Discovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological Evaluation.

Bromodomain-containing protein 4 (BRD4) represents a promising drug target for anti-inflammatory therapeutics. Herein, we report the design, synthesis, and pharmacological evaluation of novel chromone derivatives via scaffold hopping to discover a new class of orally bioavailable BRD4-selective inhibitors. Two potent BRD4 bromodomain 1 (BD1)-selective inhibitors 44 (ZL0513) and 45 (ZL0516) have been discovered with high binding affinity (IC50 values of 67-84 nM) and good selectivity over other BRD family proteins and distant BD-containing proteins. Both compounds significantly inhibited the expression of Toll-like receptor-induced inflammatory genes in vitro and airway inflammation in murine models. The cocrystal structure of 45 in complex with human BRD4 BD1 at a high resolution of 2.0 Å has been solved, offering a solid structural basis for its binding validation and further structure-based optimization. These BRD4 BD1 inhibitors demonstrated impressive in vivo efficacy and overall promising pharmacokinetic properties, indicating their therapeutic potential for the treatment of inflammatory diseases.

Pollen-induced oxidative DNA damage response regulates miRNAs controlling allergic inflammation.

A mucosal oxidative burst is a hallmark response to pollen exposure that promotes allergic inflammatory responses. Reactive species constituents of oxidative stress signal via the modification of cellular molecules including nucleic acids. One of the most abundant forms of oxidative genomic base damage is 8-oxo-7,8-dihydroguanine (8-oxoG), which is removed from DNA by 8-oxoguanine DNA glycosylase 1 (OGG1). OGG1 in complex with 8-oxoG acts as a GDP-GTP exchange factor and induces acute inflammation; however, the mechanism(s) by which OGG1 signaling regulates allergic airway inflammation is not known. Here, we postulate that the OGG1 signaling pathway differentially altered the levels of small regulatory RNAs and increased the expression of T helper 2 (Th2) cytokines in ragweed pollen extract (RWPE)-challenged lungs. To determine this, the lungs of sensitized mice expressing or lacking OGG1 were challenged with RWPE and/or with OGG1's excision product 8-oxoG. The responses in lungs were assessed by next-generation sequencing, as well as various molecular and histological approaches. The results showed that RWPE challenge induced oxidative burst and damage to DNA and activated OGG1 signaling, resulting in the differential expression of 84 micro-RNAs (miRNAs), which then exacerbated antigen-driven allergic inflammation and histological changes in the lungs. The exogenous administration of the downregulated let-7b-p3 mimetic or inhibitors of upregulated miR-23a or miR-27a decreased eosinophil recruitment and mucus and collagen production via controlling the expression of IL-4, IL-5, and IL-13. Together, these data demonstrate the roles of OGG1 signaling in the regulation of antigen-driven allergic immune responses via differential expression of miRNAs upstream of Th2 cytokines and eosinophils.

Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment.

Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that ragweed pollen extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induce allergic airway inflammation. Blocking RWPE-induced neutrophil recruitment in wild-type mice by administration of a CXCR2 inhibitor inhibits the ability of repeated exposures to RWPE to stimulate allergic sensitization and airway inflammation. Administration of neutrophils derived from naive donor mice into the airways of Tlr4 knockout recipient mice after each repeated RWPE challenge reconstitutes allergic sensitization and inflammation in these mice. Together these observations indicate that pollen-induced recruitment of neutrophils is TLR4 and CXCR2 dependent and that recruitment of neutrophils is a critical rate-limiting event that stimulates induction of allergic sensitization and airway inflammation. Inhibiting pollen-induced recruitment of neutrophils, such as by administration of CXCR2 antagonists, may be a novel strategy to prevent initiation of pollen-induced allergic airway inflammation.

Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation.

BACKGROUND: The National Health and Nutrition Examination Survey identified several pollens and cat dander as among the most common allergens that induce allergic sensitization and allergic diseases. We recently reported that ragweed pollen extract (RWPE) requires Toll-like receptor 4 (TLR4) to stimulate CXCL-mediated innate neutrophilic inflammation, which in turn facilitates allergic sensitization and airway inflammation. Myeloid differentiation protein 2 (MD2) is a TLR4 coreceptor, but its role in pollen- and cat dander-induced innate and allergic inflammation has not been critically evaluated. OBJECTIVE: We sought to elucidate the role of MD2 in inducing pollen- and cat dander-induced innate and allergic airway inflammation. METHODS: TCM(Null) (TLR4(Null), CD14(Null), MD2(Null)), TLR4(Hi), and TCM(Hi) cells and human bronchial epithelial cells with small interfering RNA-induced downregulation of MD2 were stimulated with RWPE, other pollen allergic extracts, or cat dander extract (CDE), and activation of nuclear factor κB (NF-κB), secretion of the NF-κB-dependent CXCL8, or both were quantified. Wild-type mice or mice with small interfering RNA knockdown of lung MD2 were challenged intranasally with RWPE or CDE, and innate and allergic inflammation was quantified. RESULTS: RWPE stimulated MD2-dependent NF-κB activation and CXCL secretion. Likewise, Bermuda, rye, timothy, pigweed, Russian thistle, cottonwood, walnut, and CDE stimulated MD2-dependent CXCL secretion. RWPE and CDE challenge induced MD2-dependent and CD14-independent innate neutrophil recruitment. RWPE induced MD2-dependent allergic sensitization and airway inflammation. CONCLUSIONS: MD2 plays an important role in induction of allergic sensitization to cat dander and common pollens relevant to human allergic diseases.