H. Fiaux et al. / Bioorg. Med. Chem. 16 (2008) 7337–7346
7345
5.8. Evaluation of inhibitory activity on dGMII
References and notes
1. Dennis, J. W.; Granovski, M.; Warren, C. E. BioEssays 1999, 21, 412.
2. Helenius, A.; Aebi, M. Ann. Rev. Biochem. 2004, 73, 1019.
3. Jacob, G. S. Curr. Opin. Struct. Biol. 1995, 5, 605.
4. Bernacki, R. J.; Niedbala, M. J.; Korytnyk, W. Cancer Meta Rev. 1985, 4, 81.
5. Dube, D. H.; Bertozzi, C. R. Nat. Rev. Drug Discov. 2005, 4, 477.
6. Hakomori, S. Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 10231.
7. Dennis, J. W.; Granovsky, M.; Warren, C. E. Biochim. Biophys. Acta 1999, 1473,
21.
For the determination of Ki values for the inhibition of dGMII, ali-
quots of dGMII were diluted to 5 g/ml in 10 mM sodium phosphate,
pH 6.8, 100 mM NaCl, and 10 g/ml bovine serum albumin. Inhibitor
dissolved in DMSO was added and incubated with the enzyme for
30 min at 20 °C. The reaction was initiated by the addition of para-
l
l
nitrophenyl
a-manno-pyranoside (Fluka) dissolved in DMSO, and
8. Scanlin, T. F.; Glick, M. C. Glycoconjugate J. 2000, 17, 617.
9. Liu, F. T.; Rabinovich, G. A. Nat. Rev. Cancer 2005, 5, 29.
10. Fuster, M. M.; Esko, J. D. Nat. Rev. Cancer 2005, 5, 526.
11. Gerber-Lemaire, S.; Juillerat-Jeanneret, L. Mini Rev. Med. Chem. 2006, 6, 1043.
12. Winchester, B. Glycobiology 2005, 15, 1R.
13. Ito, Y.; Hagihara, S.; Matsuo, I.; Totani, K. Curr. Opin. Struct. Biol. 2005, 15,
481.
14. Hanessian, S.; Zhan, L.; Bovey, R.; Saavedra, O. M.; Juillerat-Jeanneret, L. J. Med.
Chem. 2003, 46, 3600.
the incubation was done at 20 °C. The final concentration of DMSO
in all cases was 10%. Aliquots were removed at various time points,
put into a microtitre plate, and an equal volume of 0.5 M sodium car-
bonate was added to stop the reaction. Absorbance at 405 nm was
measuredina microplatereader (MolecularDevices). Thelinear por-
tion of the reaction time course was used to determine inhibition
constants. The anemona.xlt Excel spreadsheet58 was used for deter-
mination of Ki values using nonlinear regression analysis.
15. Gerber-Lemaire, S.; Popowycz, F.; Rodriguez-Garcia, E.; Carmona Asenjo, A. T.;
Robina, I.; Vogel, P. ChemBioChem 2002, 5, 466.
16. Popowycz, F.; Gerber-Lemaire, S.; Demange, R.; Rodriguez-Garcia, E.; Carmona
5.9. Evaluation of cellular
inhibition
a-mannosidase activity and
Asenjo, A. T.; Robina, I.; Vogel, P. Bioorg. Med. Chem. Lett. 2001, 11, 2489.
´
17. Popowycz, F.; Gerber-Lemaire, S.; Rodriguez-Garcıa, E.; Schütz, C.; Vogel, P.
Helv. Chim. Acta 2003, 86, 1914.
18. Popowycz, F.; Gerber-Lemaire, S.; Schütz, C.; Vogel, P. Helv. Chim. Acta 2004, 87,
800.
19. Fiaux, H.; Popowycz, F.; Favre, S.; Schütz, C.; Vogel, P.; Gerber-Lemaire, S.;
Juillerat-Jeanneret, L. J. Med. Chem. 2005, 48, 4237.
20. Fiaux, H.; Schütz, C.; Vogel, P.; Gerber-Lemaire, S.; Juillerat-Jeanneret, L. Chimia
2006, 60, 185.
21. Favre, S.; Fiaux, H.; Schütz, C.; Vogel, P.; Juillerat-Jeanneret, L.; Gerber-Lemaire,
S. Heterocycles 2006, 69, 179.
22. Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem. Rev. 2002, 102, 515.
23. Olden, K.; Breton, P.; Grzegorzewski, K.; Yasuda, Y.; Gause, B. L.; Oredipe, O. A.;
Newton, S. A.; White, S. L. Pharmacol. Ther. 1991, 50, 285.
24. Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J. Tetrahedron Asymetr. 2000,
11, 1645.
Human cell layers were extracted in phosphate buffers contain-
ing 0.1% Triton X-100 (Fluka) pH 5.0, and incubated at room tem-
perature for 10–30 min in the absence or presence of the synthetic
inhibitors at the concentration indicated. The activity of
nosidase(s) was determined by the addition of the fluorigenic sub-
strate 4-methyl-umbelliferyl- -manno-pyranoside (Fluka, final
concentration 150 M) by measuring the fluorescence increase
a-man-
a-D
l
for 30 min in a fluorescence multiwell plate reader (Cytofluor,
PerSeptive BioSystems) thermostated at 37 °C, kex = 360 nm;
kem = 460 nm, essentially as previously described.19 Swainsonine
(Fluka) was used as a reference inhibitor.
