1580
M. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1578–1580
4. Sawin, K. E.; Mitchison, T. J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4289.
Compound 3-fixed beads (3d)
5. Goodson, H. V.; Kang, S. J.; Endow, S. A. J. Cell Sci. 1994, 107, 1875.
6. Walczak, C. E.; Mitchison, T. J. Cell 1996, 85, 943.
7. Tao, W.; South, V. J.; Zhang, Y.; Davide, J. P.; Farrell, L.; Kohl, N. E.; Sepp-
Lorenzino, L.; Lobell, R. B. Cancer Cell 2005, 8, 49.
8. Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R. W.; Schreiber, S. L.;
Mitchison, T. J. Science 1999, 286, 971.
9. Haque, S. A.; Hasaka, T. P.; Brooks, A. D.; Lobanov, P. V.; Baas, P. W. Cell Motil.
Cytoskeleton 2004, 58, 10.
Compound 4
Veh.
Compound 5
10 30 100
Veh.
-
10
10
-
10
10
30
100
(µmol/L)
10. Matsuno, K.; Sawada, J.; Asai, A. Expert Opin. Ther. Patents 2008, 18, 253.
11. Ogo, N.; Oishi, S.; Matsuno, K.; Sawada, J.; Fujii, N.; Asai, A. Bioorg. Med. Chem.
Lett. 2007, 17, 3921.
KSP
12. DeBonis, S.; Skoufias, D. A.; Lebeau, L.; Lopez, R.; Robin, G.; Margolis, R. L.;
Wade, R. H.; Kozielsk, F. Mol. Cancer Ther. 2004, 3, 1079.
Figure 5. Competitive inhibition of the binding between 3d and KSP by various KSP
13. Synthesis of 3b: To a solution of 3a (493 mg, 1.00 mmol) in 10 mL of CH2Cl2 was
added carbodiimidazole (211 mg, 1.30 mmol). After the mixture was stirred for
5 min at room temperature, tert-butyl 2-[2-(2-aminoethoxy)ethoxy]ethyl-
carbamate (496 mg, 2.00 mmol) was added. After the mixture was stirred
overnight at room temperature, the mixture was partitioned between CH2Cl2
and H2O, and the organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated. The residue was purified by silica gel
chromatography (AcOEt/n-hexane = 2/3, v/v) to afford 3b (461 mg, 64%) as
amorphous material. 1H NMR (CDCl3) d: 1.41–1.44 (18H, m), 2.50–2.54 (1H, m),
2.69–2.73 (1H, m), 3.28–3.31 (2H, m), 3.40–3.55 (10H, m), 3.79 (3H, s), 3.88–
3.91 (1H, m), 4.90 (1H, br s), 5.09 (1H, br s), 6.48 (1H, br, s), 6.81 (2H, d,
J = 8.8 Hz), 7.19–7.41 (12H, m); MS (ESI, +) m/z 746 [M+Na]+. Synthesis of 3c
inhibitors.
thylenastron (DMEN; KSP ATPase IC50 = 1.1
derivatives 4 (KSP ATPase IC50 = 0.70 mol/L) and 5 (KSP ATPase
IC50 = 0.12 mol/L). These biaryl derivatives showed similar ATP-
ase activity to STLC derivative 1 in vitro.18 However, as shown in
Figure 5, the KSP band was attenuated by 10 mol/L STLC deriva-
tive 1 or DMEN, while no decrease in the band was observed when
10 mol/L biaryl derivative 4 or 5 was added. These results demon-
lmol/L), and biaryl
l
l
l
(KSP ATPase IC50 = 0.29 lM): To a solution of 3b (183 mg, 0.252 mmol) in
l
1.0 mL of AcOEt was added a solution of 1 N HCl in AcOEt (1.3 mL, 1.30 mmol).
After the mixture was stirred for 7 h at room temperature, the mixture was
concentrated. The residue was purified by a reversed phase HPLC to afford 3c
(73 mg, 56%) as amorphous material. 1H NMR (DMSO-d6) d: 2.38–2.42 (2H, m),
2.92–2.96 (2H, m), 3.24–3.28 (2H, m), 3.46–3.57 (8H, m), 3.75 (3H, s), 3.75–
3.77 (1H, m), 6.91 (2H, d, J = 8.8 Hz), 7.17 (2H, d, J = 8.8 Hz), 7.27–7.38 (10H, m),
7.83 (2H, br s), 8.28 (2H, br s), 8.58 (1H, br s); MS (ESI, +) m/z 546 [M+Na]+.
