Smart molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen

Science

Volumn 286, Issue 5441, 1999, Pages 971-974

  Mayer, Thomas U ^a   Kapoor, Tarun M ^a   Haggarty, Stephen J ^a,b   King, Randall W ^a   Schreiber, Stuart L ^a,b   Mitchison, Timothy J ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

^b HARVARD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTINEOPLASTIC AGENT; MONASTROL; NUCLEOLIN; TUBULIN; UNCLASSIFIED DRUG; ACTIN; EG5 PROTEIN, XENOPUS; KINESIN; MOLECULAR MOTOR; PHOSPHOPROTEIN; PYRIMIDINE DERIVATIVE; RNA BINDING PROTEIN; THIOKETONE; XENOPUS PROTEIN;

ANIMAL CELL; ARTICLE; CELL MEMBRANE PERMEABILITY; DRUG MECHANISM; IMMUNOBLOTTING; MITOSIS INHIBITION; MITOSIS SPINDLE; NONHUMAN; PHENOTYPE; PRIORITY JOURNAL; ANIMAL; CATTLE; CELL CULTURE; CELL LINE; CYTOSKELETON; DRUG EFFECT; GOLGI COMPLEX; METABOLISM; MICROTUBULE; MITOSIS; PROTEIN PROCESSING; XENOPUS;

ACTINS; ANIMALS; CATTLE; CELL LINE; CYTOSKELETON; GOLGI APPARATUS; KINESIN; MICROTUBULES; MITOSIS; MITOTIC SPINDLE APPARATUS; MOLECULAR MOTOR PROTEINS; PHENOTYPE; PHOSPHOPROTEINS; PROTEIN PROCESSING, POST-TRANSLATIONAL; PYRIMIDINES; RNA-BINDING PROTEINS; THIONES; TUMOR CELLS, CULTURED; XENOPUS; XENOPUS PROTEINS;

EID: 0033615357     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5441.971     Document Type: Article

References (30)

1. E. Hamel, Med. Res. Rev. 16, 207 (1996).
2. B. R. Stockwell, S. J. Haggarty, S. L. Schreiber, Chem. Biol. 6, 71 (1999).
3. H. J. Anderson et al., Exp. Cell. Res. 238, 498 (1998).
4. Library compounds (Diverset E, Chembridge Corporation) were tested at a final concentration of ∼45 μM. This library consists of a set of structurally diverse small molecules that vary in functional groups and charge. A549 (lung carcinoma) cells were treated with compound for 18 hours, then stained with a monoclonal antibody, TG-3, that recognizes a phosphorylated form of nucleolin (2). Cells in mitosis show a dramatic increase in TG-3 staining (3). We quantified TG3 binding by coupling secondary antibodies to an enzymatic luminescent reporter. We used the percentage of cells in mitosis after treatment with 0.1% dimethyl sulfoxide (DMSO) (control) to normalize the data.
5. Purification of bovine tubulin and labeling of tubulin with fluorescent dyes were performed as described [A. Desai et al., Cell 96, 69 (1999)]. We determined the critical concentration for microtubule polymerization [T. Mitchison and M, Kirschner, Nature 312, 237 (1984)] in the presence of each compound at ∼50 μM.
6. Purification of bovine tubulin and labeling of tubulin with fluorescent dyes were performed as described [A. Desai et al., Cell 96, 69 (1999)]. We determined the critical concentration for microtubule polymerization [T. Mitchison and M, Kirschner, Nature 312, 237 (1984)] in the presence of each compound at ∼50 μM.
7. Supplementary material is available at www. sciencemag.org/feature/data/1043253.shl
8. S. J. Haggarty, unpublished data.
9. Images of live BS-C-1 cells were taken as described [L. Cramer, T. Mitchison, J. Theriot, Curr. Opin. Cell Biol. 6, 82 (1994)]. For immunofluorescence, we stained BS-C-I cells with the Golgi-specific antibody (anti-Golgi 58K protein antibody) (Sigma) or with DM1 A, an antibody to α-tubulin (Sigma). We used tetramethyl rhodamine isothiocyanate-conjugated phalloidin (Sigma) to visualize actin. Lysosomes were stained with LysoTracker (Molecular Probes). Images were obtained with a Nikon E-800 microscope or an Olympus IX70 inverted-microscope.
10. T. U. Mayer, data not shown
11. Monastrol used in all experiments after the screen was synthesized according to published protocols [K. Lewandowski et al., J. Comb. Chem. 1, 105 (1999)] and characterized by nudear magnetic resonance imaging and mass spectroscopy.
12. C E. Walczak, I. Vernos, T. J. Mitchison, E. Karsenti, R. Heald, Curr. Biol. 8, 903 (1998).
13. R. D. Vale and R. J. Fletterick, Annu. Rev. Cell Dev. Biol. 13, 745 (1997).
14. A. P. Enos and N. R. Morris, Cell 60, 1019 (1990); I. Hagan and M. Yanagida, Nature 356, 74 (1992); M. A. Hoyt et al., J. Cell Biol. 118, 109 (1992).
15. A. P. Enos and N. R. Morris, Cell 60, 1019 (1990); I. Hagan and M. Yanagida, Nature 356, 74 (1992); M. A. Hoyt et al., J. Cell Biol. 118, 109 (1992).
16. A. P. Enos and N. R. Morris, Cell 60, 1019 (1990); I. Hagan and M. Yanagida, Nature 356, 74 (1992); M. A. Hoyt et al., J. Cell Biol. 118, 109 (1992).
17. A. Blangy et al., Cell 83, 1159 (1995).
18. K. E. Sawin et al., Nature 359, 540 (1992).
19. T. M. Kapoor and T. J. Mitchison, Proc. Natl. Acad. Sci. U.S.A. 96, 9106 (1999).
20. The plasmids encoding Xenopus Eg5 motor domain (residues 1 to 591) and human conventional kinesin (residues 1 to 560) have been described (16) [G. Woehlke et al., Cell 90, 207 (1997)]. Purification of the expressed motor proteins and in vitro motility experiments were carried out as described (76). Monastrol and DHP2 were dissolved in DMSO (final concentration 2.5%). All velocity data represent results from two or more independent experiments and the mean velocity is shown with the SE. Dose response data are best fit to hyperbola.
21. BS-C-1 cells treated with 200 μM monastrol underwent cell lysis after a few hours. This is probably due to nonspecific cytotoxic effects.
22. N. Hirokawa, Science 279, 519 (1998).
23. C. L. Rieder et al., J. Cell Biol 103, 581 (1986).
24. T. J. Mitchison, Chem. Biol. 1, 3 (1994).
25. S. L. Schreiber, Bioorg. Med. Chem. 6, 1127 (1998).
26. W. M. Saxton, Methods Cell Biol. 44, 279 (1994).
27. R. Sakowicz et al., Science 280, 292 (1998).
28. M. A. Jordan and L. Wilson, Methods Enzymol. 298, 252 (1998).
29. C. E. Walczak et al., Curr. Biol. 8, 903 (1998).
30. The Institute of Chemistry and Cell Biology is supported by NIH (CA78048) and Merck & Co. This work was also supported by grants from the Human Frontier Science Program and National Institute of General Medical Sciences (39565). We thank T. Rapoport for the p58 antibody and the lysotracker dye, P. Davies for the TG3 antibody, and members of the Mitchison and Schreiber laboratory and R. Ward for comments on the manuscript. T.U.M. is a fellow of the Deutsche Forschungsgemeinschaft. T.M.K. is a Runyon-Winchell Fellow.