Coumarin‐30 Enables Site‐Resolved Detection of Tubulin Ligands by Microscale Thermophoresis

Tubulins are among the most successful targets for cancer chemotherapy. However, the emergence of drug resistance stimulates the continuous search for novel chemotherapeutics. We discovered that coumarin-30, a widely available laser dye, binds to the colchicine site of tubulin, inhibiting microtubule dynamics and cancer cell division at submicromolar concentrations. Exploiting the excellent fluorescent properties of coumarin-30, we developed a fast, accurate, and cost-effective coumarin-30-based microscale thermophoresis (C-MST) assay as an express method for detecting tubulin-ligand interactions and discriminating colchicine site binders from ligands targeting other protein pockets. Using this assay, we identified several potent tubulin polymerization inhibitors associating with the colchicine site and validated them through in vitro microtubule dynamics and cell cycle assays in cancer cells. Furthermore, the C-MST assay was demonstrated to detect ligands targeting a novel binding site on tubulin, recently established through crystallographic fragment screening. We confirmed detection of a small-molecule ligand targeting that site and further designed and characterized a series of its analogs. The ability of the C-MST assay to detect tubulin binders regardless of their binding site or their effect on microtubule dynamics opens new avenues for developing unconventional modulators of tubulin-tubulin and tubulin-effector interactions thereby facilitating anticancer drug discovery.