3,5-Dimethylisoxazoles Act As Acetyl-lysine-mimetic Bromodomain Ligands

Histone-lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone-bromodomain interaction is of great importance. We have identified the 3,5 dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound 4d, which has IC50 values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains

Steroid-Resistant Neutrophilic Inflammation in a Mouse Model of an Acute Exacerbation of Asthma

Neutrophilic inflammation in acute exacerbations of asthma tends to be resistant to treatment with glucocorticoids. This may be related to decreased activity and expression of histone deacetylase-2 (HDAC2), which down-regulates expression of proinflammatory genes via recruitment to the glucocorticoid receptor complex. We assessed airway inflammation and response to steroid treatment in a novel mouse model of an acute exacerbation of chronic asthma. Systemically sensitized mice received low-level challenge with aerosolized ovalbumin for 4 weeks, followed by a single moderate-level challenge to induce enhanced inflammation in distal airways. We assessed the effects of pre-treatment with dexamethasone on the accumulation of inflammatory cells in the airways, airway responsiveness to methacholine, expression and enzymatic activity of nuclear proteins including histone acetyl transferase (HAT) and HDAC2, and levels of transcripts for neutrophil chemoattractant and survival cytokines. Dexamethasone suppressed inflammation associated with eosinophil and T-lymphocyte recruitment, but did not prevent neutrophil accumulation or development of airway hyperresponsiveness. Increased activity of HAT was suppressed by steroid treatment, but the marked diminution of HDAC2 activity and increased activity of nuclear factor-κB were not reversed. Correspondingly, elevated expression of mRNA for TNF-α, granulocyte-macrophage colony-stimulating factor, IL-8, and p21waf were also not suppressed by dexamethasone. Levels of lipid peroxidation and protein nitration products were elevated in the acute exacerbation model. We conclude that impaired nuclear recruitment of HDAC2 could be an important mechanism of steroid resistance of the neutrophilic inflammation in exacerbations of asthma. Oxidative stress may contribute to decreased HDAC2 activity

CSE-induced SIRT1 nuclear translocation was associated with increases in anti-oxidant gene and protein expression.

<p>(A) The messenger RNA levels of superoxide dismutase 2 (SOD2), SOD3, NADPH quinone oxidoreductase-1 (NQO-1) and hemoxygenase-1 (HO-1), all of which were normalized to mRNA expression of a GNB2L1 housekeeping gene, were assessed by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 24 hours after 3% CSE exposure with BSO. All values were mean values ± SEM of at least three experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, compared with the values of non-treatment group; ^†† <i>p</i> < 0.01, compared between the two groups. (B) The protein levels of HO-1, NQO-1 and SOD2 were assessed by SDS-PAGE/WB. (C) SIRT1 and FOXO3a protein in cytoplasmic fraction (top) and nuclear fraction (bottom) 24 hours after 3% CSE exposure with BSO. PIK75 (0.1 μM) was also treated before CSE exposure. The band density of SIRT1 and FOXO3a were also calculated and corrected to that of Lamin A/C. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, compared with the values of BSO only group; ^† <i>p</i> < 0.05, compared between with or without PIK75.</p

PI3Kα is involved in the CSE-induced SIRT1 reduction in the presence of L-buthionine-sulfoximine (BSO) in cytoplasmic fraction.

