Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin

被引:90
作者
Gupta, K
Bishop, J
Peck, A
Brown, J
Wilson, L
Panda, D [1 ]
机构
[1] Indian Inst Technol, Sch Biosci & Bioengn, Bombay 400076, Maharashtra, India
[2] Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA
关键词
D O I
10.1021/bi036112v
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The antifungal agent benomyl [methyl-l-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 muM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 muM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC50 of 5 muM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.
引用
收藏
页码:6645 / 6655
页数:11
相关论文
共 62 条
[1]   NEW BROAD SPECTRUM ANTHELMINTIC METHYL 5(6)-BUTYL-2-BENZIMIDAZOLECARBAMATE [J].
ACTOR, P ;
ANDERSON, EL ;
DICUOLLO, CJ ;
FERLAUTO, RJ ;
HOOVER, JRE ;
PAGANO, JF ;
RAVIN, LR ;
SCHEIDY, SF ;
STEDMAN, RJ ;
THEODORI.VJ .
NATURE, 1967, 215 (5098) :321-&
[2]  
BAI R, 1990, J BIOL CHEM, V265, P17141
[3]   Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine [J].
Bai, RL ;
Pei, XF ;
Boye, O ;
Getahun, Z ;
Grover, S ;
Bekisz, J ;
Nguyen, NY ;
Brossi, A ;
Hamel, E .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (21) :12639-12645
[4]   PROMOTION OF FLUORESCENCE UPON BINDING OF COLCHICINE TO TUBULIN [J].
BHATTACH.B ;
WOLFF, J .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1974, 71 (07) :2627-2631
[5]   INTERACTION OF 1-ANILINO-8-NAPHTHALENE SULFONATE WITH TUBULIN - SITE INDEPENDENT OF COLCHICINE-BINDING SITE [J].
BHATTACHARYYA, B ;
WOLFF, J .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1975, 167 (01) :264-269
[6]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[7]  
CLEVELAND DW, 1985, ANNU REV BIOCHEM, V54, P331, DOI 10.1146/annurev.bi.54.070185.001555
[8]   DIFFERENTIAL BINDING OF METHYL BENZIMIDAZOL-2-YL CARBAMATE TO FUNGAL TUBULIN AS A MECHANISM OF RESISTANCE TO THIS ANTIMITOTIC AGENT IN MUTANT STRAINS OF ASPERGILLUS-NIDULANS [J].
DAVIDSE, LC ;
FLACH, W .
JOURNAL OF CELL BIOLOGY, 1977, 72 (01) :174-193
[9]   Microtubule polymerization dynamics [J].
Desai, A ;
Mitchison, TJ .
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, 1997, 13 :83-117
[10]  
Downing K.H., 2000, Emerging Therapeutic Targets, V4, P219, DOI [10.1517/14728222.4.2.219, DOI 10.1517/14728222.4.2.219]