Discovery of a novel microtubule destabilizing agent targeting the colchicine site based on molecular docking

Abstract

Although a number of microtubule-targeting agents have been used in tumour therapy, their resistance and drug toxicity pose clinical challenges. Microtubule destabilizing agents (MDAs) targeting the colchicine site were found to have the advantage of being able to overcome drug resistance. In our previous studies, we identified a novel MDA from the compound database based on virtual screening methods. Its chemical formula is C₂₃H₁₉N₃O₅S, abbreviated as C10. In this study, molecular docking methods confirmed that the binding pattern of C10 to tubulin is similar to that of colchicine. Immunofluorescence staining and tubulin polymerization experiments showed that C10 disrupts the microtubule network and reduces the polymerization efficiency of tubulin. Cell proliferation and toxicity assay showed that C10 could effectively inhibit the growth of tumour cells. After 72 h treatment, the semi-inhibitory concentrations of A549, MCF-7 and HepG2 were 18.83 μM, 16.32 μM and 16.92 μM. Colony formation assay and EdU staining also showed that C10 significantly inhibited the proliferative capacity of tumour cells. Meanwhile, it was found by wound healing and transwell assay that the migration and invasive ability of tumour cells were relatively weakened after treatment with C10. Furthermore, flow cytometry analysis and western blot revealed that C10 reduced the expression of the B-cell lymphoma 2 (BCL-2) and upregulated the level of the Bcl-2-associated X protein (BAX), which in turn activated caspase-3 to promote apoptosis. Finally, the vivo studies in animal showed that C10 significantly inhibited the growth of tumour in nude mice without significant drug toxicity. Thus, C10 may be a colchicine binding site inhibitor with anticancer potential.

Abbreviations: BAX, bcl-2-associated X protein; BCL-2, the B-cell lymphoma 2; BSA, bovine albumin; CBSIs, colchicine binding site inhibitors; CCK-8, cell counting kits-8; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EdU, 5-ethynyl-2′-deoxyuridine; FITC, fluorescein Isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H-E, hematoxylin-eosin; HRP, horseradish Peroxidase; IHC, immunohistochemistry; MDAs, microtubule destabilizing agents; MSAs, microtubule stabilizing agents; MTAs, microtubule-targeting agents; PBS, phosphate buffered saline; PI, propidium Iodide; PVDF, polyvinylidene fluoride.