Repurposing the USFDA-approved small molecules for their affinity against the colchicine binding site (CBS) in the tubulin: Corroborating the in-silico findings through biological assessment.

The microtubules are known to possess inimitable characteristics termed 'exquisite dynamic behavior,' which allows cells to maintain their shape and assist in cellular division. This exquisite dynamicity is lost in the case of cancer, and considering their pivotal role, they are considered as central drug targets in cancer chemotherapy. To target them, microtubule-targeting agents (MTAs) are unique small molecules that inhibit one of the seven unique druggable pockets of microtubules, impacting their dynamicity and leading to cell cycle arrest and apoptosis. In the present work, we employ a drug repurposing strategy to identify plausible leads targeting the colchicine binding site (CBS) from a pool of 2787 FDA-approved drugs. CBS is a unique and flexible druggable pocket site in microtubules that has been targeted for both cancer and non-cancer diseases. Additionally, the inhibition of this site is reported to reverse drug resistance and exhibit synergistic anti-inflammatory and anti-angiogenic properties, further enhancing the anticancer effects. The cumulative analysis led to the identification of Tafenoquine, a well-known antimalarial FDA-approved molecule, as a potential tubulin-targeting agent with predicted binding to the colchicine site. The in-silico analysis was further corroborated using in vitro investigation on breast (MCF-7 and MDA-MB-231), lung (A549), and colon (HCT-116) cancer cells. Tafenoquine was found to exert broad-spectrum antiproliferative effects, with greatest potency against MDA-MB-231 cells (IC₅₀ = 4.75 ± 0.18 μM), a model for aggressive and treatment-resistant triple-negative breast cancer (TNBC). Considering the tubulin polymerization assay, the identified hit was found to inhibit microtubule assembly (low Vmax) in a manner similar to colchicine, and exhibited a similar G2/M arrest, indicating a mode of inhibition similar to that of colchicine.