BACKGROUND Human mitotic kinesins play a crucial role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention pharmaceutically in cancer chemotherapy. Spindle segregation is carried out mainly by Eg5 kinesin, and currently, it has many inhibitors in different phases of clinical trials. All the current drug candidates bind un-competitively with ATP/ADP at allosteric site 1 (formed by loop L5, helix α2 and helix α3). Recent experiments show that inhibitors that bind to the site 2 (formed by helix α4 and helix α6) are either competitive or uncompetitive to ATP/ADP. OBJECTIVES To identify suitable lead compounds that target the mitotic kinesin Eg5, using in silico screening and their validation using in vitro and cell-based assays. METHODOLOGY Potential inhibitors were screened for human Eg5 (kinesin-5) through structurebased virtual screening and the top-scoring compounds were validated using steady-state ATPase assay, differential scanning fluorimetry, and microscale thermophoresis. The anti-cancer activity of the compounds was evaluated in the epithelial (A549) and chronic myelogenous leukemia (K562) cancer cell lines. A known strong binding inhibitor, S-trityl-L-cystine, is used as a reference compound. RESULTS Out of the many compounds tested, MM01 and MM03 showed good cell-based activity against the cancer cell lines A549 and K562 and can be further studied in animal models. CONCLUSION In this study, a structure-based approach was used to identify the potential inhibitors and validate them using different in-vitro and cell-based assays.

中文翻译：

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使用基于结构的虚拟筛选和通过体外和基于细胞的测定进行验证的人类有丝分裂驱动蛋白 Eg5 的先导生成。

背景人类有丝分裂驱动蛋白在有丝分裂细胞分裂中起关键作用。靶向有丝分裂的纺锤体分离阶段在癌症化学疗法中已引起药物学的广泛关注。纺锤体分离主要由Eg5驱动蛋白进行，目前在临床试验的不同阶段有多种抑制剂。所有当前的候选药物在变构位点 1（由环 L5、螺旋 α2 和螺旋 α3 形成）与 ATP/ADP 非竞争性结合。最近的实验表明，与位点 2（由螺旋 α4 和螺旋 α6 形成）结合的抑制剂对 ATP/ADP 具有竞争性或非竞争性。目的 使用计算机筛选并使用体外和基于细胞的测定法对其进行验证，以确定靶向有丝分裂驱动蛋白 Eg5 的合适的先导化合物。方法学 通过基于结构的虚拟筛选对人 Eg5 (kinesin-5) 的潜在抑制剂进行筛选，并使用稳态 ATP 酶测定法、差示扫描荧光法和微尺度热泳法验证得分最高的化合物。在上皮 (A549) 和慢性髓性白血病 (K562) 癌细胞系中评估了化合物的抗癌活性。一种已知的强结合抑制剂 S-三苯甲基-L-胱氨酸被用作参考化合物。结果 在许多测试的化合物中，MM01 和 MM03 对癌细胞系 A549 和 K562 表现出良好的细胞活性，可以在动物模型中进一步研究。结论 在本研究中，使用基于结构的方法来识别潜在的抑制剂并使用不同的体外和基于细胞的测定法对其进行验证。