A coumarin-30-based method for identification of tubulin ligands with diverse binding sites

Tubulins are among the most successful targets for cancer chemotherapy. However, the emergence of drug resistance stimulates the continuous search for novel chemotherapeutics. Here, we discover that coumarin-30, a widely available laser dye, binds to the colchicine site of tubulin and inhibits microtubule dynamics and cancer cell division at submicromolar concentrations. By combining coumarin-30 as a fluorescent probe with the microscale thermophoresis approach, we develop a versatile assay for detecting tubulin–ligand interactions and simultaneously sorting ligands into binders of the colchicine site versus other protein pockets. The assay’s performance is demonstrated on a wide panel of compounds. Using this methodology, we identify several potent tubulin polymerization inhibitors and determine their binding sites. The results are verified with studies of microtubule dynamics in vitro and the cell cycle in cancer cell culture. Thus, the coumarin-30-based assay is a fast, accurate, and cost-effective method for characterizing tubulin ligands with diverse binding pockets.