E. S. Koltun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3727–3731
3731
4. Bonte, D.; Lindvall, C.; Liu, H.; Dykema, K.; Furge, K.; Weinreich, M. Neoplasia
2008, 10, 920.
600
500
400
300
200
100
0
Vehicle:1, 10 ml/kg, PO bid
3 mg/kg, PO qd
5. (a) Menichincheri, M.; Albanese, C.; Alli, C.; Ballinari, D.; Bargiotti, A.; Caldarelli,
M.; Ciavolella, A.; Cirla, A.; Colombo, M.; Colotta, F.; Croci, V.; D’Alessio, R.;
D’Anello, M.; Ermoli, A.; Fiorentini, F.; Forte, B.; Galvani, A.; Giordano, P.;
Isacchi, A.; Martina, K.; Molinari, A.; Moll, J. K.; Montagnoli, A.; Orsini, P.; Orzi,
F.; Pesenti, E.; Pillan, A.; Roletto, F.; Scolaro, A.; Tatò, M.; Tibolla, M.; Valsasina,
B.; Varasi, M.; Vianello, P.; Volpi, D.; Santocanale, C.; Vanotti, E. J. Med. Chem.
2010, 53, 7296; (b) Menichincheri, M.; Bargiotti, A.; Berthelsen, J.; Bertrand, J.
A.; Bossi, R.; Ciavolella, A.; Cirla, A.; Cristiani, C.; Croci, V.; D’Alessio, R.; Fasolini,
M.; Fiorentini, F.; Forte, B.; Isacchi, A.; Martina, K.; Molinari, A.; Montagnoli, A.;
Orsini, P.; Orzi, F.; Pesenti, E.; Pezzetta, D.; Pillan, A.; Poggesi, I.; Roletto, F.;
Scolaro, A.; Tato, M.; Tibolla, M.; Valsasina, B.; Varasi, M.; Volpi, D.;
Santocanale, C.; Vanotti, E. J. Med. Chem. 2009, 52, 293; (c) Zhao, C.; Tovar, C.;
Yin, X.; Xu, Q.; Todorov, I. T.; Vassilev, L. T.; Chen, L. Bioorg. Med. Chem. Lett.
2009, 19, 319; (d) Vanotti, E.; Amici, R.; Bargiotti, A.; Barthelsen, J.; Bosotti, R.;
Ciavolella, A.; Cirla, A.; Cristiani, C.; D’Alessio, R.; Forte, B.; Isacchi, A.; Martina,
K.; Molinari, A.; Menichincheri, M.; Montagnoli, A.; Orsini, P.; Pillan, A.; Roletto,
F.; Scolaro, A.; Tibolla, M.; Valsasina, B.; Varasi, M.; Volpi, D.; Santocanale, C. J.
Med. Chem. 2008, 51, 486; (e) Shafer, C. M.; Lindvall, M.; Bellamacina, C.;
Gesner, T. G.; Yabannavar, A.; Jia, W.; Lin, S.; Walter, A. Bioorg. Med. Chem. Lett.
2008, 18, 4482.
6. Tsuhako, A.L.; Brown, D.S.; Koltun, E.S.; Aay, A.; Arcalas, A.; Chan, V.; Du, H.;
Engst, S.; Franzini, F.; Galan, A.; Huang, P.; Johnston, S.; Kane, B.; Kim, M.H.;
Stott, G.; Stout, T.J.; Yu, P.;Zaharia, Z.A.; Zhang, W.; Zhou, P.; Nuss, J.M.;
7. The CDC7 homology model was built using MOE 2009 (Chemical Computing
Group, Inc.).
8. (a) Niefind, K.; Guerra, B.; Ermakowa, I.; Issinger, O. EMBO J. 2001, 20, 5320; (b)
Niefind, K.; Guerra, B.; Pinna, L.; Issinger, O.; Schomburg, D. EMBO J 1998, 17,
2451. The X-ray crystal structure coordinates of 3 in CK2 have been deposited
in the Protein Data Bank (PDB code 4ANM).
10 mg/kg, PO qd
30 mg/kg, PO qd
100 mg/kg, PO qd
30 mg/kg, PO bid
100 mg/kg, PO q2d
0
5
10
15
20
25
Days Post Implantation
Regression BW loss
Dose
skips
TGI (%)
(%)
(%)
-0.5
3 mg/kg qd
10 mg/kg qd
30 mg/kg qd
100 mg/kg qd
30 mg/kg bid
100 mg/kg q2d
8.7 (ns)
36 (ns)
83
1
1
5
7
6
5
-3.4
-8.3
-4.7
-6.4
-4.5
>100
>100
>100
70
32
21
9. Detailed experimentals for this manuscript can be found in the following
patent: Brown, S. D.; Du, H.; Franzini, M.; Galan, A. A.; Huang, P.; Kearney, P.C.;
Kim, M. H.; Koltun, E. S.; Richards, S. J.; Tsuhako, A.L.; Zaharia, C.A. WO
2009086264 A1 2009.
Figure 5. Colo-205 nude mouse xenograft study with 14 (XL413).
