Fig 1.
The development of a structure-based pharmacophore model for in silico screen of NBD mimetics.
(A) Interacting residues extracted from the X-ray structure of the NEMO/IKKβ complex (PDB ID: 3BRV) used in the generation of the structure-based pharmacophore model [21]. (B) A 3D representation of the structure-based pharmacophore model. Three hydrophobic groups (F2, F4, and F7: yellow spheres), 1 hydrogen-bond acceptor features (F3: red sphere), 1 hydrogen-bond donor (F5: green sphere), 1 positive ionizable area (F8: blue sphere), 1 negative ionizable (F6: red sphere), and 13 excluded volumes (gray spheres) are shown. (C) Chemical structures of 3 compounds that entered biological testing [24]. 3D, three-dimensional; IKK, IκB kinase; NBD, NEMO-binding domain; NEMO, NF-κB essential modulator; PDB, Protein Data Bank.
Fig 2.
Identification of small molecules inhibiting TNF-α-induced NF-κB activation.
(A) HEK293 cells stably expressing NF-κB luciferase reporter were pretreated with indicated small molecules at 100 μM for 30 min, followed by stimulation with TNF-α (10 ng/ml) for 3 h. Luciferase activity was normalized to untreated stimulated control cells. Data show the mean values of 2 independent experiments +/− SD. (B) The dose-dependent response of ZINC12909780 was tested by NF-κB luciferase assay in HEK293 cells. Three independent experiments were performed, and data shown represent mean +/− SD. (C) HEK293 cells were treated with derivatives of ZINC12909780 at 100 μM, and NF-κB luciferase assay was performed to screen for small molecules with greater NF-κB inhibitory effect. Data show the pooled results of 5 independent experiments and are the mean +/− SD. (D) HEK293 cells were cultured in the presence of 100 μM of indicated derivatives for 24 h, and cell survival was determined by MTT assay. Cell viability was calculated by normalizing to untreated cells. (E) Dose-dependent inhibitory effects were determined for ZINC5 at 0, 25, 50, and 100 μM by using NF-κB luciferase assay. (F) HEK293 were cultivated in the presence of ZINC5 at listed concentrations for 24 h, and cell survival was assessed by MTT assay. Data represent the mean +/− SD from 4–5 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Underlying data can be found in S1 Data. HEK293, human embryonic kidney 293 cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide colormetric assay for assessing cell metabolic activity; NF-κB, nuclear factor κB; TNF-α; tumor necrosis factor α; SD, standard deviation.
Fig 3.
Two identified small molecules reduce NF-κB DNA binding activity in vivo and in vitro.
(A) Levels of p-IκBα with or without treatment in response to TNF-α were determined by western blot analysis. HEK293 cells were pretreated with DMSO, ZINC12909780 (100 μM), and ZINC5 (100 μM) for 30 min followed by the stimulation of 10 ng/ml of TNF-α for 0, 5, and 10 min. Cell lysates were prepared for immunoblot against p-IκBα. GAPDH was used as a loading control. (B) EMSA analysis detecting NF-κB DNA binding activity was performed in C2C12 myoblasts. Cells were pretreated with 200 μM of DMSO, ZINC12909780, and ZINC5 in serum-free media for 1 h. TNF-α was then added to a final concentration of 10 ng/ml and incubated for 15 min. Nuclear extract was obtained for EMSA analysis. (C) EMSA analysis assessing NF-κB DNA binding activity in vivo was performed with quadriceps from mdx mice. Single-dose ZINC12909780 and ZINC5 at 10 mg/kg was given by i.p. Quadriceps was harvested at 1 h and 2 h post injection for EMSA analysis. (D) The time-course pharmacokinetics of ZINC5 and ZINC12909780 in FBS media or FBS media containing acetonitrile. Underlying data can be found in S1 Data. EMSA, electrophoretic mobility shift assay; HEK293, human embryonic kidney 293 cells; i.p., intraperitoneal; NF-κB, nuclear factor κB; t1/2, half-life; TNF-α; tumor necrosis factor α.
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.
Table 1.
Inhibitory effects of NBD mimetics on NF-κB activation were measured by luciferase assays at multiple concentrations: 0, 25, 50, 100, and 150 μM. IC50 of NBD mimetics was determined based on the dose-dependent curve by using GraphPad.
Fig 5.
NBD mimetics selectively inhibit canonical NF-κB signaling by targeting the association of NEMO with IKKβ.
