The phosphorylation of JNK is known to induce insulin resistance in insulin target tissues. The inhibition of JNK-JIP1 interaction, which interferes JNK phosphorylation, becomes a potential target for drug development of type 2 diabetes. To discover the inhibitors of JNK-JIP1 interaction, we screened out 30 candidates from 4320 compound library with
The role of c-Jun N-terminal kinase (JNK) in diabetes has been studied over the past decade. Activated JNK contributes to the inactivation of TNFα-induced insulin receptor subunit 1 (IRS1) by Ser307 phosphorylation, leading to insulin resistance (Aguirre
There have been considerable efforts to develop JNK inhibitors, initially focused on ATP-competitive inhibitors like SP600125 and its derivatives, then expanded to the other small compound groups, natural product sources, and peptide inhibitors (Bogoyevitch and Arthur, 2008). Peptide inhibitors were designed to interfere the interaction of JNK with c-Jun, its downstream substrate, or JIP1, a cytoplasmic scaffold protein that interacts with JNK and activates JNK phosphorylation (Bogoyevitch and Arthur, 2008). Even though several roles of JNK in diabetes mechanisms, only a few JNK inhibitors have been verified for antidiabetic activities. Several competitive peptide inhibitors were developed for JNK inhibitors targeting the JNK interacting site of JIP1 (Barr
To expand antidiabetic drug candidates targeting JNK-JIP1 interaction, we screened 4320 library compounds including 1280 pharmacologically active compounds and 1920 approved drugs with
Total 4320 screening compound libraries were purchased from Sigma-Aldrich (St. Louis, MO, USA) (1280 compounds of LOPAC), Enzo Life Sciences (Seoul, Korea) (640 compounds of FDA approved drug library), Tocris Bioscience (Bristol, UK) (1120 compounds of Tocriscreen compound library collection) and Microsource Discovery Systems (Gaylordsville, CT, USA) (1280 compounds of US drug collection). Full-length of JNK cDNA (GenBank Accession No. NM_002750.2) and JIP1 cDNA (GenBank Accession No. BC068470) were obtained from Open Bio (Seoul, Korea) and Imagene (Seoul, Korea) respectively. BI-78D3 was purchased from EMD Millipore (Billerca, MA, USA). TNFα was purchased from R&D Systems (Minneapolis, MN, USA) and insulin was purchased from Roche (Seoul, Korea). Lipofectamine 2000, 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) and 293A cell were purchased from ThermoFisher Scientific (Waltham, MA, USA). HeLa, HepG2 and 3T3L1 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Anti-phosphor-JNK was purchased from Abcam (Cambridge, MA, USA), anti-JNK was purchased from Santa Cruz (Dallas, TX, USA) and anti-actin was purchased from Sigma-Aldrich. Secondary mouse and rabbit antibody were purchased from The Jackson Lab (Farmington, CT, USA).
Full-length of JNK and JIP1 cDNA were cloned into pEGFP-N1 and pmRFP-C3 mammalian expression vectors by conventional molecular cloning for the
HeLa and HepG2 cells were maintained in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% Fetal Bovine Serum (FBS). 293A Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS. Pre-differentiation of 3T3L1 cells were maintained in DMEM with 10% Bovine Serum (BS). Differentiation of 3T3L1 cells were induced by DMEM containing 10% FBS with 0.5 mM 3-isobutyl-1-methylxanthine, 0.5 μM dexamethasone, and 10 μg/ml insulin and were grown for 3 days. And then, the media was changed to DMEM containing 10% FBS with 10 μg/ml insulin for two more days. All cells were grown in 5% CO2 at 37°C in a humidified environment. Cells were transiently transfected using Lipofectamine 2000 according to the manufacturer’s instructions.
For FRET screening, 293A cells were cultured in 96-well plate for 24 h. Then, these cell were co-transfected with JNK-CFP and JIP1-mRFP expression vectors for 24 h. And then, Positive compounds by
Whole cell lysate proteins were made by sonication with lysis buffer (20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 150 mM NaCl, 10% Glycerol, 1 mM Na3VO4, 50 mM NaF, 1 mM Phenylmethylsulfonyl fluoride (PMSF) and 0.05% SDS) containing a protease-inhibitor cocktail and incubated on ice for 30 min. For the western blot analysis, the supernatants were separated by SDS-PAGE using 10% gels and blotted transferred onto a polyvinylidenedifluoride (PVDF) membranes. The blots were then probed with primary antibodies (1:1000) for 1 h. Blots were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000) for 45 min followed by additional washing. Signal was detected by chemiluminescence (ECL, GE healthcare, Little Chalfont, UK) and recoded by imaging analyzer (ImageQuant LAS 4000 mini, GE healthcare).
2-Deoxyglucose uptake was measured using fluorescent 2-NBDG reagent according to the manufacturer’s instructions. Briefly, cells were cultured without serum for 16 h and then treated TNFα (20 ng/ml) for 6 h. After that, these cells were stimulated by positive compounds (1 μM) for 1 h respectively. Before measure of Deoxyglucose level, media was changed to phenol red-free media with 80 μM 2-NBDG for 10 min. The fluorescence was measured by using a fluorescence micro-reader at excitation of 485 nm and emission of 535 nm.
A docking simulation between JNK and Acebutolol was performed in Discovery Studio (version 3.1, BIOVIA, San Diego, CA, USA). The template structure of JNK (PDB ID: 4IZY) was downloaded from Protein Data Bank (Berman
To find inhibitors of JNK-JIP1 interaction, we screened 4320 compounds in HeLa cells using
Depending on inhibitory efficiency from FRET assay, we narrow down the candidates to top five positive inhibitors for further study. Selected candidates were Acebutolol, Lithium chloride, Vincristine sulfate, Niacinamide and Valproic acid. FRET images of final selected candidates together with controls were shown in the Fig. 1C.
