Inhibitors of bromodomain and extra-terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti-inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB-binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC-CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin-induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF-κB and MAPK pathways. CN210 thus represents a new mode of therapy for non-steroidal anti-inflammatory drug-induced ileitis and inflammatory bowel disease.

SK) ¶ These authors contributed equally to this work 1 Abstract 2 3 Inhibitors of bromodomain and extra-terminal motif proteins ("BET inhibitors") are emerging 4 epigenetic therapeutics that exert their effects by suppressing the expression of genes that drive 5 cancer and inflammation. The present study examined the anti-inflammatory effects of a 6 quinazoline-based BET inhibitor, CN210, which also inhibits bromodomains of two paralogous 7 histone acetyltransferases (HATs), such as cAMP-responsive element binding protein-binding 8 protein (CBP) and p300. To assess its effectiveness against inflammatory bowel disease (IBD), 9 CN210 was tested in an experimentally-induced murine Crohn's disease (CD)-like ileitis model.10 Ileitis was induced in mice by subcutaneous administration of indomethacin and CN210 was 11 given orally 30 min before and 24 h after the indomethacin administration. Whilst indomethacin 12 produced severe ileitis accompanied by an increase in ileal mucosal myeloperoxidase (MPO) 13 activity, administration of CN210 reduced the severity of ileitis in a dose-dependent manner 14 and suppressed the MPO activity. Similarly, upregulation of inflammatory cytokines was 15 observed in ileitis mucosa after indomethacin injection but this response was significantly 16 attenuated by CN210 administration. To further characterize the effects of CN210 on 17 inflammatory pathways, monocyte/macrophage-like cell line RAW264 treated with 18 lipopolysaccharide (LPS) was exposed to CN210. CN210 significantly attenuated the 19 expression of inflammatory cytokines and reversed the activation of NF-κB and MAP kinases 20 induced by LPS. These findings suggest that CN210 ameliorates indomethacin-induced ileitis 21 due at least in part to the inhibition of inflammatory cytokine expression via attenuation of NF-22 κB and MAP kinase pathways, thus representing a new mode of therapy for CD. 23 Introduction 24 25 Bromodomain and extra-terminal motif (BET) proteins, including BRD2, BRD3, BRD4 26 and BRDT, are epigenetic 'reader' proteins that bind to acetylated lysine residues on proteins, 27 including histones, by means of their bromodomains [1, 2]. Of the four BET proteins, BRD4 28 associates with general transcription cofactor complex 'Mediator' and is a key structural 29 component of extensive transcription factor complexes formed at the genomic regions termed 30 Super-Enhancers (SEs) [2-4]. Small-molecule inhibitors of BRD4 was shown to suppress the 31 expression of MYC oncogene and the genes that encode pro-inflammatory cytokines, such as 32 IL-6, IFN-β, IL-1β, IL-12α, CXCL9 and CCL12 in mice [5-7], and showed efficacy in a wide 33 range of cancer models including pancreatic, breast, ovarian, and colon cancers [8-12]. 34 Accordingly, small molecule inhibitors of BET proteins ("BET inhibitors") are now considered 35 as promising drug candidates for both cancer and inflammatory diseases [13, 14]. 36 Whilst several BET inhibitors currently are evaluated clinically [15], early results showed 37 mixed results with undesirable s...

The bromodomain and extra-terminal (BET) inhibitor have emerged as promising new cancer agents via regulation of epigenetic mechanism. Recent studies further demonstrate that BET inhibitors exhibit anti-inflammatory effects in animal models of various inflammatory diseases. In the present study, we examined the effect of BET inhibitor on inflammatory bowel disease (IBD) in experimentally-induced murine Crohn's disease (CD)-like ileitis models. Ileitis was induced in male C57BL/6 mice by subcutaneous administration of indomethacin and the ileum was examined 48 h later. CN210 was given orally 30 min before and 24 h after indomethacin administration. Further, the effect of CN210 on LPS-stimulated cytokine expression in cultured RAW264.7 cells. The administration of CN210 reduced the severity of indomethacin-induced ileitis in a dose-dependent manner. Indomethacin-induced upregulation of inflammatory cytokines such asTNF-α, IL-1β and IL-6was also significantly attenuated by administration of CN210. In RAW264.7 cells, LPS upregulated the expression of inflammatory cytokines, and this response was potently abrogated by CN210. These findings suggest that CN210 ameliorates indomethacin-induced ileitis via inhibition of inflammatory cytokine expression. Thus, CN210 is a novel candidate for the treatment with IBD including CD.