(A) Co-IP analysis detecting the IKKβ/NEMO interaction. Raw 264.7 cells were pretreated with the indicated drugs, DMSO, 8K-NBD peptide (400 μM), SR12460 (100 μM), SR12343 (100 μM), SR12454 (100 μM), and SR11481 (100 μM) for 30 min, and the cells were then harvested for Co-IP. NEMO was probed as a loading control. (B) Raw 264.7 cells pretreated with SR12343 at indicated concentrations (0, 25, 50, 100, and 150 μM) for 30 min were subjected to Co-IP assay. NEMO-binding products were then analyzed for levels of IKKβ, IKKα, TRAF2, and IκBα (negative control), and levels of NEMO were used as a loading control (left panel). Right panel shows levels of proteins in input controls (10% of cell extract used for Co-IP). (C) GST-NEMO (15 nM), preincubated with inhibitors at indicated concentrations, was incubated with IKKβ-FLAG (15 nM) for 30 min at 30 °C and isolated using Glutathione argarose. The levels of IKKβ-FLAG binding to GST-NEMO were determined by western blot analysis with GST-NEMO used as a loading control. (D, E) Raw 264.7 cells pretreated with vehicle control or SR12343 (150 μM) for 30 min were stimulated with or without 10 ng/ml TNF-α (D) or 1 μg/ml LPS (E) for 10 min. Cell lysates were analyzed for activity of the IKK/NF-κB signaling pathway including activation of IKK complex, IκBα, and p65. GAPDH was used as a loading control. (F) Raw 264.7 cells were incubated with vehicle control or SR12343 (100 μM) for 30 min, followed by stimulation with or without anti-LTβR for 8 h. Cell lysates were harvested and analyzed for activation of noncanonical NF-κB signaling, the processing of p100 to p52. (G, H) Analysis of the effects of SR12343 (150 μM) on phosphorylation of JNK and p38MAPK in response to TNF-α (G) or LPS (H), using the same cell lysate being used in panels D and E. Co-IP, co-immunoprecipitation; GST, glutathione S-transferase; IKK, IκB kinase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; LTβR, lymphotoxin β receptor; MAPK, mitogen-activated protein kinase; NBD, NEMO-binding domain; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; TNF, tumor necrosis factor; TRAF2, TNF receptor-associated factor 2.
Fig 6.
Newly identified NBD mimetics suppress LPS-induced acute inflammation in vivo.
(A) The bioavailability property of SR12460, SR12343, SR12454, and SR11481. Concentrations of NBD mimetics were determined in plasma, brain, muscle, spleen, and liver 2 h post a single i.p. injection of 10 mg/kg of drugs; n = 3 per group. (B) Shown is the model to induce LPS-mediated acute inflammation and the treatment regimen with NBD mimetics. Mouse image via http://www.clker.com/clipart-mice-blank.html (copyright-free). (C) NBD mimetics suppressed LPS-induced acute inflammation in liver and lung by down-regulating NF-κB target gene expression. Vehicle, SR12343, SR12460, and 8K-NBD peptide were dosed at 10 mg/kg 30 min prior to the LPS treatment. Acute inflammation was induced by i.p. injection of 10 mg/kg LPS. Lung and liver were collected 2–4 h post injection, and mRNA was extracted for qRT-PCR analysis. (D, E) Western blot analysis was performed to probe for the expression of p-IκBα and COX-2 at protein level in liver and lung tissues. (F) Serum levels of IL-6 were determine by ELISA. n = 3–8 each group. *P < 0.05, **P < 0.01. Underlying data can be found in S1 Data. COX-2, cyclooxygenase 2; IL-6, interleukin 6; i.p., intraperitoneal; LPS, lipopolysaccharide; NBD, NEMO-binding domain; n.d., nondetectable; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; qRT-PCR, quantitative real-time polymerase chain reaction.
Table 2.
Hematological measures in acutely treated mice.
Fig 7.
NBD mimetics improve muscular pathology and grip strength in mdx mice.
(A) Shown is the treatment regimen with NBD mimetics in mdx mice. (B) Hematoxylin–eosin staining of TA muscle from 7-wk-old, treated or untreated mdx mice. Images were taken at magnification of 20×. Representative images were shown for each treatment group. (C) Quantitation of the percentage of area exhibiting necrosis or infiltration by using Image J. (D) qRT-PCR analysis of eMyHC and Pax7 in TA muscle. (E) TA muscle from 7-wk-old, treated or untreated mdx mice was stained with laminin by IHC to outline myofibers. Overall images of TA muscle were shown on the left side, and the inset panel images were shown on the right side. (F) Quantitation of minimum Feret diameter in centrally and noncentrally nucleated myofibers. (G) Forelimb force determined by grip strength test. Five-wk-old (2 wk post treatment) and 7-wk-old (4 wk post treatment) mdx mice, treated or untreated, were tested for forelimb grip strength. Five sequential tests were performed, and the force was normalized to body weight. Data were shown as mean +/− SEM. Dosages used are as follows: SR12343 (30 mg/kg), SR12460 (30 mg/kg), and 8K-NBD peptide (10 mg/kg). n = 6–7 each group. *P < 0.05, **P < 0.01. Underlying data can be found in S1 Data. eMyHC, embryonic myosin heavy chain; IHC, immunohistochemistry; NBD, NEMO-binding domain; NEMO, NF-κB essential modulator; Pax7, paired box protein 7; qRT-PCR, quantitative real-time polymerase chain reaction; TA, tibialis anterior.
Fig 8.
Chronic treatment with NBD mimetics reduces muscle fibrosis by decreasing macrophage infiltration in mdx mice.
(A, B) Trichrome and CD68 staining of (A) diaphragm and (B) TA muscle from 7-wk-old, SR12343-treated or -untreated mdx mice. CD68 (red): marker of tissue macrophage; utrophin (green): stain for myofiber; DAPI (blue): nuclei. Trichrome images were taken at the magnification of 4× for TA and 10× for diaphragm. IHC images were taken at 10× for TA and 20× for diaphragm. Representative images were shown for each treatment group. Data were shown as mean +/− SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Underlying data can be found in S1 Data. CD68, cluster of differentiation 68; IHC, immunohistochemistry; NBD, NEMO-binding domain; NEMO, NF-κB essential modulator; TA, tibialis anterior.