Lead Generation for Human Mitotic Kinesin Eg5 Using Structure-based Virtual Screening and Validation by In-vitro and Cell-based Assays

Abstract

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: We have screened for potential inhibitors for human Eg5 (kinesin-5) through structure-based virtual screening and validated the top-scoring compounds using steady-state ATPase assay, differential scanning fluorimetry and microscale thermophoresis. The anticancer activity of the compounds was evaluated in the epithelial (A549) and chronic myelogenous leukaemia (K562) cancer cell lines. A known strong binding inhibitor S-trityl-L-cystine is used as a reference compound. Results & Conclusion: 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.