Hiroshima Research Center for Healthy Aging, Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan; Laboratory of Molecular and Chemical Cell Biology, Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan.
Laboratory of Molecular and Chemical Cell Biology, Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan.
Fungal Genet Biol. 2018 Jul;116:33-41. doi: 10.1016/j.fgb.2018.04.006. Epub 2018 Apr 21.
Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest.
许多人类癌细胞含有两个以上的中心体，但这些癌细胞可以通过一种称为中心体聚集的机制形成假二极纺锤体，并存活下来，而不是进行致命的多极有丝分裂。驱动蛋白-14/HSET 是一种指向微管负端的运动蛋白，在中心体聚集过程中发挥着关键作用。因此，HSET 被认为是一种有前途的化疗靶点，可以选择性地杀死癌细胞。最近，已经报道了三种 HSET 抑制剂（AZ82、CW069 和 SR31527），但其特异性和功效尚未得到严格评估。这在一定程度上源于缺乏用于评估这些药物的强大系统。酵母和丝状真菌不仅为基础和应用生物学提供了强大的模型，而且还为药物发现和评估提供了多功能工具。在这里，我们表明这三种抑制剂本身对裂殖酵母具有细胞毒性，这表明它们在体内除了驱动蛋白-14 之外还有其他靶点。然而，有趣的是，AZ82 可以中和其他毒性过表达的 HSET；这包括 HSET 驱动的异常有丝分裂细胞的百分比显著降低，并部分抑制其致死性。SR31527 也显示出适度的中和活性，而我们在 CW069 中未检测到这种活性。作为实验性原理验证研究，我们用从各种植物物种中制备的提取物处理 HSET 过表达的裂殖酵母细胞，发现来自 Chamaecyparis pisifera 和 Toxicodendron trichocarpum 的提取物具有挽救 HSET 驱动的致死性的活性。因此，这种在裂殖酵母中过度表达蛋白质的方法提供了一种方便的功能测定系统，不仅可以筛选选择性的人类驱动蛋白-14 抑制剂，还可以筛选其他感兴趣的分子的抑制剂。
