J. Cao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5812–5818
5817
Kovacs, I.; Park, B.; Druker, B. J.; Deininger, M. W.
Blood 2005, 106, 2128.
The fixed varied concentrations of compounds were
generated by adding the appropriate amount in 2.5 lL
of DMSO; the DMSO present in each assay was constant
at 5%. The reaction was initiated by the addition of ATP
to a final concentration of 0.5 mM. After the reaction was
allowed to proceed for 60 min, 50 lL of Kinase-Glo
reagent (Promega) was added to terminate the reaction.
The solution was then allowed to proceed for an
additional 10 min to maximize the luminescence reaction.
Values were then measured using an Ultra 384 instrument
(Tecan, Charlotte, NC, USA) set for luminosity measure-
ments. A control reaction containing no peptide substrate
was used for a zero point. Enzyme reaction rates were
derived by calculating the difference between kinase
catalyzed and uncatalyzed reactions at a specific com-
9
. Noronha, G.; Barrett, K.; Cao, J.; Dneprovskaia, E.; Fine,
R.; Gong, X.; Gritzen, C.; Hood, J.; Kang, X.; Klebansky,
B.; Lohse, D.; Mak, C. C.; McPherson, A.; Palanki, M. S.
S.; Pathak, V. P.; Renick, J.; Soll, R.; Splittgerber, U.;
Wrasidlo, W.; Zeng, B.; Zhao, N. Bioorg. Med. Chem.
Lett. 2006, 16, 5546.
0. Noronha, G.; Barrett, K.; Boccia, A.; Brodhag, T.; Chow,
J.; Cao, C. P.; Dneprovskaia, E.; Doukas, J.; Fine, R.;
Gong, X.; Gritzen, C.; Gu, H.; Hanna, E.; Hood, J. D.;
Hu, S.; Kang, X.; Key, J.; Klebansky, B.; Kousba, A.; Li,
G.; Lohse, D.; Mak, C. C.; McPherson, A.; Palanki, M. S.
P.; Pathak, V. P.; Renick, J.; Shi, F.; Soll, R.; Splittgerber,
U.; Stoughton, S.; Tang, S.; Yee, S.; Zeng, B.; Zhao, N.;
Zhu, H. Bioorg. Med. Chem. Lett. 2007, 17, 602.
1
i
pound concentration. K values were derived from rate
1
1. Warmuth, M.; Simon, N.; Mitina, O.; Mathes, R.;
Fabbro, D.; Manley, P. W.; Buchdunger, E.; Forster,
K.; Moarefi, I.; Hallek, M. Blood 2003, 101, 664.
data using the non-competitive enzyme kinetics curve
fitting capabilities of Prism (Version 4; GraphPad Soft-
ware, San Diego, CA, USA).
1
2. We have obtained crystal structures of the same TargeGen
inhibitor from a closely related series with both inactive
and active Src family kinases clearly showing the Glu310
H-bonding interaction occurring in the case of the active
Src structure validating this design concept. In the case of
the inactive kinase, the Glu310 on the aC-helix is not close
to the inhibitor, as expected, since the Glu310 moves
toward the hydrophobic pocket only upon activation. This
work has been presented previously as a poster. Pathak, V.
P.; Barrett, K.; Cao, J.; Fine, R. M.; Gritzen, C.; Hood, J.;
Kang, X.; Klebansky, B.; Lohse, D.; Mak, C. C.;
McPherson, A.; Moarefi, I.; Noronha, G.; Renick, J.;
Soll, R.; Splittgerber, U.; Zeng, B.; Zhu, H. Abstracts of
Papers, 232nd ACS National Meeting, San Francisco, CA,
United States, September 10–14, 2006.
19. Cao, J.; Barrett, K.; Fine, R. M.; Gritzen, C.; Hood, J.;
Kang, J.; Lohse, D.; Mak, C. C.; McPherson, A.;
Noronha, G.; Pathak, V. P.; Renick, J.; Soll, R.; Splitt-
gerber, U.; Zeng, B.; Zhu, H. Abstracts of Papers, 231st
ACS National Meeting, Atlanta, GA, United States,
March 26–30, 2006.
20. Mak, C. C.; Barrett, K.; Cao, J.; Gritzen, C.; Hood, J.;
McPherson, A.; Noronha, G.; Pathak, V. P.; Renick, J.;
Soll, R.; Splittgerber, U.; Zeng, B. Abstracts of Papers,
231st ACS National Meeting, Atlanta, GA, USA, March
26–30, 2006.
