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4. Biological activity
References and notes
1. (a) Welsch, M. E.; Snyder, S. A.; Stockwell, B. R. Curr. Opin. Chem. Biol. 2010, 14,
347–361; (b) Zolova, O. E.; Mady, A. S.; Garneau-Tsodikova, S. Biopolymers
2010, 93, 777–790; (c) Dawson, S.; Malkinson, J. P.; Paumier, D.; Searcet, M. Nat.
Prod. Rep. 2007, 24, 109–126; (d) Abdelfattah, M. S.; Kazufumi, T.; Ishibashi, M.
J. Nat. Prod. 2010, 73, 1999–2002.
2. (a) Greenfield, D. S.; Liebmann, J. M.; Ritch, R. J. Glaucoma 1997, 6, 250–258; (b)
Smith, J. T.; Hamilton-Miller, J. M.; Knox, R. Nature 1964, 203, 1148–1150; (c)
Flore, M. C.; Baker, T. B. N. Engl. J. Med. 2011, 365, 1222–1231.
4.1. Cell death assays
Doxycyline inducible HeLa cell lines (Dox-GFP, Dox-Noxa, Dox-
BadS3A)19,20 were induced with doxycycline (Dox = 1
lg/mL) for
3 h. Cells were then treated as indicated for 12 h. Cells were fixed
and stained with Hoechst dye and the number of condensed nuclei
was counted for each treatment to determine percent cell death.
3. (a) Burke, J. R.; Pattoli, M. A.; Gregor, K. R.; Brassil, P. J.; MacMaster, J. F.;
McIntyre, K. W.; Yang, X.; Iotzova, V. S.; Clarke, W.; Strnad, J.; Qiu, Y.; Zusi, F. C.
J. Biol. Chem. 2003, 278, 1450–1456; (b) Baffert, F.; Régnier, C. H.; De Pover, A.;
Pissot-Soldermann, C.; Tavares, G. A.; Blasco, F.; Brueggen, J.; Chéne, P.;
Drueckes, P.; Erdmann, D.; Furet, P.; Gerspacher, M.; Lang, M.; Ledieu, D.;
Nolan, L.; Ruetz, S.; Trappe, J.; Vangrevelinghe, E.; Wartmann, M.; Wyder, L.;
Hofmann, F.; Radimerski, T. Mol. Cancer Ther. 2010, 9, 1945–1955.
4. Undevia, S. D.; Innocenti, F.; Ramirez, J.; House, L.; Desai, A. A.; Skoog, L. A.;
Singh, D. A.; Karrison, T.; Kindler, H. L.; Ratain, M. J. Eur. J. Biol. Cancer 2008, 44,
1684–1692.
5. (a) Johnston, P. A.; Foster, C. A.; Tierno, M. B.; Shun, T. Y.; Shinde, S. N.; Paquette,
W. D.; Brummond, K. M.; Wipf, P.; Lazo, J. S. Assay Drug Dev. Technol. 2009, 7,
250–265; (b) Johnston, P. A.; Soares, K. M.; Shinde, S. N.; Foster, C. A.; Shun, T.
Y.; Takyi, H. K.; Wipf, P.; Lazo, J. S. Assay Drug Dev. Technol. 2008, 6, 505–518.
6. Simeonov, A.; Yasgar, A.; Jadhav, A.; Lokesh, G. L.; Klumpp, C.; Michael, S.;
Austin, C. P.; Natarajan, A.; Inglese, J. Anal. Biochem. 2008, 375, 60–70.
7. (a) Cavazzuti, A.; Paglietti, G.; Hunter, W. N.; Gamarro, F.; Piras, S.; Loriga, M.;
Allecca, S.; Corona, P.; Mcluskey, K.; Tulloch, L.; Gibellini, F.; Ferrari, S.; Costi, M.
P. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 1448–1453; (b) Chen, Q.; Bryant, V. C.;
Lopez, H.; Kelly, D. L.; Luo, X.; Natarajan, A. Bioorg. Med. Chem. Lett. 2011, 21,
1929–1932; (c) You, L.; Cho, E. J.; Leavitt, J.; Ma, L. C.; Montelione, G. T.; Anslyn,
E. V.; Krug, R. M.; Ellington, A.; Robertus, J. D. Bioorg. Med. Chem. Lett. 2011, 21,
3007–3011; (d) Chen, L. H.; Chang, C. M.; Salunke, D. B.; Sun, C. M. ACS Comb.
Sci. 2011, 13, 391–398.
4.2. Cell growth inhibition assay
Human cervical tumor cells (HeLa) were cultured in RPMI-1640
medium containing 10% FBS and maintained in a 37 °C incubator
with 5% CO2. Cells were plated at 2000 cells/well in 96 well plates
and incubated overnight. The next day, cells were treated as indi-
cated. The treated cells were assayed for viability using the alamar-
Blue assay. Briefly, 10 lL reagent was added to each well and the
plate was returned to the incubator for 3 h after which fluorescence
at 544ex/590em was measured using a SpectraMax M5e (Molecular
Devices) plate reader. The alamarBlue assay was repeated each
day for 5 days. Growth is expressed as raw fluorescent units (RFU).
4.3. Caspase 3/7 activation
HeLa cells (2000 cells/well) were treated in 96 well plates as
indicated for 6 h. Caspase Glo reagent (Promega, Inc.) was added
and luminescence was measured using a SpectraMax M5 (Molecu-
lar Devices) plate reader after 1 h. Raw luminescence values (RLU)
were normalized to alamarBlue (RFU).
8. Danial, N. N.; Korsmeyer, S. J. Cell 2004, 116, 205–219.
9. Roucou, X.; Montessuit, S.; Antonsson, B.; Martinou, J. C. Biochem. J. 2002, 368,
915–921.
10. Korsmeyer, S. J.; Wei, M. C.; Saito, M.; Weiler, S.; Oh, K. J.; Schlesing, P. H. Cell
Death Differ. 2000, 7, 1166–1173.
11. Nuñez, G.; Bendict, M. A.; Hu, Y.; Inohara, N. Oncogene 1998, 17, 3237–3245.
12. Youle, R. J.; Strasser, A. Nat. Rev. Mol. Cell Biol. 2008, 9, 47–59.
13. (a) Awan, F. T.; Kay, N. E.; Davis, M. E.; Wu, W.; Geyer, S. M.; Leung, N.; Jelinek,
D. F.; Tschumper, R. C.; Secreto, C. R.; Lin, T. S.; Grever, M. R.; Shanafel, T. D.;
Zent, C. S.; Call, T. G.; Heerema, N. A.; Lozanski, G.; Byrd, J. C.; Lucas, D. M. Blood
2009, 113, 535–537; (b) Fennell, D. A. Clin. Lung Cancer 2003, 4, 307–313; (c)
Kausch, I.; Jiang, H.; Thode, B.; Doehn, C.; Krüger, S.; Jocham, D. Eur. Urol. 2005,
47, 703–709.
4.4. PARP cleavage
HeLa cells were treated as indicated for 6 h. Cells were har-
vested by collecting media, trypsinizing cells, and centrifuging to
obtain a combined cell pellet from all steps. Cells were lysed in
radio immuno precipitation assay (RIPA) buffer (150 mM NaCl,
1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate, 50 mM Tris, pH 8.0) and protein content was subjected to
SDS–PAGE. PARP cleavage was determined via Western blotting
using anti-PARP antibody (Calbiochem #AM30).
14. Muchmore, S. W.; Sattler, M.; Liang, H.; Meadows, R. P.; Harlan, J. E.; Yoon, S.
H.; Nettesheim, D.; Chang, B. S.; Thompson, C. B.; Wong, S. L.; Ng, S. L.; Fesik, S.
W. Nature 1996, 381, 335–341.
15. (a) Petros, A. M.; Dinges, J.; Augeri, D. J.; Baumeister, S. A.; Betebenner, D. A.;
Bures, M. G.; Elmore, S. W.; Hajduk, P. J.; Joseph, M. K.; Landis, S. K.;
Nettesheim, D. G.; Rosenberg, S. H.; Shen, W.; Thomas, S.; Wang, X.; Zanze, I.;
Zhang, H.; Fesik, S. W. J. Med. Chem. 2006, 49, 656–663; (b) Oltersdorf, T.;
Elmore, S. W.; Shoemaker, A. R.; Armstrong, R. C.; Augeri, D. J.; Belli, B. A.;
Bruncko, M.; Deckwerth, T. L.; Dinges, J.; Hajduk, P. J.; Joesph, M. K.; Kitada, S.;
Korsmeyer, S. J.; Kunzer, A. R.; Letai, A.; Li, C.; Mitten, M. J.; Nettesheim, D. G.;
Ng, S.; Nimmer, P. M.; O’Connor, J. M.; Oleksijew, A.; Petros, A. M.; Reed, J. C.;
Shen, W.; Tahir, S. K.; Thompson, C. B.; Tomaselli, K. J.; Wang, B.; Wendt, M. D.;
Zhang, H.; Fesik, S. W.; Rosenberg, S. H. Nature 2005, 435, 677–681.
