S. K. De et al. / Bioorg. Med. Chem. 18 (2010) 590–596
595
enhancement solution (Perkin–Elmer) was added to each well and
fluorescence measured after 10 min incubation (excitation wave-
length, 340 nm; emission wavelength, 615 nm). Controls include
unlabeled peptide and blanks receiving no compounds. Protein
and peptide solutions were prepared in DELFIA buffer (Perkin–
Elmer).
was used to define the binding site for docking of small molecules.
The docked geometry of the indazole ATP mimetic reported in Fig-
ure 3 was simply derived from the X-ray coordinates of the parent
compound, SP600125. The genetic algorithm (GA) procedure in the
GOLD docking software performed flexible docking of small mole-
cules whereas the protein structure was static.25–29 For each com-
pound, 20 solutions were generated and subsequently ranked
according to Chemscore.25 The protein surface was prepared with
the program MOLCAD as implemented in Sybyl and was used to ana-
lyze the binding poses for studied small molecules.25–29
2.6. In vitro kinase assay
The LanthaScreen™ assay platform from Invitrogen was uti-
lized. The time-resolved fluorescence resonance energy transfer as-
say (TR-FRET) was performed in 384 well plates. Each well received
JNK1 (35 ng/mL, JNK1 MW = 45 KDa), ATF2 (400 nM), and ATP
Acknowledgments
(0.2
l
M) in 50 mM HEPES, 10 mM MgCl2, 1 mM EGTA and 0.01%
We gratefully acknowledge financial support from the NIH
(Grants nos. DK073274 and DK080263) and Syndexa pharmaceuti-
cals (to M.P.).
Brij-35, pH 7.5 and test compounds. The kinase reaction was per-
formed at room temperature for 1 h. After which, the terbium la-
beled antibody and EDTA were added into each well. After an
additional hour incubation, the signal was measured at 520/
495 nm emission ratio on a fluorescence plate reader (Victor 2, Per-
kin–Elmer).
Supplementary data
Supplementary data associated with this article can be found, in
2.7. Cell based assays for c-Jun phosphorylation
References
The cell based kinase assays for c-Jun and ATF2 phosphorylation
were carried out using the LanthaScreen c-Jun (1–79) Hela (Invit-
rogen, Carlsbad, CA) which stably express GFP-c-Jun 1–79. Phos-
phorylation was determined by measuring the time-resolved
FRET (TR-FRET) between a terbium labeled phospho-specific anti-
body and the GFP-fusion protein.12 The cells were plated in white
tissue culture treated 384 well plates at a density of 10000 cell per
1. Manning, G. Science 2002, 298, 1912–1934.
2. Manning, A. M.; Davis, R. J. Nat. Rev. Drug Disc. 2003, 2, 554–565.
3. Bogoyevitch, M. A.; Arthur, P. G. Biochim. Biophys. Acta 2008, 1784, 76–93. and
references cited therein.
4. Gupta, S.; Barrett, T.; Whitmarsh, A. J.; Cavanagh, J.; Sluss, H. K.; Dérijard, B.;
Davis, R. J. EMBO J 1996, 15, 2760–2770.
5. Kyriakis, J. M.; Avruch, J. Physiol. Rev. 2001, 81, 807–869.
6. Pearson, G.; Robinson, R.; Gibson, T. B.; Xu, B. E.; Karandikar, M.; Berman, K.;
Cobb, M. H. Endocr. Rev. 2001, 22, 153–183.
7. Kallunki, T.; Deng, T.; Hibi, M.; Karin, M. Cell 1996, 87, 929–939.
8. Yang, S.-H.; Whitmarsh, A. J.; Davis, R. J.; Sharrocks, A. D. EMBO J 1998, 17,
1740–1749.
9. Barr, R. K.; Kendrick, T. S.; Bogoyevitch, M. A. J. Biol. Chem. 2002, 277, 10987–
10997.
**
well in 32
l
L assay medium (Opti-MEMÒ, supplemented with 1%
g/mL
charcoal/dextran-treated FBS, 100 U/mL penicillin and 100
l
streptomycin, 0.1 mM non-essential amino acids, 1 mM sodium
pyruvate, 25 mM HEPES pH 7.3, and lacking phenol red). After
overnight incubation, cells were pretreated for 60 min with com-
pound (indicated concentration) followed by 30 min of stimulation
with 2 ng/mL of TNF-alpha which stimulates both JNK and p38. The
medium was then removed by aspiration and the cells were lysed
10. Bonny, C.; Oberson, A.; Negri, S.; Sauser, C.; Schorderet, D. F. Diabetes 2001, 50,
77–82.
11. Dickens, M.; Roger, J. S.; Cavanagh, J.; Raitano, A.; Xia, Z.; Halpern, J. R.;
Greenberg, M. E.; Sawyers, C. L.; Davis, R. J. Science 1997, 277, 693–696.
12. Heo, Y.-S.; Kim, S.-K.; Seo, C. I.; Kim, Y.-K.; Sung, B.-J.; Lee, H. S.; Lee, J. I.; Park,
S.-Y.; Kim, J. H.; Hwang, K. Y.; Hyun, Y.-L.; Jeon, Y. H.; Ro, S.; Cho, J. M.; Lee, T.
G.; Yang, C.-H. EMBO J 2004, 23, 2185–2195.
