.2209

Cited by 141 publications

(129 citation statements)

References 46 publications

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“…Protein-melt temperatures correlated well with previous CETSA experiments performed on BRD4 and p38 α . 38,39 Importantly, as this experiment does not measure cellular outcomes downstream of target inhibition, these results confirm unambiguously that compound V enters cells and maintains interactions with BRD4 and p38 α in a cellular context (Figure 5A). …”

Section: Resultssupporting

confidence: 58%

Molecular Basis for the N-Terminal Bromodomain-and-Extra-Terminal-Family Selectivity of a Dual Kinase–Bromodomain Inhibitor

et al. 2018

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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.

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Section: Resultssupporting

confidence: 58%

Molecular Basis for the N-Terminal Bromodomain-and-Extra-Terminal-Family Selectivity of a Dual Kinase–Bromodomain Inhibitor

et al. 2018

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“…Human BRD4 residues 44-168 (His-BRD4(1)) in the pNIC28-Bsa4 vector (Addgene) for use in AlphaSceen assays was expressed and purified as previously described (Tanaka et al, 2016; Winter et al, 2015). Expression and purification of CRBN-DDB1 were performed as described previously in Fischer et al (2014) using Sf9 cells (Invitrogen).…”

Section: Star⋆methodsmentioning

confidence: 99%

BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment

et al. 2017

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SUMMARY Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

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“…9 Accordingly, bivalent ligand discovery to generate chromatin-targeted chemical probes has been exemplified by bivalent BRD4 inhibitors (BRD4 features two BET-family bromodomains) showing significant enhancement in functional in vivo activity compared to the constituent monomeric ligands. 10,11 …”

mentioning

confidence: 99%

Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1

et al. 2018

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Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure–affinity relationships for a series of Bromosporine-based bivalent bromodo-main ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1). Experimental techniques—Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance—were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein–ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro, due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.

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Design and characterization of bivalent BET inhibitors

Cited by 141 publications

References 46 publications

Molecular Basis for the N-Terminal Bromodomain-and-Extra-Terminal-Family Selectivity of a Dual Kinase–Bromodomain Inhibitor

Molecular Basis for the N-Terminal Bromodomain-and-Extra-Terminal-Family Selectivity of a Dual Kinase–Bromodomain Inhibitor

BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment

Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1

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