Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity
Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity
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DOI：
10.1021/acschembio.6b01040
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发表时间：
2017-04-01
影响因子：
4
通讯作者：
Hancock, William O.
中科院分区：
文献类型：
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作者：
Chen, Geng-Yuan;Kang, You Jung;Hancock, William O.
为了揭示它们的对比机制，在驱动蛋白-1 和 Eg5 马达的混合马达滑动试验中分析了抑制 Eg5（驱动蛋白-5）的抗有丝分裂药物，其中 Eg5“制动”主导运动。 Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 rigor state.生化和单分子测定表明，BRD9876 可作为 ATP 和 ADP 竞争性抑制剂，K-I 为 4 nM。与其微管聚合酶活性一致，Eg5 可以稳定微管，防止解聚。这种稳定活性在 monastrol 中被消除，但被 BRD9876 增强。最后，在中期停滞的 RPE-1 细胞中，STLC 促进纺锤体塌陷，而 BRD9876 则不然。因此，不同的 Eg5 抑制剂通过不同的机制影响纺锤体的组装和结构，而严格抑制剂可能矛盾地具有稳定细胞中微管阵列的能力。
To uncover their contrasting Mechanising, antimitotic drugs that inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays of kinesin-1 and Eg5 motors in which Eg5 "braking" dominates motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 rigor state. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4 nM K-I. Consistent with its microtubule polymerase activity, Eg5 was shown to stabilize microtubules against depolymerization. This stabilization activity was eliminated in monastrol but was enhanced by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors impact spindle assembly and architecture through contrasting mechanisms, and rigor inhibitors may paradoxically have the capacity to stabilize microtubule arrays in cells.