21. Wolf, F. J.; Pfister, K., III; Wilson, R. M., Jr; Robinson,
C. A. J. Am. Chem. Soc. 1954, 76, 3551.
22. Mason, J. C.; Tennant, G. J. Chem. Soc. 1970, 911.
23. Kudo, N.; Perseghini, M.; Fu, G. C. Angew. Chem. Int.
Ed. 2006, 45, 1282.
1
3. After we had completed our work in the benzotriazines
targeting Src, Abl and Abl-T315I, a paper from Ariad
targeting Src by utilizing the Glu310 in the back of the
hydrophobic pocket by making a donor interaction
published. Dalgarno, D.; Stehle, T.; Narula, S.; Schelling,
P.; Schravendijk, M. R. V.; Adams, S.; Andrade, L.;
Keats, J.; Ram, M.; Jin, L.; Grossman, T.; MacNeil, I.;
Metcalf, C., III; Shakespeare, W.; Wang, Y.; Keenan, T.;
Sundaramoorthi, R.; Bohacek, R.; Weigele, M.; Sawyer,
T. Chem. Biol. Drug Des. 2006, 67, 46.
24. Tundel, R. E.; Anderson, K. W.; Buchwald, S. L. J. Org.
Chem. 2006, 71, 430.
25. Full-length human Bcr and Abl cDNA were purchased
from OriGene (Rockville, MD) and ligated to mimic
Philadelphia chromosome formation. The Bcr-Abl cDNA
was subcloned into a retroviral vector, pFN-Neo (Strate-
gene, San Diego, CA). Point mutations were introduced
into Abl kinase domain by the QuickChange site-directed
mutagenesis kit (Strategene, San Diego, CA). The
recombinant retroviral vectors were transfected into 293
cells (ATCC, Manassas, VA) to produce recombinant
retroviral particles. The retroviral particles were trans-
duced into a mouse pro-B cell line, Ba/F3, (DSMZ,
Germany) and selected for by 1 mg/mL G418 in
RPMI + 10% FBS + 10 ng/mL IL3 (R&D Systems, Min-
neapolis, MN). IL3 was omitted from the growth
medium for the G418 resistant cells. 2 · 106 cells were
seeded in each 10-cm dish. Compounds were added to
1
4. Palanki, M. S. S.; Cao, J.; Chow, C. P.; Dneprovskaia, E.;
Fine, R.; Gritzen, C.; Kang, X.; Klebansky, B.; Mak, C.
C.; McPherson, A.; Noronha, G.; Pathak, V. P.; Renick,
J.; Soll, R.; Zeng, B. Poster #7, 8th Winter Conference of
Medicinal and Bioorganic Chemistry, Steamboat Springs,
Colorado, January 21–25, 2007.
1
5. Nagar, B.; Hantschel, O.; Young, M. A.; Scheffzek, K.;
Veach, D.; Bornmann, W.; Clarkson, B.; Superti-Furga,
G.; Kuriyan, J. Cell 2003, 112, 859.
1
6. Hantschel, O.; Nagar, B.; Guettler, S.; Kretzschmar, J.;
Dorey, K.; Kuriyan, J.; Superti-Furga, G. Cell 2003, 112,
2
the cells following 1 h incubation in CO incubator. Cells
were treated with compound for 4 h before being
harvested by centrifugation (890 RCF, 4 ꢂC, 5 min).
Each cell pellet was lysed with 0.5 mL RIPA buffer
(Boston BioProducts, Worcester, MA). Total protein
concentrations were obtained by BCA method (Pierce,
Rockford, IL). Fifty micrograms of each sample were
resolved by 4–12% gradient SDS–PAGE. Following
transferring proteins from the gels to nitrocellulose
membranes, the membranes were blocked with 5% non-
fat dry milk (VONS, San Diego, CA) in TBST. Primary
antibodies were incubated with the membranes in 1%
non-fat dry milk in TBST followed by incubation with
secondary antibodies in 5% non-fat dry milk in TBST.
Target proteins were visualized by chemoluminescent
reaction (Pierce, Rockford, IL).
8
7. Harrison, S. C. Cell 2003, 112, 737.
45.
1
1
8. K values for compounds were determined against Abl
i
(
Invitrogen) and the T315I mutant form of Abl (Upstate
Cell Signaling Solutions). In white, flat-bottom, 96-well
plates (Nunc) assays were run at room temperature at a
final volume of 50 lL. Each well contained 40 lL of buffer
consisting of 75 mM Tris buffer, pH 7.2 containing 95 mM
2
MgCl , 1.5 mM EGTA, 0.35 mM Triton X-100, 10 lM b-
mercaptoethanol and an appropriate amount of either Abl
or T315I-Abl such that the assay was linear over 60 min.
Varying amounts (1–100 lM) of Abltide substrate
(Upstate Cell Signaling Solutions) in water were added
in the presence of a fixed varied amount of compound.