14. Preparation of drug-immobilized beads (3d): NHS-activated Sepharose 4 Fast
strated that the binding of KSP and affinity matrix 3d was effec-
tively inhibited by compound 1 or DMEN, but not by compound
4 or 5. The addition of a higher amount of 4 or 5 (>30 lmol/L) re-
sulted in a decrease in the KSP band. These results imply that biaryl
derivatives have a different mode of binding from STLC derivatives
for abrogation of ATPase activity of KSP. We have already reported
that the biaryl derivatives inhibit KSP ATPase in a microtubule
dependently, and that STLC derivatives inhibited KSP ATPase in a
microtubule independently.18 These observations might be due
to the difference in the KSP binding sites between both chemo-
types of inhibitors. Furthermore, it was suggested that the binding
site of other biaryl derivatives was not the loop 5 allosteric site,19
as distinct from most other KSP inhibitors, including STLC.12,20 Fur-
ther investigations on the detailed mode of action by biaryl deriv-
atives are currently underway.
In conclusion, we synthesized an STLC derivative immobilized
on affinity beads 3d and firstly demonstrated the direct binding
of STLC derivatives to KSP in cancer cells by using 3d. Application
of 3d including, pull-down and competition experiments with a
variety of KSP inhibitors could be successfully performed. Detailed
investigations with 3d will provide further insight into the mode of
action of STLC derivatives and other chemotypes of KSP inhibitors.
Flow (16–23
H2O (5.0 mL), and suspended in H2O (500
(100 mmol/L, 140 L) was added, and the suspension was brought to pH 8 with
lmol/mL, 600
l
L) was washed with 1 mmol/L HCl (7.50 mL) and
l
L). A solution of 3c in DMSO
l
Et3N and gently shaken 24 h at room temperature. The resin was filtered,
washed with H2O (1 mL, three times), 1 mmol/L ethanolamine (2 mL),
50 mmol/L Tris buffer (1 mL, pH 8), and 70 mmol/L AcOH (1 mL, pH 4). Tris
buffer and AcOH washings were repeated three times and the resin was
washed with ultrapure water (2 mL, three times), and stored in ultrapure
water/ethanol (4/1, v/v) until use. For the control beads,
a
solution of
ethanolamine in DMSO (100 mmol/L, 140
lL) was used instead of the
solution of 3c in DMSO.
15. HCT116 cells were trypsinized and collected by centrifugation and washed
with ice-cold PBS. After removed supernatant, the pellet was resuspended in
binding buffer (25 mmol/L Tris-OAc, pH 7.5, 5% glycerol, 20 mmol/L KCl,
5 mmol/L b-mercaptoethanol, 0.02% NP-40, 2 mmol/L MgCl2) and lysed by
three cycles of freezing and thawing.
16. Drug-immobilized or control ethanolamine-immobilized beads were washed
with binding buffer [25 mmol/L Tris-OAc (pH 7.5), 5% glycerol, 20 mmol/L KCl,
5 mmol/L b-mercaptoethanol, 0.02% NP-40, 2 mmol/L MgCl2]. Resuspended in
binding buffer including DMSO or DMSO solution of STLC derivatives and
incubated with 50 mg/mL of BSA and the 8 mg/mL cytoplasmic cell extract
with continuous rotation for two hours at 4 °C. After centrifugation,
supernatant were removed and the beads were washed three times with
binding buffer. KSP bound on the beads or supernatant was analyzed by
Western blotting with anti-KSP antibodies.
Acknowledgment
17. Skoufias, D. A.; DeBonis, S.; Saoudi, Y.; Lebeau, L.; Crevel, I.; Cross, R.; Wade, R.
H.; Hackney, D.; Kozielsk, F. J. Biol. Chem. 2006, 281, 17559.
18. Matsuno, K.; Sawada, J.; Sugimoto, M.; Ogo, N.; Asai, A. Bioorg. Med. Chem. Lett.
2009, 19, 1058.
This work was supported by the Drug Discovery Program of the
Pharma Valley Center.
19. Luo, L.; Parrish, C. A.; Nevins, N.; McNulty, D. E.; Chaudhari, A. M.; Carson, J. D.;
Sudakin, V.; Shaw, A. N.; Lehr, R.; Zhao, H.; Sweitzer, S.; Lad, L.; Wood, K. W.;
Sakowicz, R.; Annan, R. S.; Huang, P. S.; Jackson, J. R.; Dhanak, D.; Copeland, R.
A.; Auger, K. R. Nat. Chem. Biol. 2007, 11, 722.
References and notes
1. Blangy, A.; Lane, H. A.; d’Hérin, P.; Harper, M.; Kress, M.; Nigg, E. A. Cell 1995,
83, 1159.
2. Dagenbach, E. M.; Endow, S. A. J. Cell Sci. 2004, 117, 3.
3. Enos, A. P.; Morris, N. R. Cell 1990, 60, 1019.
20. Sarli, V.; Giannis, A. ChemMedChem 2006, 1, 293.