Fig 4. Modified lead NBD mimetics inhibit TNF-α- and LPS-induced NF-κB activation by disrupting the association between NEMO and IKKβ.

(A) Structures of top NBD mimetics. (B) Measurement of NF-κB activation in response to TNF-α by using Dual-luciferase reporter assays. HEK293 cells cotransfected with NF-κB luciferase reporter and SV40-Renilla plasmids were pretreated with DMSO or listed small molecules at 0, 25, 50, 100, and 150 μM for 30 min, followed by the induction of TNF-α for 3 h. Data shown are representative of 2–3 independent experiments. (C) NBD mimetics down-regulated the expression of NF-κB target genes in response to LPS. Raw 264.7 cells were pretreated with indicated drugs for 30 min and then stimulated with 1 μg/ml of LPS for 2 h. Cells were then harvested for RNA extraction and qRT-PCR analysis. Drug concentrations used are as follows: IKKi VII (2 μM), 8K-NBD peptide (200 μM), SR12343 (50 μM), SR12460 (50 μM), SR12454 (50 μM), and SR11481 (50 μM). Data are representative of 2 independent experiments. (D) Mouse IL-6 production induced by LPS was down-regulated by NBD mimetics. Raw 264.7 cells pretreated with DMSO or drugs at indicated concentrations were exposed to 1 μg/ml of LPS for 24 h, and supernatant was collected for ELISA analysis of mouse IL-6. *P < 0.05; **P < 0.01. Underlying data can be found in S1 Data. HEK293, human embryonic kidney 293 cells; IKK, IκB kinase; IL-6, interleukin 6; LPS, lipopolysaccharide; NBD, NEMO-binding domain; n.d., nondetectable; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; qRT-PCR, qualitative real-time polymerase chain reaction; TNF-α; tumor necrosis factor α.