NanoDSF Screening for Anti-tubulin Agents Uncovers New Structure–Activity Insights

Microtubule targeting agents (MTAs) constitute a vital category of tubulin-binding compounds deployed across anticancer therapies. Despite the array of MTA drugs developed by pharmaceutical entities, the quest for novel efficacious molecules continues unabated. We unveil an innovative in vitro MTA screening methodology employing nano-differential scanning fluorimetry (nanoDSF), presenting distinct advantages over known assays. This novel approach not only assesses compound-tubulin binding but also quantitatively analyzes its impact on tubulin polymerization, facilitating structure-activity relationship discovery. The proposed nanoDSF assay was rigorously validated using the Prestwick Chemical Library, which encompasses 1520 approved compounds, successfully identifying all previously known MTAs. This screening has unearthed potential antitubulin agents among drugs currently utilized for unrelated medical conditions, offering insights into their mechanisms of action in inhibiting cancer cell proliferation and/or inducing cytotoxicity. Finally, we have identified a previously unrecognized structure-activity relationship within the carbendazim and phenothiazine drug clusters, providing valuable insights for the rational optimization of compounds from these families. These discoveries open new opportunities for drug repositioning of the newly identified MTAs and significantly streamline the screening process of large chemical libraries for MTAs with novel chemical scaffolds.