13. Kaneto, H.; Nakatani, Y.; Miyatsuka, T.; Kawamori, D.; Matsuoka, T.; Matsuhisa,
M.; Kajimoto, Y.; Ichijo, H.; Yamasaki, Y.; Hori, M. Nat. Med. 2004, 10, 1128–
1132.
14. Shin, Y.; Chen, W.; Habel, J.; Duckett, D.; Ling, Y. Y.; Koenig, M.; He, Y.;
Vojkosvsky, T.; LoGrasso, P.; Kamenecka, T. M. Bioorg. Med. Chem. Lett. 2009, 19,
3344–3347. and references cited therein.
15. Gaillard, P.; Jeanclaude-Etter, I.; Ardissone, V.; Arkinstall, S.; Cambet, Y.;
Camps, M.; Chabert, C.; Church, D.; Cirillo, R.; Gretener, D.; Halazy, S.; Nichols,
A.; Szyndralewiez, C.; Vitte, P.-A.; Gotteland, J.-P. J. Med. Chem. 2005, 48, 4596–
4607.
by adding 20 lL of lysis buffer (20 mM Tris–HCl pH 7.6, 5 mM
EDTA, 1% NP-40 substitute, 5 mM NaF, 150 mM NaCl, 1:100 prote-
ase and phosphatase inhibitor mix, SIGMA P8340 and P2850,
respectively). The lysis buffer included 2 nM of the terbium labeled
anti-pc-Jun (pSer73) detection antibodies (invitrogen). After allow-
ing the assay to equilibrate for 1 h at room temperature, TR-FRET
emission ratios were determined on a BMG Pherastar fluorescence
plate reader (excitation at 340 nm, emission 520 nm and 490 nm;
100
ls lag time, 200 ls integration time, emission ratio = Em520/
Em 490).
16. Rückle, T.; Biamonte, M.; Grippi-Vallotton, T.; Arkinstall, S.; Cambet, Y.; Camps,
M.; Chabert, C.; Church, D. J.; Halazy, S.; Jiang, X.; Martinou, I.; Nichols, A.;
Sauer, W.; Gotteland, J.-P. J. Med. Chem. 2004, 47, 6921–6934.
17. Bennett, B. L.; Sasaki, D. T.; Murray, B. W.; O’Leary, E. C.; Sakata, S. T.; Xu, W.;
Leisten, J. C.; Motiwala, A.; Pierce, S.; Satoh, Y.; Bhagwat, S. S.; Manning, A. M.;
Anderson, D. W. Proc. Natl. Acad. Sci. 2001, 98, 13681–13686.
18. Vazquez, J.; De, S. K.; Chen, L.-H.; Riel-Mehan, M.; Emdadi, A.; Cellitti, J.;
Stebbins, J. L.; Rega, M. F.; Pellecchia, M. J. Med. Chem. 2008, 51, 3460–3465.
19. Stebbins, J. L.; De, S. K.; Machleidt, T.; Becattini, B.; Vazquez, J.; Kuntzen, C.;
Chen, L.-H.; Cellitti, J. F.; Riel-Mehan, M.; Emdadi, A.; Solinas, G.; Karin, M.;
Pellecchia, M. Proc. Natl. Acad. Sci. 2008, 105, 16809–16813.
20. De, S. K.; Stebbins, J. L.; Chen, L.-H.; Riel-Mehan, M.; Machleidt, T.; Dahl, R.;
Yuan, H.; Emdadi, A.; Barile, E.; Chen, V.; Murphy, R.; Pellecchia, M. J. Med.
Chem. 2009, 52, 1943–1952.
2.8. Isothermal titration calorimetry
Titrations were done using a VP-ITC calorimeter from Microcal
(Northampton, MA). C163S JNK2 and wt-JNK2 were used at 50
in 20 mM sodium phosphate buffer (pH 7.4), 5% DMSO, and 0.01%
triton X-100. Titrants were used at 750 M in the same buffer.
Titrations were carried out at 25 °C. Data were analyzed using Mic-
rocal Origin software provided by the ITC manufacturer (Microcal,
Northampton, MA).
lM
l
21. Zhao, H.; Serby, M. D.; Xin, Z.; Szczepankiewicz, B. G.; Liu, M.; Kosogof, C.; Liu,
B.; Nelson, L. T. J.; Johnson, E. F.; Wang, S.; Pederson, T.; Gum, R. J.; Clampit, J. E.;
Haasch, D. L.; Abad-Zapatero, C.; Fry, E. H.; Rondinone, C.; Trevillyan, J. M.;
Sham, H. L.; Liu, G. J. Med. Chem. 2006, 49, 4455–4458.
22. Chen, T.; Kablauoi, N.; Little, J.; Timofeevski, S.; Tschantz, W. R.; Chen, P.;
Peng, J.; Charlton, M.; Stanton, R.; Bauer, P. Biochem. J. 2009, 420, 283–
294.
2.9. Molecular modeling
Molecular modeling studies were conducted on a Linux work-
station and a 64 3.2-GHz CPUs Linux cluster. Docking studies were
performed using the X-ray coordinates of JNK1 (PDB code
1UKH).21,25 The complexed JIP peptide and ATO mimetic com-
pound SP600125 were extracted from the protein structure and
23. Chu, C.-H.; Hui, X.-P.; Xu, P.-F.; Zhang, Z.-Y.; Li, Z.-C.; Lioa, R.-A. Indian J. Chem.
2002, 41B, 2436–2438.