3110
C. Adams et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3105–3110
a 700 component library. The library was designed
based on the docking into the homology model of the
hit 1. Low-nM inhibitors emerged from the library that
had a good correlation of JAK 3 inhibition linked to
signal related functional impact. Finally, the inhibitor 9
demonstrated an effect comparable to dexamethasone in
the ear odema model.
13. Zheng, J.; Knighton, D. R.; Xuong, N.-H.; Taylor, S. S.;
Sowadski, J. M.; Ten Eyck, L. F. Protein Sci. 1993, 2, 1559.
14. Hubbard, S. R. EMBO J. 1997, 16, 5572.
15. Mohammadi, M.; McMahon, G.; Sun, L.; Tang, C.;
Hirth, P.; Yeh, B. K.; Hubbard, S. R.; Schlessinger, J. Science
1997, 276, 955.
16. Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMa-
hon, G.; Tang, C. J. Med. Chem. 1998, 41, 2588.
17. Lawrie, M. A.; Noble, E. M.; Tunnah, P.; Brown, N. R.;
Johnson, L. N.; Endicott, J. A. Nat. Struct. Biol. 1997, 4, 796.
18. Prade, L.; Engh, R. A.; Girod, A.; Kinzel, V.; Huber, R.;
Bossemeyer, D. Structure 1997, 4, 1627.
References and Notes
1. Sridhar, R.; Hanson-Painton, O.; Cooper, Denise, R.
Pharmaceut. Res. 2000, 17, 1345.
2. Fabbro, D.; Parkinson, D.; Matter, A. Curr. Opin. Phar-
macol. 2002, 2, 374.
3. Russell, S. M.; Johnston, J. A.; Noguchi, M.; Kawamura,
M.; Bacon, C. M.; Friedmann, M.; Berg, M.; McVicar, D. W.;
Witthuhn, B. A.; Silvennoinen, O. Science 1994, 266, 1042.
4. Noguchi, M.; Rosenblatt, H. M.; Filipovich, A. H.; Adel-
stein, S.; Modi, W. S.; McBride, O. W.; Leonard, W. J. Cell
1993, 73, 147.
19. Tong, L.; Pav, S.; White, D. M.; Rogers, S.; Crane, K. M.;
Cywin, C. L.; Brown, M. L.; Pargellis, C. A. Nat. Struct. Biol.
1997, 4, 311.
20. Johnson, L. N.; Noble, M. E.; Owen, D. J. Cell 1996, 85, 149.
21. 85% by LC/MS, was set as the lower limit for acceptance
of each library component.
22. Robinson, R. P.; Donahue, K. M.; Son, P. S.; Wagy, S. D.
J. Heterocycl. Chem. 1996, 33, 287.
23. Daisley, R. W.; Hanbali, J. R. Synth. Commun. 1981, 11,
743.
5. Leonard, W. J. Annu. Rev. Med. 1996, 47, 229.
6. Leonard, W. J.; O’Shea, J. J. Annu. Rev. Immunol. 1998, 16,
293.
7. Sugamura, K.; Asao, H.; Kondo, M.; Tanaka, N.; Ishii, N.;
Ohbo, K.; Nakamura, M.; Takeshita, T. Annu. Rev. Immunol.
1996, 14, 179.
8. Sudbeck, E.; Uckun, F. IDrugs 1999, 2, 1026.
9. Cetkovic-Cvrlje, M.; Dragt, A. L.; Vassilev, A.; Liu, X. P.;
Uckun, F. M. Clin. Immunol. 2003, 106, 213.
10. The protein was expressed with an N-terminal FLAG tag
using the in-house expression vector pYOUNG and the yeast
strain Kluveromycas lactis. The recombinant protein was
expressed intracellularly and was purified using M2 affinity
resin.
11. Homogenous time resolved fluorescence (HTRF) assay: A
selective synthetic tyrosine kinase containing substrate (biot-
bA-bA-bA-L-P-L-D-K-D-Y-Y-V-V-R-E-P-G-Q[NH]2 recog-
nized by JAK3 (catalytic domain) is labeled with biotin. The
substrate (1 mM) is phosphorylated by JAK3 (50 ng/assay
point) in the presence of ATP (8 uM), DTT and Mg2+, 10 min
at room temperature. APC-labeled streptavidin and a selected
cryptate-labeled anti-phosphotyrosine are then added into the
well in order to generate the specific signal that is proportional
to the phoshorylated tyrosine concentration. After 1 h of
incubation at room temperature, plates are read and the
%dmax is determined using Packard discovery counter. The
final DMSO concentration in the assay was 8%. The standard
for the assay was oxindole 1.
24. IL-2 induced murine CTLL cell proliferation: Seed CTLL’s
(2ꢁ104 cells/well) in 96-well plate, in the absence of IL-2, for 6
h. Add 10 U/mL recombinant human IL-2 at time zero. Add
JAK 3 inhibitors in DMSO (final concentration 0.5%) at time
zero. Add 1 mCi/well [3H]-thymidine at 18 h of culture. Har-
vest cells after 24 h, on to filter plates, and count scintillations
[3H]-thymidine incorporation into DNA reflects extent of
proliferation.
25. IL-2 induced JAK 3 and STAT5 tyrosine phosphorylation:
Pretreat CTLL cells with JAK 3 inhibitors for 4 h. Stimulate
cells with 10 U/mL IL-2 for 20 min, lyse cells and immuno-
precipitate with anti-mouse JAK 3 and STAT5 antibodies.
Subject immunoprecipitated samples to SDS-PAGE electro-
phoresis. Probe electrophoresized blots with anti-phosphotyro-
sine antibody.
26. Miyazaki, T.; Kawahara, A.; Fujii, H.; Nakagawa, Y.;
Minami, Y.; Liu, Z. J.; Oishi, I.; Silvennoinen, O.; Witthuhn,
B. A.; Ihle, J. N. Science 1994, 266, 1045.
27. Johnston, J. A.; Kamura, M.; Kirken, R. A.; Chen, Y. Q.;
Blake, T. B.; Shibuya, K.; Ortalso, J. R.; McVicar, D. W.;
O’Shea, J. J. Nature 1994, 370, 151.
28. Protocol for oxazolone induced ear odema: On day 1,
female BALB/c mice were sensitized by topical application of
oxazolone (100 mL of 2% solution in acetone) to the shaved
abdomen. On day 7, acetone (negative control group) or oxaz-
olone (2% solution in acetone, remainder of animals) was
applied topically to the right ear (10 mL on each side of the ear).
Test compounds or appropriate vehicles (acetone) were admi-
nistered topically to the right ear (10 mL on each side of the ear)
0.5 h after challenge. Mice were killed 24 h after challenge. A
6-mm disc of challenged (right) ear was removed and weighed.
12. Zheng, J.; Trafny, E. A.; Knighton, D. R.; Xuong, N.-H.;
Taylor, S. S.; Ten Eyck, L. F.; Sowadsky, J. M. Acta Crystal-
logr. 1993, D49, 362.