<p>(A) After pretreatment with 100 μM BSO for 16 hours, BEAS-2B cells were exposed to the 3% CSE for up to 24 hours. SIRT1 protein and phosphorylated Akt/Akt ratio(p-Akt / Total-Akt) were assayed at different time point by SDS-PAGE / WB in cytoplasmic fraction. (B) SIRT1 protein levels were examined at different concentration of CSE with or without 100 μM BSO pretreatment. All values were mean value ± SEM of at least three experiments. ** <i>p</i> < 0.01, compared with the values of non-treatment group; ^†† <i>p</i> < 0.01 between the absence or presence of BSO. (C) Various inhibitors against PI3K signaling pathway molecules were added 30 min prior to the CSE exposure (0.1 μM of PIK75 for PI3Kα inhibitor, 10 μM of GSK2636771 for PI3Kβ inhibitor, 10 μM of AS605240 for PI3Kγ inhibitor, 5 μM of IC87114 for PI3Kδ inhibitor, and 0.02 μM of rapamycin for mTOR inhibitor). The Akt activation status (p-Akt / Total-Akt) and SIRT1 protein levels were evaluated by SDS-PAGE / WB. (D, E) The concentration dependent effects of PIK75 on the SIRT1 and Akt phosphorylation levels were examined using CSE exposure with BSO pretreatment model. The ratio of p-AkT/Total-Akt or SIRT1 levels were fitted with the sigmoid-curve, and calculated for the IC₅₀ of PIK75 (E, respectively). All values were mean value ± SEM of at least three experiments. ** <i>p</i> < 0.01, compared with the values of non-treatment group; ^†† <i>p</i> < 0.01 between the two groups.</p

Localization of SIRT1 in human bronchial epithelial cells.

<p>(A) SIRT1, SIRT6 and HDAC2 protein expressions are shown in cytoplasmic and nuclei fraction. Lamin A/C and α-tubulin was used for loading control of nuclear and cytoplasmic fractions, respectively. (B) SIRT1 localization in BEAS-2B cells was detected by immunocytochemistry. Upper panel showed SIRT1 immunoreactivity, and lower panel indicated negative control (× 40 in BD Pathway 435 bioimager). (C) SIRT1 subcellular localization was examined for the different human primary cells, such as human airway epithelial cells of bronchial origin (hAEC, passage 2 and 4), air-liquid interface cultured human bronchial epithelial cells (ALI) and human pulmonary artery endothelial cells (hPAEC).</p

Shuttling of SIRT1 to nuclei was impaired by repeated CSE priming and in COPD.

<p>(A) In CSE exposure with BSO pretreatment model, the effects of different concentration of recurrent CSE in the presence or absence of repeated 0.1 μM MG-132 were examined. (B) The effect of 0.3% CSE in the presence of 0.1 μM MG-132 on SIRT1 shuttling under the oxidative stress was confirmed. (C) Cellular oxidative stress determined as malondialdehyde in 3% CSE stimulated and in repeated CSE priming with 3% CSE stimulated cells. (D) SIRT1 proteins in nuclei [N] and cytoplasm [C] in CSE exposed primary bronchial epithelial cells from subjects with or without COPD. (E) Nuclear to cytoplasmic (N/C) ratio of SIRT1 in experiments (D). The data were shown as the relative ratio of that of non-treatment group. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, compared with the value of non-treatment group; ^† <i>p</i> < 0.05, ^†† <i>p</i> < 0.01, compared between the two group.</p

Shuttling of SIRT1 to nuclei was impaired by SIRT1 inhibitor.

<p>(A) In the 3% CSE exposure with 100 μM BSO pretreatment model, the effect of the SIRT1 inhibitor sirtinol (10 μg/ml) on SIRT1 shuttling and on the mRNA levels of SOD2 (B, left) and SOD3 (B, right) were examined. All values are mean values ± SEM of at least three experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, compared with the values of non-treatment group; ^† <i>p</i> < 0.05, ^†† <i>p</i> < 0.01, compared between two groups. (C) The various kinase inhibitors were added 30 min prior to the final 3% CSE exposure (0.1 μM of PIK75 [P] for PI3Kα, 10 μM of GSK2636771 [G] for PI3Kβ, 10 μM of AS605240 [A] for PI3Kγ, 5 μM of IC87114 [I] for PI3Kδ, 0.02 μM of rapamycin [R] for mTOR, 0.1μM of BIRB786 [B] for p38MAPK and 0.1μM of U0126 [U] for ERK signaling. SIRT1 and FOXO3a protein levels in nuclei were evaluated by SDS-PAGE / WB.</p

3,5-Dimethylisoxazoles Act As Acetyl-lysine-mimetic Bromodomain Ligands

Histone–lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone–bromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound <b>4d</b>, which has IC₅₀ values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains