L. Gong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7381–7387
7387
and the substrate final volume of 40
l
l in buffer containing 25 mM HEPES, pH
suggesting that JNK inhibition may be attractive as a novel thera-
peutic approach for the treatment of asthma.
7.5, 2 mM dithiothreitol, 150 mM NaCl, 20 mM MgCl2, 0.001% TweenÒ20, 0.1%
BSA and 10% DMSO. Human JNK2
a
2 assay contains 1 nM enzyme, 1
33P] ATP. Human JNK1a1 assay contains 2 nM enzyme,
M ATP with 1 Ci [
33P] ATP. The enzyme assay was carried
lM ATF2,
8
1
l
l
M ATP with 1
M ATF2, 6
lCi [c-
l
l
c-
References and notes
out in the presence or absence of several compound concentrations. JNK and
compound were pre-incubated for 10 min, followed by initiation of the
enzymatic reaction by adding ATP and the substrate. The reaction mixture
was incubated at 30 °C for 30 min. At the end of incubation, the reaction was
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terminated by transferring 25 ll of the reaction mixture to 150 ll of 10%
glutathione SepharoseÒ slurry (Amersham #27-4574-01) containing 135 mM
EDTA. The reaction product was captured on the affinity resin, and washed on a
filtration plate (Millipore, MABVNOB50) with phosphate buffered saline for six
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a microplate scintillation counter (Packard Topcount).
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22. c-Jun Activation Assay in TNFa-Induced Human Chondrosarcoma SW1353
Cells: SW1353 cells are grown in DMEM (Invitrogen) with 10% fetal bovine
serum (Invitrogen), ascorbic acid (15 mg/ml), penicillin (100 U/ml) and
streptomycin (100 mg/ml) (Invitrogen). Cells are plated at a density of 8000
cell per well in 100 ml of growth media for 24 h before the compound
treatment. Immediately before the treatment, media is replaced with 90 ml of
fresh media, then add 10 ml of 10ꢀ concentrated compound solution and
allowed to pre-incubate with cells for 30 min. The vehicle (DMSO) is
maintained at a final concentration of 0.5% in all samples. After 30 min, the
cells are activated with 10 ng/ml of TNF
a (Roche Biochemical) and incubated
20 min at 37 °C in 5% CO2. Cells are fixed in 4% formaldehyde/PBS and
permeabilized with 0.5% Triton-100/PBS (RocheBiochemical.), then were
incubated in blocking buffer (2% BSA/PBS) for 1 h. The cell are stained with
c-Jun monoclonal antibody (Santa Cruz Biotechnology) for 1 hour and detected
with DyLight 488 Goat anti-mouse antibody (Thermo Fisher), nuclei are
stained with Hoechst dye (Thermo Fisher). Imagines are acquired and
quantified by ArrayScan reader (Thermo Fisher). The IC50 values are
calculated as the concentration of the compound at which the c-Jun intensity
was reduced to 50% of the control value using Xlfit in Microsoft Excel program.
23. Bamborough, P.; Drewry, D.; Harper, G.; Smith, G. K.; Schneider, K. J. Med. Chem.
2008, 51, 7898.
24. Goldstein, D. M.; Gray, N. S.; Zarrinkar, P. P. Nat. Rev. Drug Discovery 2008, 7,
391.
25. Ovalbumin-induced Asthma in Rat: Male Brown Norway rats were sensitized
and boosted with Ovalbumin on days 1 and 8, and exposed by inhalation to an
Ova aerosol (5% Ova—50 mg/ml for 30 min on days 15–16). Alum-sensitized,
Ova-challenged BN rats and sham-exposed BN rats served as controls (Warheit
et al., 2000). The lungs of rats were evaluated by bronchoalveolar lavage, or
snapfrozen for JNK activity assessment at several time points (0–72 h)
postexposure. The kinase activity of JNK in lung tissue was measured ex vivo
using ATF2 substrate oligopeptide phosphorylation. Compound or vehicle
control (PEG) was administered once daily at 10 mg/kg (body wt.) 24 h prior to
intranasal challenge.
26. Warheit, D. B.; Glatt, C. M.; Janney, D. M.; Webb, T. R.; Reed, K. L.; Wiethoff, A. J.
Annals Occupational Hygiene, 2000, 46(suppl 1), 362.
20. PDB accession number: 4G1W.
21. JNK activity was measured by phosphorylation of GST-ATF2 (19-96) with
[c-
33P] ATP. The enzyme reaction was conducted at Km concentrations of ATP