Suppression of inflammation by a synthetic histone mimic

Abstract

Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

Figure 1. I-BET is a selective antagonist of BET proteins

a, Chemical structure of GSK525762A (I-BET). b, Structure of I-BET (orange) bound to the acetyl-binding pocket of BRD4-BD1 overlaid with acetylated histone H4 peptide (H4Ac, green). The ‘WPF shelf’ (W81, P82, F83) as well as the asparagine N140 essential for acetylated lysine (KAc) binding are indicated. c, I-BET binds with high affinity to BET proteins as determined by isothermal titration calorimetry (ITC) of tandem bromodomain fragments of BRD2 (1–473), BRD3 (1–434), BRD4 (1–477) interaction with I-BET or BRD4 (1–477) interaction with an inactive enantiomer of I-BET (inactive I-BET). Time courses of raw injection heats (upper panel) and normalized binding enthalpies, calculated using a single site binding model (Origin software, Microcal, lower panel), are shown. d, I-BET competes with H4Ac peptide for bromodomain binding. Displacement of tetra-acetylated histone H4 peptide from bromodomains of BRD2 (blue), BRD3 (black) and BRD4 (red) by I-BET was determined by FRET analysis.

Figure 2. I-BET suppresses a specific subset of LPS-inducible genes

a, Venn diagrams display the number of LPS-inducible (>2-fold, red circles) genes that were suppressed (>2 fold, green circles) or up-regulated (>2 fold, yellow circles) by I-BET (1 μM) treatment at 1 or 4 hours after LPS stimulation (left and right panels). b, Heatmap representation of expression levels of genes that were down-regulated by I-BET at 1 hour (left panel) and 4 hours (right panel) after LPS stimulation. Scale ranges from a signal value of 2 (64, green) to 2 (16384, red). Fold change values are listed. Table shows the distribution of down-regulated genes into functional categories.

Figure 3. Epigenetic profiles of genes suppressed or unaffected by I-BET in LPS-stimulated macrophages

a, Genome-wide epigenetic profiles of sI-BET or naI-BET genes in unstimulated or LPS-stimulated (1 hour) macrophages pre-treated with 5 μM of I-BET or a DMSO control. Analyzed epigenetic marks are indicated. Axes represent the number of reads per million mapped reads. b, Epigenetic profiles of il6 and tnf. The y axes represent the average number of tags per gene per 25 bp per 1,000,000 mapped reads. Scale values are indicated in parentheses. c, The abundance of epigenetic marks on il6 and tnf gene promoters was quantified by ChIP qPCR from four (BRD3, BRD4, Pol II and Pol II S2) or two (BRD2 and P-TEFb) independent experiments performed in triplicate. Error bars are s.e.m. of independent experiments or s.d. of representative experiments, respectively. Asterisks indicate p<0.05 as determined by an unpaired T test.

Figure 4. I-BET suppresses inflammation in vivo

a, Kaplan-Meier survival curves of: LPS-treated C57BL/6 mice (5 mg/kg, i.p., n=12 per group) that were injected i.v. with a solvent control (black squares) or 30 mg/kg of I-BET 1 hour before (blue triangles) or 1.5 hours after (blue circles) LPS administration (left panel); mice injected i.v. with heat-killed Salmonella typhimurium, strain IR71 (5×10⁹/kg, n=10 per group) (middle panel); or mice subjected to cecal ligation puncture (CLP) procedure that were administered a solvent control or 30 mg/kg of I-BET twice a day for two days (n=8 per group) (right panel) b, Serum titers of indicated cytokines were measured by ELISA (n=10 per group). Mice received a solvent control (black squares) or I-BET (blue triangles) 1 hour before LPS injection and samples were collected at 2 hours after LPS treatment. *** p<0.001, ** p<0.01, *p<0.05 as determined by unpaired T test.