Makala Himesh, Alexandar Soundarya Priya, Nagarajan Devipriya, Mahapatra Santanu Kar, Ulaganathan Venkatasubramanian
Department of Biotechnology, School of Chemical and Biotechnology, SASTRA University, Thanjavur - 613 401, Tamilnadu,India.
Curr Comput Aided Drug Des. 2021;17(6):759-772. doi: 10.2174/1573409916666200722141218.
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.
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.
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.
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.
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.
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。在癌症化疗中，靶向有丝分裂的纺锤体分离阶段在药学上备受关注。纺锤体分离主要由 Eg5 驱动蛋白完成，目前，它有许多处于不同临床试验阶段的抑制剂。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 要么是竞争性的，要么是非竞争性的。
使用计算机筛选来鉴定靶向有丝分裂驱动蛋白 Eg5 的合适先导化合物，并通过体外和基于细胞的试验对其进行验证。
通过基于结构的虚拟筛选，针对人类 Eg5（驱动蛋白 -5）筛选潜在抑制剂，并使用稳态 ATP 酶测定、差示扫描荧光法和微量热泳法对得分最高的化合物进行验证。在肺癌上皮细胞系（A549）和慢性粒细胞白血病细胞系（K562）中评估这些化合物的抗癌活性。使用一种已知的强结合抑制剂 S - 三苯甲基 -L - 胱氨酸作为参考化合物。
在测试的众多化合物中，MM01 和 MM03 对癌细胞系 A549 和 K562 显示出良好的基于细胞的活性，可在动物模型中进一步研究。
在本研究中，采用基于结构的方法来鉴定潜在抑制剂，并使用不同的体外和基于细胞的试验对其进行验证。
