A quinazoline-based bromodomain inhibitor, CN210, ameliorates indomethacin-induced ileitis in mice by inhibiting inflammatory cytokine expression

Abstract

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.

Keywords: CREB-binding protein (CBP) and p300; bromodomain and extra-terminal motif (BET) proteins; ileitis.

FIG U R E 1

Effect of CN210 on indomethacin-induced ileitis. Animals received indomethacin (10 mg/kg) subcutaneously and were sacrificed 48 h later. CN210 (20 and 50 mg/kg) and dexamethasone (3 mg/kg) were administered 30 min before and 24 h after indomethacin treatment. (a) Representative macroscopic observation of indomethacin-induced ileitis. Arrowheads indicate Evans blue-stained ileal lesions. (b) Lesion area and (c) MPO activity of indomethacin-induced ileitis. (d) Representative histological observation of indomethacin-induced ileitis at ×100 magnification. Scale bar: 50 μm. Data are shown as means ± standard errors of the mean (ns = 5 to 8). One-way ANOVA with Holm-Sidak’s test revealed a statistically significant difference between the experimental mice and the controls (vehicle alone; *P < 0.05)

FIG U R E 2

Effect of CN210 on indomethacin-induced increase in cytokine expression in the ileal mucosa. Animals received indomethacin (10 mg/kg) subcutaneously, and messenger RNA expression for TNF-α, IL-6, IFN-γ, and IL-17 was determined by quantitative real-time RT-PCR 48 h later. CN210 (50 mg/kg) and dexamethasone (3 mg/kg) were administered 30 min before and 24 h after indomethacin treatment. Data are shown as means ± standard errors of the mean (n = 8). One-way ANOVA with Holm-Sidak’s test revealed a statistically significant difference between experimental mice and controls (vehicle alone; *P < 0.05) and between experimental mice and those not given indomethacin (#P < 0.05)

FIG U R E 3

Effect of CN210 on LPS-stimulated cytokine expression in RAW264 cells. The cells were treated with LPS (1 μg/mL), and total RNA was isolated 4 h later and messenger RNA expression for TNF-α, IL-6, IFN-γ, and IL-17 was determined by quantitative real-time RT-PCR. Data are shown as means ± standard errors of the mean (n = 6). One-way ANOVA with Holm-Sidak’s test revealed a statistically significant difference between experimental mice and controls (vehicle alone; *P < 0.05) and between experimental mice and those not given indomethacin (#P < 0.05)

FIG U R E 4

Effects of CN210 on LPS-activated NF-κB and MAPK in RAW264 cells. The cells were treated with LPS (1 μg/mL) and activation of NF-κB and MAPK was determined by Western blotting 4 h later. Mice given CN210 (10 μM) were cotreated with LPS. (a) Western blot analyses and (b) quantitative results for the effect of CN210 on LPS-activated NF-κB and MAPK in RAW264 cells (5 replicates at 4 h). Data are shown as mean ± standard errors of the mean (n = 5). One-way ANOVA with Holm-Sidak’s test revealed a statistically significant difference between experimental mice and controls (vehicle alone; *P < 0.05) and between experimental mice and those not given indomethacin (#P < 0.05)

FIG U R E 5

Docking study of CN210 to the bromodomain of CBP and p300. (a) Structural comparison of BRD4 BD1 (6MAU), CBP (4NR7) and p300 (5BT3). Proteins are shown in cartoons in gray. Key residues surrounding the binding pocket are shown in sticks and the carbon atoms are colored in green BRD4 BD1/cyan (CBP)/magenta (p300), respectively. Conserved residues are highlighted in bright yellow and the varied residues are highlighted in yellow-orange in the order of BRD4 BD1/CBP/p300. CN210 in the co-crystal with BRD4 BD1 is shown in yellow, and SGC-CBP30 in the co-crystals with CBP/p300 is shown in salmon. (b) Structures of CN210 and SGC-CBP30. (c) Amino acid sequence alignment of bromodomains of three proteins; N-terminal bromodomain of BRD4 (BRD4 BD1), CBP and p300. (d) Predicted binding model of CN210 (yellow) in the binding pocket of the bromodomain of CBP (4NR7). SGC-CBP30 is shown in salmon. (e) Predicted binding model of CN210 (yellow) in the binding pocket of the bromodomain of p300 (5BT3). SGC-CBP30 is shown in salmon