Title:Lead Generation for Human Mitotic Kinesin Eg5 Using Structure-based Virtual Screening and Validation by In-vitro and Cell-based Assays
Volume: 17
Issue: 6
Author(s): Himesh Makala, Soundarya Priya Alexandar, Devipriya Nagarajan, Santanu Kar Mahapatra and Venkatasubramanian Ulaganathan*
Affiliation:
- Department of Biotechnology, School of Chemical and Biotechnology, SASTRA University, Thanjavur – 613 401, Tamilnadu,India
Keywords:
Mitotic kinesins, cell division, Eg5, anti-cancer, structure-based drug design, ATP/ADP.
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: 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.
