DeBonis Salvatore, Skoufias Dimitrios A, Lebeau Luc, Lopez Roman, Robin Gautier, Margolis Robert L, Wade Richard H, Kozielski Frank
Institut de Biologie Structurale, 41, rue Jules Horowitz, 38027 Grenoble Cedex 01, France.
Mol Cancer Ther. 2004 Sep;3(9):1079-90.
Human Eg5, a member of the kinesin superfamily, plays a key role in mitosis, as it is required for the formation of a bipolar spindle. We describe here the first in vitro microtubule-activated ATPase-based assay for the identification of small-molecule inhibitors of Eg5. We screened preselected libraries obtained from the National Cancer Institute and identified S-trityl-L-cysteine as the most effective Eg5 inhibitor with an IC50 of 1.0 micromol/L for the inhibition of basal ATPase activity and 140 nmol/L for the microtubule-activated ATPase activity. Subsequent cell-based assays revealed that S-trityl-L-cysteine induced mitotic arrest in HeLa cells (IC50, 700 nmol/L) with characteristic monoastral spindles. S-trityl-L-cysteine is 36 times more potent for inducing mitotic arrest than the well-studied inhibitor, monastrol. Gossypol, flexeril, and two phenothiazine analogues were also identified as Eg5 inhibitors, and we found that they all result in monoastral spindles in HeLa cells. It is notable that all the Eg5 inhibitors identified here have been shown previously to inhibit tumor cell line growth in the NCI 60 tumor cell line screen, and we conclude that their antitumor activity may at least in part be explained by their ability to inhibit Eg5 activity.
人驱动蛋白5（Human Eg5）是驱动蛋白超家族的成员之一，在有丝分裂中起关键作用，因为它是形成双极纺锤体所必需的。我们在此描述了首个基于体外微管激活ATP酶的检测方法，用于鉴定Eg5的小分子抑制剂。我们筛选了从美国国立癌症研究所获得的预选文库，确定S-三苯甲基-L-半胱氨酸是最有效的Eg5抑制剂，其抑制基础ATP酶活性的IC50为1.0微摩尔/升，抑制微管激活ATP酶活性的IC50为140纳摩尔/升。随后的细胞实验表明，S-三苯甲基-L-半胱氨酸可诱导HeLa细胞发生有丝分裂阻滞（IC50，700纳摩尔/升），形成特征性的单星体纺锤体。S-三苯甲基-L-半胱氨酸诱导有丝分裂阻滞的效力比经过充分研究的抑制剂莫那斯特罗高36倍。棉酚、氯唑沙宗和两种吩噻嗪类似物也被鉴定为Eg5抑制剂，我们发现它们都会导致HeLa细胞形成单星体纺锤体。值得注意的是，这里鉴定出的所有Eg5抑制剂在之前的美国国立癌症研究所60种肿瘤细胞系筛选中均已显示出抑制肿瘤细胞系生长的作用，我们得出结论，它们的抗肿瘤活性至少部分可以通过其抑制Eg5活性的能力来解释。
