Design of Highly Fluorinated Peptides for Cell-based 19F NMR.

The design of imaging agents with high fluorine content is essential for overcoming the challenges associated with signal detection limits in 19F MRI-based molecular imaging. In addition to perfluorocarbon and fluorinated polymers, fluorinated peptides offer an additional strategy for creating sequence-defined 19F magnetic resonance imaging (MRI) imaging agents with a high fluorine signal. Our previously reported unstructured trifluoroacetyllysine-based peptides possessed good physiochemical properties and could be imaged at high magnetic field strength. However, the low detection limit motivated further improvements in the fluorine content of the peptides as well as removal of nonspecific cellular interactions. This research characterizes several new highly fluorinated synthetic peptides composed of highly fluorinated amino acids. 19F NMR analysis of peptides TB-1 and TB-9 led to highly overlapping, intense fluorine resonances and acceptable aqueous solubility. Flow cytometry analysis and fluorescence microscopy further showed nonspecific binding could be removed in the case of TB-9. As a preliminary experiment toward developing molecular imaging agents, a fluorinated EGFR-targeting peptide (KKKFFKK-ßA-YHWYGYTPENVI) and an EGFR-targeting protein complex E1-DD bioconjugated to TB-9 were prepared. Both bioconjugates maintained good 19F NMR performance in aqueous solution. While the E1-DD-based imaging agent will require further engineering, the success of cell-based 19F NMR of the EGFR-targeting peptide in A431 cells supports the potential use of fluorinated peptides for molecular imaging.

Endothelial p300 Promotes Portal Hypertension and Hepatic Fibrosis Through C-C Motif Chemokine Ligand 2-Mediated Angiocrine Signaling.

BACKGROUND AND AIMS: During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. APPROACH AND RESULTS: CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC-specific p300 deletion (p300LSECΔ/Δ ) or C-C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)-p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild-type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time-of-flight. Epigenetic histone marks were modified by CRISPR endonuclease-deficient CRISPR-associated 9-fused with the Krüppel associated box domain (CRISPR-dCas9-KRAB)-mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300LSECΔ/Δ mice compared to p300fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300LSECΔ/Δ mouse livers. Ccl2 was the most up-regulated chemokine in injured LSECs, but its increase was abrogated in p300LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB-p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC-specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR-dCas9-KRAB technology repressed TNFα-induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. CONCLUSIONS: In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement*.

Bromodomain and extra-terminal (BET) family proteins, BRD2-4 and T, are important drug targets; however, the biological functions of each bromodomain remain ill-defined. Chemical probes that selectively inhibit a single BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here, we develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays, we show that our lead compound is 9-33 fold selective for BRD4 D1 over the other BET bromodomains. X-ray crystallography supports a role for the selectivity based on reorganization of a non-conserved 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, BRD4 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 may shed light on differential BET bromodomain functions.

From PROTAC to inhibitor: Structure-guided discovery of potent and orally bioavailable BET inhibitors.

An X-ray structure of a CLICK chemistry-based BET PROTAC bound to BRD2(BD2) inspired synthesis of JQ1 derived heterocyclic amides. This effort led to the discovery of potent BET inhibitors displaying overall improved profiles when compared to JQ1 and birabresib. A thiadiazole derived 1q (SJ1461) displayed excellent BRD4 and BRD2 affinity and high potency in the panel of acute leukaemia and medulloblastoma cell lines. A structure of 1q co-crystalised with BRD4-BD1 revealed polar interactions with the AZ/BC loops, in particular with Asn140 and Tyr139, rationalising the observed affinity improvements. In addition, exploration of pharmacokinetic properties of this class of compounds suggest that the heterocyclic amide moiety improves drug-like features. Our study led to the discovery of potent and orally bioavailable BET inhibitor 1q (SJ1461) as a promising candidate for further development.

A 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 26 and 30 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.

4-Methyl-1,2,3-Triazoles as N-Acetyl-Lysine Mimics Afford Potent BET Bromodomain Inhibitors with Improved Selectivity.

The bromodomain and extra terminal (BET) protein family recognizes acetylated lysines within histones and transcription factors using two N-terminal bromodomains, D1 and D2. The protein-protein interactions between BET bromodomains, acetylated histones, and transcription factors are therapeutic targets for BET-related diseases, including inflammatory disease and cancer. Prior work demonstrated that methylated-1,2,3-triazoles are suitable N-acetyl lysine mimetics for BET inhibition. Here we describe a structure-activity relationship study of triazole-based inhibitors that improve affinity, D1 selectivity, and microsomal stability. These outcomes were accomplished by targeting a nonconserved residue, Asp144 and a conserved residue, Met149, on BRD4 D1. The lead inhibitors DW34 and 26 have a BRD4 D1 Kd of 12 and 6.4 nM, respectively. Cellular activity was demonstrated through suppression of c-Myc expression in MM.1S cells and downregulation of IL-8 in TNF-α-stimulated A549 cells. These data indicate that DW34 and 26 are new leads to investigate the anticancer and anti-inflammatory activity of BET proteins.

Super enhancer regulation of cytokine-induced chemokine production in alcoholic hepatitis.

Alcoholic hepatitis (AH) is associated with liver neutrophil infiltration through activated cytokine pathways leading to elevated chemokine expression. Super-enhancers are expansive regulatory elements driving augmented gene expression. Here, we explore the mechanistic role of super-enhancers linking cytokine TNFα with chemokine amplification in AH. RNA-seq and histone modification ChIP-seq of human liver explants show upregulation of multiple CXCL chemokines in AH. Liver sinusoidal endothelial cells (LSEC) are identified as an important source of CXCL expression in human liver, regulated by TNFα/NF-κB signaling. A super-enhancer is identified for multiple CXCL genes by multiple approaches. dCas9-KRAB-mediated epigenome editing or pharmacologic inhibition of Bromodomain and Extraterminal (BET) proteins, transcriptional regulators vital to super-enhancer function, decreases chemokine expression in vitro and decreases neutrophil infiltration in murine models of AH. Our findings highlight the role of super-enhancer in propagating inflammatory signaling by inducing chemokine expression and the therapeutic potential of BET inhibition in AH treatment.

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. We further determine the selectivity profile of a new class of ligands, 1,4-acylthiazepanes, and find them to have ≥3-10-fold selectivity for the C-terminal bromodomain of both BRD4 and BRDT. Given the speed and lower protein concentration required over traditional protein-observed NMR methods, we envision that these fluorinated tandem proteins may find use in fragment screening and evaluating nucleosome and transcription factor interactions.

Systematically Mitigating the p38α Activity of Triazole-based BET Inhibitors.

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 1,2,3-triazole 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.

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

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-trisubstituted-imidazole 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.