25. Asano, N. Glycobiology 2003, 13, 93R.
26. Colegate, S. M.; Dorling, P. R.; Huxtable, C. R. Austr. J. Chem. 1979, 32, 2257.
27. Yeo, T. K.; Yeo, K. T.; Parent, J. B.; Olden, K. J. Biol. Chem. 1985, 260, 2565.
28. Przybylo, M.; Litynska, A.; Poche, E. Biochimie 2005, 87, 133.
29. Humphries, M. J.; Matsumoto, K.; White, S. L.; Olden, K. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 1752.
5.10. Calculations of results
30. Kino, T.; Inamura, N.; Nakahara, H.; Kiyoto, K.; Goto, T.; Terano, H.; Kohsaka,
M.; Aoki, H.; Imanaka, H. J. Antibiot. 1995, 38, 936.
31. Goss, P. E.; Reid, C. L.; Bailey, D.; Dennis, J. W. Clin. Cancer Res. 1997, 3,
1077.
Each experiment was repeated in triplicate wells at least three
times. Means and standard deviation were calculated.
32. Dwek, R. A.; Butters, T. D.; Platt, F. M.; Zitzmann, N. Nat. Rev. Drug Discov. 2002,
1, 65.
Acknowledgments
33. Dwek, M. V.; Brooks, S. A. Curr. Cancer Drug Target 2004, 4, 425.
34. Hembre, E. J.; Pearson, W. H. Tetrahedron 1997, 53, 11021.
35. Pearson, W. H.; Hembre, E. J. J. Org. Chem. 1996, 61, 5537.
36. Pearson, W. H.; Guo, L. Tetrahedron Lett. 2001, 42, 8267.
37. Pearson, W. H.; Hembre, E. J. Tetrahedron Lett. 2001, 42, 8273.
38. Englebienne, P.; Fiaux, H.; Kuntz, D. A.; Corbeil, C. R.; Gerber-Lemaire, S.; Kuntz,
D. A.; Rose, D. R.; Moitessier, N. Proteins 2007, 69, 160.
39. Ikota, N.; Nakagawa, H.; Ohno, S.; Noguchi, K.; Okuyama, K. Tetrahedron 1998,
54, 8985.
40. Kawatkar, S. P.; Kuntz, D. A.; Woods, R. J.; Rose, D. R.; Boons, G. J. J. Am. Chem.
Soc. 2006, 128, 8310.
41. Juillerat-Jeanneret, L.; Célérier, J.; Chapuis Bernasconi, C.; Nguyen, G.; Wostl,
W.; Maerki, H. P.; Janzer, R. C.; Corvol, P.; Gasc, J. M. Br. J. Cancer 2004, 90,
1059.
We thank Ms. Catherine Chapuis Bernasconi, Solange Gros, Syl-
viane Trepey, Mr. Martial Rey, Alain Razaname, and Francisco Sep-
ulveda for excellent technical help and the CHESS synchrotron
facility for beam time. This work is based upon research conducted
at the Cornell High Energy Synchrotron Source (CHESS), which is
supported by the National Science Foundation under award
DMR-0225180, using the Macromolecular Diffraction at CHESS
(MacCHESS) facility, which is supported by award RR-01646 from
the National Institutes of Health, through its National Center for
Research Resources. We also thank the Swiss National Foundation
(Grant No. 200020-100028/ 1), the Swiss League against cancer
(Grant No. KLS-01308-02-2003), and the Canadian Institutes of
Health Research (Grants 42452 and 79312) for financial support.
42. Vasella, A.; Davies, G. J.; Böhm, M. Curr. Opin. Chem. Biol. 2002, 6, 619.
43. Daniel, P. F.; Winchester, B.; Warren, C. D. Glycobiology 1994, 4, 551.
44. Wielgat, P.; Walczuk, U.; Szajda, S.; Bien, M.; Zimmoch, L.; Mariak, Z.; Zwier, K.
J. Neurooncology 2006, 80, 243.
45. Yue, W.; Jin, Y. L.; Shi, G. X.; Liu, Y.; Gao, Y.; Zhao, Y.; Zhu, L. Int. J. Cancer 2004,
108, 189.
Supplementary data
46. Yanagida, K.; Natsuka, S.; Hase, S. Glycobiology 2006, 16, 294.
47. Rabouille, C.; Kuntz, D. A.; Lockyer, A.; Watson, R.; Signorelli, T.; Rose, D. R.; van
den Heuvel, M.; Roberts, D. B. J. Cell Sci. 1999, 112, 3319.
48. van den Elsen, J. M. H.; Kuntz, D. A.; Rose, D. R. EMBO J. 2001, 20, 3008.
49. Kuntz, D. A.; Ghavami, A.; Johnston, B. D.; Pinto, B. M.; Rose, D. R. Tetrahedron
Asymmetr. 2005, 16, 25.
50. Numao, S.; Kuntz, D. A.; Withers, S. G.; Rose, D. R. J. Biol. Chem. 2003, 278,
48074.
51. Rose, D. R.; Kuntz, D. A.; van den Elsen, J. M. H. US Pat. Appl. Publ. US. 2002,
172670
52. Collaborative Computational Project, Number 4. Acta Cryst. 1994, D50,
760.
53. Brunger, A. T.; Adams, P. D.; Clore, G. M.; Delano, W. L.; Gros, P.; Grosse-
Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.;
Rice, L. M.; Simonson, T.; Warren, G. L. Acta Cryst. 1998, D54, 905.
Supplementary Table S1: Statistics for data collection and
refinement of dGMII in complex with 11 or 12, Table S2: Inter-
atomic distances in the crystal structure, and Table S3: Inhibition
of cell growth of human glioblastoma (LN18 and LNZ308) and
endothelial (HCEC) cells by functionalized pyrrolidinones. Supple-
mental Figure S1: Interaction of 2 with residues in the active site of
dGMII. Experimental synthesis procedures and characterization of
the compounds 4, 5, 6, 7, 9, 10, 11, 12, 13, and 14. Supplementary
data associated with this article can be found, in the online version,