Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5
Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5
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DOI：
10.1021/bi049264e
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发表时间：
2004-10-19
期刊：
影响因子：
2.9
通讯作者：
Kozielski, F
中科院分区：
文献类型：
--
作者：
Brier, S;Lemaire, D;Kozielski, F
人类的卵细胞5是一个超家族的有丝分裂马达，参与有丝分裂纺锤体的形成和维持。最近发现的小分子通过结合HsEg5的催化马达结构域抑制HsEg5导致有丝分裂阻滞，这引起了人们对Eg5作为潜在抗癌药物靶点的更多兴趣。我们已经使用氢-氘交换质谱法和定向诱变来鉴定形成新的Eg5抑制剂结合位点的二级结构元件，特别是s -三酰基- l-半胱氨酸，一种在体外和基于细胞的检测中有效的Eg5活性抑制剂。该抑制剂的结合改变了确定运动域内两个区域的8个肽的氘结合率：Tyr125-Glu145和Ile202-Leu227。用粗神经孢子虫常规酪蛋白重链中的等量区域替换Tyr125-Glu145区域可以防止s -三酰基- l-半胱氨酸对Eg5 atp酶活性的抑制。我们在这里发现，s -三酰基- l-半胱氨酸和monastrol都通过诱导配合结合到Eg5上的同一区域，该区域由螺旋α 3-链β 5和环l5 -螺旋α 2形成的口袋中，并且这两种抑制剂都在相互作用位点引发类似的局部构象变化。s -三辛基- l-半胱氨酸和monastrol可能通过类似的机制抑制HsEg5。常见的抑制剂结合区似乎代表了HsEg5的“热点”，可以用于进一步的抑制剂筛选。
Human Eg5, a mitotic motor of the kinesin superfamily, is involved in the formation and maintenance of the mitotic spindle. The recent discovery of small molecules that inhibit HsEg5 by binding to its catalytic motor domain leading to mitotic arrest has attracted more interest in Eg5 as a potential anticancer drug target. We have used hydrogen-deuterium exchange mass spectrometry and directed mutagenesis to identify the secondary structure elements that form the binding sites of new Eg5 inhibitors, in particular for S-trityl-L-Cysteine, a potent inhibitor of Eg5 activity in vitro and in cell-based assays. The binding of this inhibitor modifies the deuterium incorporation rate of eight peptides that define two areas within the motor domain: Tyr125-Glu145 and Ile202-Leu227. Replacement of the Tyr125-Glu145 region with the equivalent region in the Neurospora crassa conventional kinesin heavy chain prevents the inhibition of the Eg5 ATPase activity by S-trityl-L-Cysteine. We show here that S-trityl-L-Cysteine and monastrol both bind to the same region on Eg5 by induced fit in a pocket formed by helix alpha3-strand beta5 and loop L5-helix alpha2, and both inhibitors trigger similar local conformational changes within the interaction site. It is likely that S-trityl-L-Cysteine and monastrol inhibit HsEg5 by a similar mechanism. The common inhibitor binding region appears to represent a "hot spot" for HsEg5 that could be exploited for further inhibitor screening.