10. Biochemical assay: N-terminally Myc-tagged human CDC7 (amino acids: E2–
L574) and N-terminally His-tagged human ASK (N2–F674) were co-expressed
the cell cycle, a cell phenotype indicative of CDC7 inhibition. In
addition, XL413 is relatively CYP clean (CYP3A4, 2C9, 2D6, 2C19
in E. coli and purified using nickel affinity chromatography. Human CK2
a
isoform A (R8–R333) and full-length b subunits were expressed separately as
N-terminally MBP-tagged proteins in E. coli and purified using amylose
Sepharose chromatography. The purified subunits were reconstituted to form
IC50 >50
l
M, CYP1A2 IC50 = 6.9
l
M) among the major isoforms
M). The attrac-
tested; and was inactive against hERG (IC50 > 30
l
the tetrameric
a2b2 CK2 holoenzyme. Human PIM1 (E32–D292) was
tive profile of XL413 resulted in its selection for preclinical devel-
opment and subsequent advancement into Phase 1 clinical trials.
expressed as N-terminally His-tagged proteins in E. coli and purified using
nickel affinity chromatography. Protein concentration was determined by the
Bradford assay and identification was confirmed by trypsin digestion and mass
spectrometry. Kinase activity and compound inhibition were determined using
the luciferase-luciferin-coupled chemiluminescence assay and measured as
the percentage of ATP utilized following the kinase reaction in a 384-well
format as described previously The final CDC7 kinase assay condition was 6 nM
CDC7/ASK, 1 lM ATP, 50 mM Hepes pH 7.4, 10 mM MgCl2, 0.02% BSA, 0.02%
brij 35, 0.02% tween 20 and 1 mM DTT. It is worthy to note that the CDC7/ASK
protein exhibited substrate-independent ATP utilization. The final PIM1 kinase
Acknowledgments
The authors greatly appreciate contributions from Jason Chew,
Leanne Goon, Stuart Johnson, Eun Ok Kim, Iris Ngan, Yongchang
Shi, Scott Detmer, Richard Venture, Rui Lin, Nicole Miller, Tim Heu-
er, Douglas Den Otter and the departments of Exelixis Genome Bio-
chemistry, New Lead Discovery, Cell facility, and Compound
Repository.
assay condition was 2.4 nM PIM1, 0.5
(AKRRRLSA), 20 mM Hepes pH 7.4, 10 mM MgCl2, 0.03% Triton, and 1 mM DTT.
The final CK2 kinase assay condition was 4 nM CK2 holoenzyme, 2 M ATP,
M casein, 20 mM Hepes pH 7.5 10 mM MgCl2, 0.03% Triton, 1 mM DTT and
lM ATP, 10 lM peptide substrate
l
2
l
0.1 mM NaVO3. All kinase reactions were incubated at room temperature for
1–2 h. Munagala, N. Nguyen S., Lam W., Lee J., Joly A., McMillan K. and Zhang
W. Assay Drug Dev Technol., 2007, 5, 65–73.
References and notes
11. Cell
assays:
Endogenous
MCM2
phosphorylation
fixed
cell
1. For general reviews see: (a) Sawa, M.; Masai, H. Drug Des. Dev. Ther. 2008, 2,
255; (b) Montagnoli, A.; Moll, J.; Colotta, F. Clin. Cancer Res. 2010, 16, 4503.
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Fangman, W. L. Mol. Cell Biol. 1986, 276, 1376; (c) Sclafani, R. A. J. Cell Sci. 2000,
13, 2111; (d) Snaith, H. A.; Brown, G. W.; Forburg, S. L. Mol. Cell Biol. 2000, 20,
7922; (e) Takeda, T.; Ogino, K.; Tatebayashi, K.; Ikeda, H.; Akai, Ki.; Masai, H.
Mol. Biol. Cell 2001, 12, 1257; (f) Fung, A. D.; Ou, J.; Bueler, S.; Brown, G. W. Mol.
Cell Biol. 2002, 22, 4477; (g) Matsumoto, S.; Ogino, K.; Noguchi, E.; Russell, P.;
Masai, H. J. Biol. Chem. 2005, 280, 42536; (h) Sommariva, E.; Pellny, T. K.;
Karahan, N.; Kumar, S.; Huberman, J. A.; Dalgaard, J. Z. Mol. Cell Biol. 2005, 25,
2770; (i) Montagnoli, A.; Moll, J.; Colotta, F. Clin. Cancer Res. 2010, 16, 4503.
3. (a) Montagnoli, A.; Tenca, P.; Sola, F.; Carpani, D.; Brotherton, D.; Albanese, C.;
Santocanale, C. Cancer Res. 2004, 64, 7110; (b) Kim, J. M.; Kakusho, N.; Yamada,
M.; Kanoh, Y.; Takemoto, N.; Masai, H. Oncogene 2008, 27, 3475.
immunofluorescence assay was conducted with MDA-MB-231T cells (from
ATCC) seeded at 1.0 ꢁ 104 cells/well onto 96-well plates in complete DMEM
containing 10% FBS. Cells were treated serial dilutions of test compound in 3%
DMSO and incubated for 4 h. Cells were then fixed for 20 min treated with
Triton X-100 in PBS for 5 min, followed by overnight incubation with pMCM2
(S53) antibody (#AN3011, custom produced for Exelixis by Anaspec, Inc.).
pMCM2 (S53) was read on a Cellomics Arrayscan after incubation with Alexa
Fluor 546 Goat anti-rabbit IgG (H+L) (Cat#A11010, Invitrogen). IC50 values
were determined based on pMCM2 (S53) intensity with compound treatment
versus pMCM2 (S53) intensity with DMSO treatment alone. The cell
proliferation was measured by BrdU incorporation assay, viability was
assayed by Cell Titer–Glo kits, and the apoptosis assay was measured by
Apo-ONE Homogeneous Caspase-3/7 Assay kit (Promega).