Although five candidates showed more than 40% inhibitory effect on JNK-JIP1 interaction from FRET assay, their contributions to JNK inhibition is still unknown. To identify direct relationship of candidates to JNK inhibition, phosphorylation level of JNK was tested in TNFα-treated 3T3L1 adipocyte and HepG2 liver cells, diabetic mimic cells.
To compare the effect of all drug candidates fairly and keep the consistence of all experiment condition, we continued to use the single does condition (1 μM) for second round selection. The does condition for drug candidates is decided as the same as optimized condition for control drug BI-78D3 to provide a guide of candidate selection. Treatment of TNFα increased phosphorylation of JNK in these cells (Fig. 2). Acebutolol and Lithium chloride decreased TNFα induced JNK phosphorylation and Valproic acid and Niacinamide reduced JNK phosphorylation but small inhibition was achieved compared to Acebutolol or Lithium chloride in both cells (Fig. 2). However, phosphorylation of JNK was not inhibited but increased slightly by Vincristine sulfate in these cells (Fig. 2). Along with this result, Vincristine sulfate was also known to promote JNK phosphorylation in chronic lymphocytic leukaemia (CLL) cells (Bates
Based on the effect of both cells, we concluded that Acebutolol and Lithium chloride is more effective in inhibit of JNK activity among the candidates.
Because Acebutolol contributes to improve hypoglycemia in diabetic patients as well as it is an FDA-approved drug with clinical safety guarantee, Acebutolol was selected between effective candidates for further study (Deacon
To determine whether Acebutolol stimulates the glucose uptake, one of the most important phenotype in diabetes, we performed glucose analog, 2-NBDG, uptake assay in both TNFα-induced diabetic model cells and non-induced normal cells (Fig. 3). Without TNFα pretreatment, insulin increased glucose uptake about 30% (1.28 ± 0.13). BI-78D3 increased glucose uptake about 90% (1.97 ± 0.11) and Acebutolol increase glucose uptake about 50% (1.48 ± 0.17) comparing with control respectively. With TNFα treatment, insulin increased glucose uptake about 20% (1.18 ± 0.12). BI-78D3 increased glucose uptake about 50% (1.54 ± 0.16) and Acebutolol increase glucose uptake about 45% (1.46 ± 0.02) comparing with control respectively. Because relatively short time incubation of TNFα (6 h) in our experimental condition, glucose uptake by insulin treatment was slightly decreased (Fig. 3A). The effect of BI-78D3 on glucose uptake is higher in normal cells than TNFα treated cells. Interestingly, in both cell conditions, ability of Acebutolol on glucose uptake is consistent (Fig. 3A). This can be interpreted as the effect of Acebutolol on glucose uptake is more specific to the model condition of insulin resistance: researchers use TNFα treatment for this model condition as TNFα inhibits insulin-stimulated glucose uptake.
Acebutolol and BI-78D3 enhanced approximately 50% glucose uptake but insulin increased approximately 20% glucose uptake compared with TNFα only treated 3T3L1 cells (Fig. 3B). With similar result of TNFα-treated 3T3L1 cells, glucose uptake was also enhanced in Acebutolol or BI-78D3 treated HepG2 cells compared with TNFα only treated HepG2 cells (Fig. 3C). These results show that Acebutolol increases glucose uptake effectively in these cells.
To figure out the relationship between Acebutolol effect and concentration, we performed dose-dependent study. In TNFα-induced diabetic model cells, Acebutolol showed dose-dependent response on glucose uptake that it significantly increases the glucose uptake as its concentration increased from 0.5 μM, 1.0 μM to 5.0 μM (Fig. 3D). On the other hand, in the absence of TNFα treatment, the glucose uptake at low concentration (0.5 μM) of Acebutolol reached to the similar level of high concentration (5.0 μM) of Acebutolol with TNFα-induced cells, and the effects were increased slightly at higher doses. This results explains well that Acebutolol can recover the glucose uptake from the suppressed level of TNFα-induced diabetic model cells to the level of TNFα-untreated normal condition.
Together with inhibitory effect of JNK phosphorylation, Acebutolol is a good candidate of JNK regulation in diabetic model cells.
To compare the binding regions and affinities between JNK and Acebutolol with BI-78D3, we performed the docking simulation by using LibDock analysis. The structure of Acebutolol and BI-78D3 showed in Fig. 4A and 4C respectively. The docking pose between Acebutolol and JNK shows that Acebutolol formed H-bonds with both Arg127 and Cys163 (Fig. 4B), same as that are shown in BI-78D3 (Fig. 4D). The average LibDock score of top 10 binding poses also implies that Acebutolol (65.1) has similar binding affinities with BI-78D3 (67.7) and these scores are ∼2.9 higher than Cyproterone acetate (22.6) (Fig. 4E).
These results indicated that Acebutolol inhibits phosphorylation of JNK by the similar binding regions and affinities of BI-78D3 that acting as competitive binding component of JIP1.
In the present study, we identified
Acebutolol (trade name Sectral) is a cardio-selective beta-adrenergic blocker currently in use for hypertension and cardiac arrhythmias without serious side effects since it was first introduced in the clinics more than 30 years ago (Charoenlarp and Jaroonvesama, 1978). Interestingly, in the early stage of its clinical use, there was an issue of possible adverse effect of Acebutolol on glucose and lipid metabolism due to the adrenergic receptor signaling (Kumar
Acebutolol could expand the flexibility of antidiabetic treatment by increasing the target diversity since there are only a few
This work was supported by the Bio-Synergy Research Project (NRF-2012M3A9C4048759) of the Ministry of Science, ICT and Future Planning through the National Research Foundation.