16. Van Delft, M. F.; Wei, A. H.; Mason, K. D.; Vandenberg, C. J.; Chen, L.; Czabotar,
P. E.; Willis, S. N.; Scott, C. L.; Day, C. L.; Cory, S.; Adams, J. M.; Roberts, A. W.;
Huang, D. C. Cancer Cell 2006, 10, 389–399.
17. Tahir, S. K.; Yang, X.; Anderson, M. G.; Morgan-Lappe, S. E.; Sarthy, A. V.; Chen,
J.; Warner, R. B.; Ng, S. C.; Fesik, S. W.; Elmore, S. W.; Rosenberg, S. H.; Tse, C.
Cancer Res. 2007, 67, 1176–1183.
18. (a) Zheng, L.; Yang, W.; Zhang, C.; Ding, W. J.; Zhu, H.; Lin, N. M.; Wu, H. H.; He,
Q. J.; Yang, B. Cancer Lett. 2011, 309, 27–36; (b) Tromp, J. M.; Geest, C. R.; Breij,
E. C.; Elias, J. A.; van Laar, J.; Luijks, D. M.; Kater, A. P.; Beaumont, T.; Van Oers,
M. H.; Eldering, E. Clin. Cancer Res. 2011, 18, 487–489.
4.5. Apoptosis protein expression analysis
HeLa cells were treated as indicated for 24 h. Cells were har-
vested by collecting media, trypsinizing cells, and centrifuging to
obtain a combined cell pellet from all steps. Cells were lysed in
RIPA buffer and protein content was subjected to SDS–PAGE.
Mcl-1 and XIAP expression levels were determined via Western
blotting using anti-Mcl-1 antibody (Santa Cruz Biotechnology,
Inc. sc-819) and anti-XIAP antibody (Santa Cruz Biotechnology,
Inc. sc-58537). Anti-a-tubulin antibody (Cell Signaling Technology,
Inc. #3873) was used as a control.
Acknowledgments
19. Zhang, L.; Lopez, H.; George, N. M.; Liu, X.; Pang, X.; Luo, X. Cell Death Differ.
2011, 18, 864–873.
20. Lopez, H.; Zhang, L.; George, N. M.; Liu, X.; Pang, X.; Evans, J. J.; Targy, N. M.;
Luo, X. J. Biol. Chem. 2010, 285, 15016–15026.
21. (a) Azmi, A. S.; Wang, Z.; Philip, P. A.; Mohammad, R. M.; Sarkar, F. H. Expert
Opin. Emerg. Drugs 2011, 16, 59–70; (b) Dai, Y.; Grant, S. Cancer Res. 2007, 67,
2908–2911.
22. Thomas, L. W.; Lam, C.; Edwards, S. W. FEBS Lett. 2010, 584, 2981–2989.
23. (a) Grande, F.; Aiello, F.; De Grazia, O. D.; Brizzi, A.; Garofalo, A.; Neamati, N.
Bioorg. Med. Chem. 2007, 15, 288–294; (b) Gavara, L.; Saugues, E.; Alves, G.;
Debiton, E.; Anizon, F.; Moreau, P. Eur. J. Med. Chem. 2010, 5520–5526.
24. Bryant, V. C.; Kishore Kumar, G. D.; Nyong, A. M.; Natarajan, A. Bioorg. Med.
Chem. Lett. 2012, 22, 245–248.
This project was supported in part by NIH R01CA127239 and
the Eppley Cancer Center pilot Grant. We would like to thank the
Eppley NMR facility, Dr. Srikumar Raja for his help with determin-
ing CI, Smitha Kizhake for carrying out LC–MS analyses and the
Natarajan lab members for helpful discussions.
Supplementary data
25. (a) Murata, T.; Shimada, M.; Sakakibara, S.; Yoshino, T.; Masuda, T.; Shintani, T.;
Sato, H.; Koriyama, Y.; Fukushima, K.; Nunami, N.; Yamauchi, M.; Fuchikami,
Supplementary data associated with this article can be found, in