Fig. 6 Cell viability assay using WST-8. After incubation of each cell with the indicated compounds for 48 hours, the cell viability was measured using
WST-8.
2
3
.
.
Yeh, E. S.; Means, A. R. Nat. Cell Biol. 2007, 7, 381.
Brown, N. R.; Noble, M. E. M.; Endicott, J. A.; Johnson, L. N.
Nat. Cell Biol. 1999, 1, 438.
Yaffe, M. B.; Schutkowski, M.; Shen, M.; Zhou, X. Z.;
Stukenberg, P. T.; Rahfeld, J. U.; Xu, J.; Kuang, J.; Kirschner, M.
W.; Fischer, G.; Cantley, L. C.; Lu, K. P. Science 1997, 278, 1957.
whereas ITG1 was the least. (Fig. S2). After incubation of each
cell in the presence of each inhibitor for 48 hours, the cells were
treated with WST-8 at 37 ºC for 2 hours. The cell viability was
calculated from the absorption at 450 nm. As shown in Fig. 6,
VER1, compound 5, and compound 6 all showed slightly
cytotoxic activity against cancer cell lines, PC3 and HCT116. A
possible reason why 6 is less potent than VER1 may be that the
hydrolysis of 6 was rate-limiting for the inactivation reaction.
Compound 5 showed the weakest cytotoxicity because hydrolysis
of methyl ester was probably slower than that of acetoxymethyl
ester. Further, compound 6 showed slight toxicity to not only
cancer cells, PC3 and HCT116, but also human normal cells,
TIG1. Probably, compound 6 represented off-target effect after
hydrolysis even in TIG1 cells. To confirm that these compounds
inhibited intracellular Pin1, we conducted Pin1 knockdown in
PC-3 cells (PC3-siPin1), and examined the effect on the IC50
value. After knockdown of Pin1 in PC-3 cells with siRNA, cell
viability assay was conducted (Fig. S3, 6). The IC50 value of
compound 6 for PC3-siPin1 cells was 83 µM, while that of PC-3
cells treated with a control siRNA (PC3-siCtrl) was 53 µM. This
result indicated that compound 6 did not mainly affected Pin1 to
show its moderate toxicity in cellular condition.
4
.
5. Lu, K. P. Cancer Cell 2003, 4, 175.
6
.
Wulf, G. M.; Ryo, A.; Wulf, G. G.; Lee, S. W.; Niu, T.; Petkova,
V.; Lu, K. P. EMBO J. 2001, 20, 3459.
Ryo, A.; Nakamura, M.; Wulf, G.; Liou, Y. C.; Lu, K. P. Nat. Cell
Biol. 2001, 3, 793.
7
.
8. Dougherty, M. K.; Müller, J.; Ritt, M. A.; Zhou, M.; Zhou, X. Z.;
Copeland, T. D.; Conrads, T. P.; Veenstra, T. D.; Lu, K. P.;
Morrison, D. K. Mol. Cell 2005, 17, 215.
9
.
Ayala, G.; Wang, D.; Wulf, G.; Frolov, A.; Li, R.; Sowadski, J.
Wheeler, T. M.; Lu, K. P.; Bao, L. Cancer Res. 2003, 63, 6244.
1
0. Lu, K. P.; Finn, G.; Lee, T. H.; Nicholson, L. K. Nat. Chem. Biol.
2007, 3, 619.
11. Zhang, Y.; Daum, S.; Wildemann, D.; Zhou, X. Z.; Verdecia, M.
A.; Bowman, M. E.; Lücke, C.; Hunter, T.; Lu, K. P.; Fischer, G.;
Noel, J. P. ACS Chem. Biol. 2007, 2, 320.
1
2. Hennig, L.; Christner, C.; Kipping, M.; Schelbert, B.; Rücknagel,
K. P.; Grabley, S.; Küllertz, G.; Fischer, G. Biochemistry 1998,
37, 5953.
13. Tatara, Y.; Lin, Y. C.; Bamba, Y.; Mori, T.; Uchida, T. Biochem.
Biophys. Res. Commun. 2009, 384, 394.
1
4. Guo, C.; Hou, X.; Dong, L.; Dagostino, E.; Greasley, S.; Ferre, R.;
Marakovits, J.; Johnson, M. C.; Matthews, D.; Mroczkowski, B.;
Parge, H.; Vanarsdale, T.; Popoff, I.; Piraino, J.; Margosiak, S.;
Thomson, J.; Los, G.; Murray, B. W. Bioorg. Med. Chem. Lett.
In conclusion, we designed and synthesized a new covalent
(irreversible) Pin1 inhibitor, (S)-2. Its acetoxymethyl ester, 6,
suppressed cyclin D1 expression in PC-3 cells and exhibited
moderate cytotoxicity. Probably, the cellular main target of
compound 6 was not Pin1, and identification of the target is
currently in progress.
2
009, 19, 5613.
1
1
5. Dong, L.; Marakovits, J.; Hou, X.; Guo, C.; Greasley, S.;
Dagostino, E.; Ferre, R.; Johnson, M.; Kraynov, E.; Thomson, J.;
Pathak, V.; Murray, B. W. Bioorg. Med. Chem. Lett. 2010, 20,
2
210.
6. Potter, A. J.; Ray, S.; Gueritz, L.; Nunns, C.; Bryant, C. J.; Scrace,
S. F.; Matassova, N.; Baker, L.; Dokurno, P.; Robinson, D. A.;
Surgenor, A. E.; Davis, B.; Murray, J. B.; Richardson, C. M.;
Moore, J. D. Bioorg. Med. Chem. Lett. 2010, 20, 586.
Acknowledgments
This work was supported in part by JSPS KAKENHI Grant
Number 16H05103 (H. N.). The mass fingerprinting analysis of
Pin1 proteins with ESIMS was performed at the Institute of Drug
Discovery Science, Nagoya City University.
17. Potter, A.; Oldfield, V.; Nunns, C.; Fromont, C.; Ray, S.;
Northfield, C. J.; Bryant, C. J.; Scrace, S. F.; Robinson, D.;
Matossova, N.; Baker, L.; Dokurno, P.; Surgenor, A. E.; Davis,
B.; Richardson, C. M.; Murray, J. B.; Moore, J. D. Bioorg. Med.
Chem. Lett. 2010, 20, 6483.
References and notes
18. Moore, J. D.; Potter, A. Bioorg. Med. Chem. Lett. 2013, 23, 4283.
1
9. Wei, S.; Kozono, S.; Kats, L.; Nechama, M.; Li, W.; Guarnerio, J.;
Luo, M.; You, M. H.; Yao, Y.; Kondo, A.; Hu, H.; Bozkurt, G.;
1
.
Lu, K. P.; Zhou, X. Z. Nat. Rev. Mol. Cell Biol. 2007, 8, 904.