Coarse grained modelling highlights the binding differences in the two different allosteric sites of the Human Kinesin EG5 and its implications in inhibitor design.

Kinesins involved in mitotic cell division have gained prominence as promising chemotherapy targets. One such kinesin, EG5, a motor protein responsible for cell division, is a validated chemotherapy target with several compounds at various stages of clinical trials. EG5 has an active site and two different allosteric sites that are known to have ligand specificity. Upon ligand binding, EG5's motor domain will no longer undergo nucleotide-dependent conformational changes required to complete the catalytic cycle. However, there is a lack of in-depth knowledge on the mechanism of inhibitor binding to the two different allosteric sites. To understand the EG5's inhibition mechanism and interactions at allosteric sites and other functionally important regions, we generated two coarse-grained models, Gaussian Network Model (GNM) and Anisotropic Network Model (ANM), to identify the dynamics and its correlation to EG5's function. The first three slowest modes of GNM showed marked differences between the various models of EG5. In the first mode, when the inhibitor is bound at allosteric site 1, there is a presence of a hinge region around residue 166, which is not found when the inhibitor is bound at allosteric site 2 or allosteric sites 1 and 2. The third slowest mode showed a distinctive positively correlated region when the inhibitor is bound at allosteric site 2. These differences indicated that the mechanism of binding at allosteric site 1 and allosteric site 2 are unique. Further, it was observed that the simultaneous ligand binding at allosteric sites 1 and 2 shares structural dynamics and interactions that were found while ligand binds at allosteric sites 1 and 2 independently, leading to a new mechanism. Taken together, our observations suggest that there are different mechanisms at play in each inhibitor bound